| Backgroud and ObjectiveColorectal cancer (CRC) is the third most common malignancy in the world and the leading cause of cancer deaths. The incidence of CRC is continuously increasing in China. Despite recent advances in the diagnosis and management of CRC, the overall survival of CRC patients has not improved markedly over the past years. Metastasis is still the main cause of mortality and poor prognosis. Hence, it is urgent to identify important molecules during cancer progression, which might contribute significantly to the development of new diagnostic strategies and potential drugs targets.Epithelial-mesenchymal transition (EMT), which is highly involved in tumor progression, is an initiating mechanism inducing tumor metastasis. EMT is a biological process that permits a polarized epithelial cell to assume a mesenchymal cell phenotype. Cancer cells undergoing EMT acquire aggressive properties to enterthe surrounding stroma, thereby acquiring enhanced migratory capacity, invasiveness, and elevated resistance to apoptosis. As a common event in human advanced CRC, constitutive overactivation of TGFβ acts as a key factor in the EMT process. However, in early stage malignancy, downstream inhibitors of TGFβ signaling and their suppressive effects on the overactivation of TGFβ stimulated downstream signaling have long been puzzling. Therefore, key molecules in TGFβ-mediated EMT must be explored to design new diagnostic strategies and specific targeted drugs for managing CRC metastasis.MicroRNAs (miRNAs) are a family of small noncoding RNAs that process from precursors with a characteristic hairpin secondary structure. They usually function as critical gene regulators by sequence-specific targeting mRNA transcripts, thus leading to gene translational repressions. Several miRNAs are aberrantly expressed in CRC, and their dysregulations contribute widely to cancer progression and clinical outcome. Recently, miR-1/133a cluster was demonstrated as tumor-suppressive miRNAs in human CRC for its repressing effects on the expression of LIM and SH3 protein 1(LASP1), previously identified as a CRC metastasis-associated protein. Since miRNAs often act as EMT-associated downstream effectors of receptor signaling or protein kinases, miRNAs might serve as new targets for developing anti-metastatic drugs, adding to their specificity. Moreover, preliminary data on the efficacy of miRNA are available, which would undoubtedly prompt the application of miRNA-based technology in the strategy against CRC metastasis in vivo.The present study has found that suppressive expression of miR-187 is required for TGFP-induced EMT process in CRC. Restored expression of miR-187 remarkably weakened the aggressive effects of TGFβ on CRC cells, thereby preventing activation of Smad signaling and initiated EMT during tumor progression. This added to the possibility that rising of EMT during tumor metastasis in CRC might be countered by restoring expression of miR-187, a notion that can readily be tested in the clinic.Methods1. The expression of miR-187 in CRC tissues and cells(1) The expression of miR-187 in 35 CRC tissues and their paried normal colorectal mucosas,8 CRC cell lines (HT29,HCT116, LS174T, RKO, SW480 and SW620, SW480/M5, LOVO) and the normal human colon epithelial cell line NCM460 were detected by Real-Time PCR.(2) The expression of miR-187 was examined in 119 specimens of CRC with follow-up visit information by in situ hybridization(ISH) method. The relationship between the expression of miR-187 and clinical pathological factors was analyzed by SPSS19.0 software.2. Investigating the effect of miR-187 on the biological behaviors of human CRC cell lines in vivo and in vitro(1) The biological effect of miR-187 in vitro cell proliferation,colone fomation and migration ability was investigated by CCK8, plate colony formation assays, Transwell assay and wound-healing assay after transicently transfected with miR-187 inhibitor/mimics and infected with LV-miR-187.(2) A subcutaneous tumor modelwas used to assess the effect of miR-187 on tumor genesis and growth of CRC cells. The effect of endogenous miR-187 overexpression on the lung nodule formation capacity was detected by tail vein injection of nude mice.3. MiR-187 involved in the mechanism of TGFβ-induced EMT(1) We observed the morphology after transiently transfected with miR-187 mimics/inhibitor, Western Blot and immunofluorescence also use to test the change of related indicators of EMT.