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MicroRNA-21 Regulates Stemness in Pancreatic Ductal Adenocarcinoma Cells.

ABSTRACT: Pancreatic ductal adenocarcinoma (PDAC) is the most common and aggressive type of pancreatic cancer (PCa) with a low survival rate. microRNAs (miRs) are endogenous, non-coding RNAs that moderate numerous biological processes. miRs have been associated with the chemoresistance and metastasis of PDAC and the presence of a subpopulation of highly plastic "stem"-like cells within the tumor, known as cancer stem cells (CSCs). In this study, we investigated the role of miR-21, which is highly expressed in Panc-1 and MiaPaCa-2 PDAC cells in association with CSCs. Following miR-21 knockouts (KO) from both MiaPaCa-2 and Panc-1 cell lines, reversed expressions of epithelial-mesenchymal transition (EMT) and CSCs markers were observed. The expression patterns of key CSC markers, including CD44, CD133, CX-C chemokine receptor type 4 (CXCR4), and aldehyde dehydrogenase-1 (ALDH1), were changed depending on miR-21 status. miR-21 (KO) suppressed cellular invasion of Panc-1 and MiaPaCa-2 cells, as well as the cellular proliferation of MiaPaCa-2 cells. Our data suggest that miR-21 is involved in the stemness of PDAC cells, may play roles in mesenchymal transition, and that miR-21 poses as a novel, functional biomarker for PDAC aggressiveness.

SUBMITTER: Mortoglou M

PROVIDER: S-EPMC8835847 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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microRNA-21 Regulates Stemness in Pancreatic Ductal Adenocarcinoma Cells.

Mortoglou Maria M Miralles Francesc F Arisan Elif Damla ED Dart Alwyn A Jurcevic Stipo S Lange Sigrun S Uysal-Onganer Pinar P

International journal of molecular sciences 20220124 3

Pancreatic ductal adenocarcinoma (PDAC) is the most common and aggressive type of pancreatic cancer (PCa) with a low survival rate. microRNAs (miRs) are endogenous, non-coding RNAs that moderate numerous biological processes. miRs have been associated with the chemoresistance and metastasis of PDAC and the presence of a subpopulation of highly plastic "stem"-like cells within the tumor, known as cancer stem cells (CSCs). In this study, we investigated the role of miR-21, which is highly expresse ...[more]

PMID: 35163198

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Project description:IntroductionPancreatic ductal adenocarcinoma (PDAC) is projected to rise to the second leading cause of U.S. cancer-related deaths by 2020. Novel therapeutic targets are desperately needed. MicroRNAs (miRs) are small noncoding RNAs that function by suppressing gene expression and are dysregulated in cancer. miR-21 is overexpressed in PDAC tumor cells (TC) and is associated with decreased survival, chemoresistance and invasion. Dysregulation of miR regulatory networks in PDAC tumor-associated fibroblasts (TAFs) have not been previously described. In this study, we show that miR-21 expression in TAFs promotes TC invasion.MethodsIn-situ hybridization for miR-21 was performed on the 153 PDAC patient UCLA tissue microarray and 23 patient-matched lymph node metastases. Stromal and TC histoscores were correlated with clinicopathologic parameters by univariate and multivariate Cox regression. miR-21 positive cells were further characterized by immunofluorescence for mesenchymal/epithelial markers. For in vitro studies, TAFs were isolated from freshly resected human PDAC tumors by the outgrowth method. miR-21 was overexpressed/inhibited in fibroblasts and then co-cultured with GFP-MiaPaCa TCs to assess TC invasion in modified Boyden chambers.ResultsmiR-21 was upregulated in TAFs of 78% of tumors, and high miR-21 significantly correlated with decreased overall survival (P = 0.04). Stromal miR-21 expression was also significantly associated with lymph node invasion (P = 0.004), suggesting that it is driving TC spread. Co-immunofluorescence revealed that miR-21 colocalized with peritumoral fibroblasts expressing ?-smooth muscle actin. Moreover, expression of miR-21 in primary TAFs correlated with miR-21 in TAFs from patient-matched LN metastases; evidence that PDAC tumor cells induce TAFs to express miR-21. miR-21 expression in TAFs and TCs promotes invasion of TCs and is inhibited with anti-miR-21.ConclusionsmiR-21 expression in PDAC TAFs is associated with decreased overall survival and promotes TC invasion. Anti-miR-21 may represent a novel therapeutic strategy for dual targeting of both tumor and stroma in PDAC.

