The Role Of SETD5 In Regulating The Stemness Of Gastric Adenocarcinoma Through PI3K/Akt And Hedgehog Signaling Pathways

Background:SET domain-containing 5(SETD5) is a previously uncharacterized member of the protein lysine methyltransferase family.Studies have shown that SETD5 is up-regulated in prostate cancer,pancreas ductal adenocarcinoma and breast cancers.However,the expression of SETD5 in gstric adenocarcinoma(GA) and the role of SETD5 in regulating the sternness of GA cells through Phosphoinositide 3-kinase(PI3K)/serine/threonine kinase(Akt) and Hedgehog(Hh) signaling pathway has not been reported yet.Objectives:This study aimed to investigate the expression of SETD5 in GA and its clinicopathological significance.Furthermore,we investigate the role of SETD5 in regulating the sternness of GA cells through PI3K/Akt and Hh signaling pathway.Methods:Firstly,the expression of SETD5 in 177 GA tissues and 20 adjacent non-tumor gastric mucosa was detected by immunohistochemistry(IHC) method,and the correlation between SETD5 expression and clinicopathological parameters,tumor microenvironment-related factors,cancer sternness-related proteins,cell cycle-related proteins,and signal pathway-related proteins was further analyzed.Kaplan-Meier survival analysis was used to demonstrate the effective value of SETD5 in evaluating the prognosis of GA patients.Secondly,western blotting and immunofluorescent(IF) staining were used to detect the exqrression of SETD5 and cancer sternness-related proteins in different GA cell lines(MKN74,MKN28,SNU638,and AGS).Then the SETD5 gene was silenced by esiRNA transfectioa The spheroid formation assay was used to detect the changes of the spheroid formation ability of MKN28 cells after SETD5 gene was silenced.PI single staining flow cytometry,migration,and invasion assay were used to detecte the effects of SETD5 gene silencing on the cell cycle,migration,invasion ability of MKN28 and MKN74 cells.After inhibiting the expression of Akt protein in MKN28 and MKN74 cells,western blotting was used to detect the expression of PI3K/Akt signaling pathway related proteins and SETD5.After inhibiting the expression of GLI1 protein in MKN28 and MKN74 cells,western blotting was used to detect the expression of Hh signaling pathway related proteins and SETD5.Results:1.SETD5 protein is mainly located in the nuclear of GA cells and its expression in GA was higher than that in adjacent normal gastric mucosa epithelium(P<0.05).SETD5 expression was closely related to the tumor grade,lymph node metastasis,distant metastasis,and clinical stage(all P<0.05).Kaplan-Meier survival analysis showed that the positive expression of SETD5 in GA was significantly associated with a shortened overall survival(OS) compared to the negative groups(P<0.05).Univariate and multivariate cox suggested that SETD5 was an independent prognostic factor for the OS of GA patients.2.SETD5 expression was associated with the expression of Hypoxia-inducible fector-la(HIF-1?),vascular endothelial growth factor(VEGF),and higher microvessel density(MVD)(all P<0.05).3.SETD5 expression was significantly correlated with cancer stemness-related proteins LSD1,Sox9,and CD133(all P<0.05).SETD5 expression in GAcell lines(MKN74,MKN28,SNU638,AGS)were concordant with the expression of cancer stemness-related proteins.IF analysis revealed that SETD5 co-stained with LSD1,Sox9,and CD133 in MKN28 and MKN74 cells.4.Silencing of SETD5 expression in GA MKN28 and MKN74 cell lines by small interfering RNA(esiRNA)strongly inhibited the expression of stemness-related proteins LSD1,Sox9,and CD133,and cell cycle related protein p16(all P<0.05).Silencing of SETD5 expression in GA MKN28 cell lines strongly inhibited the ability of cell clonogenicity.Silencing of SETD5 expression in GAMKN28 and MKN74 cell lines inhibited the S-phase subpopulations,as well as migration and invasion ability(all P<0.05).5.SETD5 expression in GA was significantly correlated with PI3K/Akt signaling pathway related proteins pPI3K p85,pAkt-Thr308,and pAkt-Ser473(all P<0.05).Akt inhibitor Perifosine inhibited the expression of pAkt-Thr308,pAkt-Ser473,and SETD5 in MKN28 and MKN74(all P<0.05).6.SETD5 expression in GA was significantly correlated with Hh signaling pathway related proteins Smo and GLI1(all P<0.05).GLI1 inhibitor GANT61 significantly inhibited the expression of GLI1 and SETD5 in MKN28 and MKN74(all P<0.05).Conclusion:SETD5 may be a novel cancer stemness-related proteins for the prognostic evaluation of GA patients;PI3K/Akt and Hh signaling pathway may play an essential role in the regulation of SETD5.