Expression and Role of MicroRNAs from the miR-200 Family in the Tumor Formation and Metastatic Propensity of Pancreatic Cancer.
ABSTRACT: MicroRNAs from the miR-200 family are commonly associated with the inhibition of the metastatic potential of cancer cells, following inhibition of ZEB transcription factors expression and epithelial-to-mesenchymal transition. However, previous studies performed in pancreatic adenocarcinoma revealed a more complex picture challenging this canonical model. To gain better insights into the role of miR-200 family members in this disease, we analyzed the expression of miR-200a, miR-200b, miR-200c, miR-141, miR-429, and miR-205, and ZEB1, ZEB2, and CDH1 in pancreatic tumors and matching normal adjacent parenchyma and patient-derived xenografts. We found that miR-200a, miR-429, and miR-205 are frequently overexpressed in pancreatic tumors, whereas CDH1 is downregulated, and ZEB1 and ZEB2 levels remain unchanged. Furthermore, we measured a positive correlation between miR-200 family members and CDH1 expression, and a negative correlation between ZEB1 and miR-200c, miR-141, and miR-205 expression, respectively. Interestingly, we identified significant changes in expression of epithelial-to-mesenchymal transition regulators and miR-200 members in patient-derived xenografts. Lastly, functional studies revealed that miR-141 and miR-429 inhibit the tumorigenic potential of pancreatic cancer cells. Taken together, this comprehensive analysis strongly suggests that miRNAs from the miR-200 family, and in particular miR-429, may act as a tumor suppressor gene in pancreatic cancer.
Project description:Although various pharmacological activities of the shikonins have been documented, understanding the hierarchical regulation of these diverse bioactivities at the genome level is unsubstantiated. In this study, through cross examination between transcriptome and microRNA array analyses, we predicted that topical treatment of shikonin in vivo affects epithelial-mesenchymal transition (EMT) and the expression of related microRNAs, including 200a, 200b, 200c, 141, 205, and 429 microRNAs, in mouse skin tissues. In situ immunohistological analyses further demonstrated that specific EMT regulatory molecules are enhanced in shikonin-treated epidermal tissues. RT-PCR analyses subsequently confirmed that shikonin treatment downregulated expression of microRNA-205 and other members of the 200 family microRNAs. Further, expression of two RNA targets of the 200 family microRNAs in EMT regulation, Sip1 (Zeb2) and Tcf8 (Zeb1), was consistently upregulated by shikonin treatment. Enhancement of these EMT activities was also detected in shikonin-treated wounds, which repaired faster than controls. These results suggest that topical treatment with shikonin can confer a potent stimulatory effect on EMT and suppress the expression of the associated microRNAs in skin wound healing. Collectively, these cellular and molecular data provide further evidence in support of our previous findings on the specific pharmacological effects of shikonin in wound healing and immune modulation.
Project description:Background:Lung cancer is the most common and lethal malignancy worldwide. TCTP is highly expressed in various cancers including lung cancer. Epithelial-mesenchymal transition (EMT) could increase cancer cell invasion. Whether TCTP's expression is associated with EMT in lung adenocarcinoma is largely unknown. Methods:Several Gene Expression Omnibus datasets were used to analyze the correlation between TCTP expression and overall survival of lung adenocarcinoma patients by Kaplan-Meier survival analysis. Then, 24 surgically removed fresh lung adenocarcinoma tissue samples and paired paracancer tissue samples were used to analyze the correlation between TCTP expression and tumor stage by immunohistochemical analysis. Furthermore, stable cell lines were generated using lentiviral transduction systems to knock down or overexpress TCTP in A549 cells. Cell migration and invasion were measured by scratch and transwell assays, and EMT marker proteins such as ?-SMA, ZEB1, and E-cadherin were quantitated by Western blot. The expression levels of miR-200a, miR-141, and miR-429 were determined by real-time quantitative PCR, and their target genes were predicted by an online database miRTarBase. The interaction between TCTP and these genes was analyzed by String database and visualized by Cytoscape. Results:TCTP was highly expressed in tumor tissues compared to paracancer tissues. The expression of TCTP was associated with shorter overall survival. TCTP knockdown experiment in A549 cells suggested that TCTP knockdown could decrease the migration and invasion of lung cancer cells, and the expression level of ZEB1 and ?-SMA, but increase the expression of E-cadherin and p53. Vice versa, overexpression of TCTP could increase the migration and invasion of cancer cells, and the expression level of ZEB1 and ?-SMA, but decrease the expression of E-cadherin and p53. Furthermore, we found the expression of miR-200a, miR-141, and miR-429 was associated with TCTP expression. Conclusion:TCTP promotes EMT in lung adenocarcinoma, and this effect may be associated with miR-200 family members like miR-200a, miR-141, and miR-429.
