摘要
目的研究跨膜蛋白35(T MEM35)基因在肝癌细胞生长、细胞周期中的作用,探讨跨膜蛋白TMEM35作为潜在肝癌治疗靶点的可行性。方法利用跨膜蛋白二级结构预测工具SPLIT对TMEM35蛋白的二级结构进行分析;利用实时定量多聚酶链反应技术检测TMEM35基因在肝癌患者癌组织及相应癌旁组织中的表达情况;采用化学合成干扰小RNA片段(siRNA)干扰TMEM35基因在肝癌细胞MHCC-97H、MHCC-97L中的表达,通过软琼脂克隆形成实验观察肝癌细胞非贴壁依赖性克隆生长情况,流式细胞术分析细胞周期;通过蛋白功能相互作用网络数据库STRING推测TMEM35蛋白发挥相应功能的搭档基因。结果 TMEM35为具有4个跨膜结构域的四跨膜蛋白,实时定量多聚酶链反应检测发现TMEM35基因在肝癌中表达紊乱,其中与相应癌旁组织相比较,TMEM35在14例癌组织中表达显著上调,在20例癌组织中表达显著下调(P<0.05)。针对TMEM35基因设计合成siR NAs能够特异性沉默TMEM35基因在肝癌细胞株MHCC-97H、MHCC-97L中的表达,并且肝癌细胞在软琼脂培养条件下,形成的克隆显著减少。流式细胞术分析发现干扰TMEM35基因表达,能够减缓细胞周期进展,表现为细胞G_1期增多,S期减少。结论 TMEM35基因编码具有四跨膜结构域的膜蛋白,在肝癌细胞生长中发挥重要作用。
Objective To study the role of transmembrane protein 35（TMEM35） gene in cell growth,cell cycle of hepatoma cells,and to discuss the feasibility of TMEM35 gene encoding a membrane protein as a potential therapy target of hepatoma.Methods Real-time quantitative polymerase chain reaction was performed to examine TMEM35 expression in tumor tissues and non-tumor ones of patients with liver cancer.siRNAs were chemically synthesized to interfere TMEM35 in MHCC-97H and MHCC-97L cells.Soft-agar colony formation as-say was performed to evaluate cell growth,and cell cycle was analyzed by flow cytometry.TMEM35 protein was analyzed in functional interaction networks database to predict functional interaction genes of TMEM35.Results TMEM35,as a tetraspanin,expressed abnormally in liver cancers. From 34 examined patients,TMEM35 expression increased in 14 and decreased in 20 significantly （P0.05）.Two siRNAs against TMEM35 gene were designed and synthesized to specially knockdown TMEM35 in MHCC-97H and MHCC-97L cells.TMEM35 knockdown inhibited cellular soft-agar colony formation in MHCC-97H and MHCC-97L cells.And flow cytometry analysis revealed that TMEM35 knockdown by RNA interference could result in G_1 arrest and S decrease. Conclusion TMEM35 gene,which encodes a tetraspanin,plays an important role in cell growth of hepatoma cells.
出处
《兰州大学学报（医学版）》
CAS
2012年第3期17-22,26,共7页
Journal of Lanzhou University（Medical Sciences）
基金
上海市科委自然科学基金(11ZR1425300)
国家自然科学基金(81101875)
关键词
RNA干扰
四跨膜蛋白
肝癌
细胞周期
RNA interference
tetraspanin
hepatocellular carcinoma
cell cycle
