| Part1 The expression and clinical significance circPVT1in acute T-cell lymphoblastic leukemiaPurposesTo determine the expression and clinical significance of circPVT1 in acute T lymphocytic leukemia.Methods1.To determine the expression of circPVT1 in acute T-lymphocytic leukemia patients,bone marrow samples from T-ALL patients(33 cases)and healthy volunteers(14 cases)and T-ALL cell lines were collected.Then,Reverse transcription quantitative PCR(RT-qPCR)was used to detect the relative expression of circPVT1.2.To study the cellular biological function of circPVT1 in acute T lymphocytic leukemia,siRNAs of circPVT1 were transfected into T-ALL cells to knock down the expression of circPVT1.CCK-8 and flow cytometry were used to measure the cell viability,cell cycle and apoptosis,and the expression of proliferation and apoptosis-related proteins were determined by western blot.3.To study the relationship between the expression of circPVT1 and the risk stratification and prognosis of acute T lymphocytic leukemia,patients were divided into high expression group and low expression group according to the median expression of circPVT1.The clinic features of the two groups were compared.Results1.Compared with healthy donors,the expression of circPVT1 in bone marrow samples of T-ALL patients was significantly increased(2.624±0.6732 Vs 0.6529±0.3290,P<0.001).Compared with normal cells,the expression of circPVT1 in the three A-TLL cell lines were significantly up-regulated(P<0.001).2.The cell viability of Jurkat and Molt-4cellstransfected with siRNA were decreased to 72.6%±11.0%(P<0.001)and 73.2%±12.4%(P<0.01)when compared with the control group.The apoptosis ratesof the Jurkat and Molt-4 cells transfected with siRNA were significant lyincreased(3.62%±0.17%Vs1.99%±0.1 1%,P<0.001;3.55%±0.26%Vs1.70%±0.16%,P<0.001).The cell distributions of Jurkat and Molt4 cells transfected with siRNA in the S phase were significantly reduced(32.17%±0.52%Vs45.45%±0.74%,P<0.001;33.55%±1.41%Vs45.53%±0.74%,P<0.001),whereas the cell distributions in G2 phase were significantly improved(28.87%±0.33%Vs16.6 7%±0.71%,P<0.001;28.03%±1.38%Vs16.67%±0.71%,P<0.001).Westernblot results showed that after knocking down the expression of circPVT1 in Jurkat and Molt-4 cells,the expression of apoptosis-related proteins cleaved-caspase3 and cleaved-caspase9 increased significantly,while the anti-apoptotic proteins Bcl-2 and cyclin D was significantly reduced.3.The expression level of circPVT1 has no significant correlation with clinical parameters such as gender,age,white blood cell count and bone marrow blast cell ratio of T-ALL patients.However,the expression level of circPVT1 in the high-risk group(risk stratification with different ages)was significantly higher than that in the low-risk group.Statistical analysis showed that patients with high expression of circPVT1 had a higher 5-year recurrence rate,a lower 5-year survival rate and a shorter survival time.Conclusions1.The expression of circPVT1 is significantly up-regulated in bone marrow of T-ALL patients and cell lines.2.Knockdown of the expression of circPVT1 in T-ALL cells can inhibit the proliferation of leukemia cells,promote their apoptosis,and reduce the cell distribution of cells in the S phase.At the same time,it can also increase the expression of cleaved-caspase3 and cleaved-caspase9,and reduce the expression of Bcl-2 and cyclin D.3.The high expression of circPVT1 is related to the poor clinical prognosis of acute T lymphocytic leukemia.Part 2 CircPVT1 boundwithandregulated the expression of miR-30e.PurposesTo determine whether circPVT1 can bind with and regulate the expression of miR-30e in acute T lymphoblastic leukemia cells.Methods1.The bioinformatics database starbase(ENCORI:The Encyclopedia of RNA Interactomes,http://starbase.sysu.edu.cn/)was employed to predict potential binding target of circPVT1 and screen target miRNAs based on literature.RT-qPCR was used to analyze the expression of miR-30e in T-ALL patients;mimics and inhibitors of miR-30e were synthesized,and the changes in cell proliferation were detected by the CCK-8 method in response to overexpression or knocking down miR-30e.2.RT-qPCR was used to detect the changes of miR-30e expression in Jurkat and Molt4 cells with circPVT1 knocking down.The RNA hybrid online website(https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid)was used to predictmiR-30e binding with circPVT1.Dual luciferase report assay were employed to veryfy the binding of miR-30e and circPVT1.Statistical analysis of the correlation between the expression of circPVT1 and miR-30e.3.A rescueexperiment was designed.circPVT1siRNA1 and miR-30e inhibitor were co-transfected into Jurkat and molt4 cells,and cell proliferation and cell cycle were detected.Results1.The expression of MiR-30e in bone marrow samples of patients with acute T lymphocytic leukemia is significantly reduced.The increase of miR-30e expression can inhibit the proliferation of T-ALL cells,and the reduction of its expression will promote cell proliferation.2.Knockdown of the expression of circPVT1 in Jurkat and Molt4 cells can significantly increase the expression of miR-30e(6.64±1.01 times,P<0.001 and 4.98±0.88 times,P=0.0014).The results of dual luciferase reporter gene show that,only co-transfection of miR-30e mimics and a dual-luciferase reporter gene plasmid containing wild-type circPVT1 can significantly reduce the activity of the firefly enzyme reporter gene.And the expression levels of circPVTl and miR-30e were negatively correlated in patients.3.In Jurkat and Molt4 cells,transfection of miR-30e inhibitor can significantly increase cell proliferation and the distribution of cells in S