Project description:Tumor metastasis is the leading cause of death among breast cancer patients. PELP1 (proline, glutamic acid and leucine rich protein 1) is a nuclear receptor coregulator that is upregulated during breast cancer progression to metastasis and is an independent prognostic predictor of shorter survival of breast cancer patients. Here, we show that PELP1 modulates expression of metastasis-influencing microRNAs (miRs) to promote cancer metastasis. Whole genome miR array analysis using PELP1-overexpressing and PELP1-underexpressing model cells revealed that miR-200 and miR-141 levels inversely correlated with PELP1 expression. Consistent with this, PELP1 knockdown resulted in lower expression of miR-200a target genes ZEB1 and ZEB2. PELP1 knockdown significantly reduced tumor growth and metastasis compared with parental cells in an orthotopic xenograft tumor model. Furthermore, re-introduction of miR-200a and miR-141 mimetics into PELP1-overexpressing cells reversed PELP1 target gene expression, decreased PELP1-driven migration/invasion in vitro and significantly reduced in vivo metastatic potential in a preclinical model of experimental metastasis. Our results demonstrated that PELP1 binds to miR-200a and miR-141 promoters and regulates their expression by recruiting chromatin modifier histone deacetylase 2 (HDAC2) as revealed by chromatin immunoprecipitation, small interfering RNA and HDAC inhibitor assays. Taken together, our results suggest that PELP1 regulates tumor metastasis by controlling the expression and functions of the tumor metastasis suppressors miR-200a and miR-141.
Project description:MiR-200 family is an important regulator of epithelial-mesenchymal transition and has been implicated in human carcinogenesis. However, their expression and functions in human cancers remain controversial. In the work presented here, we showed that miR-200 family members were frequently down-regulated in hepatocellular carcinoma (HCC). Although all five members of miR-200 family inhibited ZEB1/2 expression in HCC cell lines, we showed that overexpression only of the miR-200b/200c/429 subfamily, but not the miR-200a/141 subfamily, resulted in impeded HCC cell migration. Further investigations led to the identification of RhoA and ROCK2 as specific down-stream targets of the miR-200b/200c/429 subfamily. We demonstrated that the miR-200b/200c/429 subfamily inhibited HCC cell migration through modulating Rho/ROCK mediated cell cytoskeletal reorganization and cell-substratum adhesion. Re-expression of miR-200b significantly suppressed lung metastasis of HCC cells in an orthotopic liver implantation model in vivo. In conclusion, our findings identified the miR-200b/200c/429 subfamily as metastasis suppressor microRNAs in human HCC and highlighted the functional discrepancy among miR-200 family members.
