RIN1过表达对人胃癌细胞MKN28增殖的影响研究

Effects of RIN1 Overexpression on Proliferation of Human Gastric Adenocarcinoma Cell Line MKN28

[目的]研究RIN1过表达对人胃癌细胞MKN28增殖和细胞周期的影响。[方法]采用PCR法扩增人RIN1基因,并构建重组真核表达质粒pcDNA3.1(+)-RIN1。采用脂质体转染法将pcDNA3.1(+)-RIN1及空载体pcDNA3.1(+)转染至人胃癌MKN28细胞,G418法筛选阳性克隆;Western blot法和免疫荧光法鉴定RIN1的高表达。CCK-8法和平板克隆实验检测RIN1高表达对MKN28细胞增殖的影响。流式细胞术检测RIN1高表达对MKN28细胞周期的影响。[结果]成功构建RIN1真核表达载体,RIN1基因全长为2 353bp。G418法筛选获得高表达RIN1的MKN28克隆细胞株MKN28/RIN1-1和MKN28/RIN1-2,Western blot结果显示这两株细胞分别为MKN28/3.1的3.21倍和3.17倍。过表达RIN1基因后,MKN28细胞增殖显著性加快,克隆数显著性增加;细胞周期改变,G0/G1期细胞减少而S期和G2/M期细胞增加。[结论]RIN1基因过表达加速MKN28细胞从G1期向S期的转化促进细胞增殖;RIN1可能成为胃癌治疗的一个潜在靶点。

[Purpose]To investigate the effects of RIN1 overexpression on proliferation and cell cycle of human gastric adenocarcinoma cell line MKN28. [Methods] The full-length coding sequence（CDS） of human RIN1 gene was amplified by PCR technique and then cloned into pcDNA3.1（＋） eukaryotic expression plasmid to construct of pcDNA3.1（＋）-RIN1. The plasmid pcDNA3.1（＋）-RIN1 and pcDNA3.1（＋） were stably transfected into MKN28 cells by liposome transfection respectively,and the positive cells were selected by G418. RIN1 overexpression were identified by Western blot and immunofluorescence.The effects of RIN1 overexpression on cell proliferation of MKN28 cells were detected by CCK-8 assay and colony-forming assay. Furthermore,flow cytometry was used to analyze the effects of RIN1 overexpression on cell cycle of MKN28 cells.[Results] pcDNA3.1（＋）-RIN1 plasmid was constructed successfully,and the full-length CDS of human RIN1 is 2 353bp. RIN1-overexpression MKN28 cells MKN28 /RIN1-1 cells and MKN28 / RIN1-2 cells were obtained through G418 screening,and the quantity of RIN1 expression on MKN28 / RIN1-1 cells and MKN28 / RIN1-2 cells were 3.22 times and 3.17 times greater than MKN28/3.1 cells,respectively. The cell proliferation was significantly accelerated and colonies were dramatically increased in RIN1-overexpression MKN28 cells. The numbers of G0/G1phase cells were decreased and S and G2/M phase cells were increased in RIN1-overexpression MKN28 cells.[Conclusion] Overexpression of RIN1 in MKN28 cells promotes cell proliferation through accelerating transformation from G1to S phase,and RIN1 may be a potential target for treatment of gastric cancer.