Published:18 July 2025 The published article titled“Inhibition of Liver Carcinoma Cell Invasion and Metastasis by Knockdown of Cullin7 In Vitro and In Vivo”has been retracted from Oncology Research,Vol.23,No.4,2015,...Published:18 July 2025 The published article titled“Inhibition of Liver Carcinoma Cell Invasion and Metastasis by Knockdown of Cullin7 In Vitro and In Vivo”has been retracted from Oncology Research,Vol.23,No.4,2015,pp.171–181.DOI:10.3727/096504016X14519995067562 URL:https://www.techscience.com/or/v23n4/57554 Following the publication,concerns have been raised about a number of figures in this article.An unexpected area of similarity was identified in terms of the cellular data,where the results from differently performed experiments were intended to have been shown,although the areas immediately surrounding this area featured comparatively different distributions of cells.In addition,the western blots in this article were presented with atypical,unusually shaped and possibly anomalous protein bands in many cases.展开更多
Objective:To investigate the prevalence,mechanisms,and trends of knockdown resistance(kdr)in Anopheles(An.)culicifacies and its impact on the efficacy of organochlorine and other insecticides.Methods:A systematic revi...Objective:To investigate the prevalence,mechanisms,and trends of knockdown resistance(kdr)in Anopheles(An.)culicifacies and its impact on the efficacy of organochlorine and other insecticides.Methods:A systematic review was conducted based on PRISMA guidelines,extracting data from biooan.org,Embase,ProQuest,PubMed,Scopus,and Web of Science without a time limit until the end of 2022.Articles meeting the inclusion criteria were assessed using the STROBE checklist.Data on kdr mutations,insecticide resistance,and effectiveness were analyzed across eight selected studies from various regions.Results:The review revealed widespread kdr-mediated resistance in An.culicifacies,primarily against dichloro-diphenyl-trichloroethane(DDT),persisting even decades after discontinued use.Key kdr mutations,including L1014F and L1014S,were identified.Resistance to deltamethrin was less stable,with increased sensitivity observed after short-term discontinuation.The findings underscore the vector's sustained resistance to organochlorine insecticides and relative sensitivity to pyrethroids.Conclusions:Stable kdr resistance in An.culicifacies to organochlorine insecticides highlights the need for periodic susceptibility assessments and strategic rotation or combination of insecticides to combat malaria effectively and prevent the development of resistance.展开更多
CRISPR-Cas endonucleases mediate prokaryotic adaptive immunity by targeting foreign nucleic acids.CRISPR/Cas13b is a class 2 type VI-B ribonuclease that targets and cleaves single-stranded RNA.It exhibits higher RNA i...CRISPR-Cas endonucleases mediate prokaryotic adaptive immunity by targeting foreign nucleic acids.CRISPR/Cas13b is a class 2 type VI-B ribonuclease that targets and cleaves single-stranded RNA.It exhibits higher RNA interference activity than Cas13a and Cas13c and causes fewer collateral effects than RxCas13d in mammalian cells.However,a programmable CRISPR/Cas13b-mediated RNA interference system for endogenous transcripts in rice has not yet been established.Here,we developed a CRISPR/Cas13b-mediated system to target endogenous transcripts in rice.Our CRISPR/Cas13b system could inhibit multiple endogenous mRNAs simultaneously.In addition,this system efficiently repressed endogenous long noncoding RNAs with more than 50% inhibition in stable transgenic plants.Furthermore,we found only weak collateral effects of the CRISPR/Cas13b-mediated system at the transcriptome-wide level,and no difference in the agronomic traits of stable transgenic rice in the field.We present a programmable CRISPR/Cas13b-mediated knockdown system for rice,offering a potential biotechnological tool for functional genomics and crop improvement.展开更多
BACKGROUND The role of Sm-like 5(LSM5)in colon cancer has not been determined.In this study,we investigated the role of LSM5 in progression of colon cancer and the potential underlying mechanism involved.AIM To determ...BACKGROUND The role of Sm-like 5(LSM5)in colon cancer has not been determined.In this study,we investigated the role of LSM5 in progression of colon cancer and the potential underlying mechanism involved.AIM To determine the role of LSM5 in the progression of colon cancer and the potential underlying mechanism involved.METHODS The Gene Expression Profiling Interactive Analysis database and the Human Protein Atlas website were used for LSM5 expression analysis and prognosis analysis.Real-time quantitative polymerase chain reaction and Western blotting were utilized to detect the expression of mRNAs and proteins.A lentivirus targeting LSM5 was constructed and transfected into colon cancer cells to silence LSM5 expression.Proliferation and apoptosis assays were also conducted to evaluate the growth of the colon cancer cells.Human GeneChip assay and bioinformatics analysis were performed to identify the potential underlying mechanism of LSM5 in colon cancer.RESULTS LSM5 was highly expressed in tumor tissue and colon cancer cells.A high expression level of LSM5 was related to poor prognosis in patients with colon cancer.Knockdown of LSM5 suppressed proliferation and promoted apoptosis in colon cancer cells.Silencing of LSM5 also facilitates the expression of p53,cyclin-dependent kinase inhibitor 1A(CDKN1A)and tumor necrosis factor receptor superfamily 10B(TNFRSF10B).The inhibitory effect of LSM5 knockdown on the growth of colon cancer cells was associated with the upregulation of p53,CDKN1A and TNFRSF10B.CONCLUSION LSM5 knockdown inhibited the proliferation and facilitated the apoptosis of colon cancer cells by upregulating p53,CDKN1A and TNFRSF10B.展开更多
Human dental pulp cells （hDPCs） possess the capacity to differentiate into odontoblast-like cells and generate reparative dentin in response to exogenous stimuli or injury. Ten-eleven translocation 1 （TET1） is a n...Human dental pulp cells （hDPCs） possess the capacity to differentiate into odontoblast-like cells and generate reparative dentin in response to exogenous stimuli or injury. Ten-eleven translocation 1 （TET1） is a novel DNA methyldioxygenase that plays an important role in the promotion of DNA demethylation and transcriptional regulation in several cell lines. However, the role of TET1 in the biological functions of hDPCs is unknown. To investigate the effect of TET1 on the proliferation and odontogenic differentiation potential of hDPCs, a recombinant shRNA lentiviral vector was used to knock down TET1 expression in hDPCs. Following TET1 knockdown, TET1 was significantly downregulated at both the mRNA and protein levels. Proliferation of the hDPCs was suppressed in the TET1 knockdown groups. Alkaline phosphatase activity, the formation of mineralized nodules, and the expression levels of DSPP and DMP1 were all reduced in the TETl-knockdown hDPCs undergoing odontogenic differentiation. Based on these results, we concluded that TET1 knockdown can prevent the proliferation and odontogenic differentiation of hDPCs, which suggests that TET1 may play an important role in dental pulp repair and regeneration.展开更多
The micro RNA（mi RNA） let-7 was one of the first mi RNAs to be discovered, and is highly conserved and widely expressed among species. let-7 expression increases in brain tissue after cerebral ischemia/reperfusion i...The micro RNA（mi RNA） let-7 was one of the first mi RNAs to be discovered, and is highly conserved and widely expressed among species. let-7 expression increases in brain tissue after cerebral ischemia/reperfusion injury; however, no studies have reported let-7 effects on nerve injury after cerebral ischemia/reperfusion injury. To investigate the effects of let-7 gene knockdown on cerebral ischemia/reperfusion injury, we established a rat model of cerebral ischemia/reperfusion injury. Quantitative reverse transcription-polymerase chain reaction demonstrated that 12 hours after cerebral ischemia/reperfusion injury, let-7 expression was up-regulated, peaked at 24 hours, and was still higher than that in control rats after 72 hours. Let-7 gene knockdown in rats suppressed microglial activation and inflammatory factor release, reduced neuronal apoptosis and infarct volume in brain tissue after cerebral ischemia/reperfusion injury. Western blot assays and luciferase assays revealed that mitogen-activated protein kinase phosphatase-1（MKP1） is a direct target of let-7. Let-7 enhanced phosphorylated p38 mitogen-activated protein kinase（MAPK） and c-Jun N-terminal kinase（JNK） expression by down-regulating MKP1. These findings suggest that knockdown of let-7 inhibited the activation of p38 MAPK and JNK signaling pathways by up-regulating MKP1 expression, reduced apoptosis and the inflammatory reaction, and exerted a neuroprotective effect following cerebral ischemia/reperfusion injury.展开更多
