| Background:Colon cancer is the third most common tumor in the world.In the past 10 years,although there have been some advances in the treatment of colon cancer patients and it has improved the quality of life in colon cancer patients,the clinical prognosis results of colon cancer patients are still poor,and the metastasis of colorectal cancer is the main cause of high morbidity and mortality.Finding reliable and sensitive diagnostic molecular markers and drug treatment targets,early diagnosis and early treatment of colon cancer,and improving the survival rate of colon cancer patients have become important in colon cancer research.The molecular mechanism of colon cancer is heterogeneous.In the past 10 years,significant efforts have been made to develop"targeted therapies"or"biological therapies"that work through specific cellular signaling pathways that affect tumor growth.The receptor for the epidermal growth factor receptor?EGFR? or ErbB family is a cell surface receptor having tyrosine kinase activity.EGFR gene expression up-regulated in up to 80% of colon cancer patients and is associated with colon cancer metastasis risk.MicroRNAs?miRNAs? are small endogenous non-coding RNAs of 19-23nucleotides that regulate gene expression by targeting the 3'untranslated region?UTR? of mRNA.miRNAs play a fundamental role in various biological processes.In the preliminary work of this experiment,the gene chip method was used and we find that miR-320d was down-regulated in human colon cancer tissues,suggesting that miR-320d may be involved in the development of colon cancer.The miR-320d has previously been reported to be down-regulated in many tumors.However,the expression,biological function and molecular mechanism of miR-320d in colon cancer have not been studied.After bioinformatics prediction and screening,we found that Tumor Suppressor Candidate3?TUSC3? is a potential target gene for miR-320d.The TUSC3 gene?N33?is localized to human chromosome 8 broken arm 8p22.The length of this gene is 224 kbp and containing11 exons.The TUSC3 protein is a subunit on the endoplasmic reticulum membrane oligosaccharide transferase complex?OST? that functions during the initial reaction of protein N glycosylation.The EGFR family members of the cell epidermal growth factor receptor are glycosylation-modified transmembrane receptor proteins,and most of them are N-glycosylation modifications.After TUSC3 protein changes,the expression and maturation of EGFR receptor protein in cells must be affected,affecting its signaling pathway and binding to other proteins.Therefore,we hypothesized that miR-320d may inhibit the development of EGFR-overexpression colon cancer by targeting TUSC3.Objective:There is no research on the function and mechanism of miR-320d in EGFR-overexpression colon cancer.Therefore,in this study,we will focus on the effect of miR-320d on the malignant biological behavior of EGFR-overexpression colon cancer,explore miR-320d expression levels in EGFR-overexpression colon cancer tissues and cell lines,and the clinicopathological features of EGFR-overexpression colon cancer,possible molecular regulatory mechanisms.In the first part we detect the expression level of miR-320d in EGFR-overexpression colon cancer tissues and analyzes the clinicopathological relationship.In the second part we studied the effect of over-expression and down-regulation miR-320d on the biological functions of EGFR-overexpression colon cancer cell lines HCT116,SW480,including proliferation,migration and invasion,and epithelial-mesenchymal transition?EMT?.In the third part we confirms and verifies that TUSC3 is the target gene of miR-320d.The functional role of TUSC3 in EGFR-overexpression colon cancer was confirmed by cytology experiments,and the molecular mechanism by which miR-320d regulates the malignant biological behavior of EGFR-overexpression colon cancer cells by targeting TUSC3 was elucidated.These experiments provide new ideas and targets for the diagnosis and treatment of EGFR-overexpression colon cancer patients.Methods:Total 60 cases of surgically resected colon cancer tissue specimens and adjacent normal tissues were collected.The expression of miR-320d was detected by real-time quantitative PCR?qRT-PCR?.Paired t-test was used to compare miR-320d expression in cancer and adjacent tissues.The chi-square test was used to compare the relationship between the expression level of miR-320d in cancer tissues and the clinicopathological factors of colon cancer.The expression of miR-320d