MiR-17-92 Cluster Regulates Biological Behavior Of Gastric Cancer Cells By Targeting TRAF3

Gastric cancer(GC)is one of the most common malignant tumors in the digestive system.Its incidence is increasing year by year,which is a serious threat to human health.Global statistics show that GC is the fourth most common cancer and the third leading cause of cancer-related death in the world,and its mortality ranks third to lung and liver cancer.Although significant advances in diagnosis and therapeutics have improved long-term survival for early gastric cancer;however,the prognosis of advanced and metastatic gastric cancer is still not satisfactory,distant metastasis is still the leading cause of death in GC patients.Numerous studies show that a variety of oncogenes and tumor suppressor genes are involved in the development of gastric cancer,but the precise molecular mechanisms underlying the progression of gastric cancer remain uncovered.Therefore,a better understanding of the molecular pathologies of metastatic gastric cancer will provide an important theoretical basis for the new strategies for the diagnosis and treatment of gastric cancer.Micro RNA(miRNA)is a class of short(about 22-24 nt in length)regulatory non-coding RNAs which has been discovered in recent years and is widely expressed in living organisms.The base pairing interactions between miRNAs and their target m RNAs,often within the 3'-untranslated region(UTR)of target genes,result in the degradation of target m RNAs or repression of their translation.Moreover,miRNAs can also affect the cellular signaling pathways in which their target genes involved and further regulate a diverse range of physiological processes,including cell differentiation and proliferation,mammalian development and human disease.Many studies have shown that one miRNA can regulate the expressions of multiple target genes,however,several miRNAs can also regulate the expression of one single target gene,indicating that miRNAs and their target molecules can form a huge and intricate biological regulatory network.To be sure,the regulations of miRNAs play an important role in the development of malignant tumors.In gastric cancer,a variety of aberrantly expressed miRNAs have been discovered which can be used as potential biomarkers for gastric cancer screening,diagnosis,prognosis,disease monitoring,as well as therapeutic targets.Therefore,the study of the role and mechanism of miRNAs in the development of gastric cancer is very significant for the prevention and treatment of gastric cancer.The miR-17-92 cluster,which is also named as oncomiR-1,is located within intron 3 of the C13orf25 gene at 13q31.3,and has six mature miRNAs including miR-17,miR-18,miR-19 a,miR-19 b,miR-20 and miR-92.Previous studies have shown that they are overexpressed in a variety of malignant tumor tissues and can induce tumorigenesis as an oncogene,and some other studies have reported that they can inhibit cancer progression as a tumor suppressor.However,the precise role of the miR-17-92 cluster in gastric cancer progression is still largely unclear.In this study,firstly we detected the expression of each subunits of the miR-17-92 cluster in gastric cancer tissues using q RT-PCR analyses,and analyzed the relationship between the miR-17-92 expression and the clinical pathological features of gastric cancer patients.Next we investigated systematically the biological functions and the underlying molecular mechanisms of the miR-17-92 cluster in gastric cancer by establishing stably miR-17-92 overexpressing MGC-803 gastric cancer cell lines.Most importantly,for the first time,we identified that TRAF3(tumor necrosis factor receptor associated factor 3)was a direct and functional target of the miR-17-92 cluster in MGC-803 gastric cancer cells using dual luciferase reporter assays.We also detected the expression of TRAF3 in gastric cancer tissues by IHC,and analyzed the relationship between its expression and clinicopathological features and prognosis of gastric cancer patients.This study provides a theoretical basis for whether the miR-17-92 cluster can be used as a new biological indicator for the clinical diagnosis,treatment and prognosis monitoring of gastric cancer in the future.Part ? The expression pattern of the miR-17-92 cluster in gastric cancertissues and their correlation to clinicopathologicalfeatures of gastric cancer patients Objective To determine the expression pattern of the miR-17-92 cluster in human gastric cancer tissues and observe the relationship between their expressions and its clinicopathological features of gastric cancer patients.Method Systematically collect the tumor specimens and clinicopathological features of gastric cancer patients who had the gastric cancer surgery in the First Affiliated Hospital of Soochow University in 09/2015-12/2016.q RT-PCR assay was used to detect the expression of the miR-17-92 cluster in human gastric cancer tissues.Chi-square test was used to analyze the relationship between the miR-17-92 expression and clinicopathological features of gastric cancer patients.Result q RT-PCR analyses showed that the individual subunits of the miR-17-92 cluster including miR-17,miR-18,miR-19 a,miR-19 b,miR-20 and miR-92 were significantly overexpressed in the gastric cancer tissues than that in the adjacent normal gastric tissues.The expression