The Functional Studies Of MiRNAs Regulating SATB2,an Important Metastasis-related Gene In Colorectal Carcinoma

BACKGROUND&OBJECTIVEColorectal carcinoma (CRC) is one of the most common cancers.The incidence rate and mortality rate have risen rapidly in recent years. Death and tumor recurrence, after radical operation for patients with CRC, always occure.Therefore, understanding of the molecular mechanisms underlying CRC metastasis is of crucial significance to the developments of therapeutic strategies for advanced CRC patients.Tumor metastasis is a series of events with multiple steps, multiple stages and ways, and each one is controlled by many genes, proteins and biological molecules. In recent years, the studies found that instead of coding genes, non-coding genes were involved in tumor invasion and metastasis, such as microRNA.Special AT-rich sequence-binding protein2(SATB2) is a DNA binding protein that specifically binds on nuclear matrix attachment regions. Being an encode protein, SATB2involves in transcription regulation and chromatin remodeling. SATB2 expressed at higher level in adult brain tissue, moderate level in human fetal brain, and displayed lower or undetectable level in the adult liver, idney, bone marrow, and certain areas of the brain including the amygdala, corpus callosum, caudate nucleus and hippocampus. loss of S ATB2leads to cranial facial mesenchymal tissue apoptosis, and gene expression patterns changes of Pax9, Alx4and Msxl, which involoved into the craniofacial development of mice and humans. In recent years, many studies showed that SATB2plays an important role in osteoblast differentiation and development of cerebral cortex.In our previous studies, the results showed that expression of SATB2was down-regulated in M5cells. Immunohistochemical analysis of146colorectal tumour samples showed that underexpression of SATB2was strongly correlated with poor prognosis, tumour invasion, lymph node metastasis, distant metastasis, and Dukes’ classification forCRC. Univariate and multivariate survival analyses further showed that SATB2expression was a potential favourable prognostic factor for CRC. These results demonstrated not only that SATB2is a potential novel prognostic factor for CRC, but also that selection of a highly metastatic clone of SW480in vivo coupled with gene expression profiling is a powerful approach to identifying prognostic markers for CRC. So far, the key factors regulate the expression of SATB2in CRC remaine unclear. Our study is based on the hypothesis that SATB2is downregulated by a certain (some) molecule.microRNA (miRNA) is a class of non-coding small single-stranded RNA,19-25nucleotides, are important post-transcriptionl regulater found in recent years.With completly or incompletly bingding on target mRNA3’UTR, miRNA suppresses the target gene. In past decade, Cancer-associated miRNA has become a hot topic on CRC metastasis. It affects occurrence and development of tumors by following ways (1) Act as an oncogene or tumor suppressor.(2) miRNAs, involved in cell signaling pathway, change tumor cell migration, invasion, proliferation, conversion of EMT, angiogenesis and apoptosis and influence metastasis of cancers.(3) miRNAs may be a specific diagnosis marker and target for treatment. Presently, miRNAs targeting on SATB2in CRC have not been reported. Our results demonstrate that SATB2and its regulator miRNAs play important roles in CRC, and suggest a potential application of them in prognosis prediction and therapeutic application in CRC.MATERIALS AND METHODS1. Prediction of miRNAs regulating SATB2By using TargetScan, Pictar and miRanda database, we get miRNA target genes prediction results, witch combined to the results of previous microRNAs chips in order to find the miRNAs binding on SATB23’UTR.2. Detection of miRNAs expression in CRC tissues and cellsBy using RT-PCR, we detecte expressions of miRNAs and SATB2in CRC tissues and cells. On the other hand, To further investigate the clinicopathological and prognostic significance of miRNAs levels, we also measured miRNAs expression in a large cohort of paraffin-embedded CRC and normal colon tissues by in situ hybridization.3. The functional studies of miRNAs in CRC cells3.1Luciferase reporter assayThe firefly luciferase construct