Fankl is exclusively expressed in the testis from the meiosis phase to the haploid phase of spermatogenesis. In this study, we examined the function of Fankl by establishing a Fankl-knockdown transgenic mouse model. T...Fankl is exclusively expressed in the testis from the meiosis phase to the haploid phase of spermatogenesis. In this study, we examined the function of Fankl by establishing a Fankl-knockdown transgenic mouse model. The apoptotic statuses of the testes of the transgenic mice were tested using the terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） method. The FANK1 consensus DNA-binding sequence was identified using cyclic amplification of sequence target （CAST） analysis. Differentially expressed genes were examined using microarray analysis. A reduction in sperm number and an increase in apoptotic spermatocytes were observed in Fankl-knockdown mice, and the apoptotic cells were found to be primarily spermatogonia and spermatocytes. The CAST results demonstrated that the consensus DNA-binding sequence was AAAAAG, in which the percentage occurrence of each base at each position ranged from 55 to 86%. This sequence was present in the promoter regions of 10 differentially expressed genes that were examined using microarray analysis. In total, 17 genes were differentially expressed with changes in their expression levels greater than twofold. The abnormal expression of Fankl target genes that were regulated directly or indirectly by Fankl reduced the number of sperm in the knockdown mice. Thus, FANK1 may Dlav a pivotal role in sDermato^enesis as a transcription factor.Fankl is exclusively expressed in the testis from the meiosis phase to the haploid phase of spermatogenesis. In this study, we examined the function of Fankl by establishing a Fankl-knockdown transgenic mouse model. The apoptotic statuses of the testes of the transgenic mice were tested using the terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） method. The FANK1 consensus DNA-binding sequence was identified using cyclic amplification of sequence target （CAST） analysis. Differentially expressed genes were examined using microarray analysis, A reduction in sperm number and an increase in apoptotic spermatocytes were observed in Fankl-knockdown mice, and the apoptotic cells were found to be primarily spermatogonia and spermatocytes. The CAST results demonstrated that the consensus DNA-binding sequence was AAAAAG, in which the percentage occurrence of each base at each position ranged from 55 to 86%. This sequence was present in the promoter regions of 10 differentially expressed genes that were examined using microarray analysis. In total, 17 genes were differentially expressed with changes in their expression levels greater than twofold. The abnormal expression of Fankl target genes that were regulated directly or indirectly by Fankl reduced the number of sperm in the knockdown mice. Thus, FANK1 may play a pivotal role in spermatogenesis as a transcription factor.展开更多
Thin-walled structures are commonly utilized in aerospace and aircraft structures,which are prone to buckling under axial compression and extremely sensitive to geometric imperfections.After decades of efforts,it stil...Thin-walled structures are commonly utilized in aerospace and aircraft structures,which are prone to buckling under axial compression and extremely sensitive to geometric imperfections.After decades of efforts,it still remains a challenging issue to accurately predict the lower-bound buckling load due to the impact of geometric imperfections.Up to now,the lower-bound curve in NASA SP-8007 is still widely used as the design criterion of aerospace thin-walled structures,and this series of knockdown factors(KDF)has been proven to be overly conservative with the significant promotion of the manufacturing process.In recent years,several new numerical and experimental methods for determining KDF have been established,which are systematically reviewed in this paper.The Worst Multiple Perturbation Load Approach(WMPLA)is one of the most representative methods to reduce the conservatism of traditional methods in a rational manner.Based on an extensive collection of test data from 1990 to 2020,a new lower-bound curve is approximated to produce a series of improved KDFs.It is evident that these new KDFs have an overall improvement of 0.1-0.3 compared with NASA SP-8007,and the KDF predicted by the WMPLA is very close to the front of the new curve.This may provide some insight into future design guidelines of axially compressed cylindrical shells,which is promising for the lightweight design of large-diameter aerospace structures.展开更多
Efficacy of 25 essential oils was screened against filarial vector, Culex quinquefasciatus, for their larvicidal and knockdown effects in a preliminary study. Of these, 8 oils viz. calamus oil, cinnamon oil, citronell...Efficacy of 25 essential oils was screened against filarial vector, Culex quinquefasciatus, for their larvicidal and knockdown effects in a preliminary study. Of these, 8 oils viz. calamus oil, cinnamon oil, citronella oil, clove oil, eucalyptus oil, lemon oil, mentha oil and orange oil exhibited 100% larvicidal activity at 1000 ppm and 100% knockdown effect at 10% concentration. These 8 oils were screened further against Cx. Quinquefasciatus, Aedes aegypti and Anopheles stephensi for their larvicial and knockdown effects at different concentrations. Mentha oil was the most promising against An. Stephensi and Ae. Aegypti recording LC50 and LC90 values of 39.74 and 115.67 ppm and 46.23 and 165.36 ppm, respectively for larvicidal activity. Calamus oil was the most effective against Cx. Quinquefasciatus with LC50 and LC90 values of 40.40, and 140.07 ppm, respectively for larvicidal activity. Orange oil showed the most potent knockdown effect with the KT50 and KT95 values of 27.44, 26.22 and 29.91 and 70.81, 65.33 and 68.57 min, against An. stephensi, Cx. Quinquefasciatus and Ae. Aegypti, respectively. The results clearly indicated that mentha oil and calamus oil were the most promising larvicides and orange oil had potent knockdown effect against the tested mosquito species. These oils could be used to develop a new formulation to control mosquitoes.展开更多
Pitx3 is strongly associated with the phenotype, differentiation, and survival of dopaminergic neurons. The relationship between Pitx3 and glial cell line-derived neurotrophic factor（GDNF） in dopaminergic neurons re...Pitx3 is strongly associated with the phenotype, differentiation, and survival of dopaminergic neurons. The relationship between Pitx3 and glial cell line-derived neurotrophic factor（GDNF） in dopaminergic neurons remains poorly understood. The present investigation sought to construct and screen a lentivirus expression plasmid carrying a rat Pitx3 short hairpin（sh）RNA and to assess the impact of Pitx3 gene knockdown on GDNF transcriptional activity in MES23.5 dopaminergic neurons. Three pairs of interference sequences were designed and separately ligated into GV102 expression vectors. These recombinant plasmids were transfected into MES23.5 cells and western blot assays were performed to detect Pitx3 protein expression. Finally, the most effective Pitx3 sh RNA and a dual-luciferase reporter gene plasmid carrying the GDNF promoter region（GDNF-luciferase） were cotransfected into MES23.5 cells. Sequencing showed that the synthesized sequences were identical to the three Pitx3 interference sequences. Inverted fluorescence microscopy revealed that the lentivirus expression plasmids carrying Pitx3-sh RNA had 40-50% transfection efficiency. Western blot assay confirmed that the corresponding Pitx3 of the third knockdown sequence had the lowest expression level. Dual-luciferase reporter gene results showed that the GDNF transcriptional activity in dopaminergic cells cotransfected with both plasmids was decreased compared with those transfected with GDNF-luciferase alone. Together, the results showed that the designed Pitx3-sh RNA interference sequence decreased Pitx3 protein expression, which decreased GDNF transcriptional activity.展开更多
