Construction Of ZAK Lentiviral Vector And Its Effect On Biological Function Of Breast Cancer Cells

Objective:Breast cancer is the most common malignant tumor in the women.Up to now,It seriously affects women's physical and mental health but the exact cause and pathogenesis of breast cancer is still unclear.According to recent statistics,the incidence rate is increasing year by year with the improvement of people's living standards and lifestyle changes.And it shows a trend of rejuvenation.Although the ultrasound,mammography and MRI examinations,radiotherapy,chemotherapy,endocrine therapy and immunotherapy strategies and surgical methods of breast cancer have been developed in recent years,but their survival rate has not improved significantly.So,it is important for us to in-depth study of the molecular mechanism of breast cancer,and it also has an important significance as the early diagnosis,treatment and clinical prognosis of breast cancer.MAPK(mitogen-activated protein kinase)is a serine-threonine protein kinase which widely expressed in cells.ZAK is located upstream of the MAPK signaling pathway,and its overexpression in cells activates the MAPK pathway.This pathway regulates cell proliferation and apoptosis,and abnormalities in signaling pathways can also lead to the cell growthuncontrolled and tumorigenesis.It can be seen that ZAK has a significant influence on the life process of the eukaryotic cells through the regulation of MAPK and other pathways.In the previous work,we used TCGA and Oncomine public database to analyze the expression of ZAK in breast cancer.It was found that ZAK was abnormally expressed in breast cancer,suggesting that ZAK may be involved in the development of breast cancer.However,the mechanism of ZAK in breast cancer is still unclear and needs to be further elaborated.Method:DNA was reverse transcribed from the total RNA which was extracted from 293T cells.According to the CDS sequence of ZAK,we designed the primer sequences which containing XhoI and BamHI restriction endonuclease sites.The target gene was amplified by PCR,and connected with the viral expression plasmid PLVX-IRES-Puro.Then we obtained a recombinant stable overexpressed ZAK vector,lentiviral interference ZAK-shRNA vector and empty load vector.We infected MDA-MB-231 cells and MCF-7 cells with recombinant virus particles which were packaged with 293T cells.We use puromycin to screen out stably transfected cell lines.We use Real-time PCR and Western Blot to detect the expression of mRNA and the protein of ZAK.Subsequently,real-time unlabeled cell analyzer(RTCA)was used to detect the proliferation and the migrationof breast cancer cell line ahout the two different expression of ZAK.And CCK-8 proliferation assay was used to further evaluate the relationship between ZAK and the cell growth of breast cancer.Finally,the effect of ZAK on apoptosis was detected by Annexin V/PI kit.Flow Jo software were used to investigate the mechanism of ZAK which may affect the apoptosis of breast cancer cells.Result:1.Compared with the control group,the mRNA of the over expression of ZAK was significantly up-regulated in the MDA-MB-231 cell line and the MCF-7 cell line,which are all statistically different from the control group(P<0.0001).The mRNA of the interfere of ZAK of the MDA-MB-231 cell line and the MCF-7 cell line were all significantly down-regulated,and all of them had statistically significant(P<0.05).2.The proliferative capacity of MDA-MB-231 cell line and MCF-7 cell line,overexpressing ZAK was significantly higher than that of the control group(P<0.001),and the mobility was significantly enhanced(P<0.001);theinterfere of ZAK of the proliferative capacity of the two breast cancer cell lines was significantly lower than that of the control group(P<0.001),and the mobility was also significantly decreased(P<0.01).3.The apoptotic rate of MDA-MB-231 cell line and MCF-7 cell line,overexpressing ZAK was significantly lower than that of the control group(P<0.001);and two breast cancer cells interfering with ZAK expression the apoptotic rate of the strain was significantly higher than that of the control group(P<0.001).Conclusion:1.ZAK overexpression promoted the proliferation and migration of breast cancer cells,while inhibited apoptosis of breast cancer cells.2.ZAK silencing inhibited the proliferation and migration of breast cancer cells,while promoted apoptosis of breast cancer cells.