miR-4496靶向LAMP3在结直肠癌细胞增殖和侵袭中的作用

Role of miR-4496 targeting LAMP3 in proliferation and invasion of colorectal cancer cells

目的探讨微小RNA(miRNA)-4496在结直肠癌细胞增殖和侵袭中的作用及分子机制。方法qRT-PCR检测结直肠癌细胞株(SW480、HT-29、LoVo、HCT116)和正常肠黏膜上皮细胞(HIEC)中miR-4496表达,选择相对表达量最低的细胞为转染对象,分别转染mimic-NC(对照组)和miR-4496 mimic(实验组)。qRT-PCR检测两组细胞中miR-4496表达。采用四甲基偶氮唑蓝(MTT)实验和Transwell实验分别检测细胞的增殖和侵袭能力。生物信息学技术预测miR-4496的靶基因。双荧光素酶报告实验验证miR-4496和靶基因的靶向关系。qRT-PCR和Western blotting检测靶基因的表达。结果SW480、HT-29、LoVo、HCT116细胞中miR-4496的相对表达量均明显低于HIEC细胞(P<0.05),HCT116细胞中miR-4496的相对表达量最低(P<0.01)。实验组miR-4496的相对表达量明显高于对照组(P<0.01)。实验组吸光度值明显低于对照组(P<0.05)。对照组和实验组的侵袭细胞数分别为(78.67±8.84)个和(24.94±4.10)个,实验组侵袭细胞数明显较少(P<0.01)。miR-4496的靶基因可能是溶酶体膜蛋白3(lysosome associated membrane protein 3,LAMP3)。miR-4496可靶向结合LAMP3 mRNA(P<0.01)。与对照组相比,实验组LAMP3基因的表达明显降低(P<0.01),细胞增殖蛋白CDK7、Cyclin H蛋白表达降低,细胞侵袭蛋白Vimentin、ZEB-2蛋白表达降低。结论miR-4496可明显抑制结直肠癌细胞的增殖和侵袭,其可能的机制是通过靶向抑制LAMP3基因表达发挥作用。

Objective To explore the role of microRNA(miRNA)-4496 in the proliferation and invasion of colorectal cancer cells and its molecular mechanism.Methods QRT-PCR was used to detect the expression of miR-4496 in colorectal cancer cell lines(SW480,HT-29,LoVo,HCT116)and normal intestinal mucosal epithelial cells(HIEC).The cells with the lowest expression of miR-4496 were selected as the transfection objects,and were transfected with mimic-NC and miR-4496 mimic in control group and experimental group,respectively.QRT-PCR was used to detect the expression of miR-4496 in the two groups.MTT experiment and Transwell experiment were used to detect the abilities of cell proliferation and invasion,respectively.Bioinformatics technology was used to predict the target gene of miR-4496.The dual luciferase reporter experiment was used to verify the targeting relationship between miR-4496 and target gene.QRT-PCR and Western blotting were used to detect the expression of target gene.Results The relative expression of miR-4496 in SW480,HT-29,LoVo and HCT116 cells was significantly lower than that in HIEC cells(P<0.05),and the relative expression of miR-4496 in HCT116 cells was the lowest(P<0.01).The relative expression of miR-4496 in experimental group was higher than that in control group(P<0.01).The absorbance value in experimental group was lower than that in control group(P<0.05).The number of invasive cells in experimental group was significantly less than that in control group(24.94±4.10 vs 78.67±8.84,P<0.01).The target gene of miR-4496 may be lysosome associated membrane protein 3(LAMP3).The miR-4496 targeted LAMP3 mRNA(P<0.01).Compared with control group,the expression of LAMP3 gene in experimental group was significantly reduced(P<0.01),the expression of cell proliferation proteins CDK7 and Cyclin H was decreased,and the expression of cell invasion proteins Vimentin and ZEB-2 was decreased.Conclusion The miR-4496 can significantly inhibit the proliferation and the invasion of colorectal cancer cells by targetedly inhibiting LAMP3 gene expression.