MicroRNA-215-3p Suppresses The Growth,Migration,and Invasion Of Colorectal Cancer By Targeting FoxM1

Background:With the continuous aging population,lifestyle change,the incidence and mortality of colorectal cancer(CRC)in the past 20 years continued to increase in China.CRC has brought heavy economic burden to the society and families,threatening the people health and life safety of people.The clinical therapies have been updated with the further research of biological mechanism of CRC and demonstrated satisfactory curative effect.However,recurrence and metastasis also played the predominant death causes of CRC patients,extremely in advanced CRC patients.Novel targeted screening and consequent drug discovery for CRC are still the critical question of current research.Small non-coding RNA and microRNAs(miRNAs)have been proven modulate the expression of specific proteins via binding to the 3?-untranslated region(3?-UTR)of target mRNAs.MiRNAs played vital functions in biological process of tumor,including cell growth,apoptosis and gene activity regulation.Recent researches have verified that overexpression of miR-215-3p could sensitize colorectal cancer to 5-fluorouracil(5-FU)induced apoptosis through regulating CXC-chemokine receptor type 1(CXCR1).Until now,the definite mechanism of miR-215-3p in the progression of CRC have not been well explored.The human forkhead box protein M1(FoxM1)has been proven to play vital roles in the initiation,promotion and progression of several cancers.Extensive investigations demonstrated that FoxM1 expresses high in tumor tissues and modulates cancer progression,angiogenesis and metastasis.Herein,we investigated that miR-215-3p decreased the growth and aggressive of CRC cell through down-regulating its target gene,FoxM1.In this research,we studied the expression of miR-215-3p in CRC tissues and its relationship with prognosis.Further,the mechanism of FoxM1 played in miRNA-215-3p signaling will be explored.This work proved that whether miR-215-3p could act as a new molecular marker of CRC progression,and served experimental and theoretical basis for the selection of new therapeutic targets in future.Part one:Expressions and significance of miRNA-215-3p in patients with CRCObjective:Detection the expressions level of miR-215-3p in CRC tissues.Besides,Analysis of relationship between miR-215-3p expression level and clinicopathological factors or prognosis in patients with CRC.Methods:48 primary CRC and paired adjacent non-tumor tissues were obtained from the Second Affiliated Hospital of Anhui Medical University.The expressions level of miR-215-3p were detected by qRT-PCR assay.The correlation was analyzed between miR-215-3p expression and clinicopathological characteristics in patients with CRC.48primary CRC patients were divided into high expression group and low expression group in terms of the average expression level of miR-215-3p.The correlation between the expression level of miR-215-3p and overall survival in CRC patients was investigated.Results:The expressions level of miR-215-3p were significantly reduced in CRC tissues when compared to those in adjacent normal tissues(P<0.01).The expressions level of miR-215-3p related to tumor differentiation,TNM staging and lymph node metastasis(P<0.01).However,there were no associated with gender,age,tumor site,tumor size and histological type(P>0.05).The median overall survival were 65 months in 30 patients with low expression of miR-215-3p,and that were 87 months in 18patients with high expression.The Kaplan-Meier survival analysis indicated that the overall survival of patients who exhibited lower level of miR-215-3p was poor than patients who had high level of miR-215-3p(?~2=6.317,P=0.012).Conclusion:The expressions of miR-215-3p in CRC tissues were negatively associated with tumor differentiation,TNM staging and lymph node metastasis.Patients with high expression of miR-215-3p exhibited better prognosis,which suggested miR-215-3p expression may play an anti-oncogene role in regulating the CRC process and up-regulated miR-215-3p expression indicated the favorable prognosis of CRC patients.Part two:High expression of miRNA-215-3p inhibit malignant biological behaviors of CRC cellsObjective:Observation influence on the malignant biological behaviors of CRC cells which regulated by miR-215-3p.Methods:The expressions level of miR-215-3p were detected by qRT-PCR assay in colon epithelial cell lines HCoEpiC and CRC cell lines SW480,HT-29,LOVO.Then,SW480 and HT-29 cells were divided into the control group,the miR-NC group(transfected with scramble),and the miR-215-3p group(transfected with MiR-215-3p)respectively.The proliferation was detected by MTT assay,the colony formation was determined by cell colony formation assay,the migration was measured by cell scratch assay,the invasion ability was tested by transwell assay in each group.SW480 cells were randomly divided