miRNA-372-3p在胃癌组织中的表达及其对胃癌细胞生物学功能的影响

Expression of miRNA-372-3p in gastric cancer tissues and its effect on the biological function of gastric cancer cells

目的探讨miRNA-372-3p(miR-372-3p)在胃癌组织及细胞中的表达及其对RAB22A表达的调控,并探讨其对胃癌细胞生物学功能的影响。方法使用实时荧光定量聚合酶链反应(RT-PCR)检测山西省肿瘤医院2018年12月至2019年12月70例胃癌确诊患者癌组织和癌旁组织中miR-372-3p的表达水平。在胃癌MGC-803和SGC-7901细胞中转染miR-372-3p抑制剂,并以miR-372-3p NC(空载体)作为对照;分别使用克隆形成实验、CCK-8法和流式细胞术检测细胞克隆形成能力、细胞增殖能力和凋亡水平。采用双荧光素酶报告基因实验检测MGC-803细胞miR-372-3p和RAB22A表达的相关性。以miRNA-21(miR-21)作为阴性对照,采用蛋白质印迹法检测MGC-803、SGC-7901细胞RAB22A蛋白的表达。结果RT-PCR结果显示,与癌旁组织比较,胃癌组织中miR-372-3p相对表达量上调,差异具有统计学意义(0.51±0.37比0.77±0.48,t=1.98,P=0.005)。不同肿瘤长径和病理分级的胃癌组织中miR-372-3p表达水平差异均有统计学意义(均P<0.05)。体外实验表明,低表达miR-372-3p能抑制MGC-803细胞和SGC-7901细胞克隆的形成[(211±4)个比(410±5)个,t=2.78,P=0.001;(244±8)个比(423±7)个,t=2.76,P=0.001],降低MGC-803和SGC-7901细胞增殖活性(MGC-803细胞48 h吸光度值0.39±0.06比0.57±0.03,t=3.18,P=0.01;MGC-803细胞72 h吸光度值0.50±0.05比0.81±0.06,t=2.78,P<0.01;SGC-7901细胞72 h吸光度值:0.50±0.09比0.79±0.09,t=2.77,P=0.01),提高MGC-803细胞早期凋亡率[(25.19±0.26)%比(20.02±0.04)%,t=4.30,P<0.05]。双荧光素酶报告基因实验发现,同miR-372-3p NC与RAB22A野生型基因共转染相比,miR-372-3p抑制剂与RAB22A野生型基因共转染后,MGC-803细胞荧光素酶活性下降,差异有统计学意义(1.00±0.04比0.53±0.06,t=3.18,P=0.01);蛋白质印迹法结果显示,敲低miR-372-3p能抑制MGC-803、SGC-7901细胞RAB22A蛋白表达。结论胃癌组织miR-372-3p表达上调,miR-372-3p可能促进RAB22A的表达并调控胃癌的发生发展,可作为潜在的治疗靶标。

Objective To investigate the expression of miRNA-372-3p(miR-372-3p)in gastric cancer tissues and cells and the regulation of RAB22A protein as well as its effect of miR-372-3p on the biological function of gastric cancer cells.Methods The expression levels of miR-372-3p in cancer tissues of 70 patients clinically diagnosed with gastric cancer and the pericarcinomatous tissues were detected by using real-time quantitative polymerase chain reaction(RT-PCR)from Shanxi Provincial Cancer Hospital between December 2018 and December 2019.miR-372-3p inhibitor was transfected in gastric cancer MGC-803 and SGC-7901 cells,and miR-372-3p NC(empty vector)was used as the control.The colony formation,proliferation and apoptosis of gastric cancer cells were detected by using the clone formation assay,CCK-8 method and flow cytometry,respectively.Dual-luciferase reporter gene assay was used to detect the expression correlation between miR-372-3p and RAB22A of MGC-803 cells.miRNA-21(miR-21)was treated as the negative control,and Western blot was used to detect the expression of protein RAB22A in MGC-803 and SGC-7901 cells when miRNA-21(miR-21)was treated as the negative control.Results RT-PCR results showed that the mRNA relative expression level of miR-372-3p was up-regulated in gastric cancer tissues compared to their adjacent normal cancer tissues,and the difference was statistically significant(0.51±0.37 vs.0.77±0.48,t=1.98,P=0.005).There were statistically significant differences in mRNA expression level of miR-372-3p in gastric cancer tissues with different tumor diameter and pathological grade(all P<0.05).The assay in vitro showed that the low expression of miR-372-3p could inhibit the clone formation of MGC-803 and SGC-7901 cells[(211±4)vs.(410±5),t=2.78,P=0.001;(244±8)vs.(423±7),t=2.76,P=0.001],cell proliferation activity(absorbance value of MGC-803 cells for 48 h:0.39±0.06 vs.0.57±0.03,t=3.18,P=0.01;absorbance value of MGC-803 cells for 72 h:0.50±0.05 vs.0.81±0.06,t=2.78,P<0.01;absorbance value of SGC-7901 cells for 72 h:0.50±0.09 vs.0.79±0.09,t=2.77,P=0.01)and increase the early apoptosis rate of MGC-803 cells[(25.19±0.26)vs.(20.02±0.04),t=4.30,P<0.05].Dual-luciferase reporter gene assay found that compared with the cotransfection of miR-372-3p NC and RAB22A wild type gene,the luciferase activity of MGC-803 cells was decreased after the cotransfection of miR-372-3p inhibitor and RAB22A wild type gene,and the difference was statistically significant[(1.00±0.04)vs.(0.53±0.06),t=3.18,P=0.01].Western blot results showed that knockdown of miR-372-3p could inhibit the expressions of MGC-803,SGC-7901 cells and RAB22A protein.Conclusion The expression of miR-372-3p in gastric cancer tissues is up-regulated,and miR-372-3p can promote the expression of RAB22A and regulate the occurrence and development of gastric cancer.It may be a potential therapeutic target.