The Effect Of PARP-1 On Proliferation,Migration,Invasion Of Human Breast Cancer Cell

PART 1 Construction of a stable breast cancer cell line with lentivirus-mediated RNA interference for PARP-1 gene silencingObjective This study aims to detect the expression of PARP-1 gene in different breast cell lines and screen out the dominant cell line,then construct PARP-1 lentivirus interference vector and establish a stably infected human breast cancer MDA-MB-231 cell line,lay the experimental foundation for the further study of the effect of PARP-1 on the biological behaviors of breast cancer cells.Methods The expression of PARP-1 m RNA in different breast cell lines was detected by real-time quantitative PCR,screen out the dominant cell line.According to genetic information of PARP-1 c DNA sequence provided by Gen Bank,targeted PARP-1 lentiviral vector was constructed.Both the lentiviral vectorand virus packaging plasmids were transfected into 293 T cells to produce recombinant lentivirus,which was infected into human breast cancer MDA-MB-231 cell line.The m RNA and protein expression of PARP-1 were measured by real-time quantitative PCR and Western blot.Results The m RNA expression levels of PARP-1 in MCF10 a,MCF7,MDA-MB-231 cell lines were 1.007±0.143?2.310±0.411?7.894±1.639,compared with the other two groups,the expression of PARP-1 m RNA in MDA-MB-231 was significantly increased(P = 0.000).PARP-1-RNAi lentiviral vector was successively constructed and infected into human breast cancer MDA-MB-231 cell line.The m RNA expression levels of PARP-1 in blank control group,negative control group and PARP-1 group were 1.001±0.001,1.022±0.519 and 0.166±0.041;the relative expression level of PARP-1 protein were 0.9629±0.0056,0.9425±0.008 and 0.5733±0.0106.Compared with the blank control group and negative control group,the expression of PARP-1m RNA and protein in PARP-1 group were significantly decreased(P < 0.05).Conclusions A stably low-express PARP-1 breast cancer cell line is established.Part 2 The effect of silencing PARP-1 on proliferation,migration and invasion of breast cancer MDA-MB-231 cell lineObjective This part mainly discusses the changes of proliferation,migration and invasion of breast cancer MDA-MB-231 cell line after PARP-1gene silencing.Methods The proliferation of MDA-MB-231 cells was detected by CCK-8method.The migration ability of MDA-MB-231 cells before and after transfection was detected by scratch healing experiment.The changes of invasive ability of MDA-MB-231 cells before and after transfection were detected by chamber method.Results CCK-8 experimental results show that the cell proliferation rate of PAPR-1 group was significantly reduced than that of blank control group and negative control group(P<0.05).Scratch test results show that the migration ability of PARP-1 cells decreased when compared with the blank control group and the negative control group(P <0.05).Transwell invasion experiments showed that the number of transmembrane cells in the blank control group was(150.75±19.89),the number of transmembrane cells in the negative control group was(136.67±20.14),the number of transmembrane cells in PARP-1 cells was(46.78±7.76).Indicating that when the PARP-1 gene was silenced,the invasion ability of MDA-MB-231 cell was decreased(P <0.05).The Western Blot results indicates that The protein expression levels of E-cadherin in blank control group,negative control group and PARP-1 group were 0.779±0.015?0.820±0.012 and 1.165±0.023.Compared with the blank control group and negative control group,the expression of E-cadherin protein in PARP-1 group were significantly increase.The relative expression level of p-AKT protein were1.064±0.026?1.042±0.008 and 0.641±0.031,Compared with the blank controlgroup and negative control group,the expression of E-cadherin protein in PARP-1 group were significantly decrease.Conclusions After the PARP-1 gene was silenced,the proliferation,migration and invasion of MDA-MB-231 cells were decreased.This mechanism may be associated with inhibition of PI3 K / AKT signaling pathway and thus inhibition of cell epithelial stromal transformation.