into miR-NC group(the SW480 cells were transfected with scramble)and miR-215-3p group(the SW480 cells were transfected with MiR-215-3p),which were inoculated into nude mices to establish the X-enograft model.The length and width of tumor tissue was measured each week,and plotted the growth curve of the X-enograft model.Five weeks after CRC cells inoculation,nude mice were sacrificed and tumor tissue was isolated and weighed.lung metastasis model was artificially created in 5-week-old nude mices by injected SW480 cells which transfected with miR-NC and miR-215-3p respectively.Mice were sacrificed 2 weeks after inoculation and lung metastatic nodules were analyzed under a dissecting microscope.Results:The expressions level of miR-215-3p were significantly reduced in CRC cell lines when compared to those in the HCoEpiC cells(P<0.01).Besides,the expressions level of miR-215-3p in the SW480 cells were 15 times than that in the control group after transfected with miR-215-3p(P<0.01).Consistently,the expressions level of miR-215-3p in HT-29 cells were 10 times than that in the control group,there was a significant statistically difference between the two groups(P<0.01).Compared to the Control group,the proliferation of SW480 cells and HT-29 cells were significantly reduced,the number of clone formation were significantly decreased,the migration and invasion were significantly lessened respectively(P<0.01).Compared to the miR-NC group,the volume and weight of X-enograft model were significantly reduced in the miR-215-3p group,and lung metastatic nodules were significantly decreased in the same way(P<0.01).Conclusion:We further confirmed that miR-215-3p exhibited lower expression in CRC cell lines comparing with human colon epithelial cell lines.Over-expressed miR-215-3p can significantly inhibit the malignant biological behaviors of CRC cells,such as proliferation,migration and invasion in vitro and in vivo.Part three:Experimental study of miRNA-215-3p Suppresses the growth,migration,and invasion of CRC by Targeting FoxM1Objective:Investigation the specific mechanism of miR-215-3p participate in regulating the malignant process of CRC.Methods:TargetScan was selected to predict the target gene of miR-215-3p.We observed that miR-215-3p hasputative binding sites with the 3?-UTR region of FoxM1.Then,wt-FoxM1 plasmid and mut-FoxM1 plasmid were constructed by the pGL3-promoter vector.HEK-293T cells were co-transfected with vectors containing either the wild-type(wt)or mutant(mut)target sites of FoxM1 and either miR-215-3p or negative control(miR-NC).Luciferase reporter assay was performed to verify the binding between miR-215-3p and FoxM1.Western blot analysis of FoxM1 expression in CRC cell with or without miR-215-3p overexpression.Detection of endogenous FoxM1expression by western blotting after transfection of siFoxM1 in SW480 cell.Similar,SW480 cells were co-transfected with pcDNA3.1 containing FoxM1 or control vector after transfection of miR-NC or miR-215-3p.The expressions of FoxM1 were assessed using western blotting assay.IN the next moment,the proliferation was detected by MTT assay,the colony formation was determined by cell colony formation assay,the migration was measured by cell scratch assay,and the invasion ability was tested by transwell assay in each group.Results:The bioinformatics algorithms,TargetScan was utilized to find the target gene of miR-215-3p and we finally demonstrated FoxM1 was the directly target gene of miR-215-3p using luciferase reporter gene assay.Compared to the miR-NC,the miR-215-3p group luciferase activity was significantly reduced in the cell that transfected with wt-FoxM1(P<0.01).On the contrary,there was no significant luciferase activity change in the cell that transfected with mut-FoxM1(P>0.05).The double luciferase reporting system verified that FoxM1 was a direct target gene of miR-215-3p.Furthermore,miR-215-3p transfection significantly decreased the expressions of FoxM1 in HT-29 and SW480 cell.There was an inversely correlation between FoxM1and miR-215-3p level in Pearson and TCGA(R~2=0.1244,P=0.0139).SiRNA against FoxM1 was transfected into SW480 to reduce the level of FoxM1.Knockdown of FoxM1 suppressed the proliferation and colony formation of SW480 cell,in addition,siRNAs against FoxM1 reduced the migration and invasion abilities in SW480 cell(P<0.01).On the other hand,re-expression of FoxM1significantly increased the level of FoxM1 in miR-215-3p transfected SW480 cell,and up-regulation of FoxM1 abrogated the inhibitory effects of miR-215-3p on the migration and invasion of SW480 cell(P<0.01).Conclusion:MiR-215-3p was proved that could directly targeting FoxM1 in a negative relationship manner.FoxM1 inhibition can significantly inhibit the malignant behavior of CRC cells.Further,miR-215-3p can specific inhibited FoxM1 expression,and re-expression of FoxM1 could inverse the malignant biology of CRC cells.