Expression Of Mir-873-5p In Colorectal Cancer And Mechanism Of Inhibiting Cell Migration And Invasion

Colorectal cancer(CRC)is the most common malignancy in the world,with more than one million new diagnoses each year.The incidence and mortality of colorectal cancer in China were ranked 5th,and statistical analysis showed that the incidence of colorectal cancer continued to increase in both men and women.Colorectal cancer caused by multi-factors,such as colorectal adenomas,intestinal inflammation,and intestinal flora.The development of this disease involves several mechanisms,including hyperproliferation,loss of apoptosis regulation,acquisition of an invasive phenotype,increased angiogenesis,and maintenance of colorectal cancer stem cells(CSC).This progression usually involves up-regulation of many oncogenes and down-regulation of important tumor suppressor genes.Among genes,MicroRNA(miRNA)has attracted more and more attention from researchers and clinicians,and has become research hotspot.MiRNA is a non-coding RNA of about 19 to 22 nt in length.It can bind to specific mRNAs and negatively regulate gene expression at the post-transcriptional level.There is increasing evidence that miRNAs can control a variety of biological processes and are key regulators of development and cellular homeostasis.The miRNA regulates the target mRNA and fine-tunes the protein output.Thus,dysregulation of miRNA function can lead to a variety of human diseases such as Alzheimer's disease,breast cancer,lung cancer,prostate cancer,ovarian cancer,and head and neck cancer,as well as colorectal cancer.miRNAs are evolutionarily conserved molecules that can participate in a variety of physiological and pathological processes,including CRC metastasis,by affecting cancer stem cell biology,angiogenesis,epithelial-mesenchymal and mesenchymal-epithelial cell transformation or drug resistance.Abnormal expression of miRNA leads to the development of CRC.This effect may be enhanced by deletion of the miRNA locus,amplification or point mutation,epigenetic silencing,transcription factor dysregulation(modification of miRNA expression)or inhibition of primary miRNA processing into its mature form.When up-regulated,certain specific miRNAs can inhibit genes responsible for growth/proliferation inhibition,and down-regulation of other specific miRNAs can enhance the expression of genes responsible for promoting growth/proliferation,leading to the development or progression of cancer,thus identifying miRNA targets and Its functional regulatory network is critical for understanding the normal biological processes of miRNAs and their role in disease progression.So far,the expression,function and mechanism of miRNA in CRC are still not completely clear,and further research is needed.Epithelial cell mesenchymal transition(EMT)is a complex cellular process in which epithelial cells acquire a mesenchymal phenotype.EMT is associated with an aggressive or metastatic phenotype of the tumor.A growing body of research provides strong evidence for the important role that EMT plays in cancer progression and metastasis of a variety of malignancies,including CRC.In CRC,EMT is thought to be beneficial for increasing the motility of tumor cells in the invasion front and increasing the resistance of cells to apoptosis,which is an important mechanism leading to cancer metastasisZEB1(zinc finger E-box binding homeobox 1)is a well-known oncogene that promotes migration and epithelial-mesenchymal transitions in a variety of malignancies,including colorectal cancer.Some miRNAs that regulate EMT have been found to interact only with one ZEB transcription factor.For example,in bladder cancer,expression of miR-23b is used to distinguish between normal and bladder cancer tissues,and the high expression of this miR-23b is positively correlated with the high overall survival rate of bladder cancer patients.In CRC,miR-147 is highly negatively correlated with the EMT gene expression signature and is assumed to be a reverse EMT(MET).A recent study showed that ZEB1 was detected in the invasion zone of the colorectal tumor and in the tumor-matrix interface:a region showing a decrease in polar components.ZEB1 induces loss of the basement membrane(BM)by