Currently,glucose transporter 4(GLUT4)has been considered as the key player for the insulin-stimulated glucose transport in the muscle and adipose tissues.The development of recombinant DNA techniques allows the creat...Currently,glucose transporter 4(GLUT4)has been considered as the key player for the insulin-stimulated glucose transport in the muscle and adipose tissues.The development of recombinant DNA techniques allows the creations of genetically knockout,knockdown and transgenic animals and cells for the study of GLUT4’s physiological functions.Here,we have used key words to search the PubMed and summarized the methods used in Slc2a4 gene knockout,GLUT4 knockdown and overexpression in the whole body and tissue specific manner.The whole body GLUT4-null mice have growth retardation,but normal glucose tolerance and basal glucose turnover rates.Compared with whole body Slc2a4 knockout mice,adipose and muscle double knockout mice have impaired insulin tolerance and glucose intolerance.The results of GLUT4 knockdown in 3T3-L1 adipocytes have shown that its expression is needed for lipogenesis after,but not during,differentiation.Transgenic mice with the whole body GLUT4 overexpression have normal body weight and lowered blood glucose level.The adipose tissue specific overexpression of GLUT4 leads to increases in mouse body weight and adipose tissue weight.The insulin-stimulated GLUT4 translocation in the skeletal muscle contributes to the regulation of glucose homeostasis.Data from both transgenic overexpression and tissue specific Slc2a4 knockout indicate that GLUT4 probably plays a role in the glucose uptake in the fasting state.More studies are warranted to use advanced molecular biology tools to decipher the roles of GLUT4 in the control of glucose homeostasis.展开更多
This study investigated the specific mechanism of knockdown of neuropeptide Y(NPY) in reducing obesity-induced insulin resistance in the white adipose tissue. Adeno-associated virus(AAV)-mediated RNAi was utilized to ...This study investigated the specific mechanism of knockdown of neuropeptide Y(NPY) in reducing obesity-induced insulin resistance in the white adipose tissue. Adeno-associated virus(AAV)-mediated RNAi was utilized to downregulate NPY expression in rats fed either regular chow or high fat diet. By investigating the differences in rat body weight and food intake, we assessed the effect of knockdown of NPY expression on insulin sensitivity and β-cell proliferation. Glucose consumption and 2-[3 H]DG uptake in 3 T3-L1 adipocytes were assessed to determine the molecular mechanisms. The results showed that knockdown of NPY expression in the dorsomedial hypothalamus(DMH) reduced obesity-induced insulin resistance, increased glucose consumption, and decreased 2-[3 H]DG uptake in 3 T3-L1 adipocytes via the PI3 K/Akt/GSK-3β signaling pathways and the NPY Y5 receptor.展开更多
Microtubule-severing proteins(MTSPs),are a family of proteins which use adenosine triphosphate to sever microtubules.MTSPs have been shown to play an important role in multiple microtubule-involved cellular processes....Microtubule-severing proteins(MTSPs),are a family of proteins which use adenosine triphosphate to sever microtubules.MTSPs have been shown to play an important role in multiple microtubule-involved cellular processes.One member of this family,fidgetin(FIGN),is also involved in male fertility;however,no studies have explored its roles in female fertility.In this study,we found mouse fidgetin is rich within oocyte zona pellucida(ZP)and is the only MTSP member to do so.Fidgetin also appears to interact with all three ZP proteins.These findings prompted us to propose that fidgetin might prevent polyspermy.Results from in vitro maturation oocytes analysis showed that fidgetin knockdown did cause polyspermy.We then deleted all three fidgetin isoforms with CRISPR/Cas9 technologies;however,female mice remained healthy and with normal fertility.Of all mouse MTSPs,only the mRNA level of fidgetin-like 1(FIGNL1)significantly increased.Therefore,we assert that fidgetin-like 1 compensates fidgetin's roles in fidgetin knockout female mice.展开更多
RNA interference(RNAi) is a powerful tool for functional gene analysis which has been successfully used to downregulate the expression levels of target genes.The goal of this research was to provide a highly robust an...RNA interference(RNAi) is a powerful tool for functional gene analysis which has been successfully used to downregulate the expression levels of target genes.The goal of this research was to provide a highly robust and concise methodology for in-vitro screening of efficient siRNAs from a bulk to be used as a tool to protect potato plants against PVY invasion.In our study,a 480bp fragment of the capsid protein gene of potato virus Y(CP-PVY) was used as a target to downregulate PVY mRNA expression in-vitro,as the CP gene interferes with viral uncoating,translation and replication.A total of six siRNAs were designed and screened through transient transfection assay and knockdown in expression of CP-PVY mRNA was calculated in CHO-k cells.CP-PVY mRNA knockdown efficiency was analyzed by RT-PCR and real-time PCR of CHO-k cells co-transfected with a CP gene construct and siRNAs.Six biological replicates were performed in this study.In our findings,one CP gene specific siRNA out of a total of six was found to be the most effective for knockdown of CP-PVY mRNA in transfected CHO-k cells by up to 80%-90%.展开更多
In small cell lung cancer cells, various autocrine stimuli lead to the parallel activation of Gq/11 and G12/13 proteins. The contribution of the Gq/11-PLC-β cascade to the mitogenic effects in SCLC cells is well esta...In small cell lung cancer cells, various autocrine stimuli lead to the parallel activation of Gq/11 and G12/13 proteins. The contribution of the Gq/11-PLC-β cascade to the mitogenic effects in SCLC cells is well established, but the relevance of G12/13 signaling is less explored. While in prostate and breast cancer, G12/13 activation has been shown previously to promote invasiveness without being involved in cellular proliferation, previous data from our group indicate anti-proliferative effects of G12/13 knockdown in small cell lung cancer (SCLC) cells. To further investigate the role of G12/13-dependent signaling in lung tumor cells, we employed shRNA-mediated targeting of Gα12, Gα13, or both, in SCLC and NSCLC cell lines. Lentiviral expression of shRNAs resulted in specific Gα12 and Gα13 knockdown. Of note, upon single knockdown of one family member, no counter-upregulation of the other one was observed. Interestingly, inhibition of proliferation was cell line dependent. In cell lines where knock-down led to antiproliferation, single knockdown of either Gα12 or Gα13 was sufficient to impair proliferation and double knockdown of Gα12 and Gα13 tended not to further increase anti-proliferative effects. Likewise, when single knockdown was insufficient for an inhibition of proliferation, no effects were observed in double knockdowns. Taken together, these findings indicate that both Gα12 and Gα13 affect cellular proliferation individually and interference with one family member is sufficient for anti-tumor effects.展开更多
