hSmarca2-b转录变体编码的hBRM-b异构体促进Hep-3B细胞凋亡

摘要

Objective To explore whether the hBRM-b protein isoforms coded by hSmarca2-b transcript variants can promote the apoptosis of Hep-3B hepatoma cells. Methods pEGFP-N1 was used to construct five types of partial cDNAs harboring the 5′ regulatory regions and the whole open-reading-frame sequence of hSmarca2-b transcript variants so as to build the over-expressing vector of hBRM-b-EGFP fusion protein. With liposome, these vectors were separately transfected into Hep-3B cells in which hSmarca2-b variants were barely detectable. The transcribed mRNAs were confirmed by RT-PCR, while the hBRM-b-EGFP fusion proteins were confirmed by Western Blot and fluorescent imaging. The flow cytometry analysis was adopted to detect Hep-3B cells transfected with each hBRM-b-EGFP over-expressing vector, and the change of cell apoptosis rates were observed and compared with that of the control vector. Results The RT-PCR results showed that the expression level of each hSmarca2-b variant in transfected Hep-3B cells rose to a relatively high level from very low. The hBRM-b-EGFP fusion proteins conforming to the theoretical sizes were observed by Western Blot and the green fluorescence of fusion proteins were observed by the florescence imaging, which indicated the successful construction of over-expressing vectors. Compared with the control vector, the over-expression of 4 variants resulted in the Hep-3B cell apoptosis ratio increased by 100%, and the results of One-Way t Test showed that the differences were statistically significant (P<0.05). Conclusion The hBRM-b protein isoforms coded by hSmarca2-b transcript variants can promote the apoptosis of Hep-3B hepatoma cells.%目的 考察hSmarca2-b转录变体编码的hBRM-b异构体蛋白是否具有促进Hep-3B肿瘤细胞凋亡的作用.方法 利用pEGFP-N1质粒构建5种包含hSmarca2-b转录变体的5′调控序列、全长开放阅读框的部分cDNA序列以及能进一步编码产生hBRM-b-EGFP融合蛋白的过表达载体;脂质体转染过表达载体分别进入hSmarca2-b表达缺失的Hep-3B肝癌细胞,通过RT-PCR 和 Western Blot分别验证其mRNA的转录及hBRM-b-EGFP融合蛋白的表达,并通过荧光成像进一步验证融合蛋白的产生;流式检测转染hBRM-b-EGFP过表达载体后的Hep-3B细胞,考察相对于空载体组凋亡情况的改变.结果 RT-PCR显示,Hep-3B细胞转染后hSmarca2-b变体表达水平从无到较高,Western Blot检测到符合理论大小的融合蛋白,荧光成像时可见转染细胞发出的绿色荧光,证明过表达载体构建成功;相对于空载体组,其中4种变体的过表达均使凋亡细胞比例增加近100%,单侧t检验显示差异有统计学意义(P<0.05).结论 hSmarca2-b转录变体编码的hBRM-b异构体蛋白具有促进Hep-3B肝癌细胞凋亡的作用.