pahse,while siRNA of circPVT1 had the oppsite effect.And co-transfection of miR-30e inhibitor in Jurkat and Molt4 cells with si-circPVT1 can abolish the effect of si-circPVT1.Conclusions1.The expression of miR-30e is significantly decreased in acute T lymphocytic leukemia,and the decrease of miR-30e expression can increase the proliferation of cancer cells.2.CircPVT1 can bind to and negatively regulate the expression of miR-30e.There is a negative correlation between the expression of miR-30e and circPVT1 in patient samples.3.In T-ALL cells,circPVT1 regulated the expression of miR-30e.Part 3 circPVT1 regulated NOTCH signaling pathway activity through miR-30e/DLL4 in T-ALLPurposesTo determine whether circPVT1 can regulate the expression of DLL4 through miR-30e in acute T lymphocytic leukemia,thereby further activating the NOTCH signaling pathway.Methods1.The targetscan was used to predict the target gene of miR-30e and DLL4 was speculated as the target geneaccording to theresults and literatures.The dual luciferase reporter assay was usedto further verify the binding of the two.RT-qPCR was used to detect the effect of miR-30e mimics and inhibitor on the expression of DLL4 in the cells.Finally,correlation between the expression levels of miR-30e and DLL4 in bone marrow samples from T-ALL patients were analized.2.RT-qPCR and western blot were used to detect the expression changes of DLL4 after knocking down the expression of circPVT1 in T-ALL cells.A circPVT1 overexpression plasmid(mutcircPVT1-OE)with mutated miR-30e binding site(mutcircPVT1-OE)was constructed.The plasmid was transfected into T-ALL cells,the changes on cell proliferation were detected by the CCK-8 method.3.The expression changes of the two genes(HES1 and DELTEX1)downstream of the NOTCH signaling pathway were detedcted by RT-qPCR in the cells tranfected with circPVT1-siRNA1 and mutcircPVT1-OE.Results1.MiR-30e mimics could significantly reduce the expression of DLL4,while miR-30e inhibitor significantly increased the expression of DLL4.The results of the dual luciferase reporter assay showed that only in the cells co-transfected with miR-30e mimics and wild-type dual luciferase reporter gene plasmids,the firefly luciferase activity was significantly reduced.In bone marrow samples from T-ALL patients,the expression levels of miR-30e and DLL4 were negatively correlated.2.Compared with the control,DLL4 mRNA and protein levels in Jurkat and Molt4 cells transfected with circPVT1-siRNA1 levels were significantly reduced.Transfection of the overexpression plasmid mutcircPVT1-OE can significantly increase the expression level of circPVT1 in Jurkat and Molt4 cells,but the expression level of DLL4 did not change significantly.More importantly,transfection of the overexpression plasmid mutcircPVT1-OE did not enhance the proliferation of Jurkat and Molt4 cells.3.Compared with the negative control,in Jurkat and Molt4 cells transfected with circPVT1-siRNA,the expression of HES1 and DELTEX1 was significantly reduced;while in the cells transfected with mutcircPVT1-OE,the expression of HES1 and DELTEX1 did not change significantly.Conclusions1.MiR-30e can bind with and negatively regulate the expression of DLL4 in acute T lymphocyte leukemia.In the bone marrow samples of T-ALL patients,the expression of miR-30e and DLL4 are negatively correlated.2.CircPVT1 regulates the expression of DLL4 by adsorbing miR-30e in acute T lymphocytic leukemia.3.CircPVT1 can activate the NOTCH signaling pathway through miR-30e/DLL4 in acute T lymphocytic leukemia.Part 4 circPVT1promotedtumor growth in T-ALLin vivoPurposesTo verify the circPVT1/miR-30e/DLL4 regulated tumor growthin vivo.Methods1.Xenograft mice model establish:circPVTl and miRNA-30e knockdown lentivirus(sh-lentivirus)were constructed.The nude mice were randomly divided into three groups:control group,interference group and rescue group.The control group was injected subcutaneously with Jurkat cells infected with empty lentivirus;the interference group was injected subcutaneously with Jurkat cells infected with circPVT1 knockdown lentivirus;the rescue group was injected subcutaneously with co-infected Jurkat cells.2.Tumor observation:The long diameter and short diameter of the xenograftswere measured.After 8 weeks,the mice were sacrificed,the tumors were taken out and weighed.3.Gene expression detection:The expression levels of circPVT1,miR-30e,DLL4,HES1 and Deltexl in tumor tissue were measured.4.HE staining:TheHE stain were performed to observe the cell morphology in the tumor tissue.Results1.The growth rate of tumors in the interference group,the control group and the recovery group increased sequentially,and the overall weight of the transplanted tumor in the interference group was significantly lower than that of the control group and the recovery group.2.The results of HE staining showed that the nucleus of tumor cells in the interference group were pyknotic,small and darkly stained,suggesting a low cell viability,while the nucleus of the control group and the recovery group were round,large and lightly stained,suggesting a high cell viability.3.Compared with the control group,the expression of circPVT1,DLL4,HES1,and DELTEX1 in the tumor tissues of the interference group were significantly reduced,and the expression of miR-30e was significantly increased;while in the recovery group,the expression of circPVTl and miR-30e were both Significantly reduced,although DLL4 increased but not statistically significant,the expression of HES1 and Deltex1 increased slightly,but neither was statistically significant.ConclusionsCircPVT1/miR-30e/DLL4 can regulate tumor growth in vivo. |
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