Preliminary Study On The Effect Of Silencing Nucleostemin Combined With Rapamycin On Autophagy And Apoptosis Of HL-60 Cells

Objective:In this study,expression level of nucleostemin(NS)in HL-60 cells was reduced using lentivirus transfection technology,and the aims were to investigate the effects of knocking down nucleostemin(NS)combined with rapamycin(RAPA)on autophagy and apoptosis in HL-60 cells.The results provide a theoretical basis for clinical treatment of leukemia with rapamycin and NS knockdown.Methods:(1)HL-60 cells were cultured.Knockdown of NS in HL-60 cells was acheved by lentiviral transfection of small hairpin RNA(NS-RNAi-GV248),according to the optimal transfected MOI value of HL-60 cells.The transfection efficiency was observed by fluorescence inverted microscope,and Western Blot was further used to detect the protein level of NS.(2)Flow cytometry was used to detect the apoptosis of HL-60 cells after NS silencing.Western blot was used to observe the expression of autophagy-related proteins microtubule-associated protein light chain 3(LC3)and p62,and the apoptosis-related proteins BCL-2 and Bax in HL-60 cells after NS silencing.(3)After combined with rapamycin,flow cytometry was used to detect the apoptosis of HL-60 cells in each group for 24h and 48h.Western blot was used to detect the expression of autophagy-related proteins microtubule-associated protein light chain 3(LC3)and p62,as well as the expression of apoptosis-related proteins BCL-2 and Bax in each group.(4)The gray values of protein strips were analyzed and calculated by Image J Statistical analyseis were performed using SPSS v.22.0 software.Dates are expressed as mean ± standard deviation((?)±s).T test was used to compare the two groups of independent samples.Student’s t test and one-way analysis of variance were used,and Bonferroni method was used for the further comparison between pairs.A P<0.05 was considered to indicate a statistically significant result.Results:(1)HL-60 cells were transfected with the optimal MOI=100,and Western blot results showed that the NS expression level of HL-60 cells in the NS silencing group was significantly reduced,the difference was statistically significant(P<0.05).(2)After the NS gene was silenced in HL-60 cells,flow cytometry found the apoptosis significantly increased compared with the blank control group and the negative control group(P<0.05).Western blot results showed that the expression of BCL2 increased,the expression of Bax decreased and the BCL-2/Bax ratio significantly decreased in HL-60 cells group with NS silencing(P<0.05).Autophagy related proteins LC3-II/LC3-I ratio increased(P<0.05),the expression of p62 protein decreased(P<0.05).(3)After treatment with rapamycin for 24h and 48h in each group,flow cytometry detection showed that the apoptosis of cells in each group increased significantly,and the apoptosis was more obvious at 48h,showing in obvious time-dependent.In addition,compared with other treatment groups,apoptosis was more obvious in the NS-knockdown combined with rapamycin group,and the difference was statistically significant(P<0.05).(4)Western blot results showed that compared with other treatment group,LC3-II/LC3-I ratio increased significantly(P<0.05)and P62 protein decreased(P<0.05)more obviously in NS silencing combined with rapamycin group on HL 60 cells;BCL-2 decreased and Bax increased in each experimental group,and the reduction of BCL2/Bax ratio was more obvious in the combination of rapamycin and knockdown NS(P<0.05).Conclusion:(1)After silencing NS,the apoptosis of HL-60 cells increased and the autophagy activity increased.(2)NS silencing combined with rapamycin can enhance the autophagy and apoptosis in HL-60 cells.(3)NS silencing combined with rapamycin induced apoptosis on HL-60 cells,related to the expression of proteins BCL2 and Bax.