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目的:检测卵巢肿瘤中自噬相关基因5(Atg5)的表达情况,公共数据库分析Atg5在mRNA表达水平与患者预后的相关性。研究人卵巢癌细胞系SKOV3中Atg5下调对细胞增殖的影响。方法:收集卵巢肿瘤患者卵巢肿瘤标本共30例,对其进行Atg5的免疫组织化学染色,检测其在不同卵巢肿瘤患者中的表达情况。应用公共数据库Kaplan-Meier plots对多组卵巢癌患者mRNA表达谱分析,预测Atg5的表达水平对患者预后的影响。SKOV3细胞分为两组,阴性对照组(转染阴性对照siRNA)和实验组(转染Atg5 siRNA)。通过蛋白印记法检测、CCK-8法及克隆形成实验检测细胞转染siRNA后Atg5的表达及增殖情况。结果:免疫组织化学检测卵巢肿瘤组织Atg5表达情况,卵巢癌组阳性率(75.00%)较交界性卵巢肿瘤(40.00%)及良性卵巢肿瘤(20.00%)表达水平高,且卵巢癌组与良性卵巢肿瘤组差异有统计学意义(P<0.05)。公共数据库KaplanMeier plots表达谱芯片分析Atg5 mRNA表达水平对患者预后的影响,结果显示高表达Atg5组较低表达组预后差,差异有统计学意义〔(P=3.1e-05,HR=1.37(1.18,1.58)〕。对人卵巢癌细胞系SKOV3进行反义siRNA干扰,干扰后细胞采用CCK8法分别在不同时间点检测细胞的增殖能力,结果阴性对照组和转染Atg5 siRNA组(实验组)A450值在24 h、48 h、72 h对照组及实验组A450值分别为1.179±0.036、1.454±0.040、1.828±0.059;0.640±0.039、1.032±0.054 1.146±0.040,实验组较阴性对照组明显降低,差异有统计学意义(均P<0.01)。结论:免疫组织化学法检测Atg5在卵巢癌中表达与良性卵巢肿瘤及交界性卵巢肿瘤相比明显升高,对公共数据库mRNA表达谱芯片的分析显示,Atg5高表达可能与卵巢癌的不良预后有关,反义干扰siRNA显著下调了Atg5水平表达的SKOV3细胞,其增殖能力受到明显抑制。
OBJECTIVE: To detect the expression of autophagy-related gene 5 (Atg5) in ovarian tumors and to analyze the relationship between the expression of Atg5 and the prognosis of patients in public databases. To investigate the effect of Atg5 downregulation on cell proliferation in human ovarian cancer cell line SKOV3. Methods: Totally 30 ovarian cancer patients were collected for immunohistochemical staining of Atg5 and their expression in different ovarian cancer patients. The public database Kaplan-Meier plots multi-group ovarian cancer patients mRNA expression profiling to predict the prognosis of patients with Atg5 expression levels. SKOV3 cells were divided into two groups, negative control group (transfected negative control siRNA) and experimental group (Atg5 siRNA transfected). The expression and proliferation of Atg5 were detected by Western blotting, CCK-8 assay and clonogenic assay. Results: The expression of Atg5 in ovarian cancer tissue was detected by immunohistochemistry. The positive rate of ovarian cancer (75.00%) was higher than that of borderline ovarian tumor (40.00%) and benign ovarian tumor (20.00%), The difference between the tumor group was statistically significant (P <0.05). Public database KaplanMeier plots expression profile analysis of Atg5 mRNA expression in patients with prognosis, the results showed that low expression of Atg5 high expression group had a poor prognosis, the difference was statistically significant (P = 3.1e-05, HR = 1.37 (1.18 , 1.58)] .After antisense siRNA was performed on human ovarian cancer cell line SKOV3, the cell proliferation was assessed by CCK8 assay at different time points after transfection. Results Negative control group and Atg5 siRNA group (experimental group) A450 The A450 values of the control group and the experimental group at 24 h, 48 h and 72 h were 1.179 ± 0.036, 1.444 ± 0.040 and 1.828 ± 0.059, 0.640 ± 0.039, 1.032 ± 0.054 and 1.146 ± 0.040, respectively. The experimental group was significantly lower than the negative control group (All P <0.01) .Conclusion: The expression of Atg5 in ovarian cancer was significantly higher than that in benign ovarian tumors and borderline ovarian tumors by immunohistochemistry, and the analysis of mRNA expression profiles in public databases Showed that Atg5 overexpression may be related to the poor prognosis of ovarian cancer, antisense siRNA significantly down-regulated Atg5 level of SKOV3 cells, its proliferation was significantly inhibited.