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Silencing lncRNAs PVT1 Upregulates miR-145 and Confers Inhibitory Effects on Viability, Invasion, and Migration in EC.

ABSTRACT: Long non-coding RNA (lncRNA) plasmacytoma variant translocation 1 (PVT1) is correlated to various malignant tumors. Consequently, we explored effects of lncRNA PVT1 on esophageal carcinoma (EC) targeting microRNA-145 (miR-145). EC tissues, adjacent normal tissues, and EC-related cell lines were collected and cultured. Expression of lncRNA PVT1, miR-145, fascin-1 (FSCN1), and related genes with intervening expression of PVT1 and miR-145 was determined. Bioinformatic website, dual-luciferase reporter assay, and RNA immunoprecipitation (RIP) were carried to verify target relationship among lncRNA PVT1, FSCN1, and miR-145. Scratch test, Transwell assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and flow cytometry were performed for detection of migration, invasion, viability, and apoptosis of transfected cells, respectively. Finally, tumor formation in nude mice was measured. After database analysis, lncRNA PVT1, miR-145, and FSCN1 were selected for study. lncRNA PVT1 and FSCN1 can bind to miR-145. After overexpressing miR-145 or inhibiting lncRNA PVT1, EC cell viability, migration, and invasion were inhibited, while volume and weight of tumor formation in nude mice decreased. Expression of lncRNA PVT1, FSCN1, Bcl-2, CD147, VEGFR2, and MTA1 decreased and expression of miR-145 and Bax increased. Silencing lncRNA PVT1 can upregulate miR-145, which is a tumor suppressor in EC via knockdown of FSCN1. Thus, we might provide a potential theoretical basis for EC treatment.

SUBMITTER: Shen SN

PROVIDER: S-EPMC6965729 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Several theories on the origin of adenomyosis (ADS) have been proposed, of which the most widely accepted is the fundamental pathogenic role of uterine eutopic endometrium. Emerging evidence suggests that circular RNAs participate in the multiple tumorgenesis. The vital importance of circular RNA PVT1 (circPVT1) in the pathological progress like malignancies has been well documented. Nevertheless, its underlying correlation with ADS remains elusive yet. The purpose of this study was to investigate the expression pattern, regulatory effect, and internal mechanism of circPVT1 in ADS. qRT-PCR was performed to detect the relative mRNA expression of circPVT1, miR-145, and Talin1 in ADS endometrial tissue and cells. The protein level of Talin1 was measured by Western blot and immunochemistry. Immunofluorescence was used to identify the primary endometrial epithelial and stromal cells. circPVT1 knockdown in vitro was achieved by transfecting with specific lentivirus vector CCK-8, and colony formation assays were utilized to assess cell proliferation; meanwhile, the transwell assay was employed for evaluating cell invasion ability. By conducting bioinformatics, dual-luciferase reporter assay, or RNA immunoprecipitation (RIP) experiment, the interaction between miR-145 and circPVT1 or Talin1 was verified. Rescue experiments further determined the regulatory effect of circPVT1/miR-145/Talin1 axis. We found both circPVT1 and Talin1 were markedly upregulated in ADS endometrial tissue and cells, whereas miR-145 was decreased. Elevated expression of circPVT1 was closely related to the severity of dysmenorrhea, menorrhagia, and uterine enlargement of patients with ADS. Knockdown of circPVT1 inhibited adenomyotic epithelial and stromal cell proliferation and invasion. Further mechanistic experiments revealed that circPVT1 negatively regulated miR-145 through serving as a molecular sponge. And the facilitating effect of circPVT1 was partially reversed by miR-145. Talin1 was demonstrated to be a down target of miR-145 and indirectly affected by circPVT1. Our findings unveiled that enhanced circPVT1 may be involved in the pathogenesis of ADS via stimulating endometrial cell proliferation and invasion. The establishment of circPVT1/miR-145/Talin1 pathway might present a novel therapeutic insight for ADS.

