逆基因NANOGP8表达与人胃癌SGC-7901细胞的生物学特性相关

The expression of retrogene Nanogp8 is related to the biological characteristics of human SGC-7901 gastric cancer cells

目的探讨p EGFP-N1-NANOGP8真核表达载体及pshRNA-NANOGP8干扰质粒转染人胃癌SGC-7901细胞后,对细胞增殖、周期、凋亡、迁移、侵袭的影响。方法构建p EGFP-NI-NANOGP8真核表达载体及针对人NANOGP8基因的干扰质粒pshRNA-NANOGP8,进行酶切、测序鉴定。将测序正确的质粒在脂质体的介导下转染入SGC-7901细胞,通过荧光显微镜、反转录PCR和Western blot法检测其表达情况,CCK-8法检测SGC-7901细胞增殖情况,流式细胞术分析胃癌细胞周期变化和凋亡情况,TranswellTM实验验证迁移和侵袭能力的变化。结果成功构建出p EGEP-N1-NANOGP8及pshRNA-NANOGP8重组质粒。将重组质粒转染胃癌细胞后,能观察到GFP的表达;反转录PCR结果显示p EGEP-N1-NANOGP8转染组高表达NANOGP8 mRNA,而pshRNA-NANOGP8转染组NANOGP8 mRNA低表达。NANOGP8高表达组能促进肿瘤细胞增殖,促使细胞进入S期,迁移侵袭能力明显增强。而低表达组细胞增殖受抑制,细胞周期停留在G0/G1期,迁移、侵袭能力受抑制。结论逆基因NANOGP8的转录表达,与胃癌的增殖、细胞周期、凋亡、迁移、侵袭有密切的关系。

Objectives To explore the effects of NANOGP8 on proliferation, apoptosis, cell cycle, invasion and migration of human SGC-7901 gastdc cancer cells. Methods Eukaryotic expression vector pEGFP-NI-NANOGP8 and recombinant plasmid shRNA-NANOGP8 were constructed and confirmed by enzyme digestion and sequencing analysis. Then, vector pEGFP-N1-NANOGP8 and recombinant plasmid shRNA-NANOGP8 were trensfected into SGC-7901 cells via liposome. The expression of NANOGP8 mRNA and protein were tested by fluorescence microscopy, reverse trenscripUon PCR and Westem blotting, respectively. The proliferation of SGC-7901 cells was detected by CCK-8 assay. The apoptosis and cell cycle were examined by flow cytometry. TrenswellTM assay proved the changes in the invasion and migration abilities of SGC-7901 cells. Results The recombinant plasmids pEGEP-N1-NANOGP8 and pshRNA-NANOGP8 were constructed successfully and transfected into gastdc cancer cells., pEGEP-NI-NANOGI~ trensfection group showed high expression of NANOGPS, conversely, pshRNA-NANOGP8 transfection group showed low expression of NANOGPS. High expression of NANOGP8 significantly promoted the proliferation of tumor cells, arrested cells cycle in the S phase, decreased apoptotic cells and increased cell invasion and migration obviously. While the low expression group presented with inhibited cell proliferation, cell-cycle arrest in the G0/G1 phase, promoted cell apoptosis and inhibited migration and invasion. Conclusion The transcription and expression of retrogene NANOGP8 has a close relationship with the proliferation, cycle, apoptosis, migration and invasion of SGC-7901 gastric cancer cells.