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LncRNA PVT1 promotes gemcitabine resistance of pancreatic cancer via activating Wnt/?-catenin and autophagy pathway through modulating the miR-619-5p/Pygo2 and miR-619-5p/ATG14 axes.

ABSTRACT: BACKGROUND:Pancreatic cancer is one of the most lethal malignancies and has an extremely poor diagnosis and prognosis. The development of resistance to gemcitabine is still a major challenge. The long noncoding RNA PVT1 was reported to be involved in carcinogenesis and chemoresistance; however, the mechanism by which PVT1 regulates the sensitivity of pancreatic cancer to gemcitabine remains poorly understood. METHODS:The viability of pancreatic cancer cells was assessed by MTT assay in vitro and xenograft tumor formation assay in vivo. The expression levels of PVT1 and miR-619-5p were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Western blotting analysis and qRT-PCR were performed to assess the protein and mRNA levels of Pygo2 and ATG14, respectively. Autophagy was explored via autophagic flux detection under confocal microscopy and autophagic vacuole investigation under transmission electron microscopy (TEM). The functional role and mechanism of PVT1 were further investigated by gain- and loss-of-function assays in vitro. RESULTS:In the present study, we demonstrated that PVT1 was up-regulated in gemcitabine-resistant pancreatic cancer cell lines. Gain- and loss-of-function assays revealed that PVT1 impaired sensitivity to gemcitabine in vitro and in vivo. We further found that PVT1 up-regulated the expression of both Pygo2 and ATG14 and thus regulated Wnt/?-catenin signaling and autophagic activity to overcome gemcitabine resistance through sponging miR-619-5p. Moreover, we discovered three TCF/LEF binding elements (TBEs) in the promoter region of PVT1, and activation of Wnt/?-catenin signaling mediated by the up-regulation of Pygo2 increased PVT1 expression by direct binding to the TBE region. Furthermore, PVT1 was discovered to interact with ATG14, thus promoting assembly of the autophagy specific complex I (PtdIns3K-C1) and ATG14-dependent class III PtdIns3K activity. CONCLUSIONS:These data indicate that PVT1 plays a critical role in the sensitivity of pancreatic cancer to gemcitabine and highlight its potential as a valuable target for pancreatic cancer therapy.

SUBMITTER: Zhou C

PROVIDER: S-EPMC7389684 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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LncRNA PVT1 promotes gemcitabine resistance of pancreatic cancer via activating Wnt/β-catenin and autophagy pathway through modulating the miR-619-5p/Pygo2 and miR-619-5p/ATG14 axes.

Zhou Cefan C Yi Changhua C Yi Yongxiang Y Qin Wenying W Yan Yanan Y Dong Xueying X Zhang Xuewen X Huang Yuan Y Zhang Rui R Wei Jie J Ali Declan William DW Michalak Marek M Chen Xing-Zhen XZ Tang Jingfeng J

Molecular cancer 20200729 1

<h4>Background</h4>Pancreatic cancer is one of the most lethal malignancies and has an extremely poor diagnosis and prognosis. The development of resistance to gemcitabine is still a major challenge. The long noncoding RNA PVT1 was reported to be involved in carcinogenesis and chemoresistance; however, the mechanism by which PVT1 regulates the sensitivity of pancreatic cancer to gemcitabine remains poorly understood.<h4>Methods</h4>The viability of pancreatic cancer cells was assessed by MTT ass ...[more]

PMID: 32727463

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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:IntroductionColon adenocarcinoma (COAD) is the third most common tumor of the digestive tract. Recent studies reported that lncRNA's abnormal expression might play a vital role in the occurrence and development of COAD.MethodsIn the present study, we investigated the expression of ILF3-AS1 in COAD cell lines, human normal colon epithelial cell line, patient tumor tissues and adjacent normal tissues by real-time quantitative PCR (RT-qPCR). Small interfering RNAs (siRNAs) were transfected into COAD cells to inhibit the expression of ILF3-AS1. The effects of ILF3-AS1 on cell proliferation, migration, invasion and apoptosis were measured by CCK-8 assay, transwell migration and invasion assay, and flow cytometry apoptosis assay, respectively. The direct binding of ILF3-AS1 and miR-619-5p/CAMK1D was validated by the luciferase reporter assay. The expression of CAMK1D and epithelial-mesenchymal transformation (EMT)-related proteins was detected by Western Blot analysis. Besides, in vivo experiments were conducted to demonstrate the oncogenic role of ILF3-AS1 in COAD.ResultsThe results showed that the expression of ILF3-AS1 was significantly higher in COAD tissue than in normal adjacent samples, and this conclusion was confirmed in the available public datasets. After ILF3-AS1 knockdown, the proliferation of COAD cell lines SW480 and HT29 was significantly inhibited. At the same time, the apoptosis was increased, and the invasion and migration abilities were decreased. After further exploring the mechanisms, we found that ILF3-AS1 serves as a competitive endogenous RNA of mir-619-5p. It can bind to mir-619-5p and reduce its expression, thus regulating the target gene CAMK1D. In addition, we found that high expression of ILF3-AS1 was significantly associated with tumor grade, tumor size, and distant metastasis in COAD samples. In vivo experiments confirmed that ILF3-AS1 promotes tumor growth in COAD models.ConclusionThe present study demonstrated that ILF3-AS1 plays an oncogenic role in COAD through regulating the miR-619-5p/CAMK1D axis, and inhibition of ILF3-AS1 may pave the way for COAD treatment.

