MiR-141通过其靶基因ZEB1、SIP1调控EMT途径在结直肠癌细胞系中的研究

MiR-141 through its target gene ZEB1 and SIP1 regulation of the EMT pathway in colorectal cancer cell lines

目的通过比较miR-141、靶基因、上皮-间质转化(EMT)标志物在不同结直肠癌细胞系中的差异性表达及相关性;进一步揭示MicroRNA-141通过EMT途径调控结直肠癌肝转移的机制。方法选取具有间质特征的细胞系SW620和RKO。构建含miR-141的重组LV3-p GLV-h1-GFP-puro慢病毒Lenti-miR-141,用空载慢病毒NC-Lenti作为对照;稳定转染及后续实验:通过qRT-PCR和Western Blot检测证实miR-141的成功转染,并比较miR-141、靶基因、EMT标志物在不同结直肠癌细胞系中的差异性表达。结果 miR-141的重组LV3-p GLV-h1-GFP-puro慢病毒Lenti-miR-141、NC-Lenti对细胞系SW620和RKO的最佳感染MOI值均为100;PCR检测证实LentimiR-141病毒的感染使SW620、RKO细胞中miR-141的表达明显增加;免疫组化检测证实miR-141的过表达有效的抑制了靶基因ZEB1和SIP1的表达,同时增加了E-cadherin的表达并减少了vimentin的表达。结论 SW620和RKO具有间质特征;miR-141的过表达有效地抑制了靶基因ZEB1和SIP1的表达,进而影响了EMT途径标志物E-cadherin和vimentin的表达;进一步验证了MicroRNA-141通过EMT途径调控结直肠癌肝转移。

Objective Through the comparison of the miR- 141 and target genes,EMT marker in different colorectal cancer cell lines in the expression and correlation,to reveal the mechanism of liver metastasis of colorectal cancer through the EMT pathway. Methods We selected cell lines SW620 and RKO,which have interstitial features. MiR- 141 containing recombinant LV3- p GLV- h1- GFP- puro lentivirus Lenti-miR- 141 was constructed,with a load lentivirus NC- Lenti as a control. Stable transfection and subsequent experiments by qRT- PCR and Western Blot detection demonstrated success miR- 141 transfection. MiR- 141,a target gene,was compared. EMT markers differentially expressed in different cell lines of colorectal cancer. Results Recombinant miR- 141 of LV3- p GLV- h1- GFP- puro lentivirus Lenti- miR-141,NC- Lenti cell lines SW620 and RKO best infection MOI values are： 100. PCR tests confirmed Lenti- miR- 141 viral infections make SW620,RKO cells was significantly increased expression of miR- 141（ P〈0. 001）. Immunohistochemistry demonstrated overexpression of miR- 141,which effectively inhibited target gene expression and SIP1 ZEB1,while increasing the expression of E- cadherin and reducing the expression of vimentin. Conclusion Overrepresented miR- 141 was able to effectively inhibit the expression of target genes ZEB1 and SIP1,increase the expression of E- cadherin and reduce expression of vimentin. These results showed that miR- 141 regulated colorecal liver metastasis through EMT.