(2) We analyzed the gene microarray and choosed the differential genes that have over 1.5 folds change to go KEGG pathway enrichment analysis the relationship between miR-187 and TGFβ. The expression change of miR-187 in CRC cells after situmulated with TGFβ is detected by RT-OCR.(3) We observed the morphology after respectively stimulated CRC cells with control,TGFβ, TGFβ+mimics. Transwell assay and Western Blot were used to detected the change of migration of CRC cells, related indicators of EMT and Smad signaling pathway.4. SOX4, NT5E and PTK6 are direct targets of miR-187(1) Three publicly available bioinformatics algorithms (TargetScan, PicTar, miRanda) and microarray-based miR-187 signaturewere used to analyze target genes of miR-187. And after RT-PCR and information retrieval we choosed SOX4, NT5E, PTK6 as the targets of miR-187. Luciferase reporter assay was performed to determine the direct effect betwwen miR-187 and the three targets. Western blot and immunofluorescence assays were used to analyze the change of the three targets after overexpression with miR-187.(2) We observed the expression change of SOX4, NT5E and PTK6 after respectively stimulated CRC cells with control,TGFβ, TGFβ+mimics by Western Blot.(3)We transfected CRC cells which stable expressed mir-187 with SOX4,NT5E,PTK6 ORF constructs without 3’UTR,and analyzed the migration of CRC cells, the change of related indicators of EMT and Smad signaling pathway by Transwell and Western Blot.5. Statistical analysisSPSS 19.0 software was used for data analysis. The expression of miR-187 in fresh paired CRC tissues was analyzed by Wilcoxon rank-sum test. The expression of miR-187 in metastatic CRC (mCRC) tissues and non-metastatic CRC (nmCRC) tissues was analyzed by Kruskal-Wallis test. The relationship between the expression of miR-187 and clinical pathological factors was analyzed by Pearson’s chi-squared (χ2) test. Survival curves was drawn by Kaplan Meier method and the statistical differences between the normal group and cancer group was analyzed by Log-Rank test. The data of RT-PCR, Transwell assay,wound-healing assay and colony formation assay were analysed through two-tailed independent-samples t-test when compared with two group of mean±standard deviation(SD), when groups over two then use One-Way ANOVA, with the LSD or Dunnett’s tests for multiple comparisons. The data of CCK8 assay was analysed by Factorial design analysis of variance.Results1. MiR-187 is down-regulated in CRC tissues and cell lines(1) MiR-187 is down-regulated in CRC tissues and cell linesThrough GEO (Gene Expression Omnibus, GSE49246) analysis of CRC tissue microRNA chip, we find the expression of miR-187 is down-regulated in 32 of 40 CRC tissues compaired with their paried normal colorectal mucosas. Real-time PCR was used to measure the expression of miR-187 transcripts in CRC tissues and cell lines. Up to 36.5-fold down change in miR-187 expression was found from25 of all 35 CRC samples when compared to the control samples. The expression of miR-187 in CRC tissues was significantly lower than the adjacent normal tissues (P<0.05). Furthermore, metastatic CRC (mCRC) tissues showed a lower miR-187 expression compared with non-metastatic CRC (nmCRC) tissues (P=0.001). Decreased expression of miR-187 was found in all eight CRC cell lines compared with normal human colon epithelial cell line NCM460 (P<0.05).(2) Low expression of miR-187 is associated with poor prognosis in CRCThe expression level and subcellular localization of miR-187 were analyzed by in situ hybridization (ISH) from a large cohort of 119 archived paraffin-embedded CRC and normal colon tissues.It shows no significant correlation between miR-187 expression and gender, age, tumor site, tumor size, tumor differentiation, T classification, N classification, M classification when we further investigated the clinicopathologic and prognostic significance of miR-187 expression in CRC patients (P=0.164; P=0.476; P=0.744; P=0.197; P=0.575; P=0.379; P=0.272; P=0.771). Kaplan-Meier survival analysis revealed that patients with low miR-187 expression levels had a worse clinical outcome (P=0.016). Multivariate survival analysis indicated that miR-187 expression, tumor differentiation, N classification and M classification were independent predictive factors for prognosis in patients with CRC (P<0.005; P<0.01; P=0.001, P<0.05).2. MiR-187 inhibit CRC cell proliferation and migration ability in vivo and vitro(1) miR-187 suppresses CRC cell proliferation and migration in vitroThe results of CCK-8 assays