Project description:ObjectivesLung adenocarcinoma (LUAD) exhibits a higher fatality rate among all cancer types worldwide, yet the precise mechanisms underlying its initiation and progression remain unknown. Mounting evidence suggests that long non-coding RNAs (lncRNAs) exert significant regulatory roles in cancer development and progression. Nevertheless, the precise involvement of lncRNA CYP4A22-AS1 in LUAD remains incompletely comprehended.MethodsBioinformatics analyses evaluated the expression level of CYP4A22-AS1 in lung adenocarcinoma and paracancer. The LUAD cell line with a high expression of CYP4A22-AS1 was constructed to evaluate the role of CYP4A22-AS1 in the proliferation and metastasis of LUAD by CCK8, scratch healing, transwell assays, and animal experiments. We applied transcriptome and microRNA sequencing to examine the mechanism of CYP4A22-AS1 enhancing the proliferation and metastasis of LUAD. Luciferase reporter gene analyses, west-blotting, and qRT-PCR were carried out to reveal the interaction between CYP4A22-AS1, miR-205-5p/EREG, and miR-34c-5p/BCL-2 axes.ResultsCYP4A22-AS1 expression was significantly higher in LUAD tissues than in the adjacent tissues. Furthermore, we constructed a LUAD cell line with a high expression of CYP4A22-AS1 and noted that the high expression of CYP4A22-AS1 significantly enhanced the proliferation and metastasis of LUAD. We applied transcriptome and microRNA sequencing to examine the mechanism of CYP4A22-AS1 enhancing the proliferation and metastasis of LUAD. CYP4A22-AS1 increased the expression of EREG and BCL-2 by reducing the expression of miR-205-5p and miR-34-5p and activating the downstream signaling pathway of EGFR and the anti-apoptotic signaling pathway of BCL-2, thereby triggering the proliferation and metastasis of LUAD. The transfection of miR-205-5p and miR-34-5p mimics inhibited the role of CYP4A22-AS1 in enhancing tumor progression.ConclusionThis study elucidates the molecular mechanism whereby CYP4A22-AS1 overexpression promotes LUAD progression through the miR-205-5p/EREG and miR-34c-5p/BCL-2 axes.

Project description:Glioma is the most common malignant tumor in the central nervous system. Altered long noncoding RNAs (lncRNAs) are playing regulatory roles in physiological and pathogenic processes in cancer. Here, we uncovered a differentially expressed lncRNA called brain cytoplasmic RNA 1 (BCYRN1), and elucidated its function and molecular mechanism in the progression and development of glioma. Three fresh tumor tissues from glioma patients and three normal brain tissues from craniocerebral trauma patients were prepared for high-throughput RNA sequencing. Differential RNA transcripts and BCYRN1 were identified by RT-qPCR in glioma samples and controls. CCK-8, colony formation assays, flow cytometry, TUNEL assays, cell migration assays, wound-healing assays, and xenograft model were established to investigate the biological function of BCYRN1 both in vitro and in vivo. Various bioinformatics analysis, dual-luciferase reporter assays, biotinylated RNA pulldown assays, and rescue experiments were conducted to reveal the underlying mechanisms of competitive endogenous RNAs (ceRNAs). 183 lncRNAs were identified with significant dysregulation in glioma and randomly selected differential RNAs were further confirmed by RT-qPCR. Among them, BCYRN1 was the most downregulated lncRNA, and its low expression positively correlated with glioma progression. Functionally, BCYRN1 overexpression inhibited cell proliferation, migration in glioma cell lines, whereas BCYRN1 depletion resulted in the opposite way. MiR-619-5p was further confirmed as the direct target of BCYRN1. Mechanistically, miR-619-5p specifically targeted the CUE domain containing protein 2 (CUEDC2), and BCYRN1/miR-619-5p suppressed glioma tumorigenesis by inactivating PTEN/AKT/p21 pathway in a CUEDC2-dependent manner. Overall, our data presented that the reduced expression of BCYRN1 was associated with poor patient outcome in glioma. BCYRN1 functioned as a ceRNA to inhibit glioma progression by sponging miR-619-5p to regulate CUEDC2 expression and PTEN/AKT/p21 pathway. Our results indicated that BCYRN1 exerted tumor suppressor potential and might be a candidate in the diagnosis and treatment of glioma.

