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MiR-374b-5p inhibits KDM5B-induced epithelial-mesenchymal transition in pancreatic cancer.

ABSTRACT: Micro(mi)RNAs play a critical regulatory role in the progression and metastasis of pancreatic cancer (PC). In this study, we aimed to reveal the mechanisms of miR-374b-5p in regulating epithelial-mesenchymal transition (EMT) in PC. Gene Expression Omnibus datasets (GSE24279 and GSE71533) and the pancreatic ductal adenocarcinoma (PDAC) cohort of The Cancer Genome Atlas were employed to screen for potential prognostic miRNAs. The expression of miR-374b-5p was measured by quantitative real-time polymerase chain reaction (qRT-PCR) in 78 paired PDAC tissue samples. The biological effects of miR-374b-5p were investigated using in vitro and in vivo assays. Luciferase reporter assays and immunohistochemical tests were conducted to verify the interaction between miR-374b-5p and its predicted direct target, KDM5B. MiR-374b-5p was downregulated in PC tissues, and a low level of miR-374b-5p was associated with poor overall survival, greater tumor size, and more lymph node metastasis in PC. In vitro assays indicated that overexpression of miR-374b-5p suppressed the proliferation, migration, and invasion of PC cells. Mechanistically, miR-374b-5p suppressed the expression of KDM5B, which inhibited E-cadherin expression but promoted N-cadherin and vimentin expression. Finally, in vivo assays demonstrated that miR-374b-5p overexpression suppressed tumor growth and lung metastasis in PANC-1 cells. Thus, our findings indicate that miR-374b-5p could be a potential prognostic biomarker and therapeutic target for KDM5B-induced EMT in PC.

SUBMITTER: Zhao X

PROVIDER: S-EPMC8414380 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:ObjectiveThis study attempts to investigate whether hsa_circRNA_001859 (circ_001859) could regulate the proliferation and invasion of pancreatic cancer through the miR-21-5p/SLC38A2 pathway.MethodsGSE79634 microarray was analyzed with R package. The expression of circ_001859 in pancreatic cancer tissues and cells was verified by qRT-PCR. After the overexpression of circ_001859, cell proliferation, cell migration and invasion were verified by colony formation and transwell assay. The targeting relationship between miR-21-5p and circ_001859 was predicted by TargetScan and was verified by dual luciferase reporter assay, RNA pull down and qRT-PCR. The effect of miR-21-5p on cell proliferation, migration and invasion were investigated by colony formation and transwell assay respectively. Similarly, the targeting relationship between miR-21-5p and SLC38A2 was predicted by TargetScan and was verified by dual luciferase reporter assay, western blot and qRT-PCR. The effect of SLC38A2 on cell proliferation was investigated by colony formation.ResultsCirc_001859 was lowly expressed in pancreatic cancer tissues and cells. In vitro assays showed that overexpression of circ_001859 could inhibit the proliferation, migration and invasion of pancreatic cancer. In addition, this effect was also confirmed in xenograft transplantation model. Circ_001859 could be bind to miR-21-5p and sponge its expression in pancreatic cancer cells. Overexpression of miR-21-5p enhanced the proliferation, migration and invasion ability of pancreatic cancer cells, while the inhibition of miR-21-5p expression suppressed these abilities. Moreover, miR-21-5p directly targeted at SLC38A2 and inhibited SLC38A2 expression levels while circ_001859 up-regulated SLC38A2 levels. SLC38A2 expression knockdown enhanced cell proliferation but SLC38A2 overexpression resulted in decreased proliferation, and effects of SLC38A2 could be rescued by miR-21-5p and circ_001859. In addition, both QRT-PCR and immunofluorescence confirmed that circ_001859 could regulate tumor epithelial-mesenchymal transition (EMT) through the miR-21-5p/SLC38A2 pathway.ConclusionsThis study suggests that circ_001859 may inhibit the proliferation, invasion and EMT of pancreatic cancer through the miR-21-5p/SLC38A2 pathway.