in EGFR-overexpression colon cancer cell lines SW480 and HCT116 was significantly lower than that in other colon cancer cell lines.The SW480 and HCT116 cell lines were selected for further experiments.The colon cancer cell lines were transfected with miR-320d mimics.The effects of miR-320d on the proliferation,migration and invasion of EGFR-overexpression colon cancer cells were observed by MTT assay,scratch assay and Transwell cell invasion assay.Bioinformatics predicts miR-320d downstream target gene,Immunohisochemical technique was used to detect the expression level of TUSC3 in colon cancer tissues and adjacent tissues,and the relationship between TUSC3 differential expression and clinical pathological feature was analyzed.Dual luciferase gene reporter assay to verify miR-320d targeting TUSC3,qRT-PCR and Western blot were used to detect the mRNA and protein expression of TUSC3 in EGFR-overexpression colon cancer cells after transfection of miR-320d mimic.The Rescue experiment further confirmed the direct regulation of miR-320d on TUSC3 in EGFR-overexpression colon cancer cells.The effect of miR-320d on the expression of EMT and PI3K-AKT-mTOR signaling pathway-related proteins was detected by Western blot in EGFR-overexpression colon cancer cells.Results:The average 2-??CT of miR-320d in EGFR-overexpression colon cancer was0.4424±0.2558,and the mean 2-??CT value of adjacent tissues was 1.0142±0.1666.The difference was statistically significant?P<0.001?.Univariate analysis showed that the expression level of miR-320d was correlated with tumor size,distant metastasis and EGFR expression status.At 48 hours and 72 hours after transfection with miR-320d mimic in EGFR-overexpression SW480 and HCT116 cells,MTT assay showed that the cell proliferation ability of the transfected group was significantly lower than that of the control group?P<0.05?.Scratch test and Transwell experiment showed that the migration and invasion ability of SW480 and HCT116 cells were significantly lower than that of the control group after transfection of miR-320d mimic?P<0.05?.TUSC3 was highly expressed in colon cancer tissues and cells,and TUSC3 was associated with clinical stage,lymph node metastasis,and distant metastasis of colon cancer patients?P<0.05?.Transfection of TUSC3 in EGFR-overexpression colon cancer cells significantly increased cell proliferation and invasion.The dual luciferase gene reporter assay showed that miR-320d mimic significantly reduced luciferase activity in the 3'UTR region of TUSC3,whereas miR-320d mimic did not reduce luciferase activity in the 3'UTR region of TUSC3with binding site mutations.Transfection of miR-320d mimic in EGFR-overexpression SW480 and HCT116 cells reduced the mRNA and protein expression levels of TUSC3,whereas transfection of miR-320d inhibitor increased the mRNA and protein expression levels of TUSC3.Rescue experiments showed that TUSC3 partially offset the inhibitory effect of miR-320d on proliferation and invasion in EGFR-overexpression colon cancer cells.Transfection with miR-320d-mimic in EGFR-overexpression colon cancer cells increased the protein expression level of E-cadherin and decreased the protein expression level of Vimentin.Transfection of miR-320d-inhibitor in EGFR-overexpression colon cancer cells reduced the protein expression level of E-cadherin and increased the protein expression level of Vimentin.Overexpression of miR-320d in EGFR-overexpression colon cancer cells reduced the degree of phosphorylation of mTOR,PI3K and AKT,while overexpression of TUSC3 increased the degree of phosphorylation of mTOR,PI3K and AKT.Conclusion:1.This study suggests that miR-320d is underexpressed in EGFR-overexpression colon cancer tissues and cell lines,and low miR-320d expression levels are significantly associated with colon tumor size and distant metastasis.2.Overexpressed miR-320d can inhibit the migration,invasion and proliferation of EGFR-overexpression colon cancer cells.TUSC3 is a downstream target gene of miR-320d and is highly expressed in colon cancer tissues.The miR-320d inhibits the proliferation and migration invasiveness of EGFR-overexpression colon cancer cells by inhibiting TUSC3 expression.3.Overexpressed miR-320d increased the expression of E-cadherin,decreased the expression of Vimentin,and inhibited the phenotype of EMT.The miR-320d inhibits the progression of colon cancer by regulating the expression of TUSC3 and affecting the PI3K/AKT signaling pathway. |
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