Project description:BackgroundNumerous studies proved that long non-coding RNA (lncRNA) is involved in the progression of multifarious diseases, especially in some carcinomas. As a potential tumor biomarker, plasmacytoma variant translocation 1 gene (PVT1) is involved in the development and progression of multifarious cancers. Nevertheless, the intrinsic and concrete molecular mechanism of PVT1 in bladder cancer still remained unclear, which is also the dilemma faced in many non-coding RNA studies.ResultsOur research revealed that PVT1 was significantly higher expression in bladder carcinoma specimens and cell lines. Further experiments indicated that knockdown or overexpression of PVT1 restrained or promoted the malignant phenotype and WNT/β-catenin signaling in bladder cancer cells. Meanwhile miR-194-5p was in contrast and miR-194-5p could partially reverse the function of PVT1 in malignant bladder tumor cells. As a microRNA sponge, PVT1 actively promotes the expression of b-cells lymphoma-2-associated transcription factor 1 (BCLAF1) to sponge miR-194-5p and subsequently increases malignant phenotypes of bladder cancer cells. Therefore, it performs a carcinogenic effect and miR-194-5p as the opposite function, and serves as an antioncogene in the bladder carcinomas pathogenesis.ConclusionPVT1-miR-194-5p-BCLAF1 axis is involved in the malignant progression and development of bladder carcinomas. Experiments revealed that PVT1 has a significant regulatory effect on bladder cancer (BC) and can be used as a clinical diagnostic marker and a therapeutic molecular marker for patients suffering from BC.MethodsIn urothelial bladder carcinoma specimens and cell lines, the relative expression levels of PVT1 and miR-194-5p were detected by quantitative reverse transcription PCR (RT-qPCR). Through experiments such as loss-function and over-expression, the biological effects of PVT1 and miR-194-5p on the proliferation, migration, apoptosis and tumorigenicity were explored in bladder cancer cells. Co-immunoprecipitation, proteomics experiments, dual luciferase reporter gene analysis, western blot and other methods were adopted to investigate the PVT1 potential mechanism in bladder carcinomas.

Project description:BackgroundNormally, one miRNA interacts with the mRNA of one gene. However, there are miRNAs that can bind to many mRNAs, and one mRNA can be the target of many miRNAs. This significantly complicates the study of the properties of miRNAs and their diagnostic and medical applications.ResultsThe search of 2,750 human microRNAs (miRNAs) binding sites in 12,175 mRNAs of human genes using the MirTarget program has been completed. For the binding sites of the miR-619-5p the hybridization free energy of the bonds was equal to 100% of the maximum potential free energy. The mRNAs of 201 human genes have complete complementary binding sites of miR-619-5p in the 3'UTR (214 sites), CDS (3 sites), and 5'UTR (4 sites). The mRNAs of CATAD1, ICA1L, GK5, POLH, and PRR11 genes have six miR-619-5p binding sites, and the mRNAs of OPA3 and CYP20A1 genes have eight and ten binding sites, respectively. All of these miR-619-5p binding sites are located in the 3'UTRs. The miR-619-5p binding site in the 5'UTR of mRNA of human USP29 gene is found in the mRNAs of orthologous genes of primates. Binding sites of miR-619-5p in the coding regions of mRNAs of C8H8orf44, C8orf44, and ISY1 genes encode the WLMPVIP oligopeptide, which is present in the orthologous proteins. Binding sites of miR-619-5p in the mRNAs of transcription factor genes ZNF429 and ZNF429 encode the AHACNP oligopeptide in another reading frame. Binding sites of miR-619-5p in the 3'UTRs of all human target genes are also present in the 3'UTRs of orthologous genes of mammals. The completely complementary binding sites for miR-619-5p are conservative in the orthologous mammalian genes.ConclusionsThe majority of miR-619-5p binding sites are located in the 3'UTRs but some genes have miRNA binding sites in the 5'UTRs of mRNAs. Several genes have binding sites for miRNAs in the CDSs that are read in different open reading frames. Identical nucleotide sequences of binding sites encode different amino acids in different proteins. The binding sites of miR-619-5p in 3'UTRs, 5'UTRs and CDSs are conservative in the orthologous mammalian genes.

Dataset Information

LncRNA PVT1 promotes gemcitabine resistance of pancreatic cancer via activating Wnt/?-catenin and autophagy pathway through modulating the miR-619-5p/Pygo2 and miR-619-5p/ATG14 axes.

Publications

LncRNA PVT1 promotes gemcitabine resistance of pancreatic cancer via activating Wnt/β-catenin and autophagy pathway through modulating the miR-619-5p/Pygo2 and miR-619-5p/ATG14 axes.

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