PRMT7 Inhibits The Proliferation And Migration Of Gastric Cancer Cells By Suppressing The PI3K/AKT Pathway Via Methylation PTEN

Gastric cancer is one of the most common malignant tumors of digestive system in the world.A series of pathogenic factors have been found,such as diet,Epstein-Barr virus,Helicobacter pylori infection and chemical carcinogenesis.According to the latest data published by the International Agency for Research on Cancer(IARC),the number of newly diagnosed cases of gastric cancer has been increasing year by year,and has been close to 1.1million.Although with the development of surgical techniques and the implementation of radiotherapy,chemotherapy and neoadjuvant therapy,great progress has been made in the diagnosis and treatment of gastric cancer.However,because early gastric cancer does not cause typical clinical symptoms,most patients are diagnosed with advanced gastric cancer,and the 5-year survival rate of advanced gastric cancer after surgery is less than 25%.The latest statistics show that the number of deaths of gastric cancer is about 770,000 per year,which is a serious threat to human health.Therefore,finding diagnostic markers for early gastric cancer has profound significance for the clinical diagnosis and corresponding treatment of patients.The mechanism of the occurrence and development of gastric cancer involves many aspects,including malignant proliferation,abnormal apoptosis,invasion and metastasis of cancer cells and other pathological processes.The latest studies have shown that the formation of gastric cancer is also closely related to protein post-translational modification in epigenetics.Protein arginine methyltransferases 7(PRMT7)is one of the important members of this protein arginine methyltransferases family(PRMTs).Protein arginine methyltransferases are a key factor of protein post-translational modification.This enzyme regulates arginine methylation modification.PRMT7 widely exists in eukaryotes and mediates cell cycle regulation,chromatin remodeling,m RNA splicing and epigenetic changes,which are closely related to the occurrence and development of cancer.A number of studies have found that PRMT7 mediates tumor factors through methylation pathway to participate in the occurrence of malignant tumors such as breast cancer,liver cancer and non-small cell lung cancer.However,the biological function and mechanism of PRMT7 in gastric cancer have not been reported.The aim of this study is to explore the expression of PRMT7 in gastric cancer and the mechanism of PRMT7 involved in the regulation of gastric cancer progression,and to provide a new way and direction for the early diagnosis and treatment of gastric cancer.Phosphatase and tensin homolog(PTEN)is a major counterregulator of the PI3K/AKT signaling pathway.PI3K/AKT signaling pathway plays an important role in maintaining physiological homeostasis and has a variety of related functions such as regulating cell cycle and growth and development process.When its mutation or abnormal activation can induce the occurrence of a variety of malignant diseases,including cancer.Under a variety of extracellular stimuli,phosphatidylinositol 3-kinases(PI3Ks)step by step transmit signals to downstream related targets to activate AKT and play biological roles.Phosphatidylinositol-3,4,5-triphosphate(PIP3)acts as an important second messenger of PI3 K to terminate signal propagation to AKT and other PIP3 effector targets,while PTEN efficiently phosphorylates phosphatidylinositol-4,5-diphosphate(PIP2)to PIP3.Therefore,PTEN is essential for PI3K/AKT signaling.Related studies have found that PRMT6 can target the methylation modification of PTEN in human osteosarcoma cells,inhibit PI3K/AKT signal transduction,and play a role in regulating m RNA splicing.However,PRMT7 and PRMT6 are highly homologous,and whether PRMT7 can regulate the PI3K/AKT signaling pathway through methylation modification of PTEN and participate in the proliferation and migration of gastric cancer cells is still unknown.Objective:To investigate the expression of PRMT7 in gastric cancer tissues and cell lines,as well as the role of PRMT7 in gastric cancer cells,and to clarify whether PRMT7 regulates the proliferation and migration of gastric cancer cells by methylating PTEN to regulate the PI3K/AKT signaling pathway,which will open up a new path for clinical diagnosis and targeted precision treatment of gastric cancer.Methods:1.Paraffin specimens of gastric cancer were collected from the Affiliated Hospital of Chengde Medical College and two paraffin tissue chips(IWLT-N-70G72 and IWLT-N-96G43)were purchased from Wuhan Shuangxuan Biotechnology Co.,LTD.,including 152 cases of gastric cancer tissues and 69 cases of adjacent normal gastric mucosa tissues.Immunohistochemical SP method was used to detect the expression of PRMT7 in gastric cancer tissues and adjacent normal gastric mucosa tissues,and the relationship between PRMT7 expression in gastric cancer tissues and clinicopathological features was further analyzed.Meanwhile,the expression of PRMT7 in 30 pairs of gastric cancer tissues and adjacent normal gastric mucosa tissues was detected by Western blot.2.The expression of PRMT7 in four gastric cancer cell lines(AGS,MGC-803,BGC-823,SGC-7901)and normal gastric epithelial cell line GES-1 was detected by Western blot.3.Two gastric cancer cell lines(MGC-803 and AGS)with moderate PRMT7 expression were selected.Western blot and RT-q PCR(Real-time fluorescence quantita-tive pcr immunohistochemistry staining)was used to detect the transfection efficiency after knocking down or overexpressing PRMT7.Cell counting kit-8(CCK-8)assay and colony formation assay were used to detect the cell proliferation ability,Transwell assay was used to detect the cell migration ability,Western blot was used to detect the downstream proliferation-related target protein-Cyclin D1 and migration-related target protein-MMP9,and to