The Effect Of SETD5 On Tumor Sternness Of Non-Small Cell Lung Cancer

Background: SET domain-containing 5(SET domain-containing 5,SETD5)is a member of the protein lysine methyltransferase family.It has been reported that SETD5 is highly expressed in various human malignant tumors,and maintains the stemness characteristics of tumors through complex mechanisms,thereby leading to the occurrence and development of tumors.Although some scholars have suggested that SETD5 is related to the disease progression of non-small cell lung cancer(NSCLC),its mechanism of regulating tumor stemness in NSCLC has not yet been clarified.Objective: To explore the expression of SETD5 in NSCLC and its effect on tumor stemness,so as to provide a new therapeutic target for the clinical treatment of NSCLC.Methods: In this study,the expression of SETD5 and tumor stemness protein in human NSCLC tissues was firstly observed by immunohistochemistry.The chi-square test was used to analyze the correlation between SETD5 and the expression of stemness-related proteins in NSCLC.Western blotting was used to detect the expression of SETD5 and stemness-related proteins in NSCLC cells(A549,H1299,H1650)and normal lung epithelial cells(BEAS-2B).The expression relationship between SETD5 and stemness-related proteins in NSCLC cells was observed by immunofluorescence staining.The GEPIA database was searched to analyze the correlation between the expression of SETD5 and stemness-related proteins in lung adenocarcinoma and lung squamous cell carcinoma.SETD5 was specifically silenced in A549 and H1299 cell lines by cell transfection experiments,and the expression of SETD5 and stemness-related proteins in A549 and H1299 cell lines was detected by western blotting.The spheroid formation experiment was further used to observe the spheroid formation on the 7th and14 th days after silencing SETD5 in A549 and H1299 cell lines.The hypoxia model of H1650 cells was established by cobalt chloride treatment for 6h and 12 h,and the expression of SETD5 and stemness-related proteins under hypoxia was examined by immunoblotting.Results:1.Immunohistochemical results showed that SETD5 was expressed in the nucleus and adjacent nucleus of non-small cell lung cancer,and the sex-determining region Y box protein9(SOX9)was expressed in the nucleus of non-small cell lung cancer.Hypoxia-inducible factor-1α(Hypoxia-inducible factor-1α,HIF1α)is mainly expressed in the nucleus,cytoplasm and adjacent nuclei of non-small cell lung cancer.2.The expression of SETD5 was significantly correlated with the expression of stemness-related protein SOX9(p<0.001).3.Both SETD5 and SOX9 were expressed in A549,H1299,H1650 and BEAS-2B cells,but the expression in non-small cell lung cancer was significantly higher than that in human normal lung epithelial cells.In non-small cell lung cancer cell samples,the expression of SETD5 and SOX9 in A549 and H1299 was significantly stronger than that in H1650.4.The results of immunofluorescence showed that SETD5 and SOX9 were co-expressed in non-small cell lung cancer cells.5.In lung adenocarcinoma and lung squamous cell carcinoma,the expression of SETD5 was positively correlated with the m RNA expression level of SOX9,and positively correlated with the m RNA expression level of HIF1α.6.After silencing SETD5 in A549 and H1299 cell lines,the expression levels of SETD5 and SOX9 decreased significantly compared with the Control group.7.After specific silencing of SETD5,the spheroid formation ability of A549 and H1299 cells was significantly reduced.8.Hypoxic conditions were induced in H1650 cells with cobalt chloride(6h and 12h).The results of western blot analysis showed that the protein expressions of SETD5,SOX9 and HIF1α increased in a time-dependent manner.Conclusion:1.SETD5 may be related to the expression of NSCLC stemness-related protein SOX9.2.SETD5 may be involved in regulating the stemness characteristics of NSCLC.