Mechanism Study Of Long Noncoding RNA PVT1 In Promoting The Angiogenesis By Targeting MiR-26b

Background:Leukemia is the first malignant disease in the blood system and one of the top ten common malignant tumors in adults.Its morbidity and mortality are increasing year by year.As the world's most populous country,China has the largest leukemia group in the world.The huge medical expenses and the enormous energy it spends to treat leukemia put huge economic and mental pressure on patients and their families.The medical burden is increasing and it has become a social problem that cannot be ignored.The pathogenesis of leukemia is very complicated,so far,it can not be fully explained.According to the existing research,the risk stratification of patients and corresponding treatment,some patients still have the problem of drug resistance and recurrence,suggesting that we need to further explore the new pathogenesis of leukemia.Recent studies have shown that changes in the bone marrow hematopoietic microenvironment are also important factors in the pathogenesis of leukemia,and bone marrow angiogenesis is an important pathological process in the changes of bone marrow hematopoietic microenvironment.A number of studies have demonstrated significant angiogenesis in leukemia.The angiogenic cell and molecular regulation mechanisms have many commonalities among various malignant tumors,which are characterized by up-regulation of inducible factors and relatively weaker expression of inhibitory factors.The molecular mechanisms involved in the angiogenesis process are extremely complex and contain numerous signaling pathways that have not yet been elucidated.We all know that the expression level of the coding gene is regulated by the upstream miRNA.Many studies have confirmed that miRNA expression abnormalities are closely related to the development of angiogenesis.Therefore,exploring the target gene-related miRNA is the core link of the research target gene.According to the competitive endogenous RNA(ceRNA)hypothesis proposed by Salmena et al.,long-chain non-coding RNAs(LncRNAs)competitively bind to miRNAs through their own miRNA response elements(MREs),regulating the expression of related genes.One of the important functions of lncRNA is to act as a miRNA sponge to regulate target gene expression.In summary,the ceRNA mechanism such as lncRNA-miRNA-mRNA is worthy of our study.Objective:Angiogenesis is essential for a variety of biological processes,including tumor blood supply delivery,cancer cell growth,invasion,and metastasis.Recently,it has been found that lncRNA PVT1 can be used in miR-26 b by ceRNA machine in esophageal cancer and melanoma,which affects tumorigenesis,metastasis,and angiogenesis.However,the specific potential molecular mechanism by which PVT1 regulates vascular endothelial cell angiogenesis remains to be elucidated.Methods:1.To determine the effect of miR-26 b on cell proliferation,migration and angiogenesis in human umbilical vein endothelial cells(HUVECs),miR-26 mimics or miR-26 b inhibitors were transfected into HUVECs.MTT assay,Transwell migration assay and in vitro vascular tubular formation assay were used to study the role of miR-26 b in cell proliferation,migration and angiogenesis in human umbilical vein endothelial cells(HUVECs).Reverse transcription-quantitative polymerase chain reaction(RT-qPCR)and western blotting were performed to quantify mRNA and protein expression levels of the target gene.The interaction of miR-26 b with the 3'-UTR of CTGF was subsequently investigated using the dual luciferase reporter gene.2.To determine the effect of PVT1 on cell proliferation,migration and angiogenesis of human umbilical vein endothelial cells(HUVECs),PVT1 overexpression and PVT1 knockdown HUVECs were constructed.MTT assay,Transwell migration assay and in vitro vascular tubular formation assay were used to study the role of PVT1 in cell proliferation,migration and angiogenesis in human umbilical vein endothelial cells(HUVECs).Reverse transcription-quantitative polymerase chain reaction(RT-qPCR)was performed to detect changes in mRNA expression levels of miR-26 b and target genes,and Western blotting was used to quantify protein expression levels of target genes.The interaction of PVT1 and miR-26 b was then investigated using the dual luciferase reporter gene.Results:1.Overexpression of miR-26 b inhibited cell migration of HUVECs in Transwell cell invasion assays(P<0.05),while inhibition of miR-26 b expression promoted this migration(P<0.05).The cell proliferation assay(MTT assay)confirmed that the cell proliferation activity of HUVECs overexpressing miR-26 b was significantly decreased(P<0.05).On the contrary,the consumption of miR-26 b increased the cell proliferation ability of HUVECs(P<0.05).By measuring and recording the tube length,number of loops,coverage area and number of nodes of different groups of HUVECs,and statistical analysis,the results showed that miR-26 b overexpression reduced the length of blood vessels(P<0.05).RT-qPCR and western blotting showed that overexpression of miR-26 b resulted in decreased mRNA and protein expression levels of CTGF and ANGPT2 compared to the mock negative control group.Dual luciferase assay confirmed that miR-26 b recognizes and binds to wild-type CTGF 3'-UTR,resulting in decreased luciferase activity(P < 0.05),while luciferase intensity was unchanged in the mutant vector group.2.Compared with the control,PVT1 overexpressing cells(pEX2 PVT1)had higher migration ability(P<0.05),while PVT1 knockdown cells(shPVT1)had decreased migration ability(P<0.05).MTT assay confirmed that PVT1 overexpression significantly promoted the proliferation of HUVECs(P<0.05),while the low expression of PVT1 impaired the cell growth rate of HUVECs(P<0.01).In vitro angiogenesis experiments showed that PVT1 overexpression(pEX2 PVT1)promoted angiogenesis in vitro compared to PVT1 knockdown cells(shPVT1)(P < 0.001).RT-qPCR results showed that PVT1 overexpression resulted in decreased expression of miR-26b(P<0.01)and increased mRNA expression levels of CTGF and ANGPT2(P<0.05 and P<0.01),while cells lacking PVT1 showed an increase.The level of miR-26b(P<0.05)and the down-regulated mRNA levels of CTGF and ANGPT2(P<0.05).Western blotting showed that the expression levels of CTGF and ANGPT2 protein were increased in HUVECs overexpressing PVT1,while the expression levels of CTGF and ANGPT2 in HUVECs with PVT1 knockdown were decreased.The dual luciferase reporter assay demonstrated that only wild-type PVT1 significantly reduced the luciferase activity of miR-26b(P < 0.05),and PVT1 after binding site mutation did not alter the luciferase activity of miR-26 b.Conclusion:1.Overexpression of miR-26 b inhibits cell proliferation,migration and angiogenesis in HUVECs cell lines.2.MicroRNA-26 b inhibits angiogenesis in HUVECs by down-regulating CTGF and ANGPT2.3.MicroRNA-26 b directly interacts with 3??UTR of CTGF,inhibiting the transcription of CTGF.4.Overexpression of PVT1 promotes cell proliferation,migration and angiogenesis in HUVECs cell lines.5.PVT1 directly interacts with miR?26b,down-regulating miR-26 b expression.6.lncRNA PVT1 acts as a miR-26 b adsorption sponge to promote the expression of CTGF and ANGPT2,and ultimately promote angiogenesis.