inhibiting the expression of the epithelial BM component laminin-3(LAMA3).It specifically binds to Z1,Z2 and E2 on the LAMA3 promoter and inhibits transcriptional activity.ZEB1 silences up-regulation of several polar genes in colorectal cancer cells,including CRB3 and LGL2.The LGL2 promoter is a direct target of ZEB 1,and loss of LGL2 is associated with increased EMT and metastasis in the CRC.MiR-873-5p was first discovered in the hippocampus of impaired temporal lobe epilepsy rats.In Alzheimer's disease,miR-873-5p targets HMOX1 to regulate its expression and thereby inhibit the apoptosis of neuronal cells.In adhesion-induced kidney injury,METTL3/m6A/miRNA-873-5p plays a protective role by modulating oxidative stress and apoptosis by modulating the Keapl/Nrf2 pathway.In lung cancer,miR-873-5p,a downstream target gene of LncRNADCGR5,regulates the expression of TUSC3 in tumor progression.miR-873-5p is underexpressed in gastric cancer,while the expression of Glil is significantly enhanced.Overexpression of miR-873-5P reduced cell viability,increased apoptosis and cell cycle arrest.At the same time,knockout of Glil significantly induced apoptosis and cell cycle arrest in SGC-7901 cells.The dual luciferase reporter assay showed that Glil is the target gene of miR-873-5p.More importantly,even in cells transfected with si-Glil,inhibition of miR-873-5p significantly reduced the protein expression of CyclinB1 and Bcl2.In conclusion,miR-873-5p acts as a tumor suppressor gene mainly in gastric cancer by targeting Gli1.Given the role of miRNAs in CRC cell activity,studies of the function of CRC-related miRNAs and their relationship to tumorigenesis have provided new targets for the precise treatment of CRC and may help to improve prognosis.There is no report on the expression of miR-873-5p in colorectal cancer and its related biological functions.This study is to investigate the expression,biological function and possible mechanism of miR-873-5p in CRC.Part I:Expression of miR-873-5p in colorectal cancer and its biological functionObjective:The aim of this study was to investigate the expression of miR-873-5p in colorectal cancer and colorectal cancer cell lines and its possible biological functions.Materials and Methods:qRT-PCR was used to detect the expression of miR-873-5p in all tissues of normal intestinal endothelial,juvenile polyps,ulcerative colitis,colorectal adenoma,colorectal cancer,adjacent non-tumor tissues and normal intestinal endothelial cell lines,colorectal cancer cell lines.The CRC cell lines were transfected with miR-873-5p mimics and Inhibitors to overexpress or silence the expression of miR-873-5p for subsequent functional testing.Cell invasion and cell migration experiments were performed after transfection.Results:1.qRT-PCR results showed that the level of miR-873-5p in CRC tissues and cells was lower than that of normal intestinal endothelial and HIEC normal human intestinal epithelial cell line,and the difference was statistically significant.2.Different CRC cell lines were transfected with miR-873-5p mimics and Inhibitors to overexpress or silence the expression of miR-873-5p.The expression of miR-873-5p was detected by RT-PCR.After transfection of miR-873-5p mimics in HCT-116 cells,the expression level of miR-873-5p was significantly increased.After transfection of miR-873-5p Inhibitors in SW-480 cells,the expression level of miR-873-5p was significantly reduced.3.After transfection of miR-873-5p mimics overexpressing miR-873-5p in HCT-116 cells,cell proliferation,migration and invasion were significantly inhibited4 Transfection of miR-873-5p Inhibitors in SW-480 cells knocked out the expression of miR-873-5p(anti-miR-873-5p).The cell proliferation,migration and invasion ability of anti,miR-873-5p SW-480 cells was significantly increased.Summary:MiR-873-5p was down-regulated in CRC tissues and cells and inhibited CRC cell proliferation,migration and invasion,and played a role as a tumor suppressor.Part II:miR-873-5p mediates EMT regulation of CRC cell migration and invasionObjective:Explore the he possible mechanism of miR-873-5p in the migration and invasion of colorectal cancer cells.Materials and Methods:The CRC cell lines were transfected with miR-873-5p mimics and Inhibitors to overexpress or silence the expression of miR-873-5p for subsequent functional testing.Western blot and immunofluorescence staining were used to determine the expression of EMT-related markers.Results:1.After overexpression of miR-873-5p in HCT-116,the levels of epithelial cell markers E-cadherin,?