Mechanism Of The Regulation Of MiR-25 In Invasion,Metastasis And Microenvironment In The Colorectal Carcer

BackgroundRecently,along with unreasonable lifestyle and diet,the incidence and mortality of colorectal cancer(CRC)keep increasing.Surgical operation is the main treatment of CRC,which is supplemented with radiotherapy and chemotherapy.However,the result is poor.CRC is the third most common cause of death worldwide.The high mortality of CRC could,in part,be due to high propensity for recurrence and metastasis.It is necessary to find the efficient and specific metastasis-related genes in favor of filtration of CRC patients with micro-environment,which improve the treatment of CRC.miRNA is an non-coding single-stranded RNAs composed with 20-25bp nucleotides.Growing evidence indicates that miRNAs are aberrantly expressed in many human cancers such as breast cancer,lung cancer,hepatocellular carcinoma,and involve in the initiation,development and metastasis of cancers.miRNA targets the 3'untranslated region(3'UTR)of its target genes,and induces either translational repression or mRNA degradation.miRNAs negatively regulate target genes and play an important role in the progression of tumor as an oncogene or suppressor.It has been reported that miRNA get involved in the progression of cancer in different ways.First,miRNA aberrant expression alters the biological behavior of tumor cells such as proliferation,invasion and metastasis.Second,cancer cells secret exosomes or miRNAs into the microenvironment of cancer.Exosomes including miRNA are ingested by mesenchymal cells that finally promote cancer cell metastasis.miR-25,a member of miR-106b-25 cluster,is located on chromosome 7 within MCM7.It has been found that aberrant expression of miR-25 in gastric cancer,liver cancer,non-small-cell lung cancer was closely associated with tumor progression.For example,miR-25 promote cell invasion in esophageal squamous cell carcinoma.miR-25 promote cell proliferation in gastric cancer and colorectal cancer.Moreover,mir-106b-25 cluster was obviously up-regulated in colorectal cancer,hepatocellular carcinoma compared with matched adjacent non-tumor tissue.Strikingly,miR-25 was also higher in cancer stromal tissue than matched adjacent non-tumor stromal tissue.miR-25 was found to be involved in self-renewal capacity of tumor cells.Therefore,we hypothesized that miR-25 is associated with the tumor microenvironment and tumor stem cell characteristics.The study is to elucidate a novel mechanism of miR-25 in the regulation of migration,metastasis and microenvironment in CRC.Methods1.Filtration and identification of miR-25(1)The miRNA array was generated in normal mucosa,primary CRC tissues without metastasis,primary CRC tissues with lymphatic metastasis and those with distance metastasis.(2)Quantitative real-time PCR was performed to quantify miR-25 in 60 matched human CRC tissues and CRC cell lines.In-situ hybridization(ISH)was carried out to the location of miR-25 in the colorectal cancer tissues.(3)Quantitative real-time PCR was perfomed to quantify miR-25 in 6 CRC cell lines.2.The function of miR-25 in the progression of CRC(1)miR-25 inhibitor or mimics were transfected into CRC cell lines,respectively.Quantitative real-time PCR was performed to detect the expression of miR-25.(2)The miR-25 lentiviral vector was infected into CRC cell lines SW620 and SW480.The miR-25 expressing stable CRC cell lines were obtained by FCM(flow cytometry),and then tested the expression of miR-25 by Quantitative real-time PCR.(3)Functional validation of miR-25 in CRC cells was examined by Proliferation,Plate colony formation,Cell migration assays,Cell cycle and cell apoptosis assay.self-renewal assay,sbcutaneous tumor growth assay and tail vein metastasis assay and so on.(4)The effect of miR-25 on the sternness of CRC cells was performed by self-renewal assay,the expression of stem cell related markers.(5)The effect of CRC cells stable expressing miR-25 on the endothelial cells was performed by CRC cell induce HUVEC migration assays and tumor conditional medium induce HUVEC migration assays.(6)Functional validation of miR-25 in CRC cells was carried out with sbcutaneous tumor growth assay and tail vein metastasis assay and so on.3.Prediction and evaluation of miR-25 target genes(1)Five commonly databases including MiRanda,TargetScan,DIANA-microT,Pictar and RNAhybrid were used to forecast the target genes of miR-25.(2)FBXW7 and KLF4,which get high predictive values,were selected as candidate targets.Luciferase assay was adopted for the target evaluation.