revealed that miR-187 could significantly decrease cell proliferation in SW480,RKO and SW620(F=198.508, P<0.001; F=630.401, P<0.001; F=3535.860, P<0.001). MiR-187-treated cells also showed a significant decrease of migration (t=-7.718, P<0.01; t=-7.448, P<0.05; t=-5.396, P<0.01) and motility (t=29.938, P<0.001; t=10.438, P<0.001; t=11.786, P<0.001) potential in transwell and wound-healing assays. Consistent with the above results, anti-miR enhanced cell proliferation potential in SW480 and HCT116 (F=198.508, P<0.001; F=191.249, P<0.001). Moreover, depletion of endogenous miR-187 significantly increased the ability of migration(t=2.922, P<0.05; t=4.509, P<0.05) and motility (t=10.532, P<0.001; t=5.227, P<0.01). LV-miR-187 was used to infect SW480 and RKO to establish CRC cell lines with stable miR-187 overexpression. SW480 and RKO cells with stable miR-187 overexpression showed an obvious decrease of clone formation (t=7.904, P=0.001; t=9.107, P=0.001), migration (t=-10.251, P<0.005; t=-4.025, P<0.05) and motility (t=-11.489, P<0.001; t=-15.461, P<0.001).(2) Endogenous overexpression of miR-187 inhibited CRC growth and progression in vivoA subcutaneous tumor modelwas used to assess the effect of miR-187 on tumor genesis and growth of CRC cells. The tumors in the SW480/LV-miR-187 group grew more slowly than those in the SW480/LV-NC group (P< 0.05). Similarly, the tumors in the RKO/LV-miR-187 group exhibited not only slower growth but also poorer ability of tumorigenesis compared with those in the RKO/LV-NC group (P> 0.05). The tumors in miR-187-overexpressing SW480 and RKO cells also showed significantly lower Ki-67 index compared with control (t=5.579, P<0.01; t=7.320, P<0.005). The resault of tail vein injection of nude mice shows, compared with the control group, less and smaller tumor nodules were found in the lung of mice with miR-187-overexpressing group. In addition, it came out that overexpression of miR-187 led to longer overall survival of the animals (P<0.05).3. miR-187 repression is essential for TGFβ/Smad-induced EMT(1) MiR-187 inhibition of EMTWe transcient transfection SW620, RKO and SW480 with miR-187 mimics, SW480, HCT116 and NCM460 with miR-187 inhibitor. Under inverted microscopy, miR-187 overexpressed cells were immotile and displayed a spheroid-shaped morphology; conversely, miR-187 knock-down cells displayed a typical morphology of aggressive cells, presenting a spindle-shaped form. Further immunoblot assays showed that miR-187 stimulated the expression of epithelial markers (E-cadherin, β-catenin, ZO-1) while suppressing that of mesenchymal markers (N-cadherin, fibronectin, vimentin) and lowering Smad2 phosphorylation level in miR-187-transfected SW480, RKO and SW620 cells compared with control cells. In contrast, suppression of miR-187 induced EMT process in SW480, HCT116 and NCM460 cells. Similarly, immunofluorescence assays observed that miR-187 might reverse the expression of EMT-associated proteins.(2) MiR-187 participate in the TGF-beta/Smad pathwayThrough analyzed the gene array of cells that infected with LV-miR-187, screened differentially expressed genes for KEGG pathway enrichment analysis, the resault uncovered that miR-187 closely contributed to TGFβ signaling pathway. we also found an apparent overlap of 53 genes between miR-187 signature and EMT signature. Treatment with miR-187 led to the suppression of EMT gene cluster, with the upregulated expression of epithelial genes (fold change> 1.2) and decreasing of mesenchymal genes (fold change< 0.85). This further supports the notion that down-regulated expression of miR-187 initiates a partial EMT. We then detected miR-187 expression in CRC cells in response to TGFP stimulation. As expected, expression of miR-187 was remarkably decreased after treatment with TGFβ in a time-dependent way in SW480, HCT116 and MDCK (F=23.622, P=0.001; F=98.036, P<0.005; F=18.157, P<0.005). The Dunnett’s T3 multiple comparison indicated the expression of miR-187 is down-regulated in response to TGFβ stimulation 24h and 48h compared with Oh (P<0.01, P<0.001; P=0.01, P<0.001; P<0.05, P=0.001). Further immunoblot assays showed that overexpressed miR-187 lead to lower Smad2 phosphorylation level in miR-187-transfected SW480, RKO and SW620 cells compared with control cells. In contrast, inhibit the expression of miR-187, the Smad2 phosphorylation level in miR-187-transfected SW480, HCT116 and NCM460 cells is increased compared with control cells.We observed the migration ability after respectively