Let-7a过表达对结肠黏液腺癌细胞LS-174T增殖及侵袭性的影响

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摘要目的：研究let-7a过表达对结肠黏液腺癌LS-174T细胞增殖及侵袭性的影响。方法：构建let-7a过表达慢病毒载体并感染结肠黏液腺癌LS-174T细胞，设立空白对照组（A组，不进行病毒转染）；转染空病毒组（B组，只含有EGFP，浓度1×107 TU/L）；C1转染组（C1组，浓度1×106 TU/L）：转染hsa-Let-7a（含let-7a病毒和EGFP）；C2转染组（C2组，浓度1×107 TU/L）；C3转染组（C3组，浓度1×108 TU/L）。通过倒置荧光显微镜观察各组感染效率；RT-qPCR检测各组细胞中let-7a基因表达水平；MTT法检测各组细胞增殖活力；Transwell侵袭和划痕实验测评估细胞的侵袭和迁移能力；RT-qPCR和Western blot分析TGF-β1 mRNA和蛋白水平的表达。结果：倒置荧光显微镜观察各组细胞48 h绿色荧光表达情况，C3组荧光表达最强，感染效率可达90%以上，与初始病毒浓度成正相关（P＜0.05）；MTT检测结果显示各组OD值均随时间延长而增高，在第0、第1天，5组OD值差异无统计学意义（P＞0.05），第2、第3、第4天，与A组比，C组的OD值增幅明显降低（P＜0.05）；各组细胞let-7a基因表达水平行RT-qPCR检测结果显示，与A组比，C组let-7a的基因表达水平显著升高，且转染细胞let-7a的表达水平与含let-7a的慢病毒浓度梯度呈正相关（P＜0.05），而RT-qPCR及Western blot检测显示let-7a过表达后TGF-β1 mRNA和蛋白水平逐渐降低（P＜0.05）。侵袭和划痕实验结果表明，let-7a的过表达降低了LS-174T细胞的侵袭和迁移能力，且与初始病毒浓度成负相关（P＜0.05）。结论：Let-7a过表达对LS-174T细胞增殖活力有明显抑制作用，可通过下调TGF-β1含量而抑制细胞的侵袭和转移。

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关键词 ： Let-7a, 侵袭转移, 上皮-间质转化, 结肠黏液腺癌

Abstract：Objective: To study the effect of let-7a overexpression on the proliferation and invasiveness of colonic mucinous adenocarcinoma cell line LS-174T. Methods: The let-7a overexpression lentiviral vector was constructed and infected with colonic mucinous adenocarcinoma LS-174T cells. A blank control group was established: no virus transfection; B transfected empty virus group; C1 transfection group (concentration 1× 106 TU/L): transfection of hsa-let-7a [containing let-7a virus and enhanced green fluorescent protein (EGFP)]; C2 transfection group (concentration 1×107 TU/L); C3 transfection group (concentration 1×108 TU/L). The infection efficiency of each group was observed by fluorescence microscope. The expression of let-7a was detected by RT-qPCR. The proliferation of each groupcells was detected by MTT assay. The invasion and migration ability of cells were evaluated by Transwell invasion and scratch test. RT-qPCR and Western blot analysis of TGF-β1 mRNA and protein levels were expressed. Results: The expression of green fluorescence protein in each group was observed by inverted fluorescence microscope. The fluorescence expression of C3 transfection group was the strongest, with transfection efficiency over 90%, which was positively correlated with the initial virus concentration (P<0.05). The expression levels of let-7a in each group were detected by RT-qPCR. The results showed that the expression level of let-7a in the transfected group was significantly higher than that in the negative control group, and the expression level of let-7a in the transfected cells was positively correlated with the gradient of lentivirus concentration (P<0.05). The results of Western blot and RT-qPCR showed that the expression of TGF-β1 mRNA and protein decreased gradually after let-7a overexpression (P<0.05). The results of invasion and scratching showed that the overexpression of let-7a reduced the invasion and migration ability of LS-174T cells, and was negatively correlated with the initial virus concentration (P<0.05). Conclusion: Overexpression of let-7a can inhibit the invasion and metastasis of LS-174T cells by down-regulating the content of TGF-β1.

Key words： let-7a invasion and metastasis epithelial-mesenchymal transition colonic mucinous aden-
ocarcinoma

收稿日期: 2019-02-20

基金资助:温州市科技局科研基金资助项目（Y20140107）。

通讯作者: 刘长宝，主任医师，硕士生导师，Email：liuchangbao2008@aliyun.com。

作者简介: 黄崇杰（1987-），男，浙江温州人，主治医师，硕士。

链接本文:

https://xb.wmu.edu.cn/CN/ 或 https://xb.wmu.edu.cn/CN/Y2019/V49/I10/724

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