AIM To determine whether cell division cycle(Cdc)42 is regulated by microRNA(miR)-15 a in the development of pediatric inflammatory bowel disease(IBD).METHODS We cultured 293 T cells, used plasmids and performed dual-...AIM To determine whether cell division cycle(Cdc)42 is regulated by microRNA(miR)-15 a in the development of pediatric inflammatory bowel disease(IBD).METHODS We cultured 293 T cells, used plasmids and performed dual-luciferase assay to determine whether Cdc42 is a miR-15 a target gene. We cultured Caco-2 cells, and stimulated them with tumor necrosis factor(TNF)-α. We then employed lentiviruses to alter the expression of miR-15 a and Cdc42. We performed quantitative reverse transcription polymerase chain reaction(qRTPCR) and immunofluorescence to determine whether Cdc42 is regulated by miR-15 a in Caco-2 cells. Finally, we collected ileocecal tissue by endoscopy from patients and performed qRT-PCR to examine the expression of miR-15 a and Cdc42 in pediatric IBD patients.RESULTS Target Scan and dual-luciferase assay revealed thatCdc42 was a miR-15 a target gene. MiR-15 a expression increased(P = 0.0038) and Cdc42 expression decreased(P = 0.0013) in cells stimulated with TNF-α, and the expression of the epithelial junction proteins zona occludens(ZO)-1(P < 0.05) and E-cadherin(P < 0.001) decreased. Cdc42 levels decreased in miR-15 a-mimic cells(P < 0.001) and increased in miR-15 a inhibitor cells(P < 0.05). ZO-1 and E-cadherin decreased in mi R-15 a-mimic cells(P < 0.001) but not in the miR-15 a inhibitor + TNF-α cells. In Lv-Cdc42 + TNF-α cells, ZO-1 and E-cadherin expression increased compared to the Lv-Cdc42-NC + TNF-α(P < 0.05) or miR-15 a-mimic cells(P < 0.05). Fifty-four pediatric IBD patients were included in this study, 21 in the control group, 19 in the Crohn's disease(CD) active(AC) group, seven in the CD remission(RE) group, and seven in the ulcerative colitis(UC) group. MiR-15 a increased and Cdc42 decreased in the CD AC group compared to the control group(P < 0.05). miR-15 a decreased and Cdc42 increased in the CD RE group compared to the CD AC group(P < 0.05). miR-15 a was positively correlated with the Pediatric Crohn's disease Activity Index(PCDAI)(P = 0.006), while Cdc42 was negatively correlated with PCDAI(P = 0.0008). Finally, miR-15 a expression negatively correlated with Cdc42 in pediatric IBD patients(P = 0.0045).CONCLUSION Mi R-15 a negatively regulates epithelial junctions through Cdc42 in Caco-2 cells and pediatric IBD patients.展开更多
Objective Several studies have indicated that miR-15a,miR-15b and miR-16 may be the important regulators of apoptosis.Since attenuate apoptosis could protect myocardium and reduce infarction size,the present study was...Objective Several studies have indicated that miR-15a,miR-15b and miR-16 may be the important regulators of apoptosis.Since attenuate apoptosis could protect myocardium and reduce infarction size,the present study was aimed to find out whether these miRNAs participate in regulating myocardial ischemia reperfusion （I/R） injury.Methods Apoptosis in mice hearts subjected to I/R was detected by TUNEL assay in vivo,while flow cytometry analysis followed by Annexin V/PI double stain in vitro was used to detect apoptosis in cultured cardiomyocytes which were subjected to hypoxia/reoxygenation （H/R）.Taqman real-time quantitative PCR was used to confirm whether miR-15a/15b/16 were involved in the regulation of cardiac I/R and H/R.Results Compared to those of the controls,I/R or H/R induced apoptosis of cardiomyocytes was significantly iucreased both in vivo （24.4％ ± 9.4％ vs.2.2％ ± 1.9％,P ＜ 0.01,n =5） and in vitro （14.12％ ±0.92％ vs.2.22％ ± 0.08％）.The expression of miR-15a and miR-15b,but not miR-16,was increased in the mice I/R model,and the results were consistent in the H/R model.Conclusions Our data indicate miR-15 and miR-15b are up-regulated in response to cardiac I/R injury,therefore,down-regulation of miR- 15a/b may be a promising strategy to reduce myocardial apoptosis induced by cardiac I/R injury.展开更多