We have studied the expression of a subset of genes encoding important tumor growth related factors in U87 glioma cells with IRE1 (inositol requiring enzyme-1) knockdown as well as their hypoxic regulation. It was sho...We have studied the expression of a subset of genes encoding important tumor growth related factors in U87 glioma cells with IRE1 (inositol requiring enzyme-1) knockdown as well as their hypoxic regulation. It was shown that the expression levels of activating transcription factor 6 (ATF6), clusterin (CLU), adhesion G protein-coupled receptor E5 (ADGRE5), transglutaminase?2, C polypeptide (TGM2), leukemia inhibitory factor (LIF), phosphoserine aminotransferase 1 (PSAT1), glyoxalase I (GLO1) and tetraspanin 13 (TSPAN13) are significantly down-regulated in glioma cells with the knockdown of IRE1 signaling enzyme. It was also shown that in glioma cells subjected to hypoxia, the expression levels of PSAT1, TSPAN13, EIF2AK3, and TGM2 genes were up-regulated, whereas the expression of ATF6 gene was down-regulated. At the same time, the expression levels of LIF, CLU, and ADGRE5 genes did not change in response to hypoxic treatment.?Furthermore, inhibition of IRE1, a key effector of an unfolded protein response pathway, modified the effect of hypoxia on the expression of most studied genes. Present study demonstrates that IRE1 knockdown down-regulated the expression of most studied genes and modified their hypoxic regulation and that these changes possibly contributed to the suppression of glioma growth in cells without IRE1 signaling enzyme function.展开更多
Background The CREG is an important lysosomal protein involved in a variety of cellular functions including promoting cell differentiation,sustaining mature homeostasis, and antagonizing apoptosis.Deficiency of CREG i...Background The CREG is an important lysosomal protein involved in a variety of cellular functions including promoting cell differentiation,sustaining mature homeostasis, and antagonizing apoptosis.Deficiency of CREG in cell and tissue results in a pathologic apoptosis.The present study aimed to elucidate the mechanism of CREG regulation apoptosis.Methods We firstly generated stable NIH3T3 fibroblasts by transfection of pDS_shCREGs vectors.Furthermore, PI-Annexin V and TUNEL staining were used to identify that CREG knockdown promoted the cell apoptosis in NIH3T3 fibroblasts.Western blotting and immunofluorescence staining was used to identify the expression and localization of M6P/ IGF2R and cathepsin L in cytoplasm.Results pDS_shCREGs vector transfection produced an approximately 80%decrease in CREG levels both in the lysate and in the media.The expression and localization of M6P/IGF2R and cathepsin L in cytoplasma changed obviously associated with down-regulated of CREG.In addition,the retention and secretion of cathepsin L enhanced significantly.Using the specific inhibitor or siRNA to block cathepsin L activation attenuated the apoptosis mediated by CREG downregulation.Conclusions Our findings indicated that inhibition of CREG expression in NIH3T3 fibroblasts leads to impaired cathepsin L sorting function mediated by M6P/IGF2R and subsequently promotes pathological cell apoptosis.展开更多
The spatiotemporal knockdown of genes through genome editing heralds a new frontier in molecular breeding,yet it remains largely unexplored.Recognizing the intricate regulatory networks of endogenous microRNAs(miRNAs)...The spatiotemporal knockdown of genes through genome editing heralds a new frontier in molecular breeding,yet it remains largely unexplored.Recognizing the intricate regulatory networks of endogenous microRNAs(miRNAs),we posited that integration of specific miRNA target sequences into the 30 untrans-lated region(UTR)of a gene could construct artificial miRNA-dependent regulatory circuits,facilitating pre-cise spatiotemporal gene suppression.To test this hypothesis,we selected three endogenous miRNAs with unique expression profiles by analyzing rice miRNA expression profiles.Results from both transient assays and stably-edited rice plants confirmed that in-locus incorporation of miRNA targets into the 30 UTR of target genes can substantially reduce their expression in a spatiotemporal manner.Using GID1 as a target gene,we found that knockin of the miR156a target led to a remarkable 97%constitutive reduc-tion;knockin of the tissue-specifically expressed miR396c target significantly reduced its expression in shoots alone;and knock-in of the long-day-induced miR528 target triggered a dramatic and temporal decrease of 95%specifically under such light exposure.These findings underscore the viability of miRNA-mediated,in-locus knockdown(MiRKD)as a convenient approach for crop breeding,leveraging miRNA expression traits and genome editing for conditional gene suppression.展开更多
Background The cytochrome P450s-mediated metabolic resistance and the target site insensitivity caused by the knockdown resistance(kdr)mutation in the voltage-gated sodium channel(vgsc)gene were the main mechanisms co...Background The cytochrome P450s-mediated metabolic resistance and the target site insensitivity caused by the knockdown resistance(kdr)mutation in the voltage-gated sodium channel(vgsc)gene were the main mechanisms conferring resistance to deltamethrin in Culex quinquefasciatus from Thailand.This study aimed to investigate the expression levels of cytochrome P450 genes and detect mutations of the vgsc gene in deltamethrin-resistant Cx.quinquefasciatus populations in Thailand.Methods Two field-collected strains of Cx.quinquefasciatus,Cq_SP and Cq_NiH,were selected with deltamethrin to generate the resistant strains Cq_SP-R and Cq_NiH-R,respectively.Bioassays were tested on larvae and adults of each strain according to WHO methods.Eight cytochrome P450 genes were analyzed for the expression level using quantitative real time-PCR.The cDNA of mosquitoes was amplified and sequenced for four fragments of vgsc gene.The kdr L1014F mutation and the haplotype of the CYP9M10 gene were detected in survivor and dead mosquitoes after exposure to the deltamethrin WHO test paper.Statistical analyses were performed using Fisher’s exaction test.Results Bioassay tests revealed a significantly higher resistance level in Cq_SP-R than in Cq_NiH-R strains in both larvae and adults.All eight cytochrome P450 genes were significantly overexpressed in larvae of Cq_NiH-R strain compared to the parent and susceptible Cq_Sus strains.The CYP6AA7 and CYP9J34 genes had the highest expression ratios,exceeding 24-fold in Cq_NiH-R larvae.In Cq_SP-R strain,the CYP4H34 and CYP9J34 genes were overexpressed in both stages.The kdr L1014F mutation was found in Cq_SP-R and its parent Cq_SP strains with a significantly higher mutant allele frequency in the survivor mosquitoes than in dead mosquitoes(P<0.0001).The V240M and novel L925F mutations were found only in Cq_SP-R strain.Heterozygous genotype for the D-Cu(+)/Cu(-)of CYP9M10 gene was detected in Cq_NiH and Cq_NiH-R strains but other strains were mostly homozygous for the Cu(-)/Cu(-).Conclusions Overexpression of multiple cytochrome P450 genes alone has a relatively minor impact on resistance.The combined mechanisms of cytochrome P450-and kdr-mediated resistance result in significantly higher resistance to deltamethrin in Cx.quinquefasciatus.This study supports sustainable public health initiatives in Thailand to address the evolving challenges of insecticide resistance.展开更多
文摘Published:18 July 2025 The published article titled“Inhibition of Liver Carcinoma Cell Invasion and Metastasis by Knockdown of Cullin7 In Vitro and In Vivo”has been retracted from Oncology Research,Vol.23,No.4,2015,pp.171–181.DOI:10.3727/096504016X14519995067562 URL:https://www.techscience.com/or/v23n4/57554 Following the publication,concerns have been raised about a number of figures in this article.An unexpected area of similarity was identified in terms of the cellular data,where the results from differently performed experiments were intended to have been shown,although the areas immediately surrounding this area featured comparatively different distributions of cells.In addition,the western blots in this article were presented with atypical,unusually shaped and possibly anomalous protein bands in many cases.
