Research On The Mechanism Of MiR-613 Targeting At ATOH1 In Regulating Biological Function Of Colon Cancer

Colorectal cancer is one of the most common malignant tumors of the digestive tract in the world,ranking third in incidence and ranking second in mortality.In 2018,there were more than 1.8 million new cases and 0.8 million deaths worldwide,accounting for about one-tenth of cancer cases and deaths.Overall,the global incidence of colorectal cancer ranks third,but mortality ranks second.The incidence of colorectal cancer varies widely from country to country,and the incidence of colorectal cancer in developed countries is about three times that of developing countries.This disease can be regarded as a sign of socio-economic development.As countries increase with the Human Development Index(HDI),the incidence tends to rise evenly.The trends in morbidity and mortality over the past decade were evaluated and three different patterns associated with development levels in developing countries such as China and Brazil were identified.With the changes in lifestyles and diet patterns and obesity,the mortality and incidence of colorectal cancer rates are on the rise;developed countries,such as Canada,the United Kingdom,and Singapore,have reduced mortality in colorectal cancer by implementing optimal cancer treatment and management models.In the United States,Japan,and France,the mortality and incidence of colorectal cancer have declined through early testing and screening.But the mechanism of colon cancer is still unclear,and biomarkers that can be used for early diagnosis,treatment and prediction of prognosis are limited.Therefore,it is of great significance to explore the occurrence and development mechanism of colon cancer and find effective biological targets for early diagnosis,early treatment and prognosis of colorectal cancer.Micro RNAs(miRNAs)are a kind of single-stranded non-coding RNA encoded by endogenous genes(about 20 nucleotides in length).They can bind to target gene m RNA,affect its transcription process and then affect cell function,and play a pro-cancer or anti-cancer role.In addition,miRNA can also be regulated by gene silencing through degraded m RNA.Each miRNA can have multiple target genes,and several miRNA canalso regulate the same gene.This complex regulatory network can regulate the expression of multiple genes through a single miRNA,or fine-regulate the expression of a gene through the combination of several miRNA.It has been found that multiple miRNAs are expressed abnormally in multiple types of tumors,which is often related to patient diagnosis,staging,progression,prognosis and clinical efficacy.The ATOH1 gene is located on chromosome 4q22 and is a homolog of the Drosophila gene,regulating neuronal expression in the central nervous system and peripheral nervous system.In colon cancer cells,upregulation of atoh1 inhibits proliferation by prolonging G0/G1 phase,promoting cell migration and drug resistance.In this study,we first studied the role of mir-613 in colon cancer,proving that mir-613 is the upstream miRNA of ATOH1.Through co-transfection of mir-613 and ATOH1,it was confirmed that ATOH1 could reverse the promoting effect of mir-613 on colon cancer cells.Then,Western Blot was used to study the downstream proteins regulated by ATOH1,and it was speculated that mir-613 regulated the expression of ATOH1 by targeting,then supressed the JNK1 pathway,inhibited the expression of MUC2 which is a cancer-suppressor protein,and eventually led to the progression of colon cancer.Therefore,as an oncogene,mir-613 regulates the progression of colon cancer by targeting ATOH1 and has the potential to become a target for clinical therapeutic intervention.Part ? The expression and biological function of miR-613 in colon cancerObjective: The effect of mir-613 on the proliferation,migration and invasion of colon cancer was explored and analyzed in vitro.Methods: 20 cases of colon cancer and adjacent tissues were collected.The expression of miR-613 in colon cancer tissues and adjacent tissues was detected by qRT-PCR.The expression of miR-613 in colon cancer cell lines was detected by qRT-PCR.Plasmid transfection was used to construct a colon cancer cell model that downregulated miR-613 and overexpressed miR-613.The expression of colon cancer cell line miR-613 was detected by qRT-PCR.Cell proliferation was detected by MTT assay,the trace assay was used to detect the migration ability of cells,and cell invasion ability was detected by Transwell assay.The biological function of miR-613 in colon cancer cells was identified.Results: The results of qRT-PCR showed that miR-613 expression was up-regulatedin colon cancer tissues.The expression of miR-613 in colon cancer cells was significantly higher than that in normal human colon epithelial cell.MTT assay showed that overexpression of miR-613 could effectively promote the proliferation of colon cancer cells,while the proliferation of colon cancer cells down-regulated by miR-613 was significantly decreased.The Transwell test demonstrated that the invasive ability of colon cancer cell lines down-regulated by miR-613 was significantly reduced,while