Project description:The aim of the presented study was to define tissue and plasma miRNA signatures, which could potentially serve as diagnostic and prognostic markers in endometrioid endometrial cancer (EEC), and to investigate miRNA profiles in regard to clinicopathological characteristics of the tumors. Results: qPCR validation revealed regulation of 14 miRNAs in EEC tissues (miR-9, miR-141,miR-205,miR-203,miR-183,miR-200a*,miR-200b*,miR-200a,miR-200c,miR-429,miR-200b,miR-410,miR-92a,miR-1305) and 9 in plasma samples (miR-449a,miR-1290,miR-1228,miR-203,miR-200a,miR-141,miR-92a, miR-9, miR-301b). Expression of certain miRNAs showed association with FIGO stage, grade and relapse. Two miRNA signatures, miR-205/miR-410 and miR-410/miR-429/miR-92a, classified tumor tissues with higher accuracy in comparison to single miRNAs (AUC 0.972, 95% CI 0.919-0.995 and 0.991, 95% CI 0.948-1.000, resepctively). miRNA signature composed of miR-205 and miR-200a predicted relapse with AUC of 0.854 (95% CI 0.691-0.951). Tissue miRNA signatures were independent prognostic markers of overall (miR-1228/miR-200c/miR-429, HR 2.98) and progression-free survival (miR-1228/miR-429, HR 4.149). Plasma miRNA signatures classified EEC plasma samples with high accuracy. Conclusions: We conclude that miRNA signatures hold great promise for becoming non-invasive biomarkers for early EEC detection and prognosis. Tissue samples were collected from 122 women (77 EEC and 45 controls). Expression profiling of 866 human miRNAs and 89 human viral miRNAs was performed in 24 samples and was followed by qPCR validation in 104 patients. Of 24 samples analyzed by microarrays, 22 (18 EEC, 4 normal controls) were available for final analysis. Expression of 14 miRNAs was analysed in 48 plasma samples by qPCR.
Project description:Fibroblasts can be reprogrammed to induced pluripotent stem cells (iPSCs) by application of transcription factors octamer-binding protein 4 (Oct4), SRY-box containing gene 2 (Sox2), Kruppel-like factor 4 (Klf4), and c-Myelocytomatosis oncogene (c-Myc) (OSKM), but the underlying mechanisms remain unclear. Here, we report that exogenous Oct4 and Sox2 can bind at the promoter regions of mir-141/200c and mir-200a/b/429 cluster, respectively, and induce the transcription activation of miR-200 family during the OSKM-induced reprogramming. Functional suppression of miR-200s with specific inhibitors significantly represses the OSKM-caused mesenchymal-to-epithelial transition (MET, an early event in reprogramming of fibroblasts to iPSCs) and iPSC generation, whereas overexpression of miR-200s promotes the MET and iPSC generation. Mechanistic studies showed that miR-200s significantly repress the expression of zinc finger E-box binding homeobox 2 (ZEB2) through directly targeting its 3' UTR and direct inhibition of ZEB2 can mimic the effects of miR-200s on iPSC generation and MET process. Moreover, the effects of miR-200s during iPSC generation can be blocked by ZEB2 overexpression. Collectively, our findings not only reveal that members of the miR-200 family are unique mediators of the reprogramming factors Oct4/Sox2, but also demonstrate that the miR-200/ZEB2 pathway as one critical mechanism of Oct4/Sox2 to induce somatic cell reprogramming at the early stage.
Project description:Ascl2, a basic helix-loop-helix transcription factor, is a downstream target of WNT signaling that controls the fate of intestinal cryptic stem cells and colon cancer progenitor cells. However, its involvement in colon cancer and downstream molecular events is largely undefined; in particular, the mechanism by which Ascl2 regulates the plasticity of epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) programs in colon cancer cells remains unknown. In this study, we systematically demonstrate that Ascl2 loss of function in colon cancer cells promotes MET by derepressing the expression of microRNA (miR)-200s (i.e. miR-200b, miR-200a, miR-429, miR-200c, and miR-141) and further activating their expression through a transcriptional mechanism that involves direct binding to the most proximal E-box (E-box2) in the miR-200b-a-429 promoter. Activation of miR-200s due to Ascl2 deficiency led to the inhibition of ZEB1/2 expression and the alteration of epithelial and mesenchymal features. Transfection of miR-200b, miR-200a, and miR-429 inhibitors into Ascl2-deficient colon cancer cells promoted the epithelial-mesenchymal transition in a reversible manner. Transfection of miR-200a or miR-429 inhibitors into Ascl2-deficient colon cancer cells increased cellular proliferation and migration. Ascl2 mRNA levels and the miR-200a, miR-200b, miR-200c, miR-141, or miR-429 levels in the colon cancerous samples were inversely correlated. These results provide the first evidence of a link between Ascl2 and miR-200s in the regulation of EMT-MET plasticity in colon cancer.