文摘Objective:To investigate the prevalence,mechanisms,and trends of knockdown resistance(kdr)in Anopheles(An.)culicifacies and its impact on the efficacy of organochlorine and other insecticides.Methods:A systematic review was conducted based on PRISMA guidelines,extracting data from biooan.org,Embase,ProQuest,PubMed,Scopus,and Web of Science without a time limit until the end of 2022.Articles meeting the inclusion criteria were assessed using the STROBE checklist.Data on kdr mutations,insecticide resistance,and effectiveness were analyzed across eight selected studies from various regions.Results:The review revealed widespread kdr-mediated resistance in An.culicifacies,primarily against dichloro-diphenyl-trichloroethane(DDT),persisting even decades after discontinued use.Key kdr mutations,including L1014F and L1014S,were identified.Resistance to deltamethrin was less stable,with increased sensitivity observed after short-term discontinuation.The findings underscore the vector's sustained resistance to organochlorine insecticides and relative sensitivity to pyrethroids.Conclusions:Stable kdr resistance in An.culicifacies to organochlorine insecticides highlights the need for periodic susceptibility assessments and strategic rotation or combination of insecticides to combat malaria effectively and prevent the development of resistance.
基金supported by the Natural Science Foundation of Zhejiang Province,China(Grant Nos.LZ22C150002 and LR24C150001)the National Key Research and Development Program of China(Grant Nos.2021YFF1000402 and 2022YFD1401600)+1 种基金the National Natural Science Foundation of China(Grant No.32170262)the Fundamental Research Funds for the Central Universities,China(Grant No.K20240124).
文摘CRISPR-Cas endonucleases mediate prokaryotic adaptive immunity by targeting foreign nucleic acids.CRISPR/Cas13b is a class 2 type VI-B ribonuclease that targets and cleaves single-stranded RNA.It exhibits higher RNA interference activity than Cas13a and Cas13c and causes fewer collateral effects than RxCas13d in mammalian cells.However,a programmable CRISPR/Cas13b-mediated RNA interference system for endogenous transcripts in rice has not yet been established.Here,we developed a CRISPR/Cas13b-mediated system to target endogenous transcripts in rice.Our CRISPR/Cas13b system could inhibit multiple endogenous mRNAs simultaneously.In addition,this system efficiently repressed endogenous long noncoding RNAs with more than 50% inhibition in stable transgenic plants.Furthermore,we found only weak collateral effects of the CRISPR/Cas13b-mediated system at the transcriptome-wide level,and no difference in the agronomic traits of stable transgenic rice in the field.We present a programmable CRISPR/Cas13b-mediated knockdown system for rice,offering a potential biotechnological tool for functional genomics and crop improvement.
基金Supported by Natural Science Basic Research Program of Shaanxi Province,No.2021JM-256.
文摘BACKGROUND The role of Sm-like 5(LSM5)in colon cancer has not been determined.In this study,we investigated the role of LSM5 in progression of colon cancer and the potential underlying mechanism involved.AIM To determine the role of LSM5 in the progression of colon cancer and the potential underlying mechanism involved.METHODS The Gene Expression Profiling Interactive Analysis database and the Human Protein Atlas website were used for LSM5 expression analysis and prognosis analysis.Real-time quantitative polymerase chain reaction and Western blotting were utilized to detect the expression of mRNAs and proteins.A lentivirus targeting LSM5 was constructed and transfected into colon cancer cells to silence LSM5 expression.Proliferation and apoptosis assays were also conducted to evaluate the growth of the colon cancer cells.Human GeneChip assay and bioinformatics analysis were performed to identify the potential underlying mechanism of LSM5 in colon cancer.RESULTS LSM5 was highly expressed in tumor tissue and colon cancer cells.A high expression level of LSM5 was related to poor prognosis in patients with colon cancer.Knockdown of LSM5 suppressed proliferation and promoted apoptosis in colon cancer cells.Silencing of LSM5 also facilitates the expression of p53,cyclin-dependent kinase inhibitor 1A(CDKN1A)and tumor necrosis factor receptor superfamily 10B(TNFRSF10B).The inhibitory effect of LSM5 knockdown on the growth of colon cancer cells was associated with the upregulation of p53,CDKN1A and TNFRSF10B.CONCLUSION LSM5 knockdown inhibited the proliferation and facilitated the apoptosis of colon cancer cells by upregulating p53,CDKN1A and TNFRSF10B.
基金supported by the National Nature Science Foundation of China (grant no.81570971)
文摘Human dental pulp cells （hDPCs） possess the capacity to differentiate into odontoblast-like cells and generate reparative dentin in response to exogenous stimuli or injury. Ten-eleven translocation 1 （TET1） is a novel DNA methyldioxygenase that plays an important role in the promotion of DNA demethylation and transcriptional regulation in several cell lines. However, the role of TET1 in the biological functions of hDPCs is unknown. To investigate the effect of TET1 on the proliferation and odontogenic differentiation potential of hDPCs, a recombinant shRNA lentiviral vector was used to knock down TET1 expression in hDPCs. Following TET1 knockdown, TET1 was significantly downregulated at both the mRNA and protein levels. Proliferation of the hDPCs was suppressed in the TET1 knockdown groups. Alkaline phosphatase activity, the formation of mineralized nodules, and the expression levels of DSPP and DMP1 were all reduced in the TETl-knockdown hDPCs undergoing odontogenic differentiation. Based on these results, we concluded that TET1 knockdown can prevent the proliferation and odontogenic differentiation of hDPCs, which suggests that TET1 may play an important role in dental pulp repair and regeneration.
