Effects Of Targeted Knocdown Nucleostemin And Combined With Autophagy Modulators On Biological Characteristics Of HL-60 Leukemia Cells

Objective:It is proposed to construct p53 null HL-60 leukemia cells targeting knockdown nucleostemin(NS,also known as GNL3)by CRISPR/Cas9 technology.We investigated the effects of knockdown NS on proliferation,apoptosis,differentiation and autophagy activity of HL-60 cell,to further study the possible effects of NS non-p53 signaling pathway on the autophagic activity of HL-60 cells,and clarify its biological significance.Through the combination of autophagy modulators rapamycin(RAPA)and 3-methyladenine(3-MA),we analyzed the effect of autophagy activity on biological characteristics related to knockdown NS in HL-60 cells.Which laid the foundation for the study of targeted therapy strategy for acute leukemia based on inhibiting NS signaling pathway and enhancing autophagy.Methods:(1)Human myeloid leukemia cell line HL-60 was cultured.Using HL-60 cell genome as template,NS gene was amplified by PCR,and the amplifications were Sanger sequencing and analyzed in comparison with NCBI sequence.(2)Targeted small guide RNA(sg RNA)was designed,and then we selected the highest editing efficient sg RNA through the in vitro and intracellular studies.The single cells were isolated by flow cytometry.Survival monoclonal cell lines were collected for Sanger sequencing and we observed the changes of NS gene.(3)QRT-PCR and western blot were used to detect the expression of NS m RNA and protein level in HL-60 cells.(4)MTT colorimetry and flow cytometry were used to detect the effects of knockdown NS on proliferation,apoptosis and cell cycle of HL-60 leukemia cells.The expression of microtubule-associated protein 1 light chain 3(LC3)protein and P62 protein were detected by using western blot,which reflected the changes of autophagic activity.(5)HL-60 cells were treated with RAPA and 3-MA with different concentrations.The effects of knockdown NS and combined with autophagy modulators on the proliferation of HL-60 cells were measured by MTT method.Cell apoptotic rate were detected by flow cytometry.(6)The gray values of target protein were analyzed and calculated by using Image J image processing software.The datas were analyzed by SPSS 22.0 statistical software and were expressed as x ± s.One-way ANOVA was used to compare the multigroup independent samples.Bonferroni method was used to further compare the two groups,and the calibration test level was ??=0.0167.T test was used to compare the two independent samples.The difference was statistically significant at P<0.05.Results:(1)Sanger sequencing results showed that the NS gene sequence of HL-60 cells was identical with that of NCBI.(2)The gene editing ability of sg RNA 3-5 is superior to other sg RNAs.The obtained monoclonal cell lines were sequenced,however,monoclonal cell lines that completely deleted NS gene was not obtained.Two heterozygous mutant cell lines were selected for subsequent experiments.The results of q RT-PCR and western blot showed that the expression of NS m RNA and protein was significantly decreased(P < 0.05)in two heterozygous mutant HL-60 cell lines.(3)The results of MTT assay showed that the proliferation ability of knockdown groups was significantly decreased,compared with the control group.Flow cytometry showed that the apoptotic rate increased in knockdown groups.Cell cycle analysis showed that the percentage of G1 phase cells increased and the percentage of S phase cells decreased in knockdown groups.Compared with the control group,the difference was statistically significant(P < 0.05).(4)Werstern blot analysis showed that the LC3 II/LC3 I ratio in knockdown groups were higher than that in control group(P < 0.05),and the relative expression of P62 protein was lower compared with control group(P < 0.05).(5)HL-60 cells were treated with RAPA at different concentrations for 24 hours.MTT assay showed that cell proliferation was inhibited in each group,and there was a dose-dependent effect.Flow cytometry showed that the apoptotic rate increased with the increase of rapamycin concentration.(6)HL-60 cells were treated with 3-MA at different concentrations for 24 hours.MTT assay showed that the proliferation of cells was inhibited in a certain concentration range,and there was a dose-dependent effect.Flow cytometry showed that there was no significant change in apoptotic rate within the concentration range(P > 0.05).Conclusion:(1)Down-regulation of NS in human acute myeloid leukemia HL-60 cells can inhibit cell proliferation,induce cell apoptosis,block cell cycle in G0/G1 phase,and enhance autophagic activity.NS may be a potential target for the treatment of acute myeloid leukemia.(2)RAPA-activated autophagy can inhibit the growth and proliferation of HL-60 cells,induce autophagic cell death,and significantly enhance the effect of knockdown NS on proliferation and apoptosis.To some extent,inhibiting autophagy by 3-MA can slow the growth and proliferation of HL-60 cells,but has no significant effect on cell apoptosis.This study lays a foundation for the study of targeted therapy strategy based on inhibiting NS signaling pathway and enhancing autophagy in acute myeloid leukemia.