目的探讨超声微泡介导miR-545-3p调节HOXA3对结直肠癌(colorectal cancer,CRC)细胞增殖、凋亡、侵袭的影响。方法采用实时荧光定量聚合酶链式反应(quantitative real time polymerase chain reaction,qRT-PCR)检测SW620、SW480、LoVo、C...目的探讨超声微泡介导miR-545-3p调节HOXA3对结直肠癌(colorectal cancer,CRC)细胞增殖、凋亡、侵袭的影响。方法采用实时荧光定量聚合酶链式反应(quantitative real time polymerase chain reaction,qRT-PCR)检测SW620、SW480、LoVo、CACO-2及正常结肠上皮细胞系NCM460中miR-545-3p、同源盒基因3(homeobox geneA3,HOXA3)mRNA表达水平;将生长较好的SW480细胞分为空白组、微泡组、miR组、过表达miR-545-3p组、微泡+miR组、微泡+过表达miR-545-3p组、微泡+过表达miR-545-3p+空载体(pcDNA6.2/nLumio-DEST,pcDNA)组、微泡+过表达miR-545-3p+HOXA3组,采用qRT-PCR法检测各组miR-545-3p和HOXA3表达,采用MTT检测细胞增殖,采用流式细胞术检测细胞凋亡,采用Transwell实验检测细胞侵袭,采用Western blotting检测HOXA3、增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)、金属基质蛋白酶9(matrix metalloproteinase,MMP-9)、(Bcl-2-associated X protein,Bax)蛋白表达水平。通过皮下注射SW480细胞悬液,建立裸鼠移植瘤模型,并分为对照组、微泡组、miR微泡组、miR-545-3p微泡组,每组10只,检测裸鼠移植瘤瘤重。结果CRC细胞中miR-545-3p表达下调,HOXA3 mRNA表达上调(P<0.05),以SW480细胞变化最为显著,故作为后续研究对象。过表达miR-545-3p与HOXA3-WT共转染的荧光素酶活性低于miR NC与HOXA3-WT共转染(P<0.05)。过表达miR-545-3p组可上调miR-545-3p表达,下调HOXA3表达,抑制细胞增殖、侵袭数及PCNA、MMP-9蛋白表达,促进细胞凋亡及Bax蛋白表达(P<0.05),微泡+过表达miR-545-3p组上调miR-545-3p表达,下调HOXA3表达,抑制细胞增殖、侵袭数及PCNA、MMP-9蛋白表达,促进细胞凋亡及Bax蛋白表达效果优于过表达miR-545-3p组(P<0.05)。miR-545-3p微泡组裸鼠移植瘤瘤重显著低于对照组、微泡组、miR微泡组(P<0.05)。结论超声微泡介导miR-545-3p可通过下调HOXA3抑制CRC细胞增殖、侵袭,诱导其凋亡,为临床治疗CRC提供新的给药方式及潜在靶点,为CRC分子靶向治疗技术提供依据。展开更多
Micro ribonucleic acids(miRNAs)are a highly conserved class of single-stranded non-coding RNAs.Within the miR-545/374a cluster,miR-545 resides in the intron of the long non-coding RNA(lncRNA)FTX on Xq13.2.The precurso...Micro ribonucleic acids(miRNAs)are a highly conserved class of single-stranded non-coding RNAs.Within the miR-545/374a cluster,miR-545 resides in the intron of the long non-coding RNA(lncRNA)FTX on Xq13.2.The precursor form,pre-miR-545,is cleaved to generate two mature miRNAs,miR-545-3p and miR-545-5p.Remarkably,these two miRNAs exhibit distinct aberrant expression patterns in different cancers;however,their expression in colorectal cancer remains controversial.Notably,miR-545-3p is affected by 15 circular RNAs(circRNAs)and 10 long non-coding RNAs(lncRNAs),and it targets 27 protein-coding genes(PCGs)that participate in the regulation of four signaling pathways.In contrast,miR-545-5p is regulated by one circRNA and five lncRNAs,it targets six PCGs and contributes to the regulation of one signaling pathway.Both miR-545-3p and miR-545-5p affect crucial cellular behaviors,including cell cycle,proliferation,apoptosis,epithelial-mesenchymal transition,invasion,and migration.Although low miR-545-3p expression is associated with poor prognosis in three cancer types,studies on miR-545-5p are yet to be reported.miR-545-3p operates within a diverse range of regulatory networks,thereby augmenting the efficacy of cancer chemotherapy,radiotherapy,and immunotherapy.Conversely,miR-545-5p enhances immunotherapy efficacy by inhibiting T-cell immunoglobulin and mucin-domain containing-3(TIM-3)expression.In summary,miR-545 holds immense potential as a cancer biomarker and therapeutic target.The aberrant expression and regulatory mechanisms of miR-545 in cancer warrant further investigation.展开更多
基金supported by the Qiantang Scholars Fund of the Hangzhou City University(No.210000-581835).
