| Background:Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide, and its overall incidence is～5% and the 5 years survival rate ranges from 40 to 60%. Many progresses made in the treatment of CRC in the past decades, but the overall survival of patients with CRC is still low. Poor prognosis and survival rate are mainly due to metastasis, thus more than one-third of patients with CRC will ultimately develop metastatic diseases. Due to the fact that the molecular mechanisms of CRC cell migration and invasion are highly complex, further identification of new candidate molecules that take part in these processes is crucial for developing targeted therapy for CRC.MicroRNAs (miRNAs), a class of 17-25 nucleotides small non-coding RNAs(ncRNA), associate with 3’-untranslated regions (3’-UTR) of specific target messenger RNAs (mRNAs) to suppress translation and occasionally also induce their degradation. Dsyregulated miRNAs play critical roles in many human physiological and pathological processes, such as cellular growth, differentiation, apoptosis, malignant transformation and metastasis. Recently, miR-149 is reported to be downregulated in many human cancers and functions as a tumor suppressor in non-small cell lung cancer, glioma and gastric cancer. The correlations of miR-149 with CRC are also reported. Wang, et al showed that SP1 mediates the link between methylation of the tumour suppressor miR-149 and outcome in CRC. Vinci, et al investigated the distribution of sequence variants of miR-146a, miR-196a, miR-499 and miR-149 in colorectal cancer (CRC) and their effects on miRNA expression. Also, Du, et al showed that the miR-149 and miR-196a2 polymorphisms may contribute to susceptibility to CRC. These results indicated that dysregulation of miR-149 might play an important role in CRC progression and development.Forkhead Box Ml (FOXM1) is a typical proliferation of specific transcription factor that belongs to the Forkhead Box family, which is evolutionarily conserved and is defined by having a common DNA-binding domain called Forkhead or Winged-helix domain. This protein is an important regulator of both Gl-S and G2-M phases of the cell cycle and mitotic spindle integrity, and its overexpression could lead to malignant transformation and tumor development. The overexpression of FOXM1 and other forkhead transcription factors is found in a variety of human cancers, and has been reported to play important roles in tumor angiogenesis, invasion, and metastasis. In previous study, we have reported that FOXM1 overexpression is a molecular marker predicting increased invasive/metastatic potential of CRC and a poorer prognosis. However, the molecular mechanisms involved in FOXM1 overexpression are unknown.Although it has been reported that miR-149 inhibits non-small cell lung cancer cells EMT by targeting FOXM1, whether miR-149 targets FOXM1 to suppress CRC migration and invasion is not unclear. In this study, we first investigated the expression of miR-149 in CRC cell lines and tissue samples, and analyzed its clinicopathological and prognostic values. Next, we comprehensively explored its roles in regulation of FOXM1 expression, migration and invasion of CRC cells and the possible molecular mechanisms by Real time-PCR, Western blot, bioinformatics analysis and luciferase activity assay.Objective:The purpose of this study was to investigate the clinicopathological and prognostic values of miR-149 in CRC, and explore roles of miR-149/FOXM1 signaling in migration and invasion of CRC cells and the possible molecular mechanisms, and to elucidate the mechanisms of miR-149 dysregulation and lay a foundation for formulating novel therapeutic target for treatment of CRC.Methods and Results:Part I The Analysis of Expression and Clinical Significance of miR-149 in CRCMethods:1.Real time RT-PCR analysis was performed to detect the expression of miR-149 in all collected CRC tissues, the adjacent normal tissues, normal colon tissues, a panel of human CRC cell lines with different metastatic potentials (HCT116, LoVo, SW480) and a normal colonic cell line (NCM460).2. The correlations between miR-149 expression levels and the clinicopathological factors as well as prognosis were determined in the whole 78 CRC tissues.3. Real time RT-PCR analysis was performed to detect the expression of FOXM1 in all collected CRC tissues, the adjacent normal tissues, normal colon tissues. And analyse the correlation between miR-149 expression and FOXM1.Results:1. The relative expression level of miR-149 in CRC tissues (n=78) was significantly lower than that in normal colon tissues (n=20)(P<0.01). In comparison with 20 paired normal colon tissues, the relative expression level of miR-149 was also significantly lower in the tumor tissues (P<0.01). Interestingly, the relative expression level of miR-149 in CRC tissues with lymph node metastasis or distant metastasis (mCRC, n=40) was significantly lower than that in tumor tissues without metastasis (nmCRC, n=38) (P<0.01). MiR-149 was significantly downregulated in CRC cell lines in comparison with the normal colonic cell line (NCM460). Also, among the three CRC cell lines, HCT116 (high-metastatic potential) exhibited the lowest miR-149 level (P<0.01).2. MiR-149 expression level was associated with clinicopathological characteristics including lymph node metastasis (P=0.024), distant metastasis (P=0.045) and TNM stage (P=0.033) in CRC tissues. The Kaplan-Meier survival analysis and Cox proportional hazards regression model indicated that the reduced expression of miR-149 was significantly correlated with shorter OS (P<0.01) and might be an independent prognostic factor for CRC patients(P=0.011; HR=2.658; 95% CI: 1.308-3.087).3. The relative expression level of FOXM1 mRNA in CRC tissues (n=78) was significantly lower than that in normal colon tissues (n=20)(P<0.01). In comparison with 20 paired normal colon tissues, the relative expression level of FOXM1 mRNA was also significantly lower in the tumor tissues (P<0.01). Interestingly, the relative expression level of FOXM1 mRNA in CRC tissues with lymph node metastasis or distant metastasis (mCRC, n=40) was significantly lower than that in tumor tissues without metastasis (nmCRC, n=38) (P<0.01). By linear regression analysis, it was found that there was a significant inverse association between the expression of miR-149 and that of FOXM1 mRNA (r=-0.553, P<0.01)Part II The Impacts of MiR-149 on Biological Behaviors of CRC Cells And Normal Colonic CellMethods:1. MiR-149 mimics and inhibitor (anti-miR-149)was introduced into human CRC cell lines (HCT116 and LoVo) and normal colonic cell line (NCM460) through transient transfection. MTT assays, Wound healing assays and Transwell invasion assays were performed to detect the cell proliferation, migration and invasion abilities.Results:1. Compared with miR-NC mimics, the transfection with miR-149 mimics in HCT116 cells led to a significant decrease in proliferation, migration and invasion abilities (P<0.05). Conversely, downregulation of miR-149 could lead to a increased in proliferation, migration and invasion abilities of LoVo cells (P<0.05).2. Downregulation of miR-149 could lead to a increase in migration and invasion abilities of normal colonic cell (NCM460) (P<0.05).Part III FOXM1 was identified as a direct target of miR-149 in CRCMethods:1. Three different online miRNA databases (TargetScanHuman 6.0, PicTar 5.0 and microRNA Targets) were employed for prediction of miRNAs that directly target the 3’-UTR of FOXM1.2. MiR-149 mimics, inhibitor and luciferase reporter vectors that contain FOXM1 -3’UTR-wt and FOXM1-3’UTR-mut were transient co-transfected into HCT116 cell respectly. Luciferase reporter assay was performed to further confirm the results.3. Real time RT-PCR and Western blot was performed to determine the effect of miR-149 on FOXM1 expression.Results:1.3’UTR of human FOXM1 contains a potential miR-149 binding site.2.MiR-149 could significantly decrease the fluorescence intensity through combined with 3’UTR of FOXM1.3. MiR-149 could target and regulate the FOXM1 expression.Part IV The Analysis of Molecular Mechanisms of miR-149 in Regulating the Motility and Metastasis of CRC CellsMethods:1. HCT116 and LoVo cells were stably transfected with pSil/shFOXM1 vector, and then, MTT assays, Wound healing assays and Transwell invasion assays were performed to detect the cell proliferation, migration and invasion abilities.2. A plasmid vector carrying WT FOXM1 (pEGFP/FOXM1) or the control plasmid vector (pEGFP/control) was introduced into HCT116/miR-149 cells through transient transfection. Real time RT-PCR and Western blot assay were performed to detect the expression of FOXM1. MTT assays, Wound healing assays and Transwell assays were performed to detect the cell proliferation, migration and invasion abilities.3. Real time RT-PCR and Western blot assay were performed to detect the expression of MMP-2, MMP-9, VEGF-A and uPAR in HCT116/miR-149 cells, HCT116/shFOXM1 cells and the control cells. pEGFP/FOXM1 or pEGFP/control introduced into HCT116/miR-149 cells through transient transfection. Real time RT-PCR and Western blot assay were performed to detect the expression of MMP-2, MMP-9, VEGF-A and uPAR.4. Real time RT-PCR was performed to detect the expression of MMP-2, MMP-9, VEGF-A and uPAR mRNA in high-miR-149 group (n=33) and low-miR-149 group (n=45).Results:1. FOXM1 knockdown in both HCT116 and LoVo cells could significantly inhibit the cell proliferation, migration and invasion. These results clearly indicate that silencing of FOXM1 mimics the effects of miR-149 overexpression on malignant phenotypes of CRC cells.2. Overexpression of FOXM1 could partially reverse the decreased capacity of proliferation, migration and invasion in HCT116 cells induced by miR-149 mimics. These findings further show that FOXM1 is a functional target of miR-149 in CRC cells.3. The mRNA and protein expression levels of MMP-2, MMP-9, VEGF-A and uPAR in miR-149 mimics or pSil/shFOXMl-transfected HCT116 cells were significantly downregulated in comparison with miR-NC mimics or pSil/shcontrol-transfected cells. Overexpression of FOXM1 could reverse the changes of MMP-2, MMP-9, VEGF-A and uPAR mRNA and protein in HCT116 cells induced by miR-149 upregulation.4. The relative mRNA expression levels of MMP-2, MMP-9, VEGF-A and uPAR in CRC tissues with low miR-149 expression (n=45) were significantly higher than those in tissues with high miR-149 expression (n=33).Conclusions:1. MiR-149 was significantly downregulated in CRC tissues, especially mCRC tissues, while the upregulation of FOXM1 was correlated with. Downregulation of miR-149 was correlated with lymph node metastasis, distant metastasis, TNM stage and poor survival in CRC patients.2. MiR-149 could inhibit CRC cells proliferation, migration and invasion abilities.3. MiR-149 could directly target the 3’UTR of FOXM1 mRNA and regulate its expression.4. MiR-149 inhibited migration and invasion of CRC cells via downregulation of MMP2, MMP-9, VEGF-A and uPAR, at least partially by targeting FOXM1. This finding showed FOXM1 is a functional target of miR-149 in CRC cells, and MMP2, MMP-9, VEGF-A and uPAR may be important effector molecules of miR-149/FOXM1.5. In this study, we have showed the dysregulation of miR-149 in CRC and its association with the clinicalpathological factors and prognosis, the impacts of miR-149 on the proliferation, migration and invasion process of CRC cells, and moreover, the molecular mechanisms of miR-149 have been further explored. This study indicated that target miR-149/FOXM1 signaling will be a potential strategy for the treatment of metastatic CRC. |
PDF Full Text Request