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目的:探讨慢病毒介导的N-乙酰氨基葡萄糖转移酶Ⅳa(N-acetylglucosaminyltransferaseⅣa,GnTⅣa)表达下调对小鼠肝癌Hca-F细胞体外增殖及侵袭能力的影响。方法:针对GnTⅣa的编码基因Mgat4a,构建含shRNA干扰序列的慢病毒表达载体pll3.7/Mgat4a-shRNA并包装慢病毒颗粒,感染Hca-F细胞。RT-PCR及流式细胞术分别检测Mgat4a-shRNA重组慢病毒感染对Hca-F细胞Mgat4a mRNA及GnTⅣa表达的影响,CCK-8法和Transwell侵袭实验分别检测Mgat4a-shRNA重组慢病毒感染在体外对Hca-F细胞增殖和侵袭能力的影响。结果:慢病毒能够高效感染小鼠肝癌细胞Hca-F,Mgat4a-shRNA组感染效率达72%。与Hca-F细胞和Mock-shRNA组相比,Mgat4a-shRNA组细胞内Mgat4a mRNA[(0.53±0.2)vs(2.97±0.2)、(2.90±0.1);均P<0.05]和GnTⅣa[(47.80±2.25)vs(394.61±5.27)、(378.61±4.38);均P<0.05]表达显著降低;增殖能力[细胞增殖抑制率:(46.9±0.02)%vs(0.82±0.03)%、0,P<0.01]和体外侵袭能力[穿过滤膜的细胞数:(18±3)vs(42±4)、(38±3)个,均P<0.05]显著下降。结论:慢病毒介导shRNA下调GnTⅣa的表达能够抑制小鼠肝癌细胞Hca-F的增殖及体外侵袭。
AIM: To investigate the effect of lentivirus-mediated down-regulation of N-acetylglucosaminyltransferaseⅣa (GnTⅣa) on the proliferation and invasion of mouse hepatoma Hca-F cells in vitro. Methods: The lentiviral expression vector pll3.7 / Mgat4a-shRNA containing shRNA interference sequence was constructed based on the gene encoding GatT1a of GnTⅣa and packaged into lentiviral particles to infect Hca-F cells. The effects of Mgat4a-shRNA lentivirus infection on the expression of Mgat4a mRNA and GnTⅣa in Hca-F cells were detected by RT-PCR and flow cytometry. The expression of Mgat4a-shRNA lentivirus was detected by CCK-8 assay and Transwell invasion assay in vitro Hca-F cells proliferation and invasion ability. Results: Lentivirus could efficiently infect Hca-F mouse hepatoma cells, and the infection efficiency of Mgat4a-shRNA group was 72%. Compared with Hca-F cells and Mock-shRNA group, the expression of Mgat4a mRNA in [(0.53 ± 0.2) vs (2.97 ± 0.2), (2.90 ± 0.1), all P <0.05] and GnTⅣa [(47.80 ± 2.25) vs (394.61 ± 5.27) and (378.61 ± 4.38), respectively (all P <0.05). The proliferation rate was significantly lower than that of the control group (46.9 ± 0.02% vs 0.82 ± 0.03% <0.01] and in vitro invasive ability [the number of cells passing through the filter: (18 ± 3) vs (42 ± 4), (38 ± 3), all P <0.05]. Conclusion: The lentivirus-mediated shRNA downregulation of GnTⅣa can inhibit the proliferation and invasion of Hca-F in mouse hepatoma cells.