OBJECTIVE To investigate the effects of microRNA-15a （miR- 15a） on the growth of the lymphoma Raji cell line. METHODS A eukaryotic expression vector of precursor mLR-15a （pre-miR-15a） was constructed. Paired oligo...OBJECTIVE To investigate the effects of microRNA-15a （miR- 15a） on the growth of the lymphoma Raji cell line. METHODS A eukaryotic expression vector of precursor mLR-15a （pre-miR-15a） was constructed. Paired oligonucleotides for pre- miR-15a expression were designed and synthesized chemically. Annealed oligonucleotides were inserted into a pGCSIL- GFP vector under a U6 promoter to construct a pre-miR-15a expression plasmid. Oligonucleotides with a scrambled sequence which were used as a negative control were also constructed into the pGCSIL-GFP vector. A recombinant expression vector was identified through PCR and sequencing. A pre-miR-15a expression plasmid and a negative control plasmid were all transferred into Raji cells with lipofectamine 2000. Quantitative reverse-transcription polymerase chain reaction （qRT-PCR） was employed to explore expression of miR-15a. The expression levels of the protein were assayed via immunofluorescence. The growth effect of Raji cells was measured by CCK8 assay. Apoptosis was determined using Hoechst dyeing and flow cytometry. The growth-inhibitory effect on Raji cells was measured using a CCK8 assay. RESULTS The results showed that the insertion sequence was correct. The expression level of miR-15a was obviously higher in Raji cells transferred with pre-miR-15a plasmid than in the blank group and in the negative control group. After Raji cells were transferred with the pre-miR-15a expression plasmid for 48 h, Bcl-2 protein expression levels were obviously decreased, which indicated that there was a significant difference as compared with the negative control group and blank group （P 〈 0.05）. The CCK8 assay showed that transfection of pre-miR-15a expression plasmid decreased the cell growth at 24, 48 and 72 h post-transfection. After Raji cells were transferred with pre-miR-15a expression plasmid, apoptotic cells can be seen with Hoechst dyeing, and the cell apoptotic rate was obviously higher than that in blank group and negative control group. CONCLUSION Pre-miR-15a can induce apoptosis and inhibit the growth of Raji cells.展开更多
Osteoarthritis(OA)is a chronic joint disease characterized by cartilage degradation,synovial inflammation,and subchondral bone remodelling.Despite its increasing prevalence,effective diagnostic,disease-limiting,and th...Osteoarthritis(OA)is a chronic joint disease characterized by cartilage degradation,synovial inflammation,and subchondral bone remodelling.Despite its increasing prevalence,effective diagnostic,disease-limiting,and therapeutic strategies remain unattainable.Recent studies have recognized the involvement of microRNA-155(miR-155)in the pathogenesis of OA and most of its risk factors while also identifying the antidiabetic drug metformin as a potential modulator of disease progression.MiR-155,a key endogenous regulator of the immune system,mechano-transduction,and multiple genetic pathways,interacts with OA targets of cellular energetic and circadian homeostasis,promoting systemic and local articular inflammation,cartilage matrix degradation,and chondrocyte apoptosis.Metformin,widely used for type 2 diabetes,has demonstrated anti-inflammatory,anti-oxidative,and chondroprotective properties in OA,mainly through its activation of adenosine monophosphate-activated protein kinase and inhibition of nuclear factor kappa-B signalling.Enthrallingly,metformin targets the same cellular pathways as miR-155 with emerging evidence also suggesting miR-155 expression modulation,indicating synergistic,potentially disease-modifying effects in OA.This review highlights the central role of miR-155 in OA pathophysiology and its potential as a biomarker for disease diagnosis and progression.MiR-155 targeting-through microRNA therapeutics(mimics/antagomiRs)and/or metformin-could pave the way for innovative treatments,including novel articular delivery systems and cell-based therapies.展开更多
