The Role And Mechanism Of MiR-21-5p In The Development Of Colon Adenocarcinoma Through Targeting CHL1

BackgroundColorectal cancer(CRC)is the most common cancer.Cases of colon cancer have experienced a dramatic uprush during the past decades in china,while the rate of rectal cancer descends at the speed of 3%every year in several western countries.Although surgery,radiotherapy,chemotherapy and targeted therapy are used,but the prognosis isn’t ideal,colon cancer mortality has not decreased significantly in the past decade.Many patients were in advanced stages when being diagnosed.Postoperative recurrence、metastasis and chemotherapy resistance are common clinically.Therefore,for early diagnosis of colon cancer,and according to the molecular characteristics of colon cancer,carry out individualized and precise treatment,further understanding the pathogenesis of colon cancer is currently a problem to be solved.Similar to the pathogenesis of other malignant tumors,the pathogenesis of colon cancer is also a complex disease caused by multiple factors such as environmental factors and genetic factors,and its mechanism is not fully understood.In the past decade,with the development of molecular biology,genomics and epigenetics,we have a deeper understanding of the molecular mechanism of colon cancer,and found that colon cancer is a heterogeneous complex disease involving genetic and epigenetic changes.MicroRNAs are an important branch of epigenetics.They belong to endogenous non-coding regulatory RNA and are widely involved in the growth and development,immunity,inflammation,cardiovascular and cerebrovascular diseases and carcinogenesis.microRNA-21(miR-21),located at 17q23.1,is a carcinogenic microRNA that has been found to be up-regulated in a variety of tumor cells.Researchers found that the expression of miR-21 was higher in colorectal cancer tissues than in adjacent tissues.However,there are few studies on specific types of cancer cells in colorectal cancer,such as colon adenocarcinoma(COAD),and the mechanism of how miR-21-5p influences progression of COAD still remain unclear.Therefore,it is essential to carry out deeper analyses of miR-21-5p and COAD.CHL1(Close homolog of L1)is a member of the immunoglobulin superfamily neural adhesion molecule LI family(L1-CAMs).L1-CAMs are composed of L1、L1 close homolog(CHL1,L1CAM2)、NrCAM and Neurofascin.The CHL1 gene is homologous to LI CAM and is located at chromosome 3p26.3,also known as L1CAM2 or cell-like adhesion LI(CALL),which is involved in cell adhesion and migration.CHL1 is closely related to the development and regeneration of the mouse nervous system which cloned in mice by Senchenko et al.initially.Studies have found that CHL1 is not only found in the nervous system,but also in other normal tissues and tumor tissues,and its silencing contributes to the in situ growth of tumors.Therefore,CHL1 may play an important role in the occurrence and progression of tumors.Study found that CHL1 is down-regulated in colon cancer tissues compared with surrounding normal tissues,and the expression of CHL1 is also different between colorectal cancer tissues with metastasis and colorectal cancer tissues without metastasis,suggesting that CHL1 may inhibit the development of colorectal cancer and inhibits further metastatic spread(Senchenko et al.,2011).Another study found that miR-21 inhibits the growth of neuroblastoma cells by targeting CHL1(Li et al.,2016).However,there are few studies on the role of CHL1 and miR-21-5p in the development of COAD and its molecular mechanism.Purpose and significanceThis study aims to investigate the effect of miR-21-5p on process of colon adenocarcinoma(COAD)cells and its connection with CHL1.The findings of our study may assist researchers in discovering the mechanisms of progression of COAD,and may even help in developing antitumor treatment.Part Ⅰ:the Relationship Between miR-21-5p and Clinical and Pathological Features of Colon Adenocarcinoma and the Regulation of miR-21-5p on the Biological Behavior of Colon