文摘Micro ribonucleic acids(miRNAs)are a highly conserved class of single-stranded non-coding RNAs.Within the miR-545/374a cluster,miR-545 resides in the intron of the long non-coding RNA(lncRNA)FTX on Xq13.2.The precursor form,pre-miR-545,is cleaved to generate two mature miRNAs,miR-545-3p and miR-545-5p.Remarkably,these two miRNAs exhibit distinct aberrant expression patterns in different cancers;however,their expression in colorectal cancer remains controversial.Notably,miR-545-3p is affected by 15 circular RNAs(circRNAs)and 10 long non-coding RNAs(lncRNAs),and it targets 27 protein-coding genes(PCGs)that participate in the regulation of four signaling pathways.In contrast,miR-545-5p is regulated by one circRNA and five lncRNAs,it targets six PCGs and contributes to the regulation of one signaling pathway.Both miR-545-3p and miR-545-5p affect crucial cellular behaviors,including cell cycle,proliferation,apoptosis,epithelial-mesenchymal transition,invasion,and migration.Although low miR-545-3p expression is associated with poor prognosis in three cancer types,studies on miR-545-5p are yet to be reported.miR-545-3p operates within a diverse range of regulatory networks,thereby augmenting the efficacy of cancer chemotherapy,radiotherapy,and immunotherapy.Conversely,miR-545-5p enhances immunotherapy efficacy by inhibiting T-cell immunoglobulin and mucin-domain containing-3(TIM-3)expression.In summary,miR-545 holds immense potential as a cancer biomarker and therapeutic target.The aberrant expression and regulatory mechanisms of miR-545 in cancer warrant further investigation.
文摘目的探讨寨卡病毒(Zika virus,ZIKV)逃避子宫成纤维细胞先天免疫的分子机制。方法使用ZIKV感染子宫成纤维细胞后,检测YWHAE-RIG-I-MAVS-IFN-β通路关键分子的表达情况。通过实时荧光定量PCR检测YWHAE mRNA的表达情况。通过高通量测序检测ZIKV感染子宫成纤维细胞后的miRNA表达谱。通过荧光素酶报告实验确定调控YWHAE的miRNA。结果ZIKV感染子宫成纤维细胞后,IFN-β的分泌和IFN-β的启动子活性水平下降(P<0.05),YWHAE、p-IRF3S396的表达水平下降,MAVS与RIG-I、RIG-I与YWHAE的相互作用水平下降。但是,YWHAE启动子活性没有显著改变(P>0.05),而YWHAE mRNA水平下降(P<0.05)。通过高通量测序发现ZIKV感染后子宫成纤维细胞miRNA的表达发生改变。其中hsamiR-15a-5p、hsa-miR-6780b-3p、hsa-miR-6845-3p、hsa-miR-16-5p、hsa-miR-590-3p、hsa-miR-575、hsa-miR-4674、hsamiR-371a-5p、hsa-miR-4642、hsa-miR-4676-5p的表达差异在5倍以上。子宫成纤维细胞中过表达上述差异miRNA后,miR-545-3p能够降低YWHAE mRNA的表达水平(P<0.05)。敲低miR-545-3p后,YWHAE m RNA和蛋白的表达水平上升(P<0.05)。此外,miR-545-3p靶向YWHAE的3端非编码区(P<0.05)。ZIKV感染子宫成纤维细胞的同时过表达miR-545-3p后,YWHAE mRNA水平显著下降(P<0.05)、IFN-β分泌水平显著下降(P<0.05)、ZIKV复制水平均显著上升(P<0.05);但是,ZIKV感染子宫成纤维细胞的同时敲低miR-545-3p后,YWHAE m RNA水平显著上升(P<0.05)、IFN-β分泌水平显著上升(P<0.05)、ZIKV复制水平均显著下降(P<0.05)。结论ZIKV通过miR-545-3p靶向YWHAE促进了自身的复制。