of miR-17 was correlated with the tumor differentiation(P=0.0031)and TNM stage(P=0.0020).The expression of miR-18 was correlated with the TNM stage(P=0.0020).The expression of miR-19 a was correlated with tumor size(P=0.0002)and TNM stage(P=0.0012).The expression of miR-19 b was correlated with tumor size(P=0.0072)and TNM stage(P=0.0002).The expression of miR-20 was correlated with tumor differentiation(P=0.0143)and TNM stage(P=0.0023).The expression of miR-92 was correlated with tumor size(P=0.0374)and TNM stage(P=0.0035).Conclusion The expression of the miR-17-92 cluster in gastric cancer tissues were significantly up-regulated compared with the corresponding normal tissues,indicating that the miR-17-92 cluster plays an important role in gastric cancer progression.The expression levels of each subunits of the miR-17-92 cluster were positively correlated with TNM stage of gastric cancer patients,while miR-17 and miR-20 were also positively correlated with tumor differentiation,and miR-19 a,miR-19 b and miR-92 were also positively correlated with tumor size of gastric cancer patients.Part ? The effect and mechanism of the miR-17-92 cluster on biological functions of gastric cancer cells Objective To investigate the biological functions and its mechanisms of the miR-17-92 cluster in human gastric cancer cells.Method q RT-PCR assay was used to detect the expression of the miR-17-92 cluster in human gastric cancer cell lines.Lentivirus transfection was used to establish stably miR-17-92 overexpressing MGC-803 gastric cancer cell lines.The cellular proliferation ability was detected by CCK-8,Bru U,Cell Titer-Glo and colony formation assays.The cellular apoptosis was detected by TUNEL assay.The cell cycle analysis and cellular DNA content measurement were examined by flow cytometry.The cellular migration and invasion abilities were detected by scratch healing assay and Transwell migration and invasion assays.The heterotopic xenograft tumor BALB/c-nude mice model was used to identify the in vivo tumorigenic ability of MGC-803 gastric cancer cells with overexpression of the miR-17-92 cluster.The functional related genes' m RNA and protein expressions of the MGC-803-control and MGC-803-miR-17-92 cells were detected by q RT-PCR and Western blotting analyses.The MMPs activities of MGC-803 cells affected by the miR-17-92 overexpression were detected by gelatinase zymography.The ELISA-based NF-?B activity assay was performed to dissect the exact contribution of the miR-17-92 cluster to the NF-?B DNA-binding capabilities in the MGC-803 cells.Western blotting analyses were used to detect the protein expressions of AKT,ERK1/2 and NF-?B signaling pathways in the two established cell lines.Result The expressions of the miR-17-92 cluster of the MGC-803 cells were the lowest among the five human gastric cancer cell lines including AGS,SGC-7901,BGC-823,MKN-45,and MGC-803.Introduction of the miR-17-92 overexpressing plasmid into the MGC-803 cells significantly promoted cellular proliferation,migration and invasion abilities,and decreased cellular apoptosis in vitro.The heterotopic xenograft tumor BALB/c-nude mice model showed that overexpression of the miR-17-92 cluster significantly promoted xenograft tumor growth in vivo.Western blotting analyses showed that the expressions of p-AKT(Thr308)and p-ERK1/2 were markedly increased in the MGC-803-miR-17-92 cells compared to that in the MGC-803-control cells.Western blotting and Trans AM assays showed that the DNA-binding capability of each NF-?B subunit including Rel A,p50,Rel B and p52 in the MGC-803-miR-17-92 cells were significantly increased as compared with that in the MGC-803-control cells.q RT-PCR and western blotting analyses showed that both the m RNA and protein expression levels of pro-apoptotic genes BIM and BID were significantly decreased in the MGC-803-miR-17-92 cells compared to those in the MGC-803-control cells,while the expressions of anti-apoptotic genes Bcl-2,Bcl-x L and Mcl-1 were increased.The activity of MMP-2 was significantly increased in MGC-803-miR-17-92 cells than MGC-803-control cells detected by the gelatinase zymography.Western blotting analyses showed that the expression of E-cadherin,Claudin1,and Ep CAM,representing the epithelial markers,were decreased in the MGC-803-miR-17-92 cells compared to those in the MGC-803-control cells,while the expression of N-cadherin and Vimentin,the mesenchymal markers,were increased in the MGC-803-miR-17-92 cells than those in the MGC-803-control cells.Conclusion Overexpression of the miR-17-92 cluster significantly increased the cellular proliferation ability of the MGC-803 gastric cancer cells,which was associated with the activation of AKT,ERK1/2 and NF-?B signaling pathways.Overexpression of the miR-17-92 cluster significantly decreased the cellular apoptosis of the MGC-803 cells,likely due to the decreased expressions of pro-apoptotic genes BIM and BID and the increased expressions of anti-apoptotic genes Bcl-2,Bcl-x L and Mcl-1.Overexpression of the miR-17-92 cluster significantly increased the migration and invasion abilities of the MGC-803 cells,which was owing to the induced activity of MMP-2 and the occurrence of EMT.Overexpression of the miR-17-92 cluster in the MGC-803 cells significantly promoted tumor growth in vivo.Part ? Prediction and verification of the target