环状RNA circVCAN对胎盘滋养细胞增殖、侵袭及自噬的影响

Effects of circular RNA circVCAN on proliferation, invasion and autophagy of placental trophoblast cells

目的探讨环状RNA circVCAN(hsa-circ-0073239)在胎儿生长受限(fetal growth restriction, FGR)胎盘组织中的表达、功能及潜在作用机制。方法收集重庆医科大学附属第二医院妇产科2018年1月~2018年12月收治的12例FGR及12例正常孕妇的胎盘组织标本,采用实时荧光定量PCR检测胎盘组织中circVCAN的表达,培养人胎盘滋养细胞系HTR8/Svneo,用circVCAN干扰及过表达质粒分别转染HTR8/Svneo细胞,采用EdU、流式细胞仪、Transwell实验检测细胞增殖、凋亡、侵袭能力的变化,用蛋白质印迹法检测自噬相关蛋白LC3Ⅱ/LC3Ⅰ、ATG5、ATG7的表达情况。结果 circVCAN、ATG5、ATG7及LC3Ⅱ/LC3Ⅰ在FGR胎盘组织中的表达均较正常组显著增高(P<0.05)。FGR组孕妇终止妊娠孕周较正常妊娠组早(P<0.05),FGR胎儿出生体质量、胎盘质量显著轻于正常妊娠组(P<0.01,P<0.05),FGR胎儿出生身长明显短于正常胎儿(P<0.01)。沉默circVCAN后HTR8/Svneo细胞增殖、侵袭能力增强(P<0.01),过表达circVCAN后HTR8/Svneo细胞增殖、侵袭能力减弱(P<0.01),凋亡差异均无统计学意义(P>0.05)。沉默circVCAN的HTR8/Svneo细胞LC3Ⅱ/LC3Ⅰ、ATG5、ATG7表达均降低(P<0.05),过表达circVCAN的HTR8/Svneo细胞LC3Ⅱ/LC3Ⅰ、ATG5、ATG7表达均升高(P<0.05)。结论 circVCAN在FGR胎盘组织中呈高表达,circVCAN对凋亡无影响,但可抑制胎盘滋养细胞的增殖、侵袭,可能通过促进ATG5、ATG7的表达而增强自噬。

Objective To investigate the expression, function and potential mechanism of circular RNA circVCAN(also known as hsa-circ-0073239)in fetal growth restriction(FGR) placenta. Methods The placenta tissue samples were collected from 24 pregnant women(12 with FGR and 12 normal pregnancy) admitted in our hospital from January to December 2018. Real-time quantitative PCR(RT-qPCR) was performed to detect the expression of circVCAN in these placenta samples. Human placental trophoblast cell line HTR8/SVneo was cultured and then transfected with circVCAN interference and overexpression plasmid, respectively. EdU assay, flow cytometer and Transwell assay were carried out respectively to verify the variations in the proliferation, apoptosis and invasion abilities of the cells. And the expression of autophagy related proteins LC3Ⅱ/LC3Ⅰ, ATG5 and ATG7 was measured by Western blotting. Results The expression levels of circVCAN, ATG5, ATG7 and LC3Ⅱ/LC3Ⅰwere significantly higher in FGR placenta tissue than in the normal group(P<0.01). The termination of pregnancy was earlier in FGR patients than in normal group(P<0.05), with newborns from the FGR group having lower birth body weight(P<0.01) and placenta weight(P<0.05) as well as shorter birth body length(P<0.01) when compared with those from the normal group. Silencing of circVCAN resulted in enhanced proliferation and invasion abilities in HTR8/SVneo cells(P<0.01), while circVCAN overexpression led to decreased proliferation and invasion abilities in the cells(P<0.01), but no statistical difference was seen in the apoptotic rate(P>0.05). The expression levels of autophagy related proteins LC3Ⅱ/LC3Ⅰ, ATG5 and ATG7 were significantly lower in circVCAN interfered cells(P<0.05), while higher in circVCAN overexpressed cells(P<0.05). Conclusion CircVCAN is highly expressed in FGR placenta tissue, though it showing no effect on apoptosis but inhibiting the proliferation and invasion of HTR8/SVneo trophoblast cells, and may enhance autophagy by promoting the expression of ATG5 and ATG7.