卵巢癌中环状RNA-101748高表达的病理意义及对增殖的影响

Pathological significance of circular RNA-101748 expression in ovarian cancer and its effect on proliferation

目的探讨浆液性卵巢癌(SOC)中环状RNA-101748(circ-101748)表达的病理意义及其通过调控微小RNA(miR)-340表达对卵巢癌细胞增殖的影响。方法收集SOC病例69例。采用RNA原位分子杂交技术(RISH)检测circ-101748在浆液性卵巢癌中的阳性表达情况,统计分析circ-101748阳性表达与SOC病理特征的关系;采用四甲基偶氮唑蓝(MTT)增殖活性实验检测circ-101748对卵巢癌细胞增殖的影响;基于生物信息学预测circ-101748的下游miRNA及其下游靶蛋白;通过实时荧光定量聚合酶链反应(qRT-PCR)和双荧光素酶报告实验验证circ-101748对miR-340的调控及miR-340对Ki-67的调控作用;通过回复实验证实circ-101748/miR-340信号轴对Ki-67的调控及对卵巢癌细胞增殖的影响。结果RISH结果显示,circ-101748阳性染色为棕黄色,定位于胞质和胞核;circ-101748阳性例数占比为73.91%(51/69),高于癌旁正常组织的11.59%(8/69),差异有统计学意义(P<0.05)。在SOC中,circ-101748的阳性表达与肿瘤直径(χ^(2)=9.763,P<0.05)、肿瘤分期(χ^(2)=9.656,P<0.05)密切相关,但与年龄、病理级别、淋巴结转移无关联(P>0.05)。MTT实验表明,过表达circ-101748后卵巢癌细胞的增殖活性显著升高,而降低circ-101748表达后卵巢癌细胞的增殖活性显著降低(P<0.05)。通过circBANK预测miR-340是circ-101748的靶基因;qRT-PCR结果表明,在卵巢癌细胞中过表达circ-101748后,miR-340表达水平显著下降(P<0.05),降低circ-101748表达后,miR-340表达水平显著升高(P<0.05)。双荧光素酶报告实验证实,circ-101748能与miR-340特异性结合,并可负性调控miR-340的表达。通过TargetScan和miRbase预测增殖蛋白Ki-67是miR-340的下游靶基因,进一步双荧光素酶报告实验验证表明,野生型Ki-673′非翻译区(3′UTR)可降低miR-340的荧光素酶活性,提示miR-340通过Ki-673′UTR抑制Ki-67蛋白的表达。回复实验结果表明,过表达miR-340可以消除circ-101748对卵巢癌细胞增殖蛋白Ki-67的促进表达作用,而过表达circ-101748或降低miR-340的表达可进一步激发卵巢癌细胞增殖蛋白Ki-67的表达;circ-101748能直接抑制miR-340的表达,而miR-340能直接抑制Ki-67的表达,circ-101748/miR-340/Ki-67信号通路在卵巢癌生长和增殖中起到显著促进作用。结论在卵巢癌中circ-101748为致癌基因,可通过调控miR-340/Ki-67信号轴促进卵巢癌细胞增殖和生长。

Objective To investigate the pathological significance of circular RNA-101748(circ-101748)expression in serous ovarian cancer(SOC)and the effect of regulating microRNA(miR)-340 expression on ovarian cancer cell proliferation.Methods A total of 69 cases of SOC were collected.The positive expression of circ-101748 in serous ovarian cancer was detected by RNA in situ hybridization(RISH),and the relationship between circ-101748 positive expression and the pathological characteristics of SOC was statistically analyzed;Methyl Thiazolyl Tetrazolium(MTT)proliferation activity assay was used to detect the effect of circ-101748 on the proliferation of ovarian cancer cells;bioinformatics predicts the circ-101748 downstream microRNA and microRNA downstream target protein;quantitative real-time polymerase chain reaction(qRT-PCR)and double luciferase report experiments were used to verify the regulatory effect of circ-101748 on miR-340 and that of miR-340 on Ki-67;the rescue test was used to confirm the effects of the circ-101748/miR-340 signal axis on regulating Ki-67 and the proliferation of ovarian cancer cells.Results RISH results showed that circ-101748 positive staining was brown yellow,located in the cytoplasm and nucleus;the number of positive circ-101748 cases was 73.91%(51/69),which was higher than 11.59%(8/69)in adjacent normal tissues,and the difference was statistically significant(P<0.05).In SOC,the positive expression of circ-101748 in SOC was closely correlated with tumor size(χ^(2)=9.763,P<0.05)and tumor stage(χ^(2)=9.656,P<0.05),but not correlated with age,pathological grade or lymph node metastasis(P>0.05).MMT assay showed that the proliferative activity of ovarian cancer cells increased after overexpression of circ-101748,while the proliferative activity of ovarian cancer cells decreased significantly after decreased expression of circ-101748(P>0.05).It was predicted by circBANK that miR-340 was the target gene of circ-101748;the result of qRT-PCR showed that the expression level of miR-340 decreased significantly after overexpression of circ-101748 in ovarian cancer cells(P<0.05),and the expression level of miR-340 increased significantly after decreasing the expression of circ-101748(P<0.05);the double luciferase report experiment confirmed that circ-101748 could specifically bind to miR-340 and negatively regulate the expression of miR-340.The proliferation protein[STBX]Ki-67 was predicted to be the downstream target gene of miR-340 by TargetScan and miRbase.Further double luciferase report experiment verified that the wild type Ki-673′untranslated region could reduce the luciferase activity of miR-340,thus miR-340 could inhibit the expression of Ki-67 protein through Ki-673′UTR.The results of rescue experiment showed that overexpression of miR-340 could eliminate the effect of circ-101748 on the expression of Ki-67 in ovarian cancer cells,while overexpression of circ-101748 or reduction of miR-340 could further stimulate the expression of Ki-67 in ovarian cancer cells;the circ-101748 could directly inhibit the expression of miR-340,while miR-340 could directly inhibit the expression of Ki-67.Circ-101748/miR-340/Ki-67 signaling pathway played a significant role in promoting the growth and proliferation of ovarian cancer.Conclusion Circ-101748 is an oncogene in ovarian cancer,which can promote the proliferation and growth of ovarian cancer cells by regulating miR-340/Ki-67 signaling axis.