miR-221-5p通过靶向调控ALDH1A2对胃癌细胞生物学行为的影响

Effect of miR-221-5p on biological behavior of gastric cancer cells by targeting regulation of ALDH1A2

目的 探讨miR-221-5p与醛脱氢酶1家族成员A2(ALDH1A2)的靶向关系及其对胃癌细胞增殖、迁移、侵袭和上皮间质转化(EMT)的影响。方法 收集2020年7月至2022年7月于岳池县人民医院接受根治性胃癌切除术的50例胃癌患者的胃癌组织及其癌旁正常组织标本,体外培养的细胞株包括人正常胃黏膜上皮细胞系GES-1和人胃癌细胞系BGC-803、AGS、SGC-7901、BGC-823、HGC-27。实时荧光定量聚合酶链反应(qRT-PCR)检测组织和细胞中miR-221-5p、ALDH1A2 mRNA的表达,Pearson相关分析胃癌组织中二者的相关性,分析miR-221-5p表达与患者临床病理参数的关系;生物信息学预测和双荧光素酶实验验证miR-221-5p对ALDH1A2的靶向调控作用;将HGC-27细胞分为对照组、抑制物-NC组、miR-221-5p抑制物组、模拟物NC组、miR-221-5p模拟物组、miR-221-5p模拟物+pc DNA3.1组和miR-221-5p模拟物+pc DNA3.1-ALDH1A2组,转染相应的质粒;分别采用MTT法、克隆形成实验、Transwell小室实验、划痕实验、流式细胞术检测细胞的增殖、侵袭、迁移、凋亡及细胞周期分布情况;蛋白质印迹法分析细胞中EMT相关蛋白表达情况。结果 miR-221-5p在胃癌组织和细胞中显著高表达,ALDH1A2 mRNA表达显著下调,二者在胃癌组织中呈明显负相关(P<0.05);miR-221-5p表达与胃癌患者的TNM分期、肿瘤分化程度、淋巴结转移有明显相关性(P<0.05);miR-221-5p靶向抑制ALDH1A2表达;与anti-miR-NC组相比,anti-miR-221-5p组细胞的OD值、划痕愈合率、N-cadherin、Vimentin蛋白、miR-221-5p表达水平、S期细胞比例明显下降,E-cadherin、α-catenin、ALDH1A2蛋白表达水平、G0/G1期细胞比例明显升高,单克隆形成、侵袭数目明显减少(P<0.05);与miR-NC组相比,miR-221-5p组细胞获得与上述相反的结果;上调ALDH1A2表达能够逆转miR-221-5p对HGC-27细胞恶性生物学行为的影响。结论 miR-221-5p过表达可能通过靶向下调ALDH1A2表达促进胃癌细胞的增殖、迁移、侵袭、EMT和细胞周期进程,抑制细胞凋亡。

Objective To investigate the targeting relationship between miR-221-5p and aldehyde dehydrogenase-1 family member A2(ALDH1A2) and its effect on proliferation, migration, invasion and epithelial-mesenchymal transformation( EMT) of gastric cancer cells. Method Gastric cancer tissues and their adjacent normal tissues were obtained from 50 patients with gastric cancer by radical gastrectomy in Yuechi People’s Hospital from July 2020 to July 2022. The cell lines cultured in vitro included human normal gastric mucosal epithelial cell line GES-1 and human gastric cancer cell lines BGC-803, AGS, SGC-7901, BGC-823 and HGC-27. The expression of miR-221-5p and ALDH1A2 m RNA in tissues and cells was detected by q RT-PCR. The correlation between miR-221-5p expression and clinicopathological parameters in gastric cancer was analyzed by Pearson test. The targeting regulation of miR-221-5p on ALDH1A2 was verified by bioinformatics prediction and dual luciferase experiment. HGC-27 cells were divided into control group, inhibitor-NC group, miR-221-5p inhibitor group, mimic NC group, miR-221-5p mimic group, miR-221-5p mimic+ pc DNA3. 1 group and miR-221-5p mimic + pc DNA3. 1-ALDH1A2 group, and the corresponding plasmids were transfected. Cell proliferation, invasion, migration, apoptosis and cell cycle distribution were detected by MTT method, clone formation test, Transwell chamber test, scratch test and flow cytometry, respectively. The expression of EMT-related proteins in cells was analyzed by Western blot. Result The expression of miR-221-5p was significantly high in gastric cancer tissues and cells, while the expression of ALDH1A2 m RNA was significantly down-regulated in gastric cancer tissues(P< 0. 05). The expression of miR-221-5p was significantly correlated with TNM stage, tumor differentiation and lymph node metastasis in patients with gastric cancer(P<0. 05). MiR-221-5p targeted inhibition of ALDH1A2 expression. Compared with anti-miR-NC group, the OD value, scratch healing rate, E-cadherin, α-catenin protein expression and the proportion of S phase cells in anti-miR-221-5p group decreased significantly, while the expression level of E-cadherin and α-catenin protein and the proportion of G0/G1 phase cells increased significantly, while the number of monoclonal formation and invasion decreased significantly(P<0. 05). The miR-221-5p group of cells obtained the opposite results as described above when compared to the miR-NC group. Up-regulation of ALDH1A2 expression can reverse the effect of miR-221-5p on the malignant biological behavior of HGC-27 cells. Conclusion Overexpression of miR-221-5p may promote proliferation, migration, invasion, EMT and cell cycle progression, and inhibit apoptosis of gastric cancer cells through targeted down-regulation of ALDH1A2 expression.