| Lnc RNAs are transcripts which more than 200 nucleotides in length,have become one of the hot spots in cancer research in recent years.They have been reported to be involving in regulating chromatin modification,RNA shearing,gene transcription and translation.They widely participate in cell proliferation,differentiation,metabolism and multi biological processes by interacting with various molecules.Renal cancer is one of the most common malignant tumors.The incidence is within the top ten of malignant tumor both in men and women.Besides that,the mortality is highest of the three malignant tumors of the urinary system,which causes a heavy social burden.Renal cancer is associated with metabolic factors such as obesity and diabetes,and genomic analysis has revealed that mutation,inactivation or overactivation of common renal cancer genes are involved in dysregulation of energy and nutrient metabolic pathways.The main effective treatment for renal cancer is surgery,followed by molecular targeted therapy,but it is insensitive to radiotherapy and chemotherapy.It is urgent that we need develop new tumor treatment targets.Our group focused on the mechanism of classical oncogene PVT1 in promoting malignant progression by regulating metabolic reprogramming in renal cancer,and we hope this could provide new ideas for future treatment of renal cancer.Part I Expression and regulation of PVT1 in renal cancerOBJECTIVE: To investigate the expression and predictive value of lnc RNA PVT1 in renal cancer and to clarify the role of PVT1 in regulating the malignant biological behavior of renal cancer.METHODS: The data of lnc RNAs in the TCGA database was downloaded and the expression of PVT1 was analyzed in renal cancer and paraneoplastic tissues.Then,a prediction model was established based on PVT1 expression,with or without clinical information of patients to predict OS.The effects of PVT1 on the proliferation,migration and glycolytic capacity of renal cancer cells were clarified by CCK-8,colony formation,wound healing,Transwell,glucose uptake,lactate production and ECAR assays,respectively.RESULTS: PVT1 was significantly upregulated in renal cancer tissues and cells.Patients with high PVT1 expression had shorter OS and DSS.The prediction model established based on PVT1 expression combining with or without the clinical information of patients’ age,gender,and TMN stage could predict OS of renal cancer patients.Knockdown of PVT1 significantly inhibited cell proliferation,invasion.RNA sequencing and KEGG pathway analysis was conducted.Results showed that PVT1-regulated genes were enriched in metabolic pathways.After transfection with PVT1-interfering lentivirus,glucose uptake,lactate production and ECAR were reduced in 786-O and A498 cells,indicating that knockdown of PVT1 could inhibit the rate of cellular glycolysis.Conclusion: PVT1 is highly expressed in renal cancer and can be a risk factor to predict patients’ OS.PVT1 affects the biological behaviors such as proliferation and migration of renal cancer cells by promoting their glycolysis.Part II PVT1 sponges mi R-532-3p to regulate Wnt signaling pathwayOBJECTIVE: To investigate PVT1 activates Wnt signaling pathway and regulates glycolysis in renal cancer cells through regulating the mi R-532-3p/BCL9 signaling axis.METHODS: The possible mi RNAs which can bind to PVT1 was predicted by online tools.Mi R-532-3p was confirmed to be regulated by PVT1,and its function was clarified in renal cancer.The downstream gene BCL9 was screened by database prediction combined with RNA sequencing results.QRT-PCR and western blot were performed to clarified the change of BCL9 m RNA and protein levels in cells after transfecting with sh-PVT1 alone or co-transfecting with mi R-532-3p inhibitor.To investigate the ability of PVT1 to affect the glycolysis of renal cancer cells and alter their proliferation and migration by regulating mi R-532-3p.RESULTS: The mi RNAs that can bind to PVT1 were predicted in two online databases star Base v2.0 and DIANA TOOL.The correlation between mi RNAs and PVT1 was analysed,and alteration of mi RNAs was also analyzed after knockdown or overexpression of PVT1.Results showed that PVT1 could bind to mi R-532-3p.Then,mi R-532-3p mimics and inhibitors were transfected in renal cancer cells.It was verified that mi R-532-3p could inhibit proliferation,migration and glycolysis.Candidate genes were obtained by using prediction tool mi RDB,mi RWALK and Target Scan.BCL9 was identified as the target gene eventually by overlapping the results of RNA sequencing.BCL9 is a co-activator of Wnt signaling pathway.q RT-PCR and western blot experiments revealed that knockdown of PVT1 could significantly inhibited the expression of BCL9,but a reversion could be observed by inhibiting mi R-532-3p expression.The same changes occurred in c-Myc and cyclin D1,which were genes downstream of Wnt pathway.Luciferase reporter gene plasmids were constructed separately and co-transfected with mi R-532-3p mimics,and results showed that wildtype PVT1 and wild-type BCL9 plasmids reduced luciferase activity,indicating that PVT1 and BCL9 could directly bind to and mi R-532-3p.Conclusion: PVT1 could competitively bind to mi R-532-3p and upregulate the expression of BCL9,which activates the Wnt signaling pathway,leads to the activation of downstream genes.Therefore,PVT1 enhances glycolysis and cell proliferation and migration of renal cancer cells.Part III PVT1 regulates glycolysis in renal cancer cells via FGFR1/PKM2OBJECTIVE: To investigate the mechanism by which PVT1 regulates glycolysis by binding to FGFR1 and PKM2.METHODS: PVT1-sense and PVT1-antisense were constructed to perform RNA pulldown assay,followed with mass spectrometry and PKM2 was screened out,which was a key enzyme of glucose metabolism.Then it is verified that PVT1 directly binds to and regulates PKM2.It was clarified that the regulation of PKM2 by PVT1 requires the participation of FGFR1.To further explore the mechanism of PVT1 regulating PKM2 through the FGFR1-dependent pathway.RESULTS: RNA pulldown followed by silver staining,western blot and RIP assay were performed to confirm that PVT1 binds directly to PKM2,whereas PVT1 does not alter PKM2 m RNA and total protein levels,but only affects its phosphorylation level.Increasing PKM2 phosphorylation level leads to upregulation of dimeric form of PKM2 in cells,which has low pyruvate kinase activity.This resulted in enhancement of cells’ glycolytic capacity.We identified another protein enriched with PVT1,which named FGFR1,a protein that involved in phosphorylation modification of PKM2.Co-IP assay confirmed FGFR1 bind to PKM2.Knockdown PVT1 only reduced FGFR1 protein level but not m RNA level,indicated that PVT1 regulated FGFR1 at post-transcriptional.It was speculated that PVT1 phosphorylated PKM2 through regulating the expression of FGFR1.Further experiments revealed that PVT1 knockdown reduced protein stability of FGFR1 and caused FGFR1 ubiquitin degradation,which resulted in a decrease of FGFR1.Conclusion: PVT1 increases the protein stability and expression of FGFR1 by reducing its protein ubiquitination degradation.The stable expression of FGFR1 increases dimeric form of PKM2 by phosphorylating it,maintains its low pyruvate kinase activity in cells,eventually promotes glycolysis in renal cancer. |
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