the invasion ability of colon cancer cell lines overexpressing miR-613 was significantly increased.The scratch test showed that the migration ability of colon cancer cell lines expressing miR-613 was significantly decreased,while the migration ability of colon cancer cell lines overexpressing miR-613 was significantly improved.Conclusion: MiR-613 is highly expressed in colon cancer and can promote the proliferation,migration and invasion of colon cancer cells.Part ? MiR-613 targeting inhibition of ATOH1 expression andthe prognostic value of ATOH1 for colon cancerObjective: To explore the target gene of miR-613 in vitro,and to explore the regulation mechanism of miR-613 on ATOH1 and the biological behavior of colon cancer cells.Methods: The dual luciferase assay reported a binding site for ATOH1 m RNA to miR-613.Plasmid transfection was used to construct a colon cancer cell model with miR-613 inhibitor and miR-613 mimic.The expression of ATOH1 in colon cancer cell line was detected by qRT-PCR and the expression of ATOH1 protein in colon cancer cell line was detected by Western Blot.Immunohistochemical staining was used to evaluate the expression of ATOH1 protein in colon cancer tissues and to explore its relationship with survival.The expression of ATOH1 protein and ATOH1 m RNA in colon cancer tissues was determined by qRT-PCR.Results: Dual luciferase assay reports confirmed that ATOH1 was the target gene of miR-613.The results of qRT-PCR showed that the expression level of ATOH1 m RNA in down-regulated miR-613 colon cancer cell lines was significantly increased,while the expression level of ATOH1 m RNA in overexpressed miR-613 colon cancer cells was significantly decreased.Western Blot confirmed that the expression level of ATOH1 protein in the down-regulated miR-613 group was significantly increased,and the expression level of ATOH1 protein in the over-expressed miR-613 group was significantly decreased.IHC staining confirmed that ATOH1 was positively expressed in both colon cancer tissues and adjacent tissues,and the expression in adjacent tissues was higher than that in cancer tissues.The high expression of ATOH1 in colon cancer tissues was significantly correlated with lower grade and lower TNM stage.Patients with positive ATOH1 expression had longer overall survival.The expressions of ATOH1 m RNA and ATOH1 protein in colon cancer tissues were confirmed by qRT-PCR.Conclusion: ATOH1 is the target gene of miR-613,and miR-613 can inhibit the expression of ATOH1 m RNA and ATOH1 protein.ATOH1 is inversely associated with colon cancer,and patients with positive ATOH1 expression have a longer overall survival.The expression of ATOH1 m RNA in colon cancer tissues was consistent with the expression of ATOH1 protein.Part ? ATOH1 inhibits the promotion of colon cancer cell line by miR-613Objective: Compensatory experiments confirmed that ATOH1 can reverse the promotion of colon cancer cell lines by miR-613.Methods: Plasmid transfection was used to construct a colon cancer cell model co-transfected with miR-613 and ATOH1.Cell proliferation was detected by MTT assay,cell invasion ability was detected by Transwell assay,cell migration ability was detected by scratch test,and marker protein expressions were detected by Western Blot.Results: miR-613 can promote the proliferation,migration and invasion of colon cancer cells,while ATOH can significantly inhibit the proliferation,migration and invasion of colon cancer cells.At the time of co-transfection of miR-613 and ATOH1,ATOH1 reversed the proliferation,migration and invasion of miR-613 on colon cancer cell lines.Western Blot results showed that miR-613 did not change the expression of JNK1,but increased the expression of its activated form p-JNK1.In addition,miR-613 can inhibit the expression of the tumor suppressor protein MUC2.Conclusion: ATOH1 expression is a good prognostic factor for colon cancer patients.The progression of colon cancer induced by miR-613 is completely reversed by the ATOH1 overexpression vector.miR-613 promotes proliferation,migration and invasion of colon cancer cells by targeting ATOH1,possibly by inhibiting the JNK1 pathway and downregulating MUC2,ultimately leading to colon cancer progression.Part ? In vivo verified the effect of miR-613 targeting ATOH1 on the progression of colon cancerObjective: To demonstrate the effect of miR-613 on the growth of colon cancer by miR-613.Methods: The cells transfected with control,miR-613 mimic,ATOH1,miR-613+ATOH1 plasmids were inoculated subcutaneously into mice to construct a tumor model,the tumor volume was measured,and the tumor weight was weighed.Results: The tumor formation ability of Balb/c Nod tumor-bearing mice was significantly higher in the colon cancer cell group overexpressing miR-613 than in the blank control group.The tumor formation ability in Balb/c Nod tumor-bearing mice in the ATOH1 overexpressing group was significantly lower than that of the blank control group.Overexpression of ATOH1 could reverse the tumorigenic ability caused by miR-613.Conclusion: miR-613 can inhibit the growth and tumor formation of colon cancer cells by regulating the ATOH1 expression.