Regulation And Mechanism Of MiR-135b-5p In Colorectal Cancer

Background and objectives:Colorectal Cancer(CRC)is one of the most common malignant tumors of the digestive tract.It has a high incidence and mortality rate,ranking third and fourth respectively among all solid cancers.In China,the number of cases of colorectal cancer patients continues to increase due to poor living and eating habits.Surgical treatment is the most effective treatment for colorectal cancer.However,the effect of surgical resection is closely related to the preoperative stage of the patient.More than83% of patients with colorectal cancer are at an advanced stage when diagnosed,and nearly half of them have metastasis to other sites with poor prognosis.Therefore,in-depth study of the molecular mechanism of colorectal cancer development and exploration of new early diagnosis or treatment markers can provide new ideas for the accurate diagnosis and current treatment effect of colorectal cancer.micro RNA(miRNA)is a kind of 18-25 nt long non-coding RNA,which regulates the expression of target genes by binding to their 3 ’untranslated region,thereby affecting a series of physiological processes.miRNA can regulate a variety of signaling pathways in cancer,and play different roles in tumor diagnosis,staging,progression,prognosis and chemotherapy.One of the main characteristics of cancer is the uncontrolled proliferation of cancer cells,which is the basis of cancer development.As regulators,miRNAs affect tumor growth by targeting key members of colorectal cancer related proliferation signaling pathways,and many miRNAs have clear differences in expression between normal and colorectal cancer tissues.Therefore,this study aims to explore the key miRNAs and their molecular regulatory mechanisms in colorectal cancer,to study the expression of miR-135b-5p in colorectal cancer and its effects on the proliferation,apoptosis,migration and invasion,epithelial-mesenchymal transformation(EMT)capacity of CRC cells,and to explore the target genes of miR-135b-5p.This study provides a new idea for the accurate diagnosis of colorectal cancer.Methods:The Gene Expression Omnibus(GEO)and the Cancer Genome Atlas(TCGA)data sets were downloaded,and differentially expressed miRNAs(DEmiRNAs)were screened in colorectal cancer tissues and adjacent normal tissues to obtain the overlapping DEmiRNAs in the two databases,followed by enrichment analysis.Quantitative real-time PCR(q RT-PCR)was used to detect the expression of DEmiRNA in colorectal cancer clinical samples and cells.After comprehensive analysis of the key miRNAs,miR-135b-5p with consistent expression in tissues and cells was finally selected for subsequent research.In vitro experiment,a CRC cell line SW480 that interfered with miR-135b-5p was constructed(miR-135b-5p-inhibitor group),and SW480 cells were transfected with its negative control(inhibitor negative control group).Then cell counting kit-8(CCK-8)method was used to detect cell proliferation,the ability of cell adhesion,proliferation and clonal formation was tested by clonal formation assay.Flow cytometry was used to detect cell apoptosis and cell cycle,and Transwell method and Wound healing assay were used to detect cell migration and invasion.QRT-PCR and Western blot was used to detect the m RNA and proteins expression levels of EMT-related markers and so on.In vivo,a nude mouse xenograft tumor model was constructed by stably transfecting cell line SW480 with lentivirus vector silencing miR-135b-5p,and the effect of silenced miR-135b-5p on subcutaneous cancer in nude mice was detected.Illumina platform was used to sequence the transcriptome of SW480 cells transfected with miR-135b-5p-inhibitor and inhibitor negative control,and the expected target genes of miR-135b-5p were screened.The expression of the obtained target genes in CRC cell lines and tissues were detected by q RT-PCR and Western blot.Results:Two hundred and twenty-three DEmiRNAs and 134 DEmiRNAs were identified from the GEO and TCGA datasets,respectively.There were a total of 26 overlapping DEmiRNAs in the two datasets,of which 17 DEmiRNAs were up-regulated and 9DEmiRNAs were down-regulated.Enrichment analysis results shown that these DEmi NRAs were enriched in 30 Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways.In the above 26 overlapping DEmiRNAs,the expression of miR-135b-5p in colorectal cancer clinical samples and SW480 cells was higher than that in the control group(all P<0.05).Compared with the inhibitor negative control group,the expression of miR-135b-5p in the miR-135b-5p-inhibitor group was decreased,and the cell proliferation,EMT,cell migration and invasion abilities were decreased,and the cell apoptosis rate and cell cycle were changed(all P<0.05).In vivo,a nude mouse xenograft tumor model was constructed,and silencing miR-135b-5p inhibits the growth of subcutaneous tumors.In addition,a total of 3,384 dem RNAs were screened from SW480 cells in the miR-135b-5p-inhibitor group and inhibitor negative control group.After enrichment analysis,25 KEGG pathways and 326 Gene Ontology biological processes(GO-BP)were significantly enriched in the up-regulated m RNAs,and 20 KEGG pathways and276 GO-BPs were enriched in the down-regulated m RNAs.In addition,352 pairs of miR-135b-5p-target gene regulatory relationships were screened,and 10 overlapping genes were obtained after crossing with DEm RNA screened previously.These include NSA2,FOXN2,DIRAS2,DESI1,SV2 C,RAB33B,MCTS1,CNIH4,SLCO5A1 and MYCBP.After prognostic analysis,FOXN2,NSA2,MYCBP,DIRAS2,DESI1 and RAB33 B genes were found to be related to the prognosis of patients(P<0.05).Compared with inhibitor negative control group,the expression of FOXN2,NSA2 and DESI1 in miR-135b-5p-inhibitor group was significantly different at m RNA level.Moreover,DESI1 was also up-regulated at protein level(all P<0.05).Conclusion:miR-135b-5p is highly expressed in colorectal cancer.miR-135b-5p promotes the proliferation,regulates EMT characteristics,invasiveness and migration capability of colorectal cancer cells,and inhibits the apoptosis of colorectal cancer cells.The target genes of miR-135b-5p,including FOXN2,NSA2,MYCBP,DIRAS2,DESI1 and RAB33 B,have prognostic significance.In addition,miR-135b-5p promotes the development of CRC by targeting and regulating DESI1.