Construction and identification of shRNA lentiviral vector targeting TPX2 gene / 基础医学与临床

ABSTRACT

Objective To construct the lentiviral RNA interference vector targeting TPX2 and to obtain the human cervical cancer HeLa cell strain stably infected by TPX2-shRNA for studying the relationship between human cervical carcinoma and TPX2 gene.Methods By targeting TPX2 gene,four double-stranded DNA hairpin structures corresponding to shRNA were designed,synthesized and connected with Pglv2-U6-Puro to construct the recombinant plasmids.Then these recombinant plasmids were transformed into DH5α competent cells.The positive clone was extracted and transfected into 293T cells for virus packages after sequenced correctly.Human cervical carcinoma HeLa cell infected by these recombinant lentiviral was screened by Puromycin,then stable cell strain was obtained.The silencing effect of TPX2 in HeLa cell was detected by RT-fluorescent quantitative PCR and Western blot.Cell cycle and cell apoptosis wer detected by Flow cytometry.Results Sequencing results confirmed that 5 lentiviral was packaged successfully.The steady cell strain transfered TPX2-shRNA was screened with 0.4 μg/mL puromycin.HeLa cells infected by recombinant lentivirus all play the gene silencing effect especially in the group of TPX2-shRNA-1.In the group of TPX2-shRNA-1,TPX2mRNA (0.21 ± 0.07) and protein (0.19 ± 0.28) rela tive expression level is lower than those in the control group (1.08±0.07) (P＜0.01) and(0.64±0.03) (P＜ 0.01)respectively;G2 and S-phase cells are higher than those in the control group (P＜0.05)and the apoptosis rate was significantly more than those in the control group (P＜0.05).Conclusions The effective TPX2 genetic interference sequence was obtained,lentiviral vectors carrying TPX2shRNA was successfully constructed,and the HeLa cell strain with TPX2 silenced was successfully screened,which lay the research foundation for the study of the role of TPX2 in cervical cancer.