Adenocarcinoma Cells.Background and purposeThe molecular mechanism for the development and progression of colon adenocarcinoma has not been fully clarified.The role of microRNAs in malignant tumors has attracted the attention of more and more researchers,and a large number of studies have shown that it plays an important role in colorectal cancer.miR-21 plays an important role in a variety of cancers,and there are few studies on specific types of cancer cells(colon adenocarcinoma,COAD)in colorectal cancer,and the mechanism for how miR-21-5p influences COAD progression isn’t completely clear.The purpose of this study was to detect the expression of miR-21-5p in COAD tissues and different COAD cell lines,and analyze its relationship with the clinicopathological features of COAD,and explore the effects of miR-21-5p on the proliferation and invasion of COAD cell.Methods1.Collect the microarray datas of COAD miRNAs from the TCGA database and analyze the differences with normal tissues.2.The cancerous and adjacent normal tissues of 43 pairs of COAD patients confirmed by postoperative pathology were collected from the Second Hospital of Shandong University.The patients hadn’t receive chemotherapy and targeted therapy before operation.The expression of miR-21-5p in COAD tissues and adjacent normal colon tissues was detected by qRT-PCR,and the relationship between miR-21-5p and clinicopathological features was analyzed.3.The expression of miR-21-5p in four COAD cell lines CW-2,T84,SW1116,LoVo and normal colonic epithelial cells NCM-460 was detected by qRT-PCR.4.The regulation of miR-21-5p on the malignant biological phenotype of COAD cell proliferation and invasion was evaluated by colony formation assay,MTT assay,flow cytometry assay,Transwell invasion assay and ELISA.Results1.TCGA microarray data analysis showed that the expression of miR-21-5p was 6.22 times(P<0.001)in COAD samples compared with adjacent normal tissues.2.Relationship between miR-21-5p and clinicopathological features:qRT-PCR results showed that miR-21-5p was highly expressed in colon cancer tissues compared with surrounding normal colonic mucosa(P<0.001);The expression of miR-21-5p was closely related to clinical stage,lymph node metastasis and distant metastasis(P<0.05).However,there was no significant correlation between miR-21-5p expression and gender or age(all P>0.05).3.Compared with human normal colonic epithelial cell NCM-460,miR-21-5p is highly expressed in COAD cells.The expression levels of miR-21-5p in four COAD cells were significantly up-regulated,especially in LoVo cells(P<0.001).Therefore,we chose the LoVo cell line as the subject of subsequent experiments.4.In vitro functional assay:colony formation assay,MTT assay and flow cytometry assay showed that overexpression of miR-21-5p in LoVo cells significantly promoted cell entry into S phase and promoted cell proliferation;while inhibiting miR-21-5p can significantly arrest cells in the G1 phase and inhibit cell proliferation.Transwell invasion assay and ELISA showed that expression of MMP-2 and MMP-9 was elevated in LoVo cells with overexpressing miR-21-5p,which promoted invasion;whereas in LoVo cells with suppressed expression of miR-21-5p,the expression of MMP-2 and MMP-9 is reduced,inhibiting invasion.Conclusions and significanceThe expression of miR-21-5p was significantly increased in COAD tissues and cancer cell lines,and its expression was closely related to the clinical stage,lymph node metastasis and distant metastasis of COAD.In vitro cell function assays showed that miR-21-5p promoted proliferation and invasion of COAD cells.This indicates that miR-21-5p overexpression plays an important role in the carcinogenesis and development of COAD,and it can be used as an effective biomarker,which will help to early diagnosis and prognosis.Part Ⅱ:Verification CHL1 as one target of miR-21-5pBackground and purposemiRNAs are a class of endogenous single-strand non-coding small RNAs that bind to the 3’UTR of the target gene mRNA and thus participate in the