(3)Western blot or quantitative real-time PCR was performed to quantify miR-25,FBXW7 and KLF4 expression in CRC cells and tissues.Spearman's correlation coefficient was used to measure the degree of the linear relationship of gene expression levels.4.The function of miR-25 target genes in the progression of CRC(1)miR-25 and FBXW7 without 3'UTR region were co-transfected into CRC cell lines SW620 and SW480.Quantitative real-time PCR was performed to detect the expression of miR-25,and then detected the expression of FBXW7 by Western blot.(2)miR-25 and KLF4 without 3'UTR region were co-transfected into CRC cell lines SW620 and SW480.Quantitative real-time PCR was performed to detect the expression of miR-25,and then detected the expression of KLF4 by Western blot.(3)Functional validation of miR-25/FBXW7 in CRC cells was examined by Proliferation,Cell invasion assays.(4)Functional validation of miR-25/KLF4 in CRC cells was examined by self-renewal assay and so on.5.Statistical analysisQuantitative values of all experiments are presented as the mean ± SD,which are calculated from 3 independent experiments.All statistical analyses were performed by SPSS 13.0 statistical software.Statistical significance among/between groups was tested using one-way ANOVA or the independent samples t-test.Relationships between miR-3 71-5p expression and clinicopathologic characteristics were performed with Fisher exact test.Pearson's or Spearman's correlation coefficient was used to measure the degree of the linear relationship of gene expression levels.P<0.05 was considered to be statistically significant.Results1.miR-25 upregulation in CRC correlates with metastatic status,tumor size,differentiation and metastasis(1)miR-25 is markedly upregulated in CRC tissues and correlates significantly with metastasis115 metastasis-related miRNAs were tested in Microarray,of which 42 miRNAs were significantly upregulated in primary CRC tissue with lymph node or distant metastasis.miR-25 was one of the most up-regulated miRNAs.Thus we examined the expression and clinical values of miR-25 in 60 cases of paired CRC tissues.The expression of miR-25 was obviously higher in primary CRC tissues than in matched adjacent non-tumor mucosa(t=6.686,p<0.01).Strikingly,miR-25 expression correlated significantly with differentiation,tumor size,invasion and metastases(t=-2.259,p=0.028;F=21.72,p<0.01;F=5.35,p=0.007;F=4.728,p=0.013),and it had nothing to do with patients' age or sex(t=-1.001,p=0.321;t=0.893,p=0.375).(2)The expression and location of miR-25 in CRC tissuesWe examined the expression and location of miR-25 in 30 cases of paired CRC tissues by ISH.The expression of miR-25 was obviously higher in primary CRC tissues than in matched adjacent non-tumor mucosa in 25 of them.At the same time,we discovered that the expression of miR-25 in endothelial cells was comparatively higher than other kind of stromal cells.It reveals that miR-25 located in colon epithelial cells and ehdothelial cells in CRC.(3)The endogenous expression of miR-25 in 6 CRC cell linesThe expression of miR-25 in HT29 was higher than SW480,Ls174t,HCT116,LOVO and SW620.There was a significant difference of miR-25 expression between them(F=2594.033,p<0.001).2.miR-25 promotes proliferation,invasion,stem cell properties in vitro and metastasis in vivo in CRC cells.(1)Quantitative PCR shown that miR-25 significantly decreased compared with Blank or NC after miR-25 inhibitor was transfected into HT29 and SW480 cells(F=230.421,p<0.001;F=36.500,p<0.001).miR-25 significantly increased compared with Blank or NC after miR-25 mimics was transfected into SW620 and SW480 cells(F=429.974,p<0.001;F=1163.997,p<0.001).We used lentiviral constructs to infect CRC cells to establish cells stably expressing miR-25.Quantitative real-time PCR shown miR-25 significantly increased compared with mock in SW620 and SW480 cells(t=10.292,p=0.009;t=15.857,p<0.001).(2)miR-25 promote cell proliferation in CRCProliferation assays showed that knockdown of miR-25 significantly inhibited the proliferation of HT29 and SW480 cells(F=433.037,p<0.001;F=1306.266,p<0.001),inhibited cell cycle to transfer from G1 to S(G1 phase:t=-23.409,p<0.001;t=-9.240,p<0.001;S phase:t=7.543,p=0.002;t=3.365,p=0.028;G2 phase:t =5.120,p=0.007;t=9.803,p<0.001),compared to Blank or NC cells.Western Blot results shown that knockdown of miR-25 markedly decreased the expression of cell cycle related markers such as CyclinD3,CyclinD1,CDK4,CDK6.Proliferation assays showed that miR-25 over-expression markedly promoted proliferation of SW620 and SW480 cells as compared with their control cells(t=435.972,p<0.001;t=521.112,p<0.001),increased colony-forming ability(t=10.276,p=0.001;t=10.315,p<0.001),promoted cell cycle to transfer from G1 to S(G1 phase:t=19.328,p<0.001;t=12.082,p<0.001;S phase:t=-4.421,p=0.012;t=4.293,p=0.013;G2 phase:t=-14.005,p<0.007;t=16.208,p<0.001).Western Blot results shown that miR-25 over-expression markedly increased the expression of cell cycle related markers such as CyclinD3,CyclinD1,CDK4,CDK6.