基金supported by the National Natural Science Foundation of China,No.81460193
文摘The micro RNA（mi RNA） let-7 was one of the first mi RNAs to be discovered, and is highly conserved and widely expressed among species. let-7 expression increases in brain tissue after cerebral ischemia/reperfusion injury; however, no studies have reported let-7 effects on nerve injury after cerebral ischemia/reperfusion injury. To investigate the effects of let-7 gene knockdown on cerebral ischemia/reperfusion injury, we established a rat model of cerebral ischemia/reperfusion injury. Quantitative reverse transcription-polymerase chain reaction demonstrated that 12 hours after cerebral ischemia/reperfusion injury, let-7 expression was up-regulated, peaked at 24 hours, and was still higher than that in control rats after 72 hours. Let-7 gene knockdown in rats suppressed microglial activation and inflammatory factor release, reduced neuronal apoptosis and infarct volume in brain tissue after cerebral ischemia/reperfusion injury. Western blot assays and luciferase assays revealed that mitogen-activated protein kinase phosphatase-1（MKP1） is a direct target of let-7. Let-7 enhanced phosphorylated p38 mitogen-activated protein kinase（MAPK） and c-Jun N-terminal kinase（JNK） expression by down-regulating MKP1. These findings suggest that knockdown of let-7 inhibited the activation of p38 MAPK and JNK signaling pathways by up-regulating MKP1 expression, reduced apoptosis and the inflammatory reaction, and exerted a neuroprotective effect following cerebral ischemia/reperfusion injury.
文摘Fankl is exclusively expressed in the testis from the meiosis phase to the haploid phase of spermatogenesis. In this study, we examined the function of Fankl by establishing a Fankl-knockdown transgenic mouse model. The apoptotic statuses of the testes of the transgenic mice were tested using the terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） method. The FANK1 consensus DNA-binding sequence was identified using cyclic amplification of sequence target （CAST） analysis. Differentially expressed genes were examined using microarray analysis. A reduction in sperm number and an increase in apoptotic spermatocytes were observed in Fankl-knockdown mice, and the apoptotic cells were found to be primarily spermatogonia and spermatocytes. The CAST results demonstrated that the consensus DNA-binding sequence was AAAAAG, in which the percentage occurrence of each base at each position ranged from 55 to 86%. This sequence was present in the promoter regions of 10 differentially expressed genes that were examined using microarray analysis. In total, 17 genes were differentially expressed with changes in their expression levels greater than twofold. The abnormal expression of Fankl target genes that were regulated directly or indirectly by Fankl reduced the number of sperm in the knockdown mice. Thus, FANK1 may Dlav a pivotal role in sDermato^enesis as a transcription factor.Fankl is exclusively expressed in the testis from the meiosis phase to the haploid phase of spermatogenesis. In this study, we examined the function of Fankl by establishing a Fankl-knockdown transgenic mouse model. The apoptotic statuses of the testes of the transgenic mice were tested using the terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） method. The FANK1 consensus DNA-binding sequence was identified using cyclic amplification of sequence target （CAST） analysis. Differentially expressed genes were examined using microarray analysis, A reduction in sperm number and an increase in apoptotic spermatocytes were observed in Fankl-knockdown mice, and the apoptotic cells were found to be primarily spermatogonia and spermatocytes. The CAST results demonstrated that the consensus DNA-binding sequence was AAAAAG, in which the percentage occurrence of each base at each position ranged from 55 to 86%. This sequence was present in the promoter regions of 10 differentially expressed genes that were examined using microarray analysis. In total, 17 genes were differentially expressed with changes in their expression levels greater than twofold. The abnormal expression of Fankl target genes that were regulated directly or indirectly by Fankl reduced the number of sperm in the knockdown mice. Thus, FANK1 may play a pivotal role in spermatogenesis as a transcription factor.
基金the National Natural Science Foundation of China(Grant Nos.U21A20429,11772078,and 11825202)the National Defense Basic Research Program(Grant No.JCKY2020110).
文摘Thin-walled structures are commonly utilized in aerospace and aircraft structures,which are prone to buckling under axial compression and extremely sensitive to geometric imperfections.After decades of efforts,it still remains a challenging issue to accurately predict the lower-bound buckling load due to the impact of geometric imperfections.Up to now,the lower-bound curve in NASA SP-8007 is still widely used as the design criterion of aerospace thin-walled structures,and this series of knockdown factors(KDF)has been proven to be overly conservative with the significant promotion of the manufacturing process.In recent years,several new numerical and experimental methods for determining KDF have been established,which are systematically reviewed in this paper.The Worst Multiple Perturbation Load Approach(WMPLA)is one of the most representative methods to reduce the conservatism of traditional methods in a rational manner.Based on an extensive collection of test data from 1990 to 2020,a new lower-bound curve is approximated to produce a series of improved KDFs.It is evident that these new KDFs have an overall improvement of 0.1-0.3 compared with NASA SP-8007,and the KDF predicted by the WMPLA is very close to the front of the new curve.This may provide some insight into future design guidelines of axially compressed cylindrical shells,which is promising for the lightweight design of large-diameter aerospace structures.
文摘Efficacy of 25 essential oils was screened against filarial vector, Culex quinquefasciatus, for their larvicidal and knockdown effects in a preliminary study. Of these, 8 oils viz. calamus oil, cinnamon oil, citronella oil, clove oil, eucalyptus oil, lemon oil, mentha oil and orange oil exhibited 100% larvicidal activity at 1000 ppm and 100% knockdown effect at 10% concentration. These 8 oils were screened further against Cx. Quinquefasciatus, Aedes aegypti and Anopheles stephensi for their larvicial and knockdown effects at different concentrations. Mentha oil was the most promising against An. Stephensi and Ae. Aegypti recording LC50 and LC90 values of 39.74 and 115.67 ppm and 46.23 and 165.36 ppm, respectively for larvicidal activity. Calamus oil was the most effective against Cx. Quinquefasciatus with LC50 and LC90 values of 40.40, and 140.07 ppm, respectively for larvicidal activity. Orange oil showed the most potent knockdown effect with the KT50 and KT95 values of 27.44, 26.22 and 29.91 and 70.81, 65.33 and 68.57 min, against An. stephensi, Cx. Quinquefasciatus and Ae. Aegypti, respectively. The results clearly indicated that mentha oil and calamus oil were the most promising larvicides and orange oil had potent knockdown effect against the tested mosquito species. These oils could be used to develop a new formulation to control mosquitoes.
