Study On The Mechanism Of MiR-221-3P Regulating The Proliferation,Spread And Tumorigenesis Of Colon Cancer Cells By Targeting PDZRN4

Purpose and background:Colon cancer is one of the most common cancers in the world in terms of mobility and mortality,but its pathogenesis remains unclear.Colon cancer is now considered to be multi-gene,multi-step process.Congenital or acquired gene mutations cause uncontrolled proliferation,differentiation and apoptosis of somatic cells leading to tumorigenesis.MicroRNAs(miRNAs)are a group of small non-coding RNAs(ncRNAs)that interfere with mRNA translation or stabilization by binding to coding sequences or seed sequences in the 3’-untranslated region(3’-UTR)region.Therefore,miRNA plays an important role in the post-transcriptional regulation of genes.MicroRNAs are involved in tumor development by regulating mRNA expression levels of target genes.PDZ domain containing ring finger 4(PDZRN4)is the Ligand of Numb protein,so it is also called Lnx(Ligand of Numb-protein X,Lnx)protein.Thus far,there are 4 family members.PDZRN family members have different expression in a variety of tumors.In human hepatic cancer cell lines,expression of PDZRN4 inhibits the proliferation of cancer cells.The expression of PDZRN3 was downregulated in both localized and advanced prostate cancer,which correlated with disease stage negatively.This suggests that the PDZRN family may be involved in tumor development through unknown mechanism.To further explore potential therapeutic targets,we investigated the abnormal expression of PDZRN4 gene in colon cancer and its regulatory mechanism.Methods:Both mRNA and miRNA expression in colon cancer tissues were studied by retrieving RNA expression array results,and PDZRN4 was selected as the potential gene.Through functional tests,we investigated the proliferation and dissemination of cancer cells overexpressed PDZRN4.Finally,we identified a potential miRNA which may regulate PZDRN4 expression using bioinformatics in silico and its relationship with PDZRN4 was confirmed by luciferase reporter analysis.Results:PDZRN4 proteins levels in para-carcinoma tissue and colon cancer tissues was compared by Western blot which showed that the expression of PDZRN4 cancer tissues was lower.Meanwhile,RT-qPCR results indicated that PDZRN4 mRNA in tumor samples was downregulated.These results suggest that the mRNA and protein levels of PDZRN4 are inhibited during development of colon cancer.CCK-8 assay and plate cloning demonstrated that PDZRN4 reduced proliferation of HCT-116 cells.We constructed HCT-116 cells with overexpressed PDZRN4 and further confirmed that PDZRN4 could inhibit proliferation,migration and invasion of HCT-116 cells by Transwell migration,invasion assays and scratch assay.In vivo tumor formation experiments in nude mice confirmed that PDZRN4 can inhibit tumor formation.Using bioinformatics methods,we selected miR-221-3p as the regulatory molecule of PDZRN4.Directly inhibition of PDZRN4 expression by miR-2213p was confirmed through luciferase reporting experiments and protein expression experiments.Similarly,the proliferation and dissemination of HCT-116 cells decreased after the inhibition of miR-221-3p expression,indicating that miR-221-3p may target PDZRN4 to regulate the development of colon cancer.Besides,inhibition of miR-221-3p inhibited activation of the PTEN/AKT signaling pathway.Conclusion:The results of this study suggest the important role of miR-221-3pPDZRN4 regulation in the development of colon cancer,providing new insights for further identification of therapeutic targets.