(3)Apoptosis assays showed that miR-25 knockdown or over-expression did not affect percentage of apoptosis cells in CRC(t=0.164,p=0.878;t=-0.284,p=0.791).(4)The migration assays showed that knockdown of miR-25 significantly inhibited the invasion of HT29 and SW480 cells(F=100.479,p<0.001;F=94.444,p<0.001),compared to Blank or NC cells.It also shown that miR-25 over-expression markedly promoted invasion of SW620 and SW480 cells as compared with their control cells(t=433.037,p<0.001;t=1306.266,p<0.001).The invasion assays yielded the similar effect.(5)miR-25 promotes CRC cells to obtain stem cell propertiesOver-expression of miR-25 results in the acquisition of sternness,which is characterized by an increased sphere-forming capacity(t=-13.258,p<0.001;t=-5.578,p<0.001).The expression of CRC stem cell markers SOX2,CD 133 and OCT4 will be carried out.(6)miR-25 over-expression promotes Human umbilical vein endothelial cells(HUVEC)migrationThe invasion assays shown that knockdown of miR-25 in HT29 and SW480 cells significantly inhibited the migration of HUVEC(t=7.256,p<0.001;t=10.061,p<0.001),compared to NC cells.It also showed that miR-25 over-expression in SW620 and SW480 cells markedly promoted migration of HUVEC as compared with their control cells(t=-11.165,p<0.001;t=-10.205,p<0.001).(7)Tumor conditional media(TCM)of miR-25 over-expression promotes Human umbilical vein endothelial cells(HUVEC)migrationThe invasion assays shown that TCM of miR-25 knockdown from HT29 and SW480 cells significantly inhibited the migration of HUVEC(t=10.735,p<0.001;t=6.242,p<0.001),compared to NC cells.It also showed that TCM of miR-25 over-expression from SW620 and SW480 cells markedly promoted migration of HUVEC as compared with their control cells(t=9.495,p<0.001;t=10.914,p<0.001).(8)miR-25 promoted tumorigenicity in nude miceSubcutaneous tumour growth assays showed that the weight of tumour in SW620/miR-25 significantly increased compared with SW620/mock cells(t=-3.526;p=0.008).(9)miR-25 enhanced their capacity to seed lung metastasesTail vein metastasis assays showed that over-expression of miR-25 in SW620 cells strikingly enhanced their capacity to seed lung metastases compared with mock cells(t=3.241;p=0.012).3.FBXW7,KLF4,KLF2 and FBXW7 are novel targets for miR-25(1)Potential targets of miR-25 were predicted by bioinformatic algorithms such as miRDB,TargetScan,microRNA.org,Pictar,DIANA-microT.TACC2,EXOC5,SOCS5,FBXW7,KLF4,KLF2 and FBXW7 were the predicted targets of miR-25.(2)In HEK293A and SW480 cell lines,Luciferase reporter assays shown that the luciferase activity was decreased significantly respectively in miR-25 group compared to Blank or NC group(MYO1B:F=51.312,p<0.001;F=28.276,p=0.001;KLF2:F=19.624,p=0.002;F=36.597,p<0.001;KLF4:F=11.169,p=0.009;F=18.676,p=0.003;FBXW7:F=15.213,p=0.004;F=19.817,p=0.002)after transfected withwild-type MYOIB,KLF4,KLF2 and FBXW7 3'UTR plasmid.However,there was no significant difference in luciferase activity between miR-25 group,Blank group and NC group after transfected with wild-type TACC2,EXOC5 and SOCS5 3'UTR plasmid(TACC2:F=1.276,p=0.345;F=4.955,p= 0.054;EXOC5:F=4.566,p=0.062;F=1.195,p=0.366;SOCS5:F=2.253,p=0.186;F=0.848,p=0.474).(3)The basal expression of miR-25 was inversely correlated with FBXW7 mRNA in the 6 CRC cell lines(p=0.049,r=-0.814).Spearman's correlation analysis showed no relationship between miR-25 and KLF4 expression levels in the 6 CRC cell lines(p>0.05).However,there was a negative relationship between miR-25 and KLF4 expression levels in the 4 CRC cell lines(p<0.001,r=-1.000).Accordingly,we chose FBXW7,KLF4 as predicted targets of miR-25.(4)FBXW7 expression in colorectal cancer was significantly up-regulated than that in the corresponding normal tissue.Spearman's correlation analysis demonstrated that miR-25 and FBXW7 were again inversely related in expression(p<0.001,r=-0.909).Spearman's correlation analysis also showed a negative relationship between miR-25 and KLF4 expression levels in the 6 matched CRC tissues(p =0.014,r=-0.899).(5)miR-25 represses FBXW7 to promote CRC migration.The migration assays showed that the up-regulation of miR-25 increased cell migration of SW620 and SW480 cells compared with control cells,the migrated abilities of SW620 and SW480 cells significantly decreased in miR-25/FBXW7 group than in miR-25 group(F=132.531,p<0.001;F=31.268,p<0.001).The migration assays of miR-25/KLF4 yielded the similar effect(F=63.586,p<0.001;F=28.376,p<0.001).ConclusionWe identified miR-25 as an important "oncomir" in the progression of CRC and discovered that KLF4,FBXW7 are direct targets of miR-25.We also elucidated a novel mechanism of miR25 in the regulation of metastasis and microenvironment through its target genes,which may be a potential therapeutic target.