基金Supported by the National Natural Science Foundation of Shanghai,No.201540068
文摘AIM To determine whether cell division cycle(Cdc)42 is regulated by microRNA(miR)-15 a in the development of pediatric inflammatory bowel disease(IBD).METHODS We cultured 293 T cells, used plasmids and performed dual-luciferase assay to determine whether Cdc42 is a miR-15 a target gene. We cultured Caco-2 cells, and stimulated them with tumor necrosis factor(TNF)-α. We then employed lentiviruses to alter the expression of miR-15 a and Cdc42. We performed quantitative reverse transcription polymerase chain reaction(qRTPCR) and immunofluorescence to determine whether Cdc42 is regulated by miR-15 a in Caco-2 cells. Finally, we collected ileocecal tissue by endoscopy from patients and performed qRT-PCR to examine the expression of miR-15 a and Cdc42 in pediatric IBD patients.RESULTS Target Scan and dual-luciferase assay revealed thatCdc42 was a miR-15 a target gene. MiR-15 a expression increased(P = 0.0038) and Cdc42 expression decreased(P = 0.0013) in cells stimulated with TNF-α, and the expression of the epithelial junction proteins zona occludens(ZO)-1(P < 0.05) and E-cadherin(P < 0.001) decreased. Cdc42 levels decreased in miR-15 a-mimic cells(P < 0.001) and increased in miR-15 a inhibitor cells(P < 0.05). ZO-1 and E-cadherin decreased in mi R-15 a-mimic cells(P < 0.001) but not in the miR-15 a inhibitor + TNF-α cells. In Lv-Cdc42 + TNF-α cells, ZO-1 and E-cadherin expression increased compared to the Lv-Cdc42-NC + TNF-α(P < 0.05) or miR-15 a-mimic cells(P < 0.05). Fifty-four pediatric IBD patients were included in this study, 21 in the control group, 19 in the Crohn's disease(CD) active(AC) group, seven in the CD remission(RE) group, and seven in the ulcerative colitis(UC) group. MiR-15 a increased and Cdc42 decreased in the CD AC group compared to the control group(P < 0.05). miR-15 a decreased and Cdc42 increased in the CD RE group compared to the CD AC group(P < 0.05). miR-15 a was positively correlated with the Pediatric Crohn's disease Activity Index(PCDAI)(P = 0.006), while Cdc42 was negatively correlated with PCDAI(P = 0.0008). Finally, miR-15 a expression negatively correlated with Cdc42 in pediatric IBD patients(P = 0.0045).CONCLUSION Mi R-15 a negatively regulates epithelial junctions through Cdc42 in Caco-2 cells and pediatric IBD patients.
文摘Objective Several studies have indicated that miR-15a,miR-15b and miR-16 may be the important regulators of apoptosis.Since attenuate apoptosis could protect myocardium and reduce infarction size,the present study was aimed to find out whether these miRNAs participate in regulating myocardial ischemia reperfusion （I/R） injury.Methods Apoptosis in mice hearts subjected to I/R was detected by TUNEL assay in vivo,while flow cytometry analysis followed by Annexin V/PI double stain in vitro was used to detect apoptosis in cultured cardiomyocytes which were subjected to hypoxia/reoxygenation （H/R）.Taqman real-time quantitative PCR was used to confirm whether miR-15a/15b/16 were involved in the regulation of cardiac I/R and H/R.Results Compared to those of the controls,I/R or H/R induced apoptosis of cardiomyocytes was significantly iucreased both in vivo （24.4％ ± 9.4％ vs.2.2％ ± 1.9％,P ＜ 0.01,n =5） and in vitro （14.12％ ±0.92％ vs.2.22％ ± 0.08％）.The expression of miR-15a and miR-15b,but not miR-16,was increased in the mice I/R model,and the results were consistent in the H/R model.Conclusions Our data indicate miR-15 and miR-15b are up-regulated in response to cardiac I/R injury,therefore,down-regulation of miR- 15a/b may be a promising strategy to reduce myocardial apoptosis induced by cardiac I/R injury.