regulation of gene expression,which is closely related to processes such as cell cycle,differentiation,apoptosis and tumorigenesis.Each miRNA can regulate multiple target genes,while the same gene can be regulated by several miRNAs.Because of its complex function,it is difficult to fully understand the function of miRNAs,so the number of target genes of miRNAs is limited.In recent years,with the development of large-scale miRNAs target gene predictive analysis databases such as TargetScan,computer-based algorithms can predict the seed region binding sites of miRNAs,which refers to the second to eighth nucleotides,highly conservative during evolution.The results of the first part showed that miR-21-5p is up-regulated in COAD tissues and cells,suggesting that miR-21-5p may promote the carcinogenesis and development of COAD.CHL1 is a gene with tumor suppressor activity discovered in recent years,which is lowly expressed in colon cancer,and studies have found that miR-21 can target CHL1 to regulate the growth of neuroblastoma.The purpose of this study was to verify that miR-21-5p can target CHL1 and regulate its expression.The prediction was verified by a dual luciferase reporter system.Previous studies have shown that the expression level of miR-21-5p is up-regulated in LoVo cells,so LoVo cell line was used to study the overexpression and inhibition of miR-21-5p.Methods1.Analysis of microarray data for differentially expressed mRNAs in COAD tissues from the Cancer Genome Atlas(TCGA)database(https://cancergenome.nih.gov/).Differentially expressed mRNA was screened by t-test(P<0.05)combined with fold change(FC).2.Using bioinformatics software(TargetScan,miRBase)to predict that CHL1 is a direct target of miR-21-5p,miR-21-5p can target the 3’UTR of CHL1 mRNA.3.Using the dual luciferase reporter assay to verify the presence of a complementary binding relationship between miR-21-5p and the 3’UTR of CHL1;we constructed the CHL1 wild-type and mutant 3’UTR Luciferase dual fluorescent reporters pmirGLO-CHLlwt and pmirGLO-CHL1 mut,co-transfected with LoVo cells(miR-21-5p mimics group and NC mimics group).After 48 hours,the changes in luciferase activity of the wild-type group(wt)and the mutant group(mut)were observed in comparison with the control group,respectively,to determine whether miR-21-5p could directly bind to the 3’UTR of CHL1 mRNA.4.qRT-PCR and Western Blotting(WB)were used to further detect the expression of CHL1 mRNA and protein under the regulation of miR-21-5p,and to verify the targeting regulation of miR-21-5p on CHL1.Results1.We analyzed the COAD microarray datas in the TCGA database and screened differentially expressed mRNAs.The results showed that the expression of CHL1 was 2.57 times lower than that of normal tissues(P<0.001).2.Targetscan and miRBase indicated that the binding site of miR-21-5p exists in the 3’UTR region of CHL1;3.In the dual luciferase reporter assay,we successfully constructed the pmirGLO-CHL1 3’UTR-wt plasmid and the pmirGLO-CHL1 3’UTR-mut plasmid,which were co-transfected with LoVo transfected by miR-21-5p mimics and NC mimics,respectively.The luciferase activity of the cells showed that the activity of luciferase in the co-transfected group of pmirGLO-CHL1 3’UTR-wt and miR-21-5p mimics was significantly lower than that of the control group(P<0.01).After the mutant plasmid pmirGLO-CHL1 3’UTR-mut was co-transfected with miR-21-5p mimics and mimics NC,the luciferase activity of the two groups did not change significantly(P>0.05).The results indicate that miR-21-5p can bind to the 3’UTR of CHL1.4.qRT-PCR and Western Blot analysis showed that the mRNA and protein levels of CHL1 were significantly down-regulated in LoVo cells transfected with miR-21-5p mimics compared with NC transfection group(P<0.001);the expression of CHL1 in LoVo cells was significantly up-regulated after transfection with miR-21-5p inhibitor(P<0.01).Conclusions and significance1.In COAD cells,CHL1 is a target gene of miR-21-5p.2.miR-21-5p regulates the expression of CHL1 mRNA and protein