was cotransfected with a control Renilla luciferase vector pRL-CMV (Promega) into293FT cells in the presence of either miRNAs or control. A dual luciferase assay (Promega) was performed48h after transfection. The experiments were performed independently in triplicate.3.2Influence of miR-31/miR-182on biological behavior and prognosis in CRC cellWe establish stable miRNAs-overexpression cell lines and mock cell lines (miR-con). Increased expression of miRNAs upon infection in CRC cells were confirmed by real-time PCR. Further investigations revealed the overexpression of miR-31in CRC results in changes of tumor cell proliferation by CCK8assay, Colony formation assay and cell-cycle analysis. Moreover detection of cells motility by Wound healing and invasion assays. Additionally, we validate whether SATB2overexpression could reverse the promote effects of miRNAs on CRC tumorigenesis and progression.4. Western blot analysisThe expression of makers in EMT, MAPK and Smad pathways were detected by westernblot and immunofluorescense staining.5. Statistical analysisAll statistical analyses were performed using the SPSS13.0statistical software package. Data were presented as mean±SD of at least three independent experiments. Differences between variables were assessed by the χ2test, Fisher’s exact test, match-samples T test, independent T test and One-way ANOVA. Survival curves for the patients with different levels of miRNA expression were plotted using the Kaplan-Meier method and compared using the log-rank test. Multivariate survival analysis was performed on all parameters that were found to be significant in univariate analysis using the Cox repression model. P value<0.05was considered significant.RESULTS1. Searching for potential regulators to SATB2in CRCThe bioinformation and microRNA arrary analysis revealed that miR-31, miR-182and miR-27b can bind on3’UTR of SATB2mRNA, and their expressions in SW480/M5are higher than in SW480. Then we observed that ectopic overexpression of miR-31and miR-182significantly reduced SATB2expression in SW480and DLD1(FSW480=35.164, PSS480=0.002; FDLD1=10.344, PDLD1=0.023). After transfected with inhibitors of miR-31and miR-182, the expression of SATB2were upregulated in M5and SW620(FM5=29.298, PM5<0.001; FSW620=24.165, PSW620<0.001).2. expressions of miR-31and miR-182in CRC cells and tissuesThe results showed that miR-31and miR-182were found to be markedly upregulated in CRC tissues compared with adjacent non-neoplastic normal tissues (tmiR-31=-2.256, PmiR-31=0.038; tmiR-182=-3.907, PmiR-182=0.001).It was observed that upregulations of miR-31and miR-182in tumor samples were significantly associated with lymph-node metastasis (tmiR-31=-2.904,PmiR-31=0.044; tmiR-182=-4.498, PmiR-182=0.011).we also measured miR-31and miR-182expressions in a large cohort of143archived paraffin-embedded CRC and normal colon tissues using in situ hybridization (ISH), and found that high-level expression of miR-31and miR-182were significantly associated with a more aggressive and poor prognostic phenotype of patients with CRC (p<0.05).3. Functional studies of miR-31and miR-182in CRC cellsWe established stable miR-31and miR-182overexpression cell lines and mock cell lines (miR-con). Increased expression of miR-31and miR-182in cells were confirmed by real-time PCR.3.1CCK8analysisthe results of cell growth assay displayed that miR-31overexpression increased cancer cell proliferation (FsW480=255.452, Psw480<0.001; FDLD1=1065.112, PDLD1<0.001); we got the same results of SW480/miR-182and DLD1/miR-182cells (FSW480=1719.538, PSW480<0.001; FDLD1=553.862, PDLD1<0.001).3.2Colony formation assay the results of cell growth assay displayed that miR-31overexpression increased cancer cells’ ability to form colonies than that in the mock cells and wild type non-infected cells. FSW480=302.890, PSW480<0.001; FDLD1=5.851, PDLD1<0.001); Cell groups of overexpression of miR-182achieved the same result (FSW480=54.157, PSW480=0.001; FDLD1=21.752, PDLD1=0.002)Cell-cycle analysisCell cycle analysis with flow cytometry revealed that a significant decrease in the percentage of miR-31upregulated cells in the G1/G0phase (FSW480=16.389, PSW480=0.004; FDLD1=11.722, PDLD1=0.008) and increase in S phase (FSW480=21.125, PSW480=0.002; FDLD1=32.967, PDLD1=0.001). and