长链非编码RNA PVT1对卵巢癌细胞的影响及其机制

Effects and Mechanism of Long Non-coding RNA PVT1 on EMT, Invasion and Migration of Ovarian Cancer Cells

目的探究长链非编码RNA(lncRNA)PVT1在卵巢癌细胞中的表达特征及其对细胞上皮-间质转化(EMT)和侵袭、迁移的影响。方法逆转录实时荧光定量PCR技术(RT-qPCR)检测正常卵巢上皮细胞IOSE80及卵巢癌细胞Skov3、ocvar3、A2780、HO8910中PVT1的表达。体外采用慢病毒介导的细胞转染技术上调A2780细胞中PVT1的表达,并干扰Skov3细胞中PVT1的表达,RT-qPCR检测PVT1过表达及干扰模型中PVT1 mRNA的表达,Transwell实验检测各组细胞的侵袭和迁移。RT-qPCR和免疫印迹法(Western blotting)检测各组细胞中U1核小核糖核蛋白SNRPA、NGF、Vimentin、ZEB-1及E-钙黏蛋白(E-cadherin)的mRNA和蛋白表达。结果与正常卵巢上皮细胞相比,各卵巢癌细胞中PVT1表达均明显升高(P<0.05),PVT1在Skov3细胞中表达水平最高,而在A2780细胞中表达相对较低(P<0.05)。在A2780细胞中成功过表达PVT1(空白组及过表达组),在Skov3细胞中成功干扰PVT1表达(对照组、干扰1组、干扰2组);过表达组侵袭及迁移细胞数量显著高于空白组,而干扰1组、干扰2组侵袭及迁移细胞数量显著低于对照组(P<0.01)。过表达组细胞SNRPA、NGF、Vimentin、ZEB-1的mRNA及蛋白表达水平显著高于空白组,E-cadherin的mRNA及蛋白表达水平显著低于空白组(P<0.01);干扰1组、干扰2组SNRPA、NGF、Vimentin、ZEB-1的mRNA及蛋白表达水平显著低于对照组,E-cadherin的mRNA及蛋白表达水平显著高于对照组(P<0.01)。结论 PVT1可通过上调SNRPA、NGF的表达,促进卵巢癌细胞的EMT过程及侵袭、迁移能力。

Objective To investigate the expression of long non-coding RNA(lncRNA) plasmacytoma variant translocation 1(PVT1) in ovarian cancer cells and its effects on cell epithelial-mesenchymal transformation(EMT), invasion and migration. Methods The expressions of PVT1 in ovarian epithelial cell IOSE80, ovarian cancer cell Skov3, ocvar3, A2780 and HO8910 were detected by reverse transcription real-time fluorescence quantitative PCR technology(RT-qPCR). Lentivirus-mediated cell transfection was used to up-regulate the expression of PVTI in A2780 cell line and interfere with the expression of PVT1 in Skov3 cell line. Then the expression of PVT1 mRNA in overexpression and interference models was detected by RT-qPCR. The invasion and migration ability of each cell line was detected by transwell assay. The expression of mRNA and protein of SNRPA, NGF, Vimentin, ZEB-1 and E-cadherin were detected by RT-qPCR and Western blotting. Results The expression levels of PVT1 in the ovarian cancer epithelial cells were significantly higher than in normal ovarian epithelial cells(P<0.05). The expression level of PVT1 was the highest in Skov3 cells, but relatively lower in A2780 cells(P<0.05). PVT1 was successfully overexpressed in A2780 cells(blank group and overexpression group). And the expression of PVT1 was successfully interfered in Skov3 cells(control group, interference group 1, interference group 2). The number of invasive and migratory cells in overexpression group was significantly higher than in blank group. The number of invasive and migratory cells in interference group 1 and 2 were significantly lower than in control group(P<0.01). Mechanism research showed that the mRNA and protein levels of SNRPA, NGF, Vimentin, ZEB-1 in overexpression group were significantly higher than in blank group, and that the mRNA and protein levels of E-cadherin were significantly lower than in blank group(P<0.01). The mRNA and protein levels of SNRPA, NGF, Vimentin, ZEB-1 were significantly lower in interference group 1 and 2 than in control group, while the mRNA and protein levels of E-cadherin were significantly higher in interference group 1 and 2 than in control group(P<0.01). Conclusion PVT1 promotes EMT, invasion and migration of ovarian cancer cells via up-regulating of SNRPA and NGF expression.