Project description:BackgroundGlioma metastasis, invasion, epithelial-mesenchymal transition (EMT) and chemoresistance indicate poor prognosis. Accumulating evidence reveals that Notch1 is an important factor in tumour progression. However, the role of Notch1 in glioma EMT and associated microRNAs (miRNAs) with the Notch pathway remain controversial.MethodsUtilizing cBioPortal database to examine the gene signature of NOTCH1 (encoding Notch1), CDH2 (encoding N-cadherin) and SNAI1 (encoding Snail-1) in disease-free survival (DFS) and overall survival (OS). We analyzed the Notch1 expression from Oncomine. We used Western blot (WB), immunohistochemistry (IHC) and immunofluorescence to determine protein levels. Transcription was evaluated by quantitative real-time (qRT)-PCR. siRNA and lentivirus were used to knock down Notch1 and overexpress miR-139-5p, respectively. The migration and invasion of glioma cells were assessed by wound healing and transwell assays. Luciferase reporter assays were utilized to verify the relationship between Notch1 and miR-139-5p. A U87-implanted intracranial model was used to study the effect of miR-139-5p on tumour growth and Notch1 suppression efficacy or EMT reversion.ResultsIt revealed the association of NOTCH1, CDH2, SNAI1 genomic alterations with decreases in DFS and OS. Notch1 was upregulated in classical and proneural subtypes of GBM, and associated with tumour grade. Notch1 inhibition suppressed the biological behaviours of metastasis, invasion and EMT. Notch1 was identified as a novel direct target of miR-139-5p. MiR-139-5p overexpression partially phenocopied Notch1 siRNA, whereas the forced expression of Notch1 reversed the effects of miR-139-5p on the invasion of glioma. Moreover, intracranial tumourigenicity and EMT behaviours were reduced by the introduction of miR-139-5p and partially mediated by the decreased Notch1 expression.ConclusionsmiR-139-5p was identified as a tumour suppressor by negatively targeting Notch1, and this work suggests a possible molecular mechanism of the miR-139/Notch1/EMT axis for glioma treatment.

Project description:Inflammatory bowel disease (IBD), which consists of Crohn's disease (CD) and ulcerative colitis (UC), is a chronic, inflammatory disorder of the gastro-intestinal tract with unknown etiology. Current evidence suggests that intestinal epithelial cells (IECs) is prominently linked to the pathogenesis of IBD. Therefore, maintaining the intact of epithelium has potential roles in improving pathophysiology and clinical outcomes of IBD. MicroRNAs (miRNAs) act as post-transcriptional gene regulators and regulate many biological processes, including embryonal development, cell differentiation, apoptosis and proliferation. In this study, we found that miR-200b decreased significantly in inflamed mucosa of IBD, especially for UC, when compared with their adjacent normal tissue. Simultaneously, we also found that the genes of E-cadherin and cyclin D1 were reduced significantly and correlated positively to the miR-200b. In addition, the upregulation of transforming growth factor-beta 1 (TGF-?1) was inversely correlated to the miR-200b in IBD. To investigate the possible roles of miR-200b in IECs maintaining, we used TGF-?1 to induce epithelial-mesenchymal transition (EMT) in IEC-6 initially. After sustained over-expressing miR-200b in IEC-6, the EMT was inhibited significantly that was characterized by downregulation of vimentin and upregulation of E-cadherin. Furthermore, we found that miR-200b enhanced E-cadherin expression through targeting of ZEB1, which encode transcriptional repressors of E-cadherin. SMAD2 was found to act as a target of miR-200b with direct evidence that miR-200b binding to the 3' UTR of SAMD2 and the ability of miR-200b to repress SMAD2 protein expression. With SMAD2 depletion, the expression of vimentin decreased correspondingly, which suggested miR-200b might reduce vimentin through regulating the SMAD2. With endogenous over-expression of miR-200b, the proliferation of IEC-6 cells increased significantly by increasing S-phase entry and promoting expression of the protein cyclin D1. Summarily, our study suggested a potential role for mir-200b in maintaining intact of intestinal epithelium through inhibiting EMT and promoting proliferation of IECs.

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MiR-374b-5p inhibits KDM5B-induced epithelial-mesenchymal transition in pancreatic cancer.

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