cells with miR-182upregulated have the same change, decrease in G1/G0phase (FSW480=47.257, PSW480<0.001; FDLD1=24.915, PDLD1<0.001) and increase in S phase (FSW480=45.507, PSW480<0.001; FDLD1=25.408, PDLD1<0.001)3.3Tumour growth in vivotumor growth in the SW480/miR-31or SW480/miR-182groups were significantly more rapid than that in the SW480/miR-con group (FSW480/miR-31=12.381,-PSW480/miR-182<0.001; FSW480/miR-182=4.977, PSW480/miR-182<0.001). IHC Staining showed that the tumors of control group displayed much lower Ki-67index than that in miR-31or miR-182overexpressed groups.3.4Tumour matastasis in vivoAfter8weeks, SW480/miR-31or SW480/miR-182cells exhibited dramatically metastasis of liver and peritoneal cavity, whereas SW480/miR-con cells only formed tumor increasing without any metastasis. H&E staining showed obviously invasion in intestinal wall and metastasis of stomach, diaphragm, abdominal wall, liver, lymph node and lung in SW480/miR-31or SW480/miR-182cells.4、SATB2is one of direct targets of miR-31and miR-182 We observed that increased expressions of miR-31and miR-182significantly reduced the luciferase activity of the reporter containing SATB23’UTR, while did not affect the luciferase activity of the empty vector control (FmiR-31=18.721, PmiR-3i <0.001; FmiR-182=123.177, PmiR-182<0.001)5、The changes of CRC cells caused by miR-31/miR-182could be attenuated by overexpression of SATB25.1CCK8analysisSATB2overexpression in SW480/miR-31and DLD1/miR-31cells could inhibit proliferation ability (FSW480=255.452, PSW480<0.001; FDLD1=1065.112, PDLD1<0.001); SATB2overexpression could also inhibit SW480/miR-182and DLD1/miR-182cells’ proliferation ability (FSW480=1719.538, PSW480<0.001; FDLD1=553.862, FDLD1<0.001).5.2Cell-cycle analysisSATB2overexpression could cause an increase the percentage of SW480/miR-31and DLDl/miR-31cells in the G1/G0phase (FSW480=255.452, PSW480<0.001; FDLD1=1065.112, PDLD1<0.001)and decrease in S phase(FSW480=1719.538, FSW480<0.001; FDLD1=553.862, FDLD1<0.001). With similar results, percentage of SW480/miR-182and DLDl/miR-182cells in the G1/G0phase increased (tSW480=-18.160; PSW480<0.001; tDLD1=-6.156, FDLD1=0.004), and cells in S phase decreased (tSW480=16.378, PSW480<0.001; tDLD1=11.718, FDLD1<0.001)5.3Wound-healing and transwell migrationSATB2overexpression in SW480/miR-31and DLD1/miR-31cells could inhibit migration ability (tSW480=9.167, FSW480<0.001; tDLD1=9.500, FDLD1<0.001); SATB2overexpression could also inhibit SW480/miR-182and DLD1/miR-182cells’ migration ability (tSW480=10.698, FSW480=0.003; tDLD1=10671, FDLd1<0.001) 6. Overexpression of miR-31and miR-182induce CRC cells metastasis though EMT PathwayWe assessed the epithelial and mesenchymal markers by western blot in miR-31/miR-182-overexpression CRC cells and control cells. The expression levels of Snail and two mesenchymal makers (N-cadherin and vimentin) were strikingly upregulated in miR-31/miR-182-overexpression cells, while SATB2and two epithelial marker (E-cadherin and β-catenin) levels were downregulated. Then, SATB2overexpression could significantly attenuate the expression changes of the above markers resulted by miR-31or miR-182.miR-182overexpression could increase Smad3protein expression in SW480/miR-182and DLD1/miR-182cells, which could amplify TGF-beta signaling. Moreover, we observied increase of SPAK/JUK protein expression in SW480/miR-182and DLD1/miR-182cells after SATB2-pCAG transfected.CONCLUSION1. miR-31and miR-182were upregulated in metastatic CRC cells and human primary CRC tissues with lymph node metastases. Also, overexpression of miR-31and miR-182were associated with aggressive phenotypes and poor prognosis of patients with CRC.2. Overexpression of miR-31and miR-182promote CRC cells growth, invasion and migration in vitro and facilitate tumour growth and matastasis in vivo.3. SATB2is one of direct targets of miR-31and miR-182directly target SATB23’UTR.4. Overexpression of miR-31and miR-182induce CRC cells metastasis though EMT pathway, and SATB2overexpression could significantly attenuate the expression changes of the above markers resulted by miR-315. Participating in the Smad signaling pathway and MAPK signal pathways, miR-182and SATB2regulated the proliferation and metastasis of colorectal cancer cells.