Project description:BackgroundLong-non-coding RNA PVT1 (lncRNA PVT1) can be used as an oncogenic regulatory non-coding RNA (ncRNA) for many cancers. However, its function and mechanism in breast cancer (BRCA) are still not clearly elucidated.ObjectiveWe attempt to explain the mechanism of PVT1's role in breast cancer from different perspectives.MethodsWe analyzed the expression of PVT1 and its correlation with the breast cancer related clinical data in the The Cancer Genome Atlas (TCGA) database. We used PVT1 overexpression and knockdown lentivirus to infect breast cancer MDA-MB-231 cell line for cell function verification, in vitro using CCK-8 to measure proliferation, flow cytometry to measure apoptosis, transwell test to measure invasion and migration ability, detecting cell extracellular acidification rate (ECAR) to assess glycolysis metabolism and explore the biological functions of PVT1 in breast cancer cells. Transcriptome sequencing was used to analyze the changes of related genes in cells after overexpression of PVT1. In vivo we used a xenograft model to study the effect of PVT1 on breast cancer.ResultsPVT1 was up-regulated in breast cancer tissues and was positively correlated with the clinical stage of breast cancer patients. Overexpression of PVT1 in vitro promoted cell proliferation, migration and invasion, and promoted tumor growth in vivo. Knockdown of PVT1 led to the opposite biological consequence. Further bioinformatics analysis showed that PVT1 changes the glycolysis metabolism of tumors through regulation of glycolysis-related genes. In addition, the expression of miR-145-5p is negatively correlated with PVT1. We consider the possibility of PVT1 promoting cell proliferation and metastasis by regulating the aerobic glucose metabolism in breast cancer cells through sponging the miR-145-5p.ConclusionOur results reveal a potential pathway for competing endogenous RNA to regulate breast cancer glucose metabolism. PVT1 regulates glycolysis related genes expression by competitively binding to endogenous miR-145-5p in breast cancer cells to change the metabolic phenotype. This may Provide new ideas for precise molecular therapy targets for breast cancer.

Project description:BackgroundMicrovascular invasion (MVI) is an independent detrimental risk factor for tumor recurrence and poor survival in hepatocellular carcinoma (HCC). Competitive endogenous RNA (ceRNA) networks play a pivotal role in the modulation of carcinogenesis and progression among diverse tumor types. However, whether the ceRNA mechanisms are engaged in promoting the MVI process in patients with HCC remains unknown.MethodsA ceRNA regulatory network was constructed based on RNA-seq data of patients with HCC from The Cancer Genome Atlas (TCGA) database. In total, 10 hub genes of the ceRNA network were identified using four algorithms: "MCC," "Degree," "Betweenness," and "Stress." Transcriptional expressions were verified by in situ hybridization using clinical samples. Interactions between ceRNA modules were validated by luciferase reporting assay. Logistic regression analysis, correlation analysis, enrichment analysis, promoter region analysis, methylation analysis, and immune infiltration analysis were performed to further investigate the molecular mechanisms and clinical transformation value.ResultsThe ceRNA regulatory network featuring a tumor invasion phenotype consisting of 3 long noncoding RNAs, 3 microRNAs, and 93 mRNAs was constructed using transcriptional data from the TCGA database. Systemic analysis and experimentally validation identified a ceRNA network (PVT1/miR-1258/DUSP13 axis) characterized by lipid regulatory potential, immune properties, and abnormal methylation states in patients with HCC and MVI. Meanwhile, 28 transcriptional factors were identified as potential promotors of PVT1 with 3 transcriptional factors MXD3, ZNF580, and KDM1A promising as therapeutic targets in patients with HCC and MVI. Furthermore, miR-1258 was an independent predictor for MVI in patients with HCC.ConclusionThe PVT1/DUSP13 axis is significantly associated with MVI progression in HCC patients. This study provides new insight into mechanisms related to lipids, immune phenotypes, and abnormal epigenetics in oncology research.

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Silencing lncRNAs PVT1 Upregulates miR-145 and Confers Inhibitory Effects on Viability, Invasion, and Migration in EC.

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