| Leukemia is a kind of malignant tumor in hematopoietic system,which has the highest incidence of cancer in adolescents.There are many types of leukemia,the pathogenesis is more complex,and leukemic cells are easy to transfer rapidly,which leads to treatment failure.Therefore,in-depth study of the specific molecular mechanism of the occurrence and development of leukemia,looking for new effective treatment targets,and then provide new strategies for the treatment of leukemia,has important clinical significance and social value.Werner（WRN）DNA helicase is an important member of the RecQ family of helicases,which plays an important role in DNA replication,transcription,repair and maintenance of telomere stability,and is essential for maintaining genomic integrity and cell homeostasis.Its mechanism is mainly related to WRN damage repair of DNA.The abnormal function of WRN gene can lead to human premature senility syndrome and is prone to a variety of cancers,but its direct function in cancer biology is not clear.In this paper,we first studied the expression of RecQ family helicases in different types of leukemic cells,screened leukemic cell lines with high expression of WRN to construct WRN deficient cell lines,and studied the effects of weak expression of WRN on the growth,cycle,differentiation and senescence of leukemic cells.Combined with transcriptome and molecular biology techniques,we studied its molecular mechanism,and analyzed the molecular mechanism of WRN regulation on the occurrence and development of leukemia.Firstly,the expression of five human RecQ helicases in different types of leukemic cells was studied.RTPCR and real-time q RT-PCR were used to detect five RecQ helicase members（WRN,BLM,RECQL1,WRN,BLM,RECQL1）in six kinds of leukemia cells:K562（chronic myeloid leukemia cell）,HEL（human erythroleukemia cell）,CCRF-CEM（human acute T lymphoblastic leukemia cell）,Jurkat（human acute T lymphoblastic leukemia cell）,THP-1（human acute monocytic leukemia cell）and Raji（human lymphoma cell）.The expression of RECQ4 and RECQ5 at the transcriptional level.The protein expression of five RecQ helicase members in the above cells was detected by Western Blot.The results of RT-PCR assay showed that the expression level of WRN was higher in K562,HEL and Raji cells（p<0.05）,and lower in the other three kinds of cells;BLM was not detected in THP-1 cells,but lower expression level was detected in the other five kinds of cells,and there was no significant difference;the expression level of RECQL1 was the highest in Raji cells,the lowest in CCRF-CEM cells,and no difference in the other four kinds of cells.The expression of RECQ4 was higher in all 6 cell lines,and there was no significant difference between them.RECQ5 was not detected in 6 kinds of leukemia cells.Western Blot detection showed that the expression of WRN protein was the highest in K562 cells,lower in HEL,Raji and THP-1 cells,and not detected in the other two human acute T lymphoid leukemia cells.Low expression of BLM protein was found in K562,HEL and Raji cells,but not in the other three kinds of cells;the expression level of RECQL1protein was higher in HEL,CCRF-CEM and THP-1 cells,but lower in the other three kinds of cells;the expression level of RECQ4 protein was consistent with the transcriptional level;no expression of RECQ5 protein was detected in six kinds of leukemia cells.Through the comprehensive analysis of transcriptional and protein levels,it was found that compared with the other four helicases,the expression level of WRN helicase was significantly different in 6leukemia cells（p<0.05）,and the expression level of WRN helicase was the highest in K562cells.Therefore,in the follow-up experiment,K562 cells were selected as the research model and WRN gene as the research object.Secondly,the effects of weak expression of WRN helicase on the proliferation,cycle,differentiation and senescence of K562 cells were studied.By constructing the target vector and empty vector of short hairpin RNA（sh RNA）interfering with the expression of WRN protein,the vector was stably transfected into K562 cells by lentivirus packaging system.K562 cells with stable and weak expression of WRN and K562 cells transfected with empty vector were screened by puromycin.The expression of WRN in transfected K562 cells was detected by q RT-PCR and Western blot,the effect of weak expression of WRN on the growth of K562 cells was detected by CCK8 cell proliferation test and cell clone formation test,the changes of cell cycle and differentiation were detected by flow cytometry,the expression of cell cycle-related proteins was detected by Western blot assay,and cell senescence was detected by cell aging staining kit.The results of q RT-PCR assay showed that the expression level of WRN gene or protein in K562 cells transfected with target vector was significantly lower than that in K562cells without transfection（p<0.01）.There was no significant difference in the expression level of WRN gene in K562 cells transfected with empty vector.The results of Western blot assay were consistent with those of q RT-PCR assay,indicating that K562 cells with stable and low expression of WRN protein were successfully constructed,and the results of cell proliferation and cell clone formation assay showed that compared with the control group,the weak expression of WRN significantly inhibited the proliferation and growth of K562 cells（p<0.01）.The results of flow cytometry showed that compared with the control group,the weak expression of WRN could block the K562 cell cycle in G2/M phase（p<0.01）,and the weak expression of WRN did not affect the erythroid differentiation of K562 cells.The results of Western blot assay showed that compared with the control group,the expression of G2/M phase marker proteins CDC2 and Cyclin B1 in K562 cells with weak expression of WRN was significantly decreased（p<0.01）,indicating that the weak expression of WRN may block the cell cycle in G2max M