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MiR-10b inhibits migration and invasion of pancreatic ductal adenocarcinoma via regulating E2F7.

ABSTRACT: BACKGROUND:Abnormal microRNAs (miRNAs) expression is closely related to the development and poor prognosis of pancreatic ductal adenocarcinoma (PDAC). We aimed to elucidate the invasive mechanism and clinical significance of miR-10b in PDAC. METHODS:The RNA sequence data of pancreatic cancer were extracted from the TCGA database. R packages were performed to analyze the differential expression of RNAs. TargetScan, picTar, and miRanda were used to predict the target gene of miRNA. The expression level of the selected candidate was tested by western blot and RT-PCR in PDAC cells and tissues. Scrape and Transwell assays were determined the effect of candidate molecules on cell migration and invasion. The gain of function and loss of function was achieved by co-culture with mimics and vector. Luciferase reporters were generated based on the psiCHECK2 vector. The relative luciferase activity was measured with the Dual-Luciferase Reporter Assay System and Infinate M200 PRO microplate reader. RESULTS:Based on the TCGA data and bioinformatics analysis, we obtained seven differentially expressed miRNAs. Both TCGA data and our center clinical date indicated that miR-10b was contributed to the poor survival of PDAC. Based on the target gene prediction database, we found that E2F7 was a target mRNA of miR-10b. In subsequent experiments in molecular biology, miR-10b expression was downregulated in PDAC cells and tissues, while E2F7 was upregulated. Scrape and Transwell assay indicated that miR-10b could inhibit the invasion and migration of PDAC. MiR-10b was confirmed to be by the E2F7 targeting site by dual-luciferase report. Moreover, rescue experiments prove that miR-10b could inhibit the invasion and migration of PDAC cells by regulating E2F7 expression. CONCLUSION:Our results suggest that miR-10b could inhibit the progression of PDAC by regulating E2F7 expression and acts as an independent prognostic risk factor for PDAC.

SUBMITTER: Xu C

PROVIDER: S-EPMC7595905 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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MiR-10b inhibits migration and invasion of pancreatic ductal adenocarcinoma via regulating E2F7.

Xu Cui C Qi Xiangxiu X

Journal of clinical laboratory analysis 20200626 10

<h4>Background</h4>Abnormal microRNAs (miRNAs) expression is closely related to the development and poor prognosis of pancreatic ductal adenocarcinoma (PDAC). We aimed to elucidate the invasive mechanism and clinical significance of miR-10b in PDAC.<h4>Methods</h4>The RNA sequence data of pancreatic cancer were extracted from the TCGA database. R packages were performed to analyze the differential expression of RNAs. TargetScan, picTar, and miRanda were used to predict the target gene of miRNA. ...[more]

PMID: 32592206

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Project description:BACKGROUND:Pancreatic ductal adenocarcinoma (PDAC) is presently one of the cancers with the worst survival rates and least effective treatments. Moreover, total deaths due to PDAC are predicted to increase in the next 15 years. Therefore, novel insights into basic mechanism of PDAC development and therapies are needed. PDAC is characterized by a complex microenvironment, in which cancer and stromal cells release different molecules, such as ATP. ATP can be transported and/or exocytosed from active cancer cells and released from dying cells in the necrotic core of the cancer. We hypothesized that one of the ATP receptors, the P2X7 receptor (P2X7R) could be an important player in PDAC behaviour. METHODS:We determined the expression (real time PCR and Western blot) and localization (immunofluorescence) of P2X7R in human PDAC cell lines (AsPC-1, BxPC-3, Capan-1, MiaPaCa-2, Panc-1) and a "normal" human pancreatic duct epithelial cell line (HPDE). The function of P2X7R in proliferation (BrdU assay), migration (wound assay) and invasion (Boyden chamber with matrigel) was characterized. Furthermore, we studied P2X7R-dependent pore formation (YoPro-1 assay) and cell death (caspase and annexin V / propidium iodide assays). RESULTS:We found higher expression of P2X7R protein in PDAC compared to HPDE cells. P2X7R had notable disparate effects on PDAC survival. Firstly, high concentrations of ATP or the specific P2X7R agonist, BzATP, had cytotoxic effects in all cell lines, and cell death was mediated by necrosis. Moreover, the P2X7R-pore antagonist, A438079, prevented ATP-induced pore formation and cell death. Second, in basal conditions and with low concentrations of ATP/BzATP, the P2X7R allosteric inhibitor AZ10606120 reduced proliferation in all PDAC cell lines. P2X7R also affected other key characteristics of cancer cell behavior. AZ10606120 reduced cell migration and invasion in PDAC cell lines compared to that of untreated/vehicle-treated control cells, and stimulation with sub-millimolar concentrations of ATP or BzATP substantially increased cell invasion. CONCLUSIONS:PDAC cell lines overexpress P2X7R and the receptor plays crucial roles in cell survival, migration and invasion. Therefore, we propose that drugs targeting P2X7R could be exploited in therapy of pancreatic cancer.

Project description:Long non-coding RNA tissue differentiation-inducing non-protein coding (TINCR) is associated with the carcinogenesis of several cancers. However, little is known about the function and mechanism of TINCR in lung adenocarcinoma (LUAD). Here, we aimed to analyze expression of TINCR and elucidate its mechanistic involvement in the progression of LUAD. The expression of TINCR was investigated according to Gene Expression Profiling Interactive Analysis at first and then detected in 29 LUAD tissues and paired adjacent normal tissues using qRT-PCR. Results indicated that TINCR was evidently downregulated in LUAD. The association between TINCR and clinicopathological parameters was analyzed by Pearson's chi-square test, suggesting TINCR was closely correlated with TNM stage and lymph mode metastasis. Subsequently, the function role of TINCR was examined by gain- and loss-of-function studies in LUAD (A549 and NCI-H292) cells. As analyzed by the scratch wound-healing and transwell assays, results revealed that TINCR suppressed the migration and invasion of A549 and NCI-H292 cells. However, TINCR exerted no effects on the cell proliferation as determined by CCK8 assay. Furthermore, we reported that loss of Sp1 could inhibit TINCR expression. Expressions of miR-107/miR-1286 were detected by qRT-PCR assay in A549 and NCI-H292 cells after TINCR knockdown or overexpression. In addition, the direct binding ability of the predicted miR-107 or miR-1286 binding site on TINCR was validated by luciferase activity assay. Results indicated TINCR could constrain the expression of miR-107/miR-1286, and was a target of them in LUAD cells. Bioinformatics analyses showed that BTRC and RAB14 was the potential target gene of miR-107 and miR-1286, respectively. These data revealed a possible regulatory mechanism in which upregulation of TINCR induced by Sp1 could constrain the migration and invasion through regulating miR-107 or miR-1286 in LUAD cells. Conjointly, our findings provide a valuable insight into the regulatory mechanism of TINCR in LUAD, supportive to its potential of therapeutic target for LUAD patients.

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MiR-10b inhibits migration and invasion of pancreatic ductal adenocarcinoma via regulating E2F7.

Publications

MiR-10b inhibits migration and invasion of pancreatic ductal adenocarcinoma via regulating E2F7.

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