MicroRNA-192 And -215 Negatively Regulate APC To Activate Wnt/β-catenin Signaling Pathway In Gastric Cancer

BackgroundGastric cancer(GC)is one of the most common malignant tumors worldwide,about 10%of the global cancer mortality,ranking second.The five-year survival rate of gastric cancer patients is only 36% in China.According to the Cancer Data Report of China in 2015,679,000 new cases of GC are found every year,with 498,000 deaths.Globally,nearly half of the new cases of GC and deaths happened in China.Because of the poor diagnosis of gastric cancer,most of the patients were diagnosed in the advanced stage thus missing the best time of therapy.However,the cure rate of early gastric cancer is 85%.Therefore,it’s necessary to elucidate the pathogenesis mechanism of gastric cancer and to identify novel biomarkers and effective therapeutic targets.MicroRNA(miRNA)is a class of non-coding single-stranded RNA with 19-22 nucleotides in length.At present,many reports have shown that miRNAs participate in many cellular processes,such as cell proliferation,differentiation and migration,by regulating the expression of target genes.The abnormal expression of miRNAs in different tumors was also reported,which revealed that miRNAs may play a significant role in tumorigenesis.MiRNAs negatively regulate the expression of target genes through their "seed sequence" in 5’-UTR,which binds to the 3’-UTR of target genes completely or incompletely.New studies have shown that a variety of miRNAs are involved in the carcinogenesis of GC.At the same time,miRNA-192 and-215 have been reported to be significantly up-regulated in gastric cancer.However,the molecular mechanism of their involvement in the genesis and development of GC still remains to be further explored.Wnt signaling pathway is a evolutionarily highly conserved signaling pathway,which is involved in the early embryonic development,cell differentiation,cell fate determination,body axis formation and other important biological processes.The Wnt/β-catenin pathway is the canonical Wnt signaling pathway,which leads to the regulation of gene transcription.Several studies have shown that Wnt/β-catenin signaling pathway is closely related to the development of cancer.Constitutive activation of Wnt/β-catenin signaling pathway has been found in about 30%of gastric cancer cases.APC(adenomatous polyposis coli)was first found in colon cancer.As a well-known tumor suppressor gene,APC is a key negative regulator of Wnt/β-catenin signaling pathway.APC is an essential component of a multiprotein destruction complex with other members of GSK-3β,CK1,Axin,mediating the ubiquitination and degradation ofβ-catenin.Abnormal expression of APC will lead to activation of Wnt/β-catenin signaling pathway.MethodsMicroRNA expression profiling chip was used to screen the up-regulated miRNAs in gastric cancer,and qRT-PCR was used to verify the expression of miRNA-192 and-215 in gastric tissue.The target genes of miRNA-192 and-215 were predicted by bioinformatics software such as miRWalk.After the transfection of miRNA-192 and-215 mimics or inhibitors,the expression of APC was preliminarily validated by qRT-PCR and Western blotting.The binding of miRNA-192 and-215 with APC was confirmed by dual-luciferase reporter assay.MiRNA-192 and-215 were found to activate Wnt/β-catenin signaling pathway via APC by Topflash/Fopflash assay,Western blotting and immunofluorescence assay.EdU detection 、scratch assay and Transwell experiment were conducted to explore the cellular biological functions of APC and miRNA192/-215 in gastric cancer.ResultsThe expression of miRNA-192/-215 and APC in 40 pairs of gastric cancer tissues and adjacent normal tissues was detected by qRT-PCR.The expression of miRNA-192 and-215 were significantly up-regulated in gastric cancer(P < 0.01),and APC was significantly down-regulated in gastric cancer(P < 0.001).qRT-PCR,Western blotting and dual-luciferase reporter assay confirmed that miRNA-192 and-215 directly bind to APC and negatively regulate its expression.Immunofluorescence analysis showed that miRNA-192 and-215 promoted β-catenin translocation into the nucleus.Topflash/Fopflash assay and Western blotting confirmed that miRNA-192 and-215 activated Wnt/β-catenin signaling pathway by targeting APC.EdU detection、scratch assay and Transwell experiment confirmed that APC was involved in cellular biological behavior induced by miRNA-192 and-215.Conclusion(1)MiRNA-192 and-215 are significantly up-regulated in GC.(2)APC is the target gene of miRNA-192 and-215.(3)MiRNA-192 and-215 activate Wnt/β-catenin signaling pathway via APC,promoting cell proliferation and cell migration.