clarify the biological function of PRMT7 in gastric cancer cells.4.Western blot was used to detect the expression of PTEN and PI3K/AKT signaling pathway key proteins PI3Kp110α,PI3Kp85α,AKT,p-AKT after knockdown or overexpression PRMT7 in MGC-803 and AGS cells to determine the signaling pathway regulated by PRMT7.5.PRMT7 overexpressed MGC-803 and AGS gastric cancer cells were treated with si PTEN.Cell counting kit-8(CCK-8)assay and colony formation assay were used to detect cell proliferation ability of the cells,and Transwell assay was used to detect cell migration ability of the cells.Western blot was used to detect whether si PTEN reversed the effect of PRMT7 overexpression on the key proteins in PTEN and PI3K/AKT signaling pathway key proteins PI3Kp110α,PI3Kp85α,AKT,p-AKT and its downstream proliferation-related target protein Cyclin D1 and migration-related target protein MMP9.Whether PRMT7 plays a tumor suppressor role in gastric cancer through PTEN is preliminarily determined.To further determine whether PRMT7 inhibits the proliferation and migration of gastric cancer cells through PI3K/AKT signaling pathway downstream of PTEN,MGC-803 and AGS gastric cancer cell lines were treated with PI3K/AKT signaling pathway inhibitor LY294002 on the basis of si PRMT7#2 treatment.To observe the effect of LY294002 on the ability of proliferation and migration,and PI3K/AKT signaling pathway key proteins PI3Kp110α,PI3Kp85α,AKT,p-AKT and its downstream proliferation-related target protein Cyclin D1 and migration-related target protein MMP9 of gastric cancer cells.6.After PRMT7 was knocked down or overexpressed in MGC-803 and AGS gastric cancer cell lines,the expression of PTEN at m RNA level was detected by RT-q PCR.Co-immunoprecipitation(COIP)was used to detect whether PRMT7 and PTEN could bind to each other in AGS cells.After PRMT7 was knocked down or overexpressed,the expression of PTEN protein was detected by methylation assay in vitro to detect whether PRMT7 affected the methylation level of PTEN protein.Results:1.Immunohistochemistry SP method and Western blot were used to detect the expression of PRMT7 in gastric cancer tissues and adjacent normal gastric mucosa tissues.The results showed that the expression of PRMT7 in gastric cancer tissues was lower than that in normal gastric mucosa tissues(P<0.05),and its expression was related to the degree of differentiation,lymph node metastasis,tumor size,TNM stage and depth of invasion(P<0.05),but not with gender and age(P<0.05);In 30 fresh paired gastric cancer tissues,the expression level of PRMT7 was significantly lower in the cancer tissues than in the paired adjacent normal gastric mucosa tissues(P<0.05).2.The results of Western blot showed that PRMT7 was down-regulated in four gastric cancer cell lines AGS,MGC-803,BGC-823 and SGC-7901 compared with normal gastric epithelial cell line GES-1(P<0.05).3.PRMT7 inhibited the proliferation and migration of gastric cancer cells.The results of Western blot and RT-q PCR showed that the transfection efficiency was successful in MGC-803 and AGS cells(P<0.05).CCK-8 and colony formation assays showed that PRMT7 inhibited the proliferation of gastric cancer cells(P<0.05).Transwell assay showed that PRMT7 inhibited the migration ability of gastric cancer cells(P<0.05).The results of Western blot showed that the expression levels of proliferation-related target protein Cyclin D1 and migration-related target protein MMP9 were down-regulated after up-regulation of PRMT7(P<0.05),while the opposite results were observed after down-regulation of PRMT7(P<0.05).4.PRMT7 regulates PTEN and its downstream PI3K/AKT signaling pathway: Western blot results showed that PRMT7 overexpression increased the expression of PTEN protein and inhibited the expression of PI3Kp110α,PI3Kp85α,p-AKT/AKT in PI3K/AKT signaling pathway(P<0.05),while the opposite effect was observed after PRMT7 knockdown(P<0.05).5.The inhibitory effect of PRMT7 on gastric cancer depends on PTEN and its downstream PI3K/AKT signaling pathway.First,overexpression of PRMT7 in gastric cancer cells followed by si PTEN treatment not only reversed the effects of PRMT7 overexpression on the proliferation and migration of gastric cancer cells(P<0.05).It also significantly reversed the expression of PTEN and PI3K/AKT signaling pathway key proteins PI3Kp110α,PI3Kp85α,pAKT/AKT and downstream proliferation-related target protein Cyclin D1 and migration-related target protein MMP9(P<0.05);Further treatment with the PI3K/AKT signaling pathway inhibitor LY294002 not only reversed the inhibitory effect of PRMT7 knockdown on cell proliferation and migration,but also reversed the inhibitory effect of PRMT7 knockdown on cell proliferation and migration(P<0.05).It also reversed the expression of PI3Kp110α,PI3Kp85α,p-AKT/AKT and its downstream proliferation-related target protein Cyclin D1 and migration-related target protein MMP9(P<0.05).6.PRMT7 interacted with PTEN protein and promoted the methylation of PTEN: after PRMT7 knockdown and overexpression,the RT-q PCR results showed that there was no change in the m RNA level of PTEN(P>0.05).Endogenous and exogenous co-immunoprecipitation assay confirmed that PRMT7 protein could interact with PTEN protein.The results of in vitro methylation experiments showed that the methylation level of PTEN was decreased after PRMT7 knockdown,while the methylation level of PTEN was significantly induced after PRMT7 overexpression.Conclusions:1.PRMT7 has a low expression trend in gastric cancer,and its expression is related to tumor size,differentiation degree,lymph node metastasis,TNM stage and depth of invasion,which is involved in the occurrence and development of gastric cancer.2.PRMT7 inhibits the proliferation and migration of gastric cancer cells.3.PRMT7 inhibits the proliferation and migration of gastric cancer cells by regulating the PI3K/AKT signaling pathway through PTEN methylation modification.