胎盘长链非编码RNA CTD-2012K14.6异常表达致胎儿巨大的初步功能研究

A preliminary study on abnormal expression of long noncoding RNA CTD- 2012K14.6 in placentas and its role in inducing macrosomia

目的探讨长链非编码RNA(long non-coding RNA,lncRNA)致妊娠糖尿病(gestational diabetes mellitu,GDM)巨大儿发生的作用机制。方法采用实时荧光定量PCR(qantitative real-timePCR,qRT-PCR)检测CTD-2012K14.6在GDM和正常胎盘组织中的表达量,并进行胎儿体重相关性分析;运用生物信息分析技术确定CTD-2K14.6调控的下游靶基因,并利用过表达及干扰RNA技术改变CTD-2K4.6在人滋养层细胞系中的表达,及其对下游靶基因表达的调控作用。结果与正常妊娠胎盘组织相比,CTD-2012K14.6在GDM孕妇的胎盘组织中的相对表达量明显上调(1.70±0.63对1.00±0.56,P<0.01),且与胎儿体重呈正相关,r=0.850,P<0.01;在线分析CTD-2012K14.6位于chr16:67,549,214-67,563,958,染色体位置分析发现,CTD-2012K14.6位于CTCF基因的内含子内;过表达CTD-2012K14.6可明显降低CTCF核酸与蛋白表达量,而使胰岛素样生长因子Ⅱ(IGF-Ⅱ)核酸与蛋白表达量显著升高;敲除CTD-2012K14.6显著升高CTCF核酸与蛋白表达量,而明显降低IGF-Ⅱ核酸与蛋白表达量。结论胎盘组织中异常表达的CTD-2012K14.6可能通过调控CTCF,IGF-Ⅱ影响巨大儿发生发展。

Objective To explore the role of long noncoding RNA (lncRNA) CTD-2012K14.6 in the development of gestational diabetes mellitus (GDM) related macrosomia. Methods The quantitative real-time PCR (qRT-PCR) was performed to measure the expression of CTD-2012K14.6 in placentas of women with or without GDM, and the quantity of CTD-2012K14.6 expression and its association with fetal weights were analyzed;Bioinformatic analysis was performed to predict the downstream molecules. CTD-2012K14.6 over-expressing lentiviral and siRNA was constructed in human trophoblastic cell line HTR-8/SVneo cells, qRT-PCR and Western blot (WB) were used to invest its effect in modulating the expression of downstream molecules. Results The expression of CTD-2012K14.6 in GDM placentas was significantly higher than that in normal controls (1.70±0.63 vs 1.00±0.56, t=3.68, P<0.01), and positively correlated with fetal weight (r=0.8501, P<0.01);on-line analysis showed that CTD-2012K14.6 was located at chr16: 67, 549, 214-67, 563, 958, which was located in the intron of CCCTC-binding factor(CTCF);Up-regulating CTD-2012K14.6 could significantly reduce the expression of CTCF mRNA and protein, and increase the expression of insulin-like growth factor-Ⅱ(IGF-Ⅱ) mRNA and protein, while down-regulating CTD-2012K14.6 could significantly increase the expression of CTCF mRNA and protein, and reduce the expression of IGF-Ⅱ mRNA and protein. Conclusion The CTD-2012K14.6 may play an important role in the pathogenesis of GDM related macrosomia by upregulating the expression of CTCF and IGF-Ⅱ.