KMT2D Regulates The Proliferation Ability Of Gastric Cancer Cells Through The PTEN/PI3K/AKT Pathway

Background:Gastric cancer(GC)is one of the most common malignant tumors in the world;it is ranked fifth and third for morbidity and mortality,respectively.Incidence of GC varies widely among different countries,but it has a particularly negative effect in less developed countries and especially in China.Surgical intervention and chemotherapy are effective measures for patients with GC.Owing to the national screening program and progress made in gastrectomy and chemotherapy,the survival of GC patients has improved significantly.However,its mortality remains relatively high in China,mostly as a result of the accumulation of genetic mutations and epigenetic alterations.Moreover,the efficacy of traditional treatments remains very limited,'especially for advanced GC.Hence,there is an urgent need to identify novel potential treatment targets that drive tumorigenesis.Histone lysine methyltransferase 2D(KMT2D/MLL2),is one of the most significant epigenetic effectors,responsible for catalyzing the aminomethylation of H3K4(H3K4mel).The KMT2D gene exhibits high mutation frequency in some types of cancer.At the same time,KMT2D acts as an enhancer for numerous genes and thus plays a significant role in regulating several signaling pathways,including the p53,cAMP-mediated,and cholestasis pathways.Importantly,KMT2D can effectivelyinhibit the occurrence and metastasis of lymphomas.In contrast,in solid tumors,such as colorectal cancer,breast cancer,prostate cancer,and pancreatic adenocarcinoma,KMT2D is indispensable for the growth of tumor cells.Indeed,knocking out the KMT2D gene has been shown to inhibit tumor growth and strengthen chemotherapeutic sensitivity.These findings demonstrate that KMT2D might act as a novel tumor marker,and a possible chemotherapeutic target that inhibits tumor cell growth.While its significance has been established for other cancers,the relationship between KMT2D and the development of GC remains unknown.We conceived and designed the present study to explore the clinical significance of KMT2D in GC.To further u,nderstand the mechanism by which KMT2D is involved in carcinogenesis,we examined the influence of KMT2D on proliferation and on the protease and tensin homolog(PTEN)/phosphoinositide-3-kinase(PI3K)/AKT serine/threonine kinase 1(AKT)pathway in GC cell lines.Part I Expression characteristics and clinical significance of KMT2Din Gastric cancer tissuesObjectiveTo detect the expression level of KMT2D both in gastric cancer tissue and its para cancerous normal tissue,then analysis its clinical implications.Method1.110 cases of gastric cancer tissue samples were obtained from patients who underwent surgery from January 2008 to January 2011.And every postoperative tissue samples were diagnosed by two experienced pathologists.2.Real-time quantitative polymerize chain reaction(RT-qPCR)was applied to detect the expression level of KMT2D in cancer tissues and para cancerous normal tissues of 110 GC patients.3.Immunohistochemistry was used to detect the expression level of KMT2D in cancer tissues and para cancerous normal tissues of 110 GC patients.Result1.Compared with para cancerous normal tissues,KMT2D in the GC tissues showed higher expression(30/40,75%,P<0.05).2.The high expression of KMT2D was closely association with the tumor size(P= 0.005),TNM stages(P = 0.004),Lymphatic infiltration(P = 0.006)and lymphatic metastasis(P<0.001).3.Immunohistochemistry staining revealed that the KMT2D protein,which localized primarily to the nucleus,was significantly over-expressed in GC tissue4.From the Kaplan-Meier analysis,the median t median overall survival time of GC patients with higher KMT2D expression had a shorter survival,including both overall survival(P = 0.025)and disease-free survival(P = 0.005).ConclusionsIn GC cancer tissues,we found that the expression of KMT2D was up-regulated,which was closely correlated to the tumor size,TNM stages,Lymphatic infiltration and lymphatic metastasis.Therefore,KMT2D had a promising application for predicting poor prognosis of GC patients.Part II The effects of KMT2D expression on proliferation abilities of GC cancer cell linesObjectiveTo detect the effects of KMT2D on cell proliferation of GC.Method1.The expression levels of KMT2D in the GC cell lines(MKN45,BGC823,MGC803,SGC-7901,and HGC-27 cell lines)and normal gastric epithelial cells(GES-1)were detected by Western blotting.2.We knocked down the expression of KMT2D in SGC-7901 and MKN45cells by lentivirus transfection.Then,we screened the cell lines with stable low expression ofKMT2D by puromycin.3.Cell Counting Kit-8(CCK-8),Plate clone formation assay and in vitro animal study were applied to detect the cell proliferation ability of SGC-7901 and MKN45cells after the depletion of KMT2D.4.After inhibiting the expression of KMT2D by siRNA,cell apoptosis rates of SGC-7901 and MKN45cells were analyzed by flow cytometry.5.After inhibiting the expression of KMT2D by siRNA,cell cycle of SGC-7901 and MKN45cells was analyzed by flow cytometry.6.The migration abilities of SGC-7901 and MKN45cells were analyzed by Transwell assays.7.Markers associated with the PTEN/PI3K/AKT pathway were evaluated by western blotting after the depletion of KMT2D.Result1.Western blot results showed that,the expression level of KMT2D in SGC-7901 and MKN45cell lines showing the highest expression(P<0.05).2.The expression of KMT2D in the SGC-7901 and MKN45cell lines decreased significantly after transfection with KMT2D specific siRNA-lentivirus.3.CCK-8,Plate clone formation assay and in vitro tumorigenicity study consistently all showed that the cell proliferation of SGC-7901 and MKN45cell lines was dramatically inhibited after knocking down the expression of KMT2D.4.The down-regulation of KMT2D induced cell apoptosis at a greater extent compared with that in the control group.5.After knocking down the expression of KMT2D by siRNA,SGC-7901 and MKN45cell lines were both arrested in G2/M phase.6.After the expression of KMT2D was down-regulated,the migration and metastasis abilities of SGC-7901 and MKN45cell lines were significantly inhibited,while the expression of EMT pathway was significantly inhibited.ConclusionsDown-regulating the expression of KMT2D could inhibit cell proliferation,induce apoptosis and arrested more cells at G2/M phages.Our findings indicate that KMT2D might be considered as a crucial molecular target for GC patients.