融合蛋白SMP30-IP10真核表达质粒的构建及其对肝癌细胞迁移、侵袭、增殖的影响

Construction of eukaryotic expression plasmid containing fusion protein SMP30-IP10 and its effect on migration,invasion,and proliferation of liver cancer cells

目的构建融合蛋白SMP30-IP10真核表达质粒,并初步探究其对肝癌细胞株SK-hep1迁移、侵袭及增殖的影响。方法将肝癌细胞株SK-hep1分为两组,实验组转染p IRES-SMP30-IP10质粒,对照组转染空质粒p IRES;通过重叠PCR方法构建p IRES-SMP30-IP10真核表达质粒;采用脂质体转染法转染肝癌细胞SK-hep1;Western blotting法检测蛋白表达;Transwell法检测细胞的迁移和侵袭能力;CCK8法检测细胞增殖能力。结果通过PCR、双酶切及测序鉴定证实成功构建了真核表达质粒p IRES-SMP30-IP10,并在肝癌细胞株SK-hep1中正确表达。与对照组相比,实验组细胞迁移、侵袭能力下降(P均<0.05),但细胞形态及增殖能力均差异无统计学意义(P均>0.05)。结论成功构建了融合蛋白SMP30-IP10真核表达质粒,融合蛋白SMP30-IP10可抑制肝癌细胞的迁移、侵袭能力,但对细胞的增殖能力影响不明显。

Objective To construct the eukaryotic expression plasmid of fusion protein SMP30-IP10,and to investigate its effects on migration,invasion,and proliferation of hepatoma cell line SK-hep1. Methods The SK-hep1 cells were divided into two groups： the experimental group which was transfected with p IRES-SMP30-IP10 plasmid,and the control group which was transfected with empty p IRES. The eukaryotic expression plasmid p IRES-SMP30-IP10 was constructed by overlapping PCR,and then we transfected the plasimd into hepatoma cell line SK-hep1 by liposome transfection; the protein expression of cells was detected by Western blotting; the migration and invasion of cells were detected by Transwell assay; the proliferation of cells was detected by CCK8. Results The eukaryotic expression plasmid p IRES-SMP30-IP10 was successfully constructed and approved by PCR,double restriction enzyme digestion and DNA sequencing,and was expressed in hepatocellular carcinoma cell line SK-hep1. Compared with the control group,the migration and invasion were inhibited to some extent in the experimental group（ both P〈0. 05）,but the morphology and proliferation had no significant change（ both P〉0. 05）. Conclusion The fusion protein SMP30-IP10 eukaryotic expression plasmid is successfully constructed,and it can inhibit the migration and invasion abilitiesof hepatoma cells,but has no effect on the cell proliferation.