目的 探讨 miR-1273g-3p 调控转化生长因子 β(transforming growth factor-β,TGF-β)/mothers against decapentaplegic homolog 4(Smad4)信号通路对结直肠癌细胞生物学功能的影响.方法 采用人结肠上皮细胞株NCM460为对照,对人结直肠癌细胞株HCT116和SW480采用qRT-PCR检测细胞的miR-1273g-3p表达.采用Western blot检测TGF-β和Smad4蛋白的表达.将miR-1273g-3p抑制剂转染至HCT116和SW480细胞中(miR-1273g-3p抑制剂组),并设立转染抑制剂对照剂的空载对照(抑制剂对照组).采用qRT-PCR验证转染效果.将转染后的HCT116细胞皮下注射至BALB/c裸鼠体内.采用qRT-PCR和Western blot检测转染后细胞中TGF-β和Smad4 mRNA和蛋白的表达.采用细胞增殖和细胞毒性分析(Cell Counting Kit-8,CCK-8)、划痕实验和transwell小室侵袭实验检测细胞增殖、迁移和侵袭能力.通过BiBiServ及TargetScan网站预测miR-1273g-3p与TGF-β的相关性,并用双荧光素酶报告基因实验验证miR-1273g-3p与TGF-β的结合情况.结果 与人结肠上皮细胞NCM460比较,人结直肠癌细胞HCT116和SW480中miR-1273g-3p、TGF-β蛋白和Smad4蛋白均高表达(均P＜0.01).瞬时转染48 h后,与抑制剂对照组比较,miR-1273g-3p抑制剂组miR-1273g-3p表达降低(P＜0.01),TGF-β和Smad4的mRNA和蛋白表达均减少(均P＜0.01),细胞的增殖、迁移和侵袭能力均降低(均P＜0.01).双荧光素酶报告基因实验结果表明,miR-1273g-3p 可以靶向结合 TGF-β;SW480 细胞中,miR-1273g-3p 抑制野生型 TGF-β-3'untranslated region(UTR)质粒转染的荧光素酶活性(P＜0.01),而对突变型TGF-β-3'UTR质粒转染的荧光素酶活性无影响(P＞0.05).结论 抑制miR-1273g-3p的表达可抑制结直肠癌细胞HCT116和SW480的增殖、迁移和侵袭能力.此作用可能与TGF-β/Smad4信号通路有关.
Objective To investigate the effect of miR-1273g-3p on the biological function of colorectal cancer cells by regulating transforming growth factor-β(TGF-β)/mothers against decapentaplegic homolog 4(Smad4)signaling pathway.Methods The expression of miR-1273g-3p in the colorectal cancer HCT116 and SW480 cells was detected by qRT-PCR,and the expression of TGF-β and Smad4 proteins in the two cell lines was detected by Western blot.Human colon epithelial cells NCM460 were used as the control.miR-1273g-3p inhibitor was transfected into HCT116 and SW480 cells(miR-1273g-3p inhibitor group),and an empty control group(inhibitor control group)was set up.qRT-PCR was used to verify the transfection effect.The transfected HCT116 cells were subcutaneously injected into BALB/c nude mice.qRT-PCR and Western blot were used to detect the mRNA and protein expressions of TGF-β and Smad4,respectively,in each group of cells after transfection.The proliferation,migration and invasion of cells were detected by Cell Counting Kit-8(CCK-8),scratch assay and transwell chamber invasion assay.The correlation between miR-1273g-3p and TGF-β was predicted by BiBiServ and TargetScan website,and the binding of miR-1273g-3p to TGF-β was verified by dual-luciferase reporter gene assay.Results Compared with human colon epithelial cells NCM460,miR-1273g-3p,TGF-β protein and Smad4 protein were all highly expressed in human col-orectal cancer cells HCT1 16 and SW480(all P＜0.01).After transfection for 48 hours,compared with the inhibitor control group,the ex-pression of miR-1273g-3p in the miR-1273g-3p inhibitor group was significantly decreased(P＜0.01),the mRNA and protein expressions of TGF-β and Smad4 were significantly decreased(all P＜0.01),and the cellular proliferation,migration and invasion were significantly reduced(all P＜0.01).The results of the dual-luciferase reporter gene assay showed that miR-1273g-3p could target and bind TGF-β,and miR-1273g-3p inhibited the luciferase activity in SW480 cells transfected with wild-type TGF-β-3'untranslated region(UTR)plasmids(P＜O.01),but had no effect on the luciferase activity in SW480 cells transfected with mutant TGF-β-3'UTR plasmids(P＞0.05).Conclusions Inhibiting the expression of miR-1273g-3p can significantly inhibit the proliferation,migration and invasion of colorectal cancer cells HCT116 and SW480,which may be related to the TGF-β/Smad4 signaling pathway.
夏添明;李朝辉;王云帅;王皇建;刘大威;武会斌;韩保卫
新乡医学院,洛阳市中心医院普外科,河南洛阳 471000
结直肠癌;miR-1273g-3p;TGF-β/Smad4信号通路;细胞增殖;细胞迁移;细胞侵袭
colorectal cancer;miR-1273g-3p;TGF-β/Smad4 signaling pathway;cell proliferation;cell migration;cell invasion
《实用肿瘤杂志》 2024 (003)
219-227 / 9
河南省医学科技攻关计划(联合共建项目)(LHGJ20210866,2018020898)
10.13267/j.cnki.syzlzz.2024.034
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