文摘OBJECTIVE To investigate the effects of microRNA-15a （miR- 15a） on the growth of the lymphoma Raji cell line. METHODS A eukaryotic expression vector of precursor mLR-15a （pre-miR-15a） was constructed. Paired oligonucleotides for pre- miR-15a expression were designed and synthesized chemically. Annealed oligonucleotides were inserted into a pGCSIL- GFP vector under a U6 promoter to construct a pre-miR-15a expression plasmid. Oligonucleotides with a scrambled sequence which were used as a negative control were also constructed into the pGCSIL-GFP vector. A recombinant expression vector was identified through PCR and sequencing. A pre-miR-15a expression plasmid and a negative control plasmid were all transferred into Raji cells with lipofectamine 2000. Quantitative reverse-transcription polymerase chain reaction （qRT-PCR） was employed to explore expression of miR-15a. The expression levels of the protein were assayed via immunofluorescence. The growth effect of Raji cells was measured by CCK8 assay. Apoptosis was determined using Hoechst dyeing and flow cytometry. The growth-inhibitory effect on Raji cells was measured using a CCK8 assay. RESULTS The results showed that the insertion sequence was correct. The expression level of miR-15a was obviously higher in Raji cells transferred with pre-miR-15a plasmid than in the blank group and in the negative control group. After Raji cells were transferred with the pre-miR-15a expression plasmid for 48 h, Bcl-2 protein expression levels were obviously decreased, which indicated that there was a significant difference as compared with the negative control group and blank group （P 〈 0.05）. The CCK8 assay showed that transfection of pre-miR-15a expression plasmid decreased the cell growth at 24, 48 and 72 h post-transfection. After Raji cells were transferred with pre-miR-15a expression plasmid, apoptotic cells can be seen with Hoechst dyeing, and the cell apoptotic rate was obviously higher than that in blank group and negative control group. CONCLUSION Pre-miR-15a can induce apoptosis and inhibit the growth of Raji cells.
文摘Osteoarthritis(OA)is a chronic joint disease characterized by cartilage degradation,synovial inflammation,and subchondral bone remodelling.Despite its increasing prevalence,effective diagnostic,disease-limiting,and therapeutic strategies remain unattainable.Recent studies have recognized the involvement of microRNA-155(miR-155)in the pathogenesis of OA and most of its risk factors while also identifying the antidiabetic drug metformin as a potential modulator of disease progression.MiR-155,a key endogenous regulator of the immune system,mechano-transduction,and multiple genetic pathways,interacts with OA targets of cellular energetic and circadian homeostasis,promoting systemic and local articular inflammation,cartilage matrix degradation,and chondrocyte apoptosis.Metformin,widely used for type 2 diabetes,has demonstrated anti-inflammatory,anti-oxidative,and chondroprotective properties in OA,mainly through its activation of adenosine monophosphate-activated protein kinase and inhibition of nuclear factor kappa-B signalling.Enthrallingly,metformin targets the same cellular pathways as miR-155 with emerging evidence also suggesting miR-155 expression modulation,indicating synergistic,potentially disease-modifying effects in OA.This review highlights the central role of miR-155 in OA pathophysiology and its potential as a biomarker for disease diagnosis and progression.MiR-155 targeting-through microRNA therapeutics(mimics/antagomiRs)and/or metformin-could pave the way for innovative treatments,including novel articular delivery systems and cell-based therapies.