Project description:Despite progress in genomic characterization, no single prognostic marker that can be evaluated using an easy-to-perform and relatively inexpensive method is available for pancreatic ductal adenocarcinoma (PDAC). MicroRNAs, which are stable, tumor- and tissue-specific molecules, are potentially ideal biomarkers, and we established an inter-laboratory validated method to investigate miR-21 as a prognostic biomarker in PDAC. The study samples of PDAC patients were recruited from a test cohort of Glasgow (n = 189) and three validation cohorts of Pisa (n = 69), Sydney (n = 249), and International Cancer Genome Consortium (ICGC) (n = 249). Tissue microarrays were used for miR-21 staining by chromogenic in situ hybridization (CISH). The patients were subdivided into no/low and high miR-21 staining groups using a specific histoscore. Furthermore, miR-21 staining was evaluated against clinicopathological variables and follow-up data by Fisher/log-rank test and Cox proportional models. The prognostic variables found to be significant in univariate analysis (P value < 0.10) were included in multivariate analysis in a backward-stepwise fashion. MiR-21 expression was cytoplasmic, with more consistent staining in the malignant ductal epithelium than in the stroma. The expression of miR-21 was significantly associated with tumor size and lymph node metastasis, whereas no association was observed with other clinicopathological variables. High miR-21 staining (histoscore ≥ 45 [median score]) was an independent predictor of survival in the Glasgow test cohort (HR 2.37, 95% CI: 1.42-3.96, P < 0.0001) and three validation cohorts (Pisa, HR 2.03, 95% CI: 1.21-3.39, P = 0.007; Sydney, HR 2.58, 95% CI (1.21-3.39), P < 0.0001; and ICGC, HR 3.34, 95% CI: 2.07-5.84, P = 0.002) when adjusted for clinical variables in a multivariate model. In comparison to the patients with low miR-21, the patients with high miR-21 expression had significant increase in OS as they benefit from gemcitabine-based adjuvant chemotherapy (Glasgow 16.5 months [with chemotherapy] vs 10.5 months [without chemotherapy]); Sydney 25.0 vs 10.6; ICGC 25.2 vs 11.9. These results indicated that miR-21 is a predictor of survival, prompting prospective trials. Evaluation of miR-21 offers new opportunities for the stratification of patients with PDAC and might facilitate the implementation of clinical management and therapeutic interventions for this devastating disease.

Project description:BackgroundPancreatic cancer stem cells are crucial for tumorigenesis and cancer metastasis. Presently, long non-coding RNAs were found to be associated with Pancreatic Ductal Adenocarcinoma stemness characteristics but the underlying mechanism is largely known. Here, we aim to explore the function of LINC00909 in regulating pancreatic cancer stemness and cancer metastasis.MethodsThe expression level and clinical characteristics of LINC00909 were verified in 80-paired normal pancreas and Pancreatic Ductal Adenocarcinoma tissues from Guangdong Provincial People's Hospital cohort by in situ hybridization. RNA sequencing of PANC-1 cells with empty vector or vector encoding LINC00909 was experimented for subsequent bioinformatics analysis. The effect of LINC00909 in cancer stemness and metastasis was examined by in vitro and in vivo experiments. The interaction between LINC00909 with SMAD4 and the pluripotency factors were studied.ResultsLINC00909 was generally upregulated in pancreatic cancer tissues and was associated with inferior clinicopathologic features and outcome. Over-expression of LINC00909 enhanced the expression of pluripotency factors and cancer stem cells phenotype, while knock-down of LINC00909 decreased the expression of pluripotency factors and cancer stem cells phenotype. Moreover, LINC00909 inversely regulated SMAD4 expression, knock-down of SMAD4 rescued the effect of LINC00909-deletion inhibition on pluripotency factors and cancer stem cells phenotype. These indicated the effect of LINC00909 on pluripotency factors and CSC phenotype was dependent on SMAD4 and MAPK/JNK signaling pathway, another downstream pathway of SMAD4 was also activated by LINC00909. Specifically, LINC00909 was localized in the cytoplasm in pancreatic cancer cells and decreased the stability the SMAD4 mRNA. Finally, we found over-expression of LINC00909 not only accelerated tumor growth in subcutaneous mice models, but also facilitated tumorigenicity and spleen metastasis in orthotopic mice models.ConclusionWe demonstrate LINC00909 inhibits SMAD4 expression at the post-transcriptional level, which up-regulates the expression of pluripotency factors and activates the MAPK/JNK signaling pathway, leading to enrichment of cancer stem cells and cancer metastasis in pancreatic cancer.

Project description:MicroRNAs (miRNAs) are 21-24 nucleotide RNA molecules that regulate the translation and stability of target messenger RNAs. Abnormal miRNA expression is a common feature of diverse cancers. Several previous studies have classified miRNA expression in pancreatic ductal adenocarcinoma (PDAC), although no uniform pattern of miRNA dysregulation has emerged. To clarify these previous findings as well as to set the stage for detailed functional analyses, we performed global miRNA expression profiling of 21 human PDAC cell lines, the most extensive panel studied to date. Overall, 39 miRNAs were found to be dysregulated and have at least two-fold or greater differential expression in PDAC cell lines compared to control nontransformed pancreatic ductal cell lines. Several of these miRNAs show comparable dysregulation in first-passage patient derived xenografts. Initial functional analyses demonstrate that enforced expression of miRNAs derived from the miR-200 family and the miR-17-92 cluster, both of which are overexpressed in PDAC cell lines, enhances proliferation. In contrast, inhibition of the miR-200 family, the miR-17-92 cluster, or miR-191 diminishes anchorage independent growth. Consistent with a known role for the miR-200 family in negatively regulating an epithelial-to-mesenchymal transition (EMT), the abundance of these miRNAs correlated positively with E-cadherin expression and negatively with the EMT-associated transcription factor and established miR-200 target ZEB1. Finally, restituted expression of miR-34a, a miRNA whose expression is frequently lost in PDAC cell lines, abrogates growth, demonstrating that the anti-proliferative activity of this miRNA is operative in PDAC. These results, and the widespread availability of PDAC cell lines wherein the aforementioned data were generated, provide a valuable resource for the pancreatic cancer research community and will greatly facilitate functional studies essential for elucidating the consequences of miRNA dysregulation in pancreatic cancer.

Dataset Information

MicroRNA-21 Regulates Stemness in Pancreatic Ductal Adenocarcinoma Cells.

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microRNA-21 Regulates Stemness in Pancreatic Ductal Adenocarcinoma Cells.

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