TRIM44沉默对肝癌细胞增殖的影响及分子机制研究

目的:研究三结构域蛋白44(tripartite motif-containing protein 44,TRIM44)对肝癌细胞增殖的影响并探讨其分子机制。方法:RT-q PCR和Western blot法分别检测TRIM44在正常肝组织、肝癌组织和癌旁组织,以及永生化肝细胞和肝癌细胞系中的mRNA和蛋白表达水平;在肝癌细胞中转染靶向沉默TRIM44的shRNA,Western blot实验检测对TRIM44的沉默效果;MTS实验分析TRIM44沉默对肝癌细胞活力的影响,Ed U标记实验检测TRIM44沉默对肝癌细胞DNA合成能力的影响;软琼脂集落形成实验检测TRIM44沉默对肝癌细胞锚定非依赖生长能力的影响;Western blot法检测TRIM44沉默对哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,m TOR)总蛋白及磷酸化水平的影响;用m TOR激活剂MHY1485处理TRIM44沉默的肝癌细胞,并通过MTS实验分析肝癌细胞的活力。结果:TRIM44的mRNA和蛋白水平在肝癌组织中高于癌旁组织和正常肝组织,TRIM44的mRNA和蛋白水平在肝癌细胞系中高于永生化肝细胞;TRIM44沉默可抑制肝癌细胞的活力、DNA合成能力及锚定非依赖生长能力;TRIM44沉默可降低m TOR磷酸化水平,MHY1485拮抗TRIM44沉默对肝癌细胞活力的抑制作用。结论:TRIM44沉默可能通过下调m TOR活性抑制肝癌细胞增殖。

Effect of TRIM44 silencing on proliferation of hepatocellular carcinoma cells and its molecular mechanism

ATM: To investigate the effect of tripartite motif-containing protein 44 (TRIM44) on the proliferation of hepatocellular carcinoma (HCC) cells and to study the molecular mechanism. METHODS: The expression of TRIM44 at mRNA and protein levels in normal liver tissues, HCC tissues, adjacent nontumor liver tissues, immortalized hepatocytes and hepatoma cell lines was determined by RT-qPCR and Western blot, respectively. The silencing of TRIM44 was conducted by transfection of vector expressing shRNA targeting TRIM44 (shTRIM44) in the HCC cells, and the protein level of TRIM44 was measured by Western blot. The viability of the HCC cells was analyzed by MTS assay. The DNA synthesis of HCC cells was detected by Click-iT EdU Imaging Kit. The ability of anchorage-independent growth was determined by the method of colony formation on the soft agar. The effects of TRIM44 on the total protein and phosphorylation of mammalian target of rapamycin (mTOR) levels were measured by Western blot. The HCC cells were transfected with shTRIM44 and treated with mTOR agonist MHY1485, and the cell viability was analyzed by MTS assay. RESULTS: The mRNA and protein levels of TRIM44 in the HCC tissues were significantly higher than those in the adjacent nontumor liver tissues and normal liver tissues. In addition, the mRNA and protein levels of TRIM44 in the hepatoma cell lines were significantly higher than those in the immortalized hepatocytes. TRIM44 silencing significantly inhibited the viability of HCC cells and reduced the abilities of DNA synthesis and anchorage-independent growth of the HCC cells. TRIM44 silencing decreased the phosphorylation level of mTOR protein. MHY1485 significantly antagonized the inhibitory effect of TRIM44 silence to the viability of HCC cells. CONCLUSION: TRIM44 silencing inhibits the proliferation of HCC cells possibly through down-regulating the activity of mTOR.