genes of the miR-17-92 cluster Objective To search and verify the target genes of the miR-17-92 cluster.Method Bioinformatics software was used to predict the downstream target genes of the miR-17-92 cluster.The expressions of TRAF3 at both the m RNA and protein levels of the MGC-803-control and MGC-803-miR-17-92 cells were detected by q RT-PCR and Western blotting analyses.The molecular cloning experiments were used to construct the recombinant plasmids of both the wild-type and mutant-type pmir GLO-TRAF3-3'UTR plasmids.Dual luciferase reporter assay was performed to detect the binding of the miR-17-92 cluster to the TRAF3-3'UTR.The sh RNA interference method was used to establish stably TRAF3-silencing MGC-803 gastric cancer cell lines.q RT-PCR and Western blotting analyses were used to determine the transfection efficiency of TRAF3-silencing in the MGC-803 cells.Western blotting analyses were used to detect the protein expressions of NF-?B signaling molecules in the two established cell lines.The ELISA-based NF-?B activity assay was performed to dissect the exact effect of TRAF3-silencing to the NF-?B DNA-binding capabilities in the MGC-803 cells.The cellular proliferation ability was detected by CCK-8 and Cell Titer-Glo cell viability assays.The cellular migration and invasion abilities were detected by Transwell migration and invasion assays.Result Target Scan Release 5.1 online software showed that the TRAF3 gene was one of the predicted target genes of the miR-17-92 cluster.The sequencing results showed that we the wild-type and mutant-type pmir GLO-TRAF3-3'UTR recombinant plasmids was constructed successfully.Dual luciferase reporter assay showed that in the experimental group transfected with the pmir GLO-TRAF3-3'UTR wild-type plasmid,the relative luciferase activity of the miR-17-92 group was significantly decreased compared to the control group.However,in the experiment group transfected with pmir GLO-TRAF3-3'UTR mutant-type plasmid,the relative luciferase activity between the miR-17-92 group and the control group showed no significant differences.q RT-PCR and western blotting analyses showed that the expressions of TRAF3 at both the m RNA and protein levels were significantly decreased in the MGC-803-si TRAF3 cells compared to that in the MGC-803-sictrl cells,indicating a successful RNA interference(RNAi)of the TRAF3 gene in the MGC-803 cells.TRAF3-silencing significantly activated both the canonical and the non-canonical NF-?B signaling in the MGC-803 cells.TRAF3-silencing significantly promoted cellular proliferation and enhanced migration and invasion abilities of the MGC-803 cells in vitro.Conclusion Dual luciferase reporter assay showed that TRAF3 was a direct and functional target of the miR-17-92 cluster.We further confirmed the targeted relationship between the TRAF3 gene and the miR-17-92 cluster in the MGC-803 gastric cancer cells.Part ? The expression of TRAF3 in gastric cancer tissues and their correlation to clinicopathological features and prognosis of gastric cancer patients Objective To determine the expression of TRAF3 in human gastric cancer tissues and observe the relationships between its expression and clinicopathological features as well as prognosis of gastric cancer patients.Method Human gastric cancer tissue chip(HStm A030PG03)was purchased from Shanghai Core Super Biotechnology Co.,Ltd.IHC assay was used to detect the expression of TRAF3 in human gastric cancer tissues.Chi-square test was used to analyze the relationship between TRAF3 expression and clinicopathological features of gastric cancer patients.Kaplan-Meier survival analysis was used to analyze the relationship between TRAF3 expression and prognosis of gastric cancer patients.Univariate and multivariate analyses were used to assess the prognostic indicators of gastric cancer.Result IHC analyses showed that the the expression of TRAF3 in gastric tumor tissues was significantly lower than that in the adjacent normal gastric mucosa.The expression of TRAF3 was negatively correlated with tumor diameter(P=0.036),depth of invasion(P=0.003)and TNM stage(P=0.009).Kaplan-Meier survival analysis results revealed that the overall survival time of gastric cancer patients with low expression of TRAF3 was significantly shorter than patients with high expression of TRAF3(P=0.002).One-way ANOVA showed that tumor size(P=0.009),tumor differentiation(P=0.033),lymph node metastasis(P=0.032),distant metastasis(P=0.000),TNM stage(P=0.001),and TRAF3 expression(P=0.002)were important indicators for the prognosis of gastric cancer patients.Multi-way ANOVA showed that TRAF3 expression(P=0.004)was not an independent prognostic indicator of gastric cancer patients.Other indicators including tumor size(P=0.010),distant metastasis(P=0.000),and TNM stage(P=0.010)were also associated with prognosis of gastric cancer patients.Conclusion The expression of TRAF3 in gastric cancer tissues was relatively lower than that in normal tissues.TRAF3 expression was negatively correlated with tumor size,depth of invasion and TNM stage of gastric cancer patients.Low expression of TRAF3 was positively correlated with the prognosis of gastric cancer patients,that is,they have shorter overall survival time compared to patients with high expression of TRAF3.TRAF3 can be used as a new prognostic indicator of gastric cancer patients.