-catenin and ZO-1 increased significantly,while the levels of interstitial cell markers N-cadherin and Vimentin decreased significantly.At the same time,the cell morphology is transformed from interstitial cells to epithelial cells.2.After knocking out miR-873-5p in SW-480 cell line,the levels of epithelial cell markers E-cadherin,(3-catenin and ZO-1 were significantly reduced,while the levels of interstitial cell markers N-cadherin and Vimentin were significantly increased.Summary:miR-873-5p regulates CRC cell migration and invasion by mediating EMT.Part III:Identification of miR-873-5p downstream target genesObjective:Explore possible downstream target genes for miR-873-5p and validate.Materials and Methods:Bioinformatics methods(miRanda and TargetScan)were used to speculate the possible targets of miR-873-5p?and the intersections were screened to screen for genes negatively regulated by miR-873-5p,which were further screened for EMT and cell migration and invasion.The gene predicts its possible target of action.The possible mechanism of action was then identified by molecular biology experiments(luciferase reporter gene,pull down experiments,rescue experiments,etc.).Results:1.Predict the target genes of miR-873-5p by using two bioinformatics prediction tools(TargetScan and miRanda),and make an intersection,a total of 77 potential target mRNAs,and 13 of 77 mRNAs were miR-873-5p Negative adjustment.Among these 13 mRNAs,ZEB1 is closely related to cell proliferation,migration and epithelial-mesenchymal transition(EMT).The putative binding sequence between miR-873-5p and ZEB1 was obtained by using the public bioinformatics tool TargetScan.2.ZEB1 was highly expressed in CRC tissues and cell lines.3.Dual luciferase reporter assay is shown in the HCT-116 cell line,miR-873-5p overexpression significantly reduced the luciferase activity of ZEB1 wild-type 3'UTR(ZEB1-WT),but did not significantly reduce ZEB1 mutation Luciferase activity of the type 3'UTR(ZEB1-MUT).In SW-480 cells,inhibition of miR-873-5p expression potently enhanced the luciferase activity of the wild-type 3'UTR of ZEB1-WT,but did not increase the luciferase of the mutant 3'UTR of ZEB1-MUT Activity,data indicate that ZEB1 expression is negatively regulated by miR-873-5p.4.Pull down test analysis showed that ZEB1 can be pulled down by Bio-miR-873-5p instead of Bio-miR-873-5p-MUT or Bio-NC.ZEB1 was confirmed to be a downstream target of miR-873-5p.5.In HCT-116 transfected miR-873-5p mimics overexpressing miR-873-5p,ZEB1 expression was down-regulated in mRNA and protein levels,while SW-480 transfected miR-873-5p inhibitors underexpressed miR-873-5p,ZEB1 increased in mRNA and protein levels.6.Remediation experiments confirmed that the overexpression of ZEB1 partially reversed the inhibitory effect of miR-873-5p on the migration and invasion of HCT-116 cells.Furthermore,overexpression of ZEB1 attenuated the reversal of EMT by miR-873-5p on HCT-116 cells.At the same time,knockdown of ZEB1 reversed anti-miR-873-5p-mediated migration,invasion and EMT of SW-480 cells.Summary:ZEB1 is a downstream target of miR-873-5p and is negatively regulated by miR-873-5p.Conclusion:Low expression of miR-873-5p in CRC tissues and cells was identified by qRT-PCR analysis.Functional experiments confirmed that miR-873-5p inhibited CRC cell proliferation,migration and invasion.miR-873-5p may affect cell proliferation,migration and invasion by mediating EMT biological processes.By bioinformatics analysis,luciferase report assay and pull-down assay,we found that ZEB1 is a downstream target gene of miR-873-5p and is negatively regulated by miR-873-5p.The rescue experiments demonstrated the effects of the miR-873-5p-ZEB 1 axis on migration,invasion,and EMT processes in colorectal cancer cells.In conclusion,we demonstrate that miR-873-5p inhibits cell migration,invasion and EMT in colorectal cancer by targeting ZEB1.