摘要
目的:研究miR-181b对人肝癌G2细胞(HepG2)中B细胞淋巴瘤/白血病-2(Bcl-2)和转录因子SP1蛋白及mRNA表达的调节作用,探讨miR-181b对HepG2细胞增殖的影响。方法:用人工合成的miR-181b相应的双链互补DNA片段,插入miRNASelectTMpEGP-miR载体中,经测序鉴定后克隆microRNA的高表达质粒;用脂质体将miR-181b高表达质粒转染进HepG2细胞,经嘌呤霉素筛选获得阳性克隆。用RT-PCR技术和Western blotting方法分别检测该细胞系中Bcl-2和SP1的mRNA和蛋白表达情况。用MTT法检测HepG2细胞增殖的变化情况。结果:Western blotting结果显示miR-181b能够减少Bcl-2和SP1蛋白质的表达,RT-PCR分析显示Bcl-2和SP1 mRNA表达减少。MTT显示转染后细胞的生长速率比未转染的细胞明显减慢。结论:miR-181b抑制HepG2肝癌细胞增殖,提示可能与其下调Bcl-2和SP1的表达有关。
AIM： To investigate the role of miR - 181 b in the expression of Bcl -2 and SP1 at mRNA and protein levels in the human hepatoma G2 cells （HepG2）, and to explore the effect of miR- 181b on the regulation of HepG2 cell proliferation. METHODS： The synthetic double - strand complementary DNA based on the sequence of miR - 181b was inserted into the vector of miRNASelectTM pEGP - miR. The microRNA high - expression plasmid was cloned, and the sequences were identified. The miR - 181b plasmid was transfected into HepG2 cells with liposomes. The stable cell line was screened by puromycin. The mRNA and protein levels of Bcl - 2 and SP1 were measured by RT - PCR and Western blotting, respectively. Methyl thiazolyl tetrazolium （MTr） method was used to analyze the proliferation of HepG2 cells. RESULTS： The Western blotting results showed that miR- 181b inhibited the protein expression of Bcl- 2 and SP1. The result of RT - PCR also indicated that the mRNA expression of Bcl - 2 and SP1 was suppressed. Compared with the con- trol, the growth rate of HepG2 with high expression of miR - 181b was significantly decreased. CONCLUSION： miR - 181b inhibits the proliferation of HepG2, which may be related to the down - regulation of Bcl -2 and SPI.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2010年第11期2106-2111,共6页
Chinese Journal of Pathophysiology