基金supported by the National Natural Science Foundation of China,No.81372698
文摘Pitx3 is strongly associated with the phenotype, differentiation, and survival of dopaminergic neurons. The relationship between Pitx3 and glial cell line-derived neurotrophic factor（GDNF） in dopaminergic neurons remains poorly understood. The present investigation sought to construct and screen a lentivirus expression plasmid carrying a rat Pitx3 short hairpin（sh）RNA and to assess the impact of Pitx3 gene knockdown on GDNF transcriptional activity in MES23.5 dopaminergic neurons. Three pairs of interference sequences were designed and separately ligated into GV102 expression vectors. These recombinant plasmids were transfected into MES23.5 cells and western blot assays were performed to detect Pitx3 protein expression. Finally, the most effective Pitx3 sh RNA and a dual-luciferase reporter gene plasmid carrying the GDNF promoter region（GDNF-luciferase） were cotransfected into MES23.5 cells. Sequencing showed that the synthesized sequences were identical to the three Pitx3 interference sequences. Inverted fluorescence microscopy revealed that the lentivirus expression plasmids carrying Pitx3-sh RNA had 40-50% transfection efficiency. Western blot assay confirmed that the corresponding Pitx3 of the third knockdown sequence had the lowest expression level. Dual-luciferase reporter gene results showed that the GDNF transcriptional activity in dopaminergic cells cotransfected with both plasmids was decreased compared with those transfected with GDNF-luciferase alone. Together, the results showed that the designed Pitx3-sh RNA interference sequence decreased Pitx3 protein expression, which decreased GDNF transcriptional activity.
文摘Currently,glucose transporter 4(GLUT4)has been considered as the key player for the insulin-stimulated glucose transport in the muscle and adipose tissues.The development of recombinant DNA techniques allows the creations of genetically knockout,knockdown and transgenic animals and cells for the study of GLUT4’s physiological functions.Here,we have used key words to search the PubMed and summarized the methods used in Slc2a4 gene knockout,GLUT4 knockdown and overexpression in the whole body and tissue specific manner.The whole body GLUT4-null mice have growth retardation,but normal glucose tolerance and basal glucose turnover rates.Compared with whole body Slc2a4 knockout mice,adipose and muscle double knockout mice have impaired insulin tolerance and glucose intolerance.The results of GLUT4 knockdown in 3T3-L1 adipocytes have shown that its expression is needed for lipogenesis after,but not during,differentiation.Transgenic mice with the whole body GLUT4 overexpression have normal body weight and lowered blood glucose level.The adipose tissue specific overexpression of GLUT4 leads to increases in mouse body weight and adipose tissue weight.The insulin-stimulated GLUT4 translocation in the skeletal muscle contributes to the regulation of glucose homeostasis.Data from both transgenic overexpression and tissue specific Slc2a4 knockout indicate that GLUT4 probably plays a role in the glucose uptake in the fasting state.More studies are warranted to use advanced molecular biology tools to decipher the roles of GLUT4 in the control of glucose homeostasis.
基金Supported by the Zhengzhou Science and Technology Innovation Team Project(131PCXTD631)
文摘This study investigated the specific mechanism of knockdown of neuropeptide Y(NPY) in reducing obesity-induced insulin resistance in the white adipose tissue. Adeno-associated virus(AAV)-mediated RNAi was utilized to downregulate NPY expression in rats fed either regular chow or high fat diet. By investigating the differences in rat body weight and food intake, we assessed the effect of knockdown of NPY expression on insulin sensitivity and β-cell proliferation. Glucose consumption and 2-[3 H]DG uptake in 3 T3-L1 adipocytes were assessed to determine the molecular mechanisms. The results showed that knockdown of NPY expression in the dorsomedial hypothalamus(DMH) reduced obesity-induced insulin resistance, increased glucose consumption, and decreased 2-[3 H]DG uptake in 3 T3-L1 adipocytes via the PI3 K/Akt/GSK-3β signaling pathways and the NPY Y5 receptor.
基金supported by the National Natural Science Foundation of China(Grant No.31671561)to Dong Zhang.
文摘Microtubule-severing proteins(MTSPs),are a family of proteins which use adenosine triphosphate to sever microtubules.MTSPs have been shown to play an important role in multiple microtubule-involved cellular processes.One member of this family,fidgetin(FIGN),is also involved in male fertility;however,no studies have explored its roles in female fertility.In this study,we found mouse fidgetin is rich within oocyte zona pellucida(ZP)and is the only MTSP member to do so.Fidgetin also appears to interact with all three ZP proteins.These findings prompted us to propose that fidgetin might prevent polyspermy.Results from in vitro maturation oocytes analysis showed that fidgetin knockdown did cause polyspermy.We then deleted all three fidgetin isoforms with CRISPR/Cas9 technologies;however,female mice remained healthy and with normal fertility.Of all mouse MTSPs,only the mRNA level of fidgetin-like 1(FIGNL1)significantly increased.Therefore,we assert that fidgetin-like 1 compensates fidgetin's roles in fidgetin knockout female mice.
文摘RNA interference(RNAi) is a powerful tool for functional gene analysis which has been successfully used to downregulate the expression levels of target genes.The goal of this research was to provide a highly robust and concise methodology for in-vitro screening of efficient siRNAs from a bulk to be used as a tool to protect potato plants against PVY invasion.In our study,a 480bp fragment of the capsid protein gene of potato virus Y(CP-PVY) was used as a target to downregulate PVY mRNA expression in-vitro,as the CP gene interferes with viral uncoating,translation and replication.A total of six siRNAs were designed and screened through transient transfection assay and knockdown in expression of CP-PVY mRNA was calculated in CHO-k cells.CP-PVY mRNA knockdown efficiency was analyzed by RT-PCR and real-time PCR of CHO-k cells co-transfected with a CP gene construct and siRNAs.Six biological replicates were performed in this study.In our findings,one CP gene specific siRNA out of a total of six was found to be the most effective for knockdown of CP-PVY mRNA in transfected CHO-k cells by up to 80%-90%.
文摘In small cell lung cancer cells, various autocrine stimuli lead to the parallel activation of Gq/11 and G12/13 proteins. The contribution of the Gq/11-PLC-β cascade to the mitogenic effects in SCLC cells is well established, but the relevance of G12/13 signaling is less explored. While in prostate and breast cancer, G12/13 activation has been shown previously to promote invasiveness without being involved in cellular proliferation, previous data from our group indicate anti-proliferative effects of G12/13 knockdown in small cell lung cancer (SCLC) cells. To further investigate the role of G12/13-dependent signaling in lung tumor cells, we employed shRNA-mediated targeting of Gα12, Gα13, or both, in SCLC and NSCLC cell lines. Lentiviral expression of shRNAs resulted in specific Gα12 and Gα13 knockdown. Of note, upon single knockdown of one family member, no counter-upregulation of the other one was observed. Interestingly, inhibition of proliferation was cell line dependent. In cell lines where knock-down led to antiproliferation, single knockdown of either Gα12 or Gα13 was sufficient to impair proliferation and double knockdown of Gα12 and Gα13 tended not to further increase anti-proliferative effects. Likewise, when single knockdown was insufficient for an inhibition of proliferation, no effects were observed in double knockdowns. Taken together, these findings indicate that both Gα12 and Gα13 affect cellular proliferation individually and interference with one family member is sufficient for anti-tumor effects.
