内质网氧化还原酶1α表达下调对结肠癌细胞增殖、凋亡、迁移和自噬的影响

Effects of endoplasmic reticulum oxidoreduclin 1α knockdown on proliferation, apoptosis, migration and autophagy of colon cancer cells

目的 :探讨敲减内质网氧化还原酶1α(endoplasmicreticulumoxidoreduclin1α,ERO 1α)基因表达对结肠癌细胞增殖、凋亡、迁移和自噬的影响,以及其潜在的作用机制。方法:利用TCGA(e Cancer Genome Atlas)数据库检索ERO1αmRNA在结肠癌组织中的表达情况;应用蛋白质印迹法检测结肠癌细胞株SW480、RKO、SW620、HCT116和HT29中ERO1α蛋白的表达水平。分别构建含ERO1α-shRNA的重组慢病毒(shERO1α组)及其对照(shCtrl组)并感染结肠癌RKO细胞,72 h后用荧光显微镜评估两组细胞的慢病毒感染效率;采用蛋白质印迹法检测病毒感染后细胞中ERO1α的表达水平;采用FCM法检测细胞凋亡,并用蛋白质印迹法检测凋亡相关分子的表达;用Cellomics细胞计数仪检测细胞增殖,并采用平板克隆实验检测细胞克隆形成能力;采用Transwell小室法检测细胞迁移能力;最后,采用蛋白质印迹法检测细胞自噬关键分子的表达。结果 :结肠癌组织中ERO1αmRNA表达水平明显高于相应的癌旁组织(P <0.001)。结肠癌RKO细胞中ERO1α蛋白表达水平相对最高。成功构建shERO1α重组慢病毒,其对RKO细胞的感染效率超过80%。与对照组shCtrl相比,shERO1α可有效敲减ERO1α基因的表达(P <0.01)。ERO1α敲减促使细胞凋亡率升高(P <0.05),Caspase-3表达水平上调(P <0.01),Bcl-2表达水平下调(P <0.01)。ERO1α敲减使结肠癌RKO细胞的增殖、克隆形成和迁移能力均明显降低(P值均<0.01)。ERO1α敲减细胞中自噬相关蛋白LC3和Beclin 1的表达水平明显升高(P值均<0.05),而P62蛋白表达水平明显下调(P <0.05)。结论 :ERO1α在结肠癌组织和细胞株中呈高丰度表达。ERO 1α基因敲减促进结肠癌细胞凋亡,抑制细胞增殖和迁移,并促使细胞自噬水平增高,表明ERO1α在结肠癌恶性进展中发挥重要作用。

Objective: To investigate the effects of knocking down endoplasmic reticulum oxidoreduclin 1α(ERO 1α) gene expression on the proliferation, apoptosis, migration and autophagy of colon cancer cells, as wells as the possible mechanism.Methods: The Cancer Genome Atlas(TCGA) database was used to verify the expression of ERO1α mRNA in colon cancer tissues. The expression of ERO1α protein in colon cancer cell lines SW480, RKO, SW620, HCT116 and HT29 was detected by Western blotting. The recombinant lentiviruses carrying ERO1α-shRNA(shERO1α) and the control(shCtrl) were constructed and infected into colon cancer RKO cells, respectively. The fluorescence microscopy was used to evaluate the infection efficiency of lentiviruses in the two groups after 72 h. The expression level of ERO1α protein in RKO cells after infection was detected by Western blotting. The apoptosis of RKO cells was assessed by flow cytometry, and the expression levels of apoptosis-related molecules were detected by Western blotting. The proliferation of RKO cells was detected by Cellomics cell counter, and the cell cloning formation ability was detected by plate cloning formation assay. The migration ability of RKO cells was detected by Transwell chamber assay. Finally, the expression of key molecules in autophagy was detected by Western blotting.Results: The expression level of ERO1α mRNA in colon cancer tissues was significantly higher than that in the adjacent tissues(P<0.001). The expression level of ERO1α protein in RKO cells was highest among the detected colon cancer cells. The recombinant lentivirus shERO1α was constructed successfully, and its infection efficiency into RKO cells was more than 80%. Compared with the control group, shERO1α could effectively knock down the expression level of ERO 1α gene(P<0.01). ERO1α knockdown promoted cell apoptosis(P<0.05), increased Caspase-3 expression(P<0.01), and decreased Bcl-2 expression(P<0.01). The proliferation, cloning formation and migration abilities of RKO cells were decreased after ERO1α knockdown(all P<0.01). The expressions of autophogy-related molecules LC3 and Beclin 1 were increased(P<0.05), and the expression of P62 was decreased(P<0.01) in ERO1α knockdown cells.Conclusion: The ERO1α is highly expressed in colon cancer tissues and cell lines. Knockdown of ERO 1α gene can promote apoptosis, inhibit proliferation and migration, promote autophagy of colon cancer cells, suggesting that ERO1α plays an important role in the malignant process of colon cancer.