stimulated CRC cells with control,TGFβ, TGFP+mimics, Transwell assay shows significant differences between the three groups on migration ability (F=28.240, P=0.001; F=32.745, P=0.001; F=14.889, P=0.005). The Dunnett’s T3 multiple comparison indicated the number of migrated cells are more in TGFβ sitimulated group than control group (P<0.005; P<0.005; P<0.01); and the number of migrated cells are less in TGFβ +mimics group than TGFβ sitimulated group (P<0.01; P<0.01; P<0.05). After TGFβ treatment for 48 h, the cells displayed a spindle-shaped and fibroblasticlike phenotype instead of the cobblestone-like phenotype.Meanwhile, concomitant TGFβ stimulation and miR-187 transfection partially abolished morphology changes induced by TGFβ, suggesting that miR-187 neutralized the influence of TGFβ on cell phenotype changes. Introduction of miR-187 in TGFβ-treated cells rescued the expression of epithelial markers and weakened the expression of mesenchymal markers. TGFβ-induced activation of Smad signaling via promotion of Smad2 phosphorylation was counteracted by restoring the expression of miR-187, suggesting that miR-187 repression was essential for TGFβ/Smad-induced EMT.4. SOX4, NT5E and PTK6 are direct targets of miR-187(1) Prediction of miR-187 targetsThree publicly available bioinformatics algorithms (TargetScan, PicTar, miRanda) and microarray-based miR-187 signaturewere used to analyze target genes of miR-187. The collection of genes with absolute fold change (FC)> 1.5 in either SW480 or RKO was taken to make an intersection with three published target prediction engines including miRanda, TargetScan, and PicTar. The results showed that 114 genes were overlapped between microarray data and bioinformatics data. To combine GO analysis with KEGG pathway analysis,27 genes were selected to validate their expression using realtime PCR assay. The results showed a steady expression decrease of SOX4, NT5E and PTK6 in SW480 and HCT116 cells transfected by miR-187 (t=5.798, P<0.005; t=3.783, P<0.05; t=5.357; P<0.01).(2) Identification of miR-187 targetsLuciferase reporter assay shows Luciferase reporter assay was performed to determine whether miR-187 could directly target the 3’UTR region of SOX4, NT5E and PTK6.3’UTR fragments (wt) of SOX4, NT5E and PTK6 containing miR-187 binding site and their mutant fragments (mt) were cloned into luciferase report vectors. The wt or mt 3’UTR vector and miR-187 mimic were co-transfected into 293T cells. Significant decrease of luciferase activity was detected in wt vectors after transfected with miR-187 mimic. A mutation in the putative binding site in the SOX4, NT5E and PTK6 3’UTR region abrogated the suppressive effect mediated by miR-187, thereby providing additional evidence of direct interaction between miR-187 and 3’UTR region of SOX4, NT5E and PTK6.(3) SOX4, NT5E and PTK6 play crucial roles in TGFβ/miR-187-induced EMT of CRC cellsTo evaluate the biological role of SOX4, NT5E and PTK6 in EMT induced by TGFβ/miR-187 pathway, we rescued SOX4, NT5E and PTK6 expressions in miR-187-transfected CRC cells by transfection of SOX4, NT5E and PTK6 ORF constructs without 3’UTRs. The resault of Transwell assay shows the migration ability among groups are significant different (F=29.368, P<0.001; F=44.690, P<0.001). The Dunnett’s T3 multiple comparison indicated the migration ability of SW480 and RKO is decreased after overexpression of miR-187 (P<0.001; P<0.001), exogenous introduction of SOX4, NT5E or PTK6 commendably removed miR-187-induced suppression of migratory potential in SW480 and RKO cells (P=0.001, P<0.001, P<0.001; P<0.001, P<0.001, P<0.001).Consistent with the results of biological phenotype, reexpression of SOX4, NT5E, and PTK6 reversed miR-187-induced alteration of EMT signature and restarted Smad pathway. The results suggested that gains of SOX4, NT5E and PTK6 lead to a molecular and phenotype change similar to that with TGFβ stimulation or miR-187 inhibition, strongly supporting that SOX4, NT5E and PTK6 were common mediators of TGFβ/miR-187-induced invasion.Conclusions:1, MiR-187 is down-regulated in CRC tissues and cell linesis, low expression of miR-187 is associated with poor prognosis in colorectal cancer, indicated miR-187 may play a tumor supress gene in CRC;2, MiR-187 inhibit CRC cell proliferation and migration ability in vivo and vitro;3, MiR-187 repression is essential for TGFβ/Smad-induced EMT;4, SOX4, NT5E and PTK6 are direct targets of miR-187... |
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