长链非编码RNA525893重组慢病毒载体的构建及其对宫颈癌细胞增殖的影响

Construction of recombinant lentivirus vector carrying lnc525893 and its effect on the proliferation of cervical cancer cells

目的:构建含长链非编码RNA525893(lnc525893)的重组慢病毒载体,观察其对宫颈癌细胞增殖的影响。方法:以人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)为模板扩增出lnc525893基因片段,并将其插入至慢病毒载体p CDH-CMV-MCS-EF1-cop GFP中,构建重组质粒p CDH-lnc525893。将重组质粒p CDHlnc525893、包装质粒ps PAX2和包膜质粒p MD2.G共同转染人胚肾上皮细胞(293 T细胞),包装含有lnc525893的重组慢病毒,采用梯度稀释法测定病毒滴度。将重组慢病毒感染宫颈癌He La细胞,通过实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)检测He La细胞中lnc525893的表达;采用细胞增殖实验(cell counting kit-8,CCK-8)检测过表达lnc525893对He La细胞增殖的影响。结果:质粒双酶切鉴定以及核酸测序比对结果证实重组质粒p CDH-lnc525893构建成功。梯度稀释法测得重组慢病毒滴度约为1×107pfu/m L。重组慢病毒感染He La细胞后,qRT-PCR结果显示细胞中lnc525893表达显著升高;CCK-8结果表明,高表达lnc525893可以抑制He La细胞的增殖。结论:成功构建含lnc525893的重组慢病毒载体,该长链非编码RNA能够抑制宫颈癌He La细胞的增殖。

Objective： To construct the recombinant lentivirus vector carrying long noncoding RNA 525893 （lne525893） , and identify its effect on the proliferation of cervical cancer cells. Methods： The sequences of lnc525893 were cloned from human umbilical vein endothelial cells （HUVECs） , and insert- ed into lentivirus vector pCDH-CMV-MCS-EFI-copGFP. Then recombinant plasmid pCDH-lne525893, package plasmid psPAX2 and envelope plasmid pMD2. G were co-transfected into the 293 T cells. Super- natant were harvested and filtered then the viral titer was determined by gradient dilution and counting cells which expressed the green fluorescent protein（GFP）. Next, lentivirus-lnc525893 and lentivirus-pC- DH were transduced into HeLa cells respectively, the expression of lnc525893 was detected by quantita- tive real-time PCR（qRT-PCR）. Cell counting kits assays were performed to identify its effect on the pro- liferation of HeLa cells. Results： The recombinant plasmid carrying 1nc525893 was successfully construc- ted. The recombinant lentiviral titer was 1 107pfu/mL. The expression of lnc525893 in inc525893-He- La cells was significantly higher than pCDH-HeLa cells. In addition, overexpression of lnc525893 in He- La cells inhibited the proliferation of HeLa cells. Conclusion： The recombinant lentivirus carryinglnc525893 was successfully constructed and lnc525893 could inhibit the proliferation of HeLa cells.