宫颈癌组织PVT1表达及其作用

Expression and its role of plasmacytoma variant translocation 1 in cervical cancer

目的中晚期宫颈癌患者因缺乏有效的治疗靶点而疗效较差,需从更深入的研究宫颈癌发病机制中寻找治疗靶点。有研究发现长链非编码RNA(long non-coding RNA,LncRNA)人浆细胞瘤转化迁移基因1(plasmacytoma variant translocation 1,PVT1)在胃癌、卵巢癌等多种恶性肿瘤中高表达并参与肿瘤进程,起着类似癌基因的作用。本研究拟检测LncRNA PVT1在宫颈癌组织及细胞系中的表达,并观察其对宫颈癌细胞增殖的影响。方法选取2015-08-30-2017-04-30在淄博市中心医院行手术切除的30例宫颈癌及相应癌旁新鲜组织(距癌组织>2cm)。采用RT-PCR检测癌及癌旁组织PVT1表达,并同时检测宫颈癌HeLa及SiHa细胞系中PVT1表达;参考PVT1基因序列设计2个PVT1 siRNA序列:si-PVT1-1、si-PVT1-2,及无关序列对照siRNA(NC-siRNA)。利用Lipofectamine 2000转染试剂盒将si-PVT1-1、si-PVT1-2、NC-siRNA序列分别转染宫颈癌HeLa及SiHa细胞株。分别采用CCK-8实验及集落形成实验检测下调PVT1表达后宫颈癌HeLa及SiHa细胞的增殖能力变化;Transwell小室检测迁移和侵袭能力。结果 PVT1在30例宫颈癌组织和HeLa及SiHa细胞系中表达上调。与癌旁组织(0.062±0.015)相比,宫颈癌组织中PVT1(0.115±0.031)的表达上调,差异有统计学意义,t=3.92,P<0.05。HeLa和SiHa细胞中PVT1的相对表达量高于正常宫颈Ect1/E6E7细胞。转染PVT1siRNA后,宫颈癌HeLa及SiHa细胞的PVT1表达被抑制。CCK-8法显示与转染NC-siRNA序列组比较,宫颈癌细胞HeLa及SiHa转染PVT1 siRNA后增殖能力降低,F=112.608,P<0.001。转染si-PVT1-1(44.23±0.52)和NC-siRNA序列(115.38±1.21)的宫颈癌HeLa细胞集落形成数差异有统计学意义,F=126.328,P<0.001;转染si-PVT1-1(48.03±0.65)和NC-siRNA序列(121.15±1.51)的宫颈癌SiHa细胞集落形成数差异有统计学意义,F=118.245,P<0.001。Transwell检测结果示,和NC-siRNA组相比较si-PVT1-1组HeLa细胞迁移能力降低为0.50±0.03,侵袭能力降低为0.55±0.08,F=29.412,P<0.001;而si-PVT1-1组SiHa细胞迁移能力、侵袭能力分别降低至0.57±0.06和0.50±0.06,F=31.326,P<0.001。提示siRNA下调PVT1表达后HeLa及SiHa细胞株增殖、迁移和侵袭能力均降低。结论 PVT1在宫颈癌发挥类似癌基因作用,这可能为宫颈癌提供新的治疗靶点。

OBJECTIVE The curative effect of patients with advanced cervical cancer is very poor because there are no effective therapeutic targets.So it is necessary to further study the pathogenesis of cervical cancer in order to find therapeutic targets.More and more studies suggests that LncRNAPVT1(plasmacytoma variant translocation gene 1)is highly expressed in gastric cancer,ovarian cancer and other malignant tumors and participates in the process of cancer,playing a role similar to oncogene.This study detected the expression of LncRNA PVT1 in cervical cancer tissues and cell lines,and observed its effect on the proliferation of cervical cancer cells.METHODS Thirty cervical cancer fresh tissues and matched paracancerous tissues were obtained form patients of Central Hospital of Zibo form 2015-08-30 to 2017-04-30.RT-PCR was used to detect the expression of PVT1 in 30 cervical cancer tissues and HeLa,SiHa cell lines.Two PVT1 siRNA sequences(si-PVT1-1 and si-PVT1-2)and scramble control sequence siRNA(NC-siRNA)were designed according to the sequence of PVT1 gene.Then all sequences were transfected to HeLa and SiHa cell lines of cervical cancer using Lipofectamine 2000 according to manufacturer’s instructions.CCK-8 kit assay and colony formation assay were used to detect the proliferative ability of HeLa and SiHa cells with down-regulation of PVT1 in cervical cancer was detected.Trasnwell was used to check the migration and invasion ability of cervical cancer cells.RESULTS PVT1 was up-regulated in30 cervical cancer tissues and both cell lines.Compared with the adjacent tissues(0.062±0.015),the expression of PVT1(0.115±0.031)in cervical cancer tissues was increased(t=3.92,P<0.05).The same results were found in cervical cancer cell lines:the relative expression of PVT1 in HeLa and SiHa cells was higher than that in Ect1/E6 E7 cells.The expression of PVT1 in HeLa and SiHa cells was inhibited after transfected of PVT1 siRNA.The proliferations of HeLa and SiHa transfected PVT1 siRNA were decreased compared with that of NC-siRNA group by CCK-8(F=112.608,P<0.01).The colony forming numbers of HeLa cells transfected with si-PVT1-1 and NC-siRNA sequence were 44.23±0.52,115.38±1.21(F=126.328,P<0.01).The colony forming numbers of SiHa cells transfected with siPVT1-1 and NC-siRNA sequence were 48.03±0.65 and 121.15±1.51(F=118.245,P<0.01).Compared with NC-siRNA group,migration ability and invasive ability of HeLa cell transfected with si-PVT1-1 group decreased to 0.50±0.03 and 0.55±0.08 respectively(F=29.412,P<0.01).The numbers of SiHa cell transfected with si-PVT1-1 were 0.57±0.06 and 0.50±0.06(F=31.326,P<0.01).The proliferation,migration and invasion ability of HeLa and SiHa cells were inhibited by PVT1 siRNA.CONCLUSIONS PVT1 plays the role of carcinoid gene in cervical cancer.This may provide new therapeutic targets for cervical cancer.