Caveolin-1RNA干扰真核表达载体的构建及其对人类胃癌细胞生物学行为的影响

Construction of Caveolin-I RNA interfering eukaryotic expressing vector and its influence on biological behavior of human gastric cancer cells

目的构建针对Caveolin-1（CAV1）基因的小干扰RNA（siRNA）真核表达载体psilence4．1-CMV-neo-CAV1并转染胃癌细胞株SGC7901，探讨siRNA抑制CAV1表达对人类胃癌细胞株SGC7901生物学行为的影响。方法根据GeneBank提供的CAVl基因核苷酸序列，设计并化学合成3对双链siRNA，瞬时转染胃癌细胞株SGC7901，通过半定量RT—PCR检测各转染细胞CAV1表达，筛选一个有效的siRNA序列。设计并合成能表达其小发卡结构RNA（shRNA）的DNA序列，构建CAV1基因的RNA干扰真核表达载体，稳定转染胃癌细胞株SGC7901，RT—PCR法鉴定SGC7901细胞中CAV1基因的表达，通过细胞生长曲线、克隆形成实验及流式细胞术检测抑制CAV1的表达对胃癌细胞生长增殖的影响。结果成功构建CAV1RNA干扰真核表达载体psilence4．1．CMV—neo—CAV1，稳定转染靶细胞后显著降低CAV1的表达，与转染空载体组比较，增生指数及集落形成率增高，差异有统计学意义（分别为t=7．98，P〈0．05；t=13．19，P〈0．01），生长速度亦增快。结论RNA干扰胃癌细胞CAV1的表达促进了胃癌细胞生长增殖能力。

Objective To design siRNA for Caveolin-1 （CAV1） and construct eukaryotic expression vector psilence4.1-CMV-neo-CAVl, which is transfected into gastric cancer cell line SGC7901 to study the effects of inhibition of CAV1 expression with siRNA on biological behavior of gastric cell line SGC7901. Methods Design and chemosynthesize three pairs of double strands siRNA based on the neucleotide sequence of CAV1 gene provided by GeneBank. The siRNAs were transient transfected into SGC7901. The CAV1 expression of transfected cells were detected respectively by semi-quantity RT-PCR to sieve a valid siRNA sequence. Design and synthesized DNA sequence which can express small hairpin RNA （shRNA）, construct RNA interfering eukaryotic expression vector of CAVI gene. The SGC7901 was stably transfected and was detected the expression of CAV1 gene by RT-PCR. The influence on the gastric cell growth and proliferation after CAV1 was inhibited was detected by cell growth curve, clone formation test and FCM. Results The CAV1 RNA interfering eukaryotic expression vector psilence4.l-CMV-neo-CAVl was successfully constructed, which can significantly reduce the expression of CAV1 after stably transfected to target cells, and in contrast to psilence4.1-CMV-neo transfection group, PI and colony for mution rate were increased（t = 7.98, P〈 0.05; t = 13.19, P〈 0.01）, the growth speed was up. Conclusion The growth and proliferation ability of tumor is remarkably accelerated by inhibiting the expression of CAVl of gastric cell by RNAi.