RNA干扰抑制雌激素受体α亚基对雌激素诱导人成骨样细胞护骨素表达的影响

Effect of estrogen receptor α subunit inhibited by RNA interference on expression of estrogen induced human osteoprotegerin

目的探讨RNA干扰抑制人成骨样细胞株MG63中雌激素受体α亚基(estrogenreceptorαsubunitERα)后,对17β雌二醇(E2)诱导护骨素(OPG)表达的影响。方法利用计算机辅助设计ERα特异性siRNA,体外合成siRNA基因,并将其定向克隆入真核表达载体pSilencer41CMV。以脂质体法转染ERαsiRNA载体至人的成骨样细胞株MG63中,鉴定后分别用不同浓度的雌激素或G蛋白抑制剂苏拉明干预经ERαsiRNA转染的MG63细胞或未经ERαsiRNA转染的MG63细胞,RTPCR法检测OPGmRNA的表达水平。结果双酶切法鉴定重组质粒后,RTPCR法鉴定ERαsiRNA载体可特异地抑制MG63细胞中ERα的表达。不同浓度的17βE2上调MG63细胞OPGmRNA的表达,其中以10-7mol/L的作用最强。但经ERαsiRNA载体抑制MG63细胞ERα亚基后,17βE2上调MG63细胞OPGmRNA表达的作用消失。结论17βE2主要经ERα亚基上调MG63细胞OPG的表达。

Objective To study estrogen receptor α (ERα) subunit in human osteoblast-like cell line MG63 cell inhibited by RNA interference on expression of osteoprotegerin (OPG) induced by 17β-estradiol. Methods With computer aided design, ERα specific siRNA gene was designed and synthesized in vitro, and cloned into the expression vector pSilencer 4.1-CMV. The constructed ERα siRNA was transfected into human osteoblast-like cell line MG63 and the inhibition effect of ERα siRNA on expression of ERα in MG63 cells was detected using the semi-quantitive RT-PCR. After transfected by the constructed ERα siRNA vector or not, MG63 cells were cultured with difference concentration of 17β-estradiol or/and suramin, a kind of G-protein inhibitor, the semi-quantitive RT-PCR was performed to detect the mRNA expression of osteoprotegerin gene. Results After identified recombinant plasmid with double enzyme digestion analysis, ERα specific siRNA expression vector inhibited specifically ERα expression in MG63 cells. 17β-estradiol with different concentration obviously up-regulated the expression of OPG mRNA and reached the maximal effect at 10-7 mol/L of 17β-estradiol. However, after the ERα subunits of MG63 cells were inhibited by ERα specific siRNA expression vector, the up-regulative action of OPG mRNA by 17β-estradiol also was fade away. Conclusions 17β-estradiol via ERα subunit up-regulated OPG mRNA expression.