文摘We have studied the expression of a subset of genes encoding important tumor growth related factors in U87 glioma cells with IRE1 (inositol requiring enzyme-1) knockdown as well as their hypoxic regulation. It was shown that the expression levels of activating transcription factor 6 (ATF6), clusterin (CLU), adhesion G protein-coupled receptor E5 (ADGRE5), transglutaminase?2, C polypeptide (TGM2), leukemia inhibitory factor (LIF), phosphoserine aminotransferase 1 (PSAT1), glyoxalase I (GLO1) and tetraspanin 13 (TSPAN13) are significantly down-regulated in glioma cells with the knockdown of IRE1 signaling enzyme. It was also shown that in glioma cells subjected to hypoxia, the expression levels of PSAT1, TSPAN13, EIF2AK3, and TGM2 genes were up-regulated, whereas the expression of ATF6 gene was down-regulated. At the same time, the expression levels of LIF, CLU, and ADGRE5 genes did not change in response to hypoxic treatment.?Furthermore, inhibition of IRE1, a key effector of an unfolded protein response pathway, modified the effect of hypoxia on the expression of most studied genes. Present study demonstrates that IRE1 knockdown down-regulated the expression of most studied genes and modified their hypoxic regulation and that these changes possibly contributed to the suppression of glioma growth in cells without IRE1 signaling enzyme function.
文摘Background The CREG is an important lysosomal protein involved in a variety of cellular functions including promoting cell differentiation,sustaining mature homeostasis, and antagonizing apoptosis.Deficiency of CREG in cell and tissue results in a pathologic apoptosis.The present study aimed to elucidate the mechanism of CREG regulation apoptosis.Methods We firstly generated stable NIH3T3 fibroblasts by transfection of pDS_shCREGs vectors.Furthermore, PI-Annexin V and TUNEL staining were used to identify that CREG knockdown promoted the cell apoptosis in NIH3T3 fibroblasts.Western blotting and immunofluorescence staining was used to identify the expression and localization of M6P/ IGF2R and cathepsin L in cytoplasm.Results pDS_shCREGs vector transfection produced an approximately 80%decrease in CREG levels both in the lysate and in the media.The expression and localization of M6P/IGF2R and cathepsin L in cytoplasma changed obviously associated with down-regulated of CREG.In addition,the retention and secretion of cathepsin L enhanced significantly.Using the specific inhibitor or siRNA to block cathepsin L activation attenuated the apoptosis mediated by CREG downregulation.Conclusions Our findings indicated that inhibition of CREG expression in NIH3T3 fibroblasts leads to impaired cathepsin L sorting function mediated by M6P/IGF2R and subsequently promotes pathological cell apoptosis.
基金supported by the National Key Research and Development Program of China(no.2021YFD1201300)the Shanghai Agricultural Science and Technology Innovation Program(no.K2023001)+2 种基金the National Natural Science Foundation of China(no.32070396)the China Postdoctoral Science Foundation(no.2024M751985)he Chongqing Natural Science Foundation(CSTB2022NSCQ-MSX0524).
文摘The spatiotemporal knockdown of genes through genome editing heralds a new frontier in molecular breeding,yet it remains largely unexplored.Recognizing the intricate regulatory networks of endogenous microRNAs(miRNAs),we posited that integration of specific miRNA target sequences into the 30 untrans-lated region(UTR)of a gene could construct artificial miRNA-dependent regulatory circuits,facilitating pre-cise spatiotemporal gene suppression.To test this hypothesis,we selected three endogenous miRNAs with unique expression profiles by analyzing rice miRNA expression profiles.Results from both transient assays and stably-edited rice plants confirmed that in-locus incorporation of miRNA targets into the 30 UTR of target genes can substantially reduce their expression in a spatiotemporal manner.Using GID1 as a target gene,we found that knockin of the miR156a target led to a remarkable 97%constitutive reduc-tion;knockin of the tissue-specifically expressed miR396c target significantly reduced its expression in shoots alone;and knock-in of the long-day-induced miR528 target triggered a dramatic and temporal decrease of 95%specifically under such light exposure.These findings underscore the viability of miRNA-mediated,in-locus knockdown(MiRKD)as a convenient approach for crop breeding,leveraging miRNA expression traits and genome editing for conditional gene suppression.
基金supported by CMU Mid-Career Research Fellowship program to J.Y.
文摘Background The cytochrome P450s-mediated metabolic resistance and the target site insensitivity caused by the knockdown resistance(kdr)mutation in the voltage-gated sodium channel(vgsc)gene were the main mechanisms conferring resistance to deltamethrin in Culex quinquefasciatus from Thailand.This study aimed to investigate the expression levels of cytochrome P450 genes and detect mutations of the vgsc gene in deltamethrin-resistant Cx.quinquefasciatus populations in Thailand.Methods Two field-collected strains of Cx.quinquefasciatus,Cq_SP and Cq_NiH,were selected with deltamethrin to generate the resistant strains Cq_SP-R and Cq_NiH-R,respectively.Bioassays were tested on larvae and adults of each strain according to WHO methods.Eight cytochrome P450 genes were analyzed for the expression level using quantitative real time-PCR.The cDNA of mosquitoes was amplified and sequenced for four fragments of vgsc gene.The kdr L1014F mutation and the haplotype of the CYP9M10 gene were detected in survivor and dead mosquitoes after exposure to the deltamethrin WHO test paper.Statistical analyses were performed using Fisher’s exaction test.Results Bioassay tests revealed a significantly higher resistance level in Cq_SP-R than in Cq_NiH-R strains in both larvae and adults.All eight cytochrome P450 genes were significantly overexpressed in larvae of Cq_NiH-R strain compared to the parent and susceptible Cq_Sus strains.The CYP6AA7 and CYP9J34 genes had the highest expression ratios,exceeding 24-fold in Cq_NiH-R larvae.In Cq_SP-R strain,the CYP4H34 and CYP9J34 genes were overexpressed in both stages.The kdr L1014F mutation was found in Cq_SP-R and its parent Cq_SP strains with a significantly higher mutant allele frequency in the survivor mosquitoes than in dead mosquitoes(P<0.0001).The V240M and novel L925F mutations were found only in Cq_SP-R strain.Heterozygous genotype for the D-Cu(+)/Cu(-)of CYP9M10 gene was detected in Cq_NiH and Cq_NiH-R strains but other strains were mostly homozygous for the Cu(-)/Cu(-).Conclusions Overexpression of multiple cytochrome P450 genes alone has a relatively minor impact on resistance.The combined mechanisms of cytochrome P450-and kdr-mediated resistance result in significantly higher resistance to deltamethrin in Cx.quinquefasciatus.This study supports sustainable public health initiatives in Thailand to address the evolving challenges of insecticide resistance.
