SH3BP4基因对肝癌细胞迁移和增殖作用的实验研究

Effects of SH3BP4 gene on the migration and proliferation of hepatocarcinoma cells

摘要:

目的：构建SH3结构域结合蛋白4（SH3-domain binding protein 4，SH3BP4）重组腺病毒载体及shSH3BP4慢病毒载体，初步探索SH3BP4对肝癌细胞迁移增殖能力的影响。方法：PCR扩增SH3BP4 cDNA序列并构建腺病毒重组质粒pAdEasy-SH3BP4，HEK293细胞包装重组腺病毒AdSH3BP4；构建SH3BP4基因短发夹型RNA干扰慢病毒载体，转染至HEK293T细胞，包装重组慢病毒shSH3BP4。采用Western blot检测SH3BP4在人正常肝脏细胞及肝癌细胞的内源性表达，将SH3BP4过表达实验部分设为3个组：Mock组、AdGFP组和AdSH3BP4组，Mock组为空白对照组，AdGFP组为感染腺病毒AdGFP的对照组，AdSH3BP4组为感染腺病毒AdSH3BP4的实验组；将SH3BP4敲低实验部分设为2个组：shControl组和shSH3BP4组，shControl组为感染慢病毒shPLL3.7的对照组，shSH3BP4组为感染慢病毒shSH3BP4的实验组。通过Transwell和划痕实验检测SH3BP4过表达和敲低对肝癌细胞迁移能力的影响；MTS和直接克隆形成实验检测SH3BP4过表达和敲低对肝癌细胞的增殖能力的影响。结果：经限制性内切酶酶切及DNA测序验证，成功构建AdSH3BP4重组腺病毒载体和shSH3BP4慢病毒载体。Transwell结果表明，AdSH3BP4组（74.800±0.544）相较于空白对照组（219.467±2.640）与AdGFP组（210.533±8.498），迁移的细胞数明显减少（P=0.000）；而shSH3BP4组（191.467±3.906））比shControl组（91.733±2.763）细胞的迁移数目明显增加（P=0.000）。划痕实验结果表明，AdSH3BP4组的细胞修复率[（10.400±0.036）%]明显低于空白对照组[（37.500±0.063）%]与AdGFP组[（50.000±0.063）%]（P=0.000）；而shSH3BP4组[（55.00±0.05）%]明显高于shControl组[（23.3±0.029）%]（P=0.000）。MTS结果表明：AdSH3BP4组在96 h的吸光度值（0.921±0.053）与空白对照组（1.091±0.043）及AdGFP组（1.062±0.024）相比明显降低（P=0.005）。shSH3BP4组在96 h的吸光度值（1.497±0.020）与shControl对照组（1.142±0.103）相比明显升高（P=0.004）。直接克隆形成实验结果表明，AdSH3BP4组的细胞克隆形成数（34.333±2.082）相较于空白对照组（86.333±4.726）与AdGFP组（87.333±1.528）明显减少（P=0.000），而shSH3BP4组（65.000±6.557）却明显高于shControl组（31.000±2.000）（P=0.001）。上述实验结果说明SH3BP4过表达能显著抑制肝癌细胞的迁移、侵袭和增殖能力，而SH3BP4敲低能明显促进肝癌细胞的迁移、侵袭和增殖能力。结论：SH3BP4可抑制肝癌细胞迁移、侵袭和增殖，可能为肝癌的诊治提供一个新的靶标。

Abstract:

Objective：To investigate the effect of SH3BP4 on the migration and proliferation of hepatocarcinoma cells by the construc－tion of SH3BP4 recombinant adenovirus vector and lentiviral vector. Methods：The SH3BP4 cDNA sequence was amplified and then the recombinant Adplasmid pAdTrack-SH3BP4 was con－structed. The recombinant adenovirus Ad-SH3BP4 was packaged and amplified by using HEK293 cells. The short hairpin RNA recombinant lentiviral vector of SH3BP4 was constructed and transfected into HEK293T cells，then the recombinant lentivi-ruses shSH3BP4 was packaged and amplified. The endogenous expression of SH3BP4 in normal human hepatocytes and hepa－tocarcinoma cells was examined by the Western blot. The SH3BP4 overexpression experiment was divided into three groups：mock group（the blank control group），AdGFP group（the control group of those were infected with adenovirus AdGFP） and AdSH3BP4 group（the experimental group of those were infected with adenovirus AdSH3BP4）；the SH3BP4 knockdown experiment was divided into two groups，shControl group，the control group of those were in－fected with lentiviruses PLL3.7 vector，and shSH3BP4 group，the experimental group of those were infected with lentiviruses shSH3BP4. The effect on the migration of hepatocarcinoma cells was observed by the Transwell and wound healing assay，and the effect on the proliferation was observed by the MTS and colony formation assay. Results：A recombinant adenovirus AdSH3BP4 and a recombinant lentiviral shSH3BP4 were successfully constructed，verified by the restriction endonuclease and DNA sequencing. The Transwell assay showed that the number of cell migration in AdSH3BP4 group（74.800±0.544） significantly decreased compared with that in mock group（219.467±2.640） and AdGFP group（210.533±8.498）（P=0.000）. In contrast，the number of cell migration in shSH3BP4 group（191.467±3.906） increased significantly compared with that in shControl group（91.733±2.763）（P=0.000）. The wound healing assay showed that the cell repair rate in AdSH3BP4 group[（10.400±0.036）%] was lower than that in mock group[（37.500±0.063）%] and AdGFP group[（50.000±0.063）%]（P<0.000），but it was significantly higher in shSH3BP4 group[（55.00±0.05）%] compared with that in shControl group[（23.300±0.029）%]（P=0.000）. The MTS assay showed that the absorbance value at 96 h in AdSH3BP4 group（0.921±0.053） decreased significantly compared with that in black control group（1.091±0.043） and AdGFP group（1.062±0.024），while it was significantly higher in shSH3BP4 group（1.497±0.020） compared with that in shControl group（1.142±0.103）（P=0.004）. The colony formation assay showed that the number of clone forming cells in AdSH3BP4 group（34.333±2.082） significantly decreased compared with that in mock group（86.333±4.726） and AdGFP control group（87.333±1.528）（P=0.000），but it was significantly higher in shSH3BP4 group（65.000±6.557） compared with that in shControl（31.000±2.000）（P=0.001）. Conclusion：SH3BP4 can inhibit the migration，invasion and proliferation of hepatocarcinoma cells，and it may provide a new target for the diagnosis and treatment of liver cancers.