phase by regulating the expression of CDC2 and Cyclin B1 proteins,and the results of cell senescence staining showed that the weak expression of WRN could significantly promote the senescence of K562 cells（p<0.01）.Then,the effect of weak expression of WRN on the sensitivity of K562 cells to DNA damage inducer Etoposide was studied.K562,K562 cells transfected with empty vector（sh Vector）and K562 cells with weak expression of WRN（sh WRN）were treated with DMSO（control group）and Etoposide with different concentrations（0.1,1,2,5,10μmol/L）for 24 h.Cell proliferation was detected by CCK8 assay,cell cycle,cell differentiation and cell senescence were detected by flow cytometry.Western blot assay was used to detect the expression of cell cycle and senescence-related proteins,and q RT-PCR assay was used to detect the expression of cell differentiation-related genes.The results showed that Etoposide significantly inhibited the proliferation of K562,sh WRN and sh Vector cells in a concentration-dependent manner（p<0.01）,and their IC50 values were（7.267±0.651）,（3.065±0.312）and（8.054±0.702）μmol/L,respectively.Compared with K562 cells,the IC50 value of Etoposide on sh WRN cells was significantly lower than that of K562 cells（p<0.01）,but there was no significant difference on sh Vector cells.The results of cell cycle assay showed that Etoposide blocked K562 cell cycle in G2max M phase,and the weak expression of WRN promoted the arrest of K562 cell cycle by Etoposide in G2max M phase.Flow cytometry showed that Etoposide induced erythroid differentiation of K562,sh WRN and sh Vector cells in a concentration-dependent manner,but the weak expression of WRN had no significant effect on erythroid differentiation of K562 cells induced by Etoposide.The results of Western blot assay showed that compared with the control group（DMSO）,Etoposide regulated G2/M phase related protein Cyclin B1 significantly up-regulated and CDC2 significantly down-regulated（p<0.05）.The results of q RT-PCR assay showed that the erythroid differentiation-related genes EKLF andβ-globin of the three cells treated with drugs were significantly up-regulated at the transcriptional level（p<0.01）.Compared with K562 cells,the weak expression of WRN had no significant effect on the expression of erythroid differentiation-related genes EKLF andβ-globin.The results of SA-β-gal activity assay showed that weak expression of WRN and Etoposide led to senescence of K562 cells in a concentration-dependent manner,while weak expression of WRN promoted cell senescence induced by Etoposide.Through Western blot assay,it was found that the senescence-related protein P16 was down-regulated and the senescence-related protein P21 was significantly up-regulated by Etoposide compared with the control group（DMSO）.Compared with K562 cells,the expression of P21 protein was up-regulated more significantly when WRN was weakly expressed（p<0.01）.Finally,the molecular mechanism of inhibitory effect of WRN weak expression on the growth of K562 cells was analyzed by transcriptome,and the results of transcriptome sequencing were verified by real-time fluorescence quantitative PCR and Westernblot experiments.The results of transcriptome sequencing showed that compared with the control group,there were 304 differentially expressed genes with weak expression of WRN（the difference multiple was more than 1.2）,of which 92 genes were up-regulated and 212 down-regulated.Through GO enrichment analysis,it was found that these differentially expressed genes were related to cell growth,mitotic cell cycle,osteoblast differentiation and DNA repair.Kegg pathway analysis showed that differentially expressed genes were mainly enriched in MAPK,PI3K-AKT,Calcium and other signal pathways,which were related to cell proliferation,cycle,differentiation,senescence and DNA damage repair,which were consistent with the above cell experimental results.The results of q RT-PCR assay showed that compared with the control group,WRN,HDAC9,TP53,FGFR1 and VEGFA genes were significantly down-regulated（p<0.05）,while PARP8,CASP4,IL1RL1,IL18RAP and TGFBR2 genes were significantly up-regulated（p<0.05）,which was consistent with the results of transcriptome sequencing.The results of Western blot assay showed that compared with the control group DMSO,the expression of WRN protein in K562 cells,sh Vector and sh WRN cells was down-regulated in a concentration-dependent manner（p<0.01）,and the expression level of CHK2protein in K562 cells and sh WRN cells decreased in a concentration-dependent manner.Compared with the control group（DMSO）,p53,RAD51 and MER11 proteins were significantly up-regulated in K562 and sh WRN cells（p<0.05）.The expression of RAD50protein was significantly up-regulated in K562 and sh Vector cells,and the expression of p53and MER11 protein in sh WRN cells was significantly higher than that in K562 cells at the same concentration,while the expression of Rad51 and Rad50 protein was significantly decreased in K562 cells（p<0.05）.To sum up,the expression of WRN helicase was significantly different in leukemia cells from different sources,and was highly expressed in chronic myeloid leukemia cell line K562.Weak expression of WRN could significantly inhibit the proliferation of K562 cells,block cell cycle,promote cell senescence,enhance the sensitivity of K562 cells to Etoposide,and promote cell cycle arrest,erythroid differentiation and cell senescence induced by Etoposide.The study of its molecular mechanism shows that the weak expression of WRN can regulate the expression of CDC2,Cyclin B1,P16,P21 and the key proteins in DNA damage repair pathway in K562 cells,including p53,Rad51,Rad50 and MER11.It is confirmed that the functional loss of DNA damage repair factor WRN can significantly promote the sensitivity of leukemia cells to DNA damage inducers,which provides a new idea for the treatment of leukemia and the development of targeted drugs. |
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