Effect Of Camk?? On The Proliferation Of Rat Liver Cell Line BRL-3A

Objective:To investigate the effect of CaMKII? expression on the proliferation of rat liver cell line BRL-3A by constructing CaMKII? lentivirus and transfecting stably passaged rat liver cell line BRL-3A.Methods:The rat liver cell line BRL-3A stably cultured in vitro was constructed,and the lentiviral system of CaMKII? overexpression(LV?CaMKII?),knockdown(LV-CaMKII? shRNA)and corresponding blank vector was constructed and transfected into rat liver cell BRL-3A and a blank control group was established.The transfection efficiency was detected by flow cytometry.The expression of CaMKII?mRNA was detected by QRT-PCR at different time points.The expression of CaMKII? protein in different time points was detected by Western blot.The cells were detected by CCK-8 method.Growth conditions and growth curves were used to detect cell counts at different times;flow cytometry was used to detect cell cycle at different time points in each group.Results:(1)Lentivirus groups were successfully transfected into rat liver cell line BRL-3A.(2)Application of QRT-PCR detection results:On the first day after infection,the relative expression of CaMKII? mRNA in LV-CaMKII? group and CON group was significant(P<0.01),and there was no significant difference between LV-CaMKII? shRNA group and CON group(P>0.05).On the 3rd,5th and 7th day after infection,the relative expression of CaMKII? mRNA in LV-CaMKII? group was significantly higher than that in CON group(P<0.05),and LV-CaMKII? shRNA group was significantly lower than CON group(P<0.05).The relative expression of CaMKII? mRNA in CaMKII?-NC group,LV-CaMKII?shRNA-NC group and CON group was not statistically significant(P>0.05).(3)Western blot results:On the first day,there was no significant difference between the groups(P>0.05).On the third day,the relative expression of CaMKII? protein in LV-CaMKII? group and LV-CaMKKII?-NC group and LV?CaMKII? shRNA group was statistically significant(P<0.05),and the CON group was not statistically significant(P>0.05).The relative expression of CaMKII? protein in LV-CaMKIIyshRNA group and CON group was statistically significant(P<0.01)compared with LV-CaMKII?shRNA-NC group(P<0.05).On the 5th and 7th day,The relative expression of CaMKII? protein was statistically significant in the LV-CaMKII? group and the CON group(P<0.01).The LV-CaMKII? shRNA group was significantly different from the CON group(P<0.01).LV-CaMKII?-NC group and LV-CaMKII? shRNA There was no statistical significance between the NC group and the CON group(P>0.05).(4)The results of CCK-8 assay showed that the proliferation of LV-CaMKII? shRNA group was the fastest,which was statistically significant compared with CON group(P<0.05).The cell proliferation of LV-CaMKII? group was the slowest,which was statistically significant compared with CON group(P<0.05);There was no significant difference between LV-CamkII?-NC group,LV-CaMKII? shRNA-NC group and CON group(P>0.05).(5)Cell cycle results were detected by flow cytometry:In G0/G1 phase,on the 1st,3rd,5th,and 7th day after transfection,the proportion of cells in LV-CaMKII?group was significantly higher than that in CON group(P<0.05),and the proportion of cells in LV-CaMKII? shRNA group was CON.The group was statistically significant(P<0.05),and there was no statistical significance among the other groups(P>0.05).In the S phase,on the 1st,3rd,5th,and 7th day after transfection,the proportion of cells in the LV-CaMKII? shRNA group was statistically significant(P<0.01),on the first day,3 days,LV.The proportion of cells in CaMKII? group and CON group was not statistically significant(P>0.05).On the 5th and 7th day,the proportion of cells in LV-CaMKII? group was lower than that in CON group(P<0.01).In the G2/M phase,on the first day after transfection,the proportion of cells in the LV-CaMKII? shRNA group was statistically significant(P<0.01),and the proportion of cells in the LV-CaMKII? group and the CON group was not statistically significant(P<0.01).P>0.05),there was no statistical significance among the other three groups(P>0.05).On the 3rd,5th and 7th day,the proportion of cells in LV-CaMKKII? group was significantly lower than that in CON group(P<0.01).The LV-CaMKII? shRNA group was significantly different from CON group(P<0.01).Conclusions:The expression level of CaMKII? protein is closely related to the proliferation of rat liver cell line BRL-3A;knockdown of CaMKII? protein expression can promote the proliferation of rat liver cell line BRL-3A,while overexpression of CaMKII? protein inhibits the proliferation of rat liver cell line BRL-3A.