by binding to the 3’UTR of CHL1 mRNA.Part III:miR-21-5p Participates in the Regulation of Proliferation and Invasion of Colon Adenocarcinoma Cells by Targeting CHL1Purpose and backgroundThe neuronal cell adhesion molecule L1(CHL1)is a neuroadhesion molecule that functions not only in neural signaling pathways but also in normal human tissues and various cancers(He et al.,2013).It has been reported that CHLI was closely related to mental retardation in 3p syndrome(Wei et al.,1998)and is associated with schizophrenia in the Chinese population(Chen et al.,2005).The researchers also found that changes in CHL1 are associated with the development of different cancers such as colon cancer.CHL1 has been shown to inhibit the invasive growth of COAD and inhibit further metastatic spread(Senchenko et al.,2011).In part two we have identified that CHL1 is a target gene of miR-21-5p.In this part,we will observe the effects of miR-21-5p on the biological behavior of COAD by CHL1 through in vitro and in vivo experiments.Methods1.Transfected NC,pcDNA3.1CHL1,CHL1 siRNA and miR-21-5p inhibitor+CHL1 siRNA in LoVo cell line,respectively.The colony formation assay and MTT assay were used to compare the transfection group and the control group to evaluate CHL1 and miR-21-5p on cell proliferation.The effects of CHL1 and miR-21-5p on cell cycle were evaluated by flow cytometry cell cycle assay comparing each transfection group and control group.The effects of CHL1 and miR-21-5p on cell invasion were evaluated by transwell invasion assay comparing each transfection group and control group.2.In vivo experiments were performed to examine the effects of CHL1 and miR-21-5p on COAD tumor growth.We divided the 4-weeks-old BALB/C athymic female nude mice into 5 groups(4 mice in each group),and injected them with cells transfected by miR-21-5p inhibitor negative control,miR-21-5p inhibitor,CHL1 siRNA,siRNA negative control and miR-21-5p inhibitor together with CHL1 siRNA respectively.1 x 107 treated cells were injected subcutaneously into the back of each nude mouse.The volume of nude mice in all groups was measured weekly using a caliper as follows:volume =(length x width2)/2,with each parameter standing for the maximum diameter and the minimum diameter,respectively.The weight of tumor was measured after 4 weeks.The tumor tissues were excised,RNA was extracted,miR-21-5p and CHL1 were detected by qRT-PCR,and the expression of CHL1 was detected by Western Blot.Results1.After cell transfection,cell proliferation was evaluated by colony formation assay and MTT assay.The results showed that pcDNA3.1 CHL1 inhibited cell viability(P<0.05);Conversely,deletion of CHL1 enhanced cell Survival ability(P<0.05).In addition,the effect of CHL1 siRNA was rescued by miR-21-5p inhibitors(P<0.05);Flow cytometry analysis showed that compared with the control group,the cells in the pcDNA3.1 CHL1 group were retained in the G0/G1 phase(P<0.05),while the number of cells in the S phase of the CHL1 siRNA group increased(P<0.01).After co-transfection with CHL1 siRNA and miR-21,5p inhibitor,cells in the S phase were reduced(P<0.05).Transwell invasion assay showed that the invasive ability of COAD cells was weakened in the pcDNA3.1 CHL1 group(P<0.01).In the CHL1 siRNA group,the invasive ability of the cells was enhanced(P<0.01),but it was rescued by miR-21-5p inhibitor(P<0.05).In summary,CHL1 inhibits the growth and invasion of the COAD cell,and the cells were retained in the G0/G1.2.miR-21-5p inhibitor can inhibit the growth of COAD tumors in vivo experiments,and knockout of CHL1 can promote the growth of COAD tumors.The expression of miR-21-5p and CHL1 in tumor tissues was detected by Western Blot and qRT-PCR.The results showed that inhibition of miR-21-5p resulted in up-regulation of CHL1 expression(P<0.01).Therefore,miR-21-5p promotes tumor growth by inhibiting the expression of CHL1 in vivo.Conclusions and significancemiR-21-5p inhibits CHL1 expression,thereby promoting the proliferation and invasion of COAD cells and the growth of COAD.