【摘要】 目的通过慢病毒介导的干扰及过表达探讨HSP90β对人骨肉瘤细胞系MG-63侵袭的影响。方法使用慢病毒构建干扰、过表达HSP90β的质粒,通过琼脂糖电泳、测序法鉴定。构建慢病毒质粒转染H1299细胞。使用嘌呤霉素对MG-63转染细胞进行筛选,计算转染效率。实验分干扰对照组(negative control-shRNA)、HSP90β干扰组(HSP90β-shRNA)、过表达对照组(negative control-pEZ)及HSP90β过表达组(HSP90β-pEZ)。以实时荧光定量PCR、Western-blot法检测mRNA和蛋白表达水平。用Transwell小室法检测MG-63侵袭能力。结果插入片段与目的基因序列一致。嘌呤霉素MG-63细胞株最小致死浓度为3μg/mL,感染复数(multiplicity of infection, MOI)为200,感染效率为80%。HSP90β干扰效率为81.15%,过表达组HSP90βmRNA及蛋白相对表达增加(P<0.01)。干扰组HSP90βmRNA及蛋白的相对表达量下降(P<0.01)。与亲本细胞相比,shRNA-HSP90β组MG-63细胞的侵袭能力下降(P<0.01),而pEZ-HSP90β组MG-63细胞侵袭能力增强(P<0.05)。shRNA-HSP90β组的MMP2、Snail表达减少,E-cadherin表达增加,而pEZ-HSP90β组的MMP2、Snail表达增加,E-cadherin表达减少。结论干扰及过表达HSP90β的人骨肉瘤细胞系MG-63构建成功,并稳定表达;HSP90β可通过抑制E-cadherin表达增加MMP2、Snail表达,增强骨肉瘤细胞MG-63的侵袭性。更多还原

【Abstract】 Objective To examine the effects of HSP90β on the invasion of osteosarcoma cell line MG-63 based on the interference and overexpression of lentivirus mediated interference.Methods Lentivirus was used to construct interference and overexpression HSP90β plasmid.Identified by agarose electrophoresis and sequencing, H1299 cells were used to package virus for plasmids system.MG-63 transfected cells were screened by Purinemycin, and the transfection efficiency was calculated.The experiment was divided into interference control group(negative control shRNA),interference group of HSP90β(HSP90β-shRNA),overexpression control group(negative control PEZ)and overexpression group of HSP90β(HSP90β-pEZ).The mRNA and protein levels were detected by real-time PCR and Western-blot.The invasion ability of MG-63 was detected by the method of Transwell Boyden cell.Results The inserted gene fragment was identical to HSP90β.The minimal lethal concentration of Purinomycin MG-63 cell line was 3 μg/mL,the multiplicity of infection(MOI)was 200 and the infection efficiency was 80%.The interference efficiency of HSP90β was 81.15%.HSP90β mRNA and relative expression of protein increased(P<0.01) in the overexpression group.HSP90β mRNA and relative expression of protein decreased(P<0.01)in the interference group.Compared with parental cells, the invasion ability of MG-63 cells of shRNA-HSP90β group decreased(P<0.01),while that of pEZ-HSP90β group increased(P<0.01).The expression of MMP2 and Snail decreased in the shRNA-HSP90β group, while the expression of E-cadherin increased.The expression of MMP2 and Snail increased, while the expression of E-cadherin decreased in the pEZ-HSP90β group.Conclusions Interference and overexpression of HSP90β osteosarcoma cell line MG-63 is successfully constructed and stably expressed; HSP90β can increase the expression of MMP2 and snail by inhibiting the expression of E-cadherin, and promote the invasion of osteosarcoma cell line MG-63.更多还原