Project description:The tumor suppressor p21 acts as a cell cycle inhibitor and has also been shown to regulate gene expression by functioning as a transcription corepressor. Here, we identified p21-regulated microRNAs (miRNAs) by sequencing small RNAs from isogenic p21(+/+) and p21(-/-) cells. Three abundant miRNA clusters, miR-200b-200a-429, miR-200c-141, and miR-183-96-182, were downregulated in p21-deficient cells. Consistent with the known function of the miR-200 family and p21 in inhibition of the epithelial-mesenchymal transition (EMT), we observed EMT upon loss of p21 in multiple model systems. To explore a role of the miR-183-96-182 cluster in EMT, we identified its genome-wide targets and found that miR-183 and miR-96 repressed common targets, including SLUG, ZEB1, ITGB1, and KLF4. Reintroduction of miR-200, miR-183, or miR-96 in p21(-/-) cells inhibited EMT, cell migration, and invasion. Conversely, antagonizing miR-200 and miR-183-96-182 cluster miRNAs in p21(+/+) cells increased invasion and elevated the levels of VIM, ZEB1, and SLUG mRNAs. Furthermore, we found that p21 forms a complex with ZEB1 at the miR-183-96-182 cluster promoter to inhibit transcriptional repression of this cluster by ZEB1, suggesting a reciprocal feedback loop.
Project description:The pancreatic adenocarcinoma (PDAC) microenvironment is largely comprised of fibrotic tumor associated stroma (TAS) that contributes to the lethal biology of PDAC. microRNA (miRNA) are small non-coding RNAs that regulate gene expression. We hypothesized that interactions between PDAC cells and TAS cells within the microenvironment modulate miRNA expression and thus, tumor biology. We observed that miR-205 and members of the miR-200 family (miR-200a, -200b, -200c, -141 and miR-429) were exclusively expressed in PDAC cells, consistent with an epithelial miRNA signature, while miR-145 and miR-199 family members (miR-199a and -199b) were solely expressed in TAS cells, consistent with a stromal miRNA signature. This finding was confirmed by qRT-PCR of RNA obtained by laser-capture microdissection of surgical specimens. Using an in vitro co-culture model, we further demonstrated regulation of miRNA expression by cell-cell contact. Forced expression in TAS cells of miR-200b/-200c and miR-205 to mimic these observed changes in miRNA concentrations induced secretion of GM-CSF and IP10, and notably inhibited migration. These data suggest interactions within the tumor microenvironment alter miRNA expression, which in turn have a functional impact on TAS.
Project description:To use stem cell therapy effectively, it is important to enhance the therapeutic potential of stem cells with soluble factors. Although sonic hedgehog (shh) is important in maintaining the stem cell, the recovery effect of mouse embryonic stem cells (mESCs) with shh has not yet been elucidated. The present study investigated the effect of mESCs with shh in skin recovery in vivo as well as the related intracellular signal pathways in vitro.The healing effect of mESCs with shh on skin wounds was examined in vivo in ICR mice. The involvement of Smads, the microRNA (miR)-200 family, zinc finger E-box-binding homeobox (ZEBs) and E-cadherin on shh-induced mESC migration and self-renewal was determined in vitro.The mESCs with shh increased re-epithelialization and VEGF expression in skin wounds. Shh-treated mESCs increased both secreted and intracellular levels of VEGF. Shh induced dephosphorylation of glycogen synthase kinase 3? through the Smoothened receptor and increased the phosphorylation of Smad1 and Smad2/3 in mESCs. Shh-induced decrease of the mmu-miR-141, -200c, -200a, -200b and -429 expression levels was significantly reversed by Smad4 siRNA. Shh increased nuclear expression of ZEB1/ZEB2 and decreased E-cadherin expression while increasing cell migration and skin wound healing. Both these effects were reversed by mmu-miR-141 and -200b mimics.Mouse ESCs accelerated skin wound healing by shh through down-regulating E-cadherin, an effect dependent on mmu-miR-141 and -200b. Our data provides evidence for the effectiveness of shh in stem cell-based therapy in vivo.