The Effects Of Hsa_circ₀043415,hsa_circ₀010358 And Hsa_circ₀009353 On The Proliferation,cycle And Apoptosis Of B-cell Lymphoma Cells

Background and ObjectiveNon-Hodgkin's lymphoma?NHL?is a common human malignant tumor.According to the cellular origin,NHL could be divided into three types:B-cell lymphoma?BCL?,T-cell lymphoma and NK-cell lymphoma.BCL is a common type of NHL,accounting for about 80%of the total number of cases.The comprehensive treatment of combination of cyclophosphamide,doxorubicin,vincristine,prednisone and targeted anti-CD20 rituximab has become the mainstream of the treatment of BCL.Comprehensive treatment has improved the quality of life and long-term prognosis of BCL patients,but there are still some BCL patients with recurrence and metastasis.At present,many studies had found that circRNAs were abnormally expressed in lung cancer,liver cancer,gastric cancer and other tumors.circ RNAs could remove regulatory effect of miRNA on its target genes and participate in occurrence and development of tumor by microRNA response elements?MREs?.For example,the expression of circ-LAMP1 was up-regulated in T-cell lymphoblastic lymphoma,which could promote cell proliferation and inhibit cell apoptosis.Circ-LAMP1 regulated DDR2by targeting miR-615-5p to participate in carcinogenesis.Combined with our previous research,we found that miR-148b-3p could directly target BCL-W to reduce the viability and colony formation of BCL cells after radiotherapy,and promote cell apoptosis.With the decrease of mitochondrial membrane potential and the release of cytochrome C,the cleavage of caspase 9 and caspase 3 increased,and the expression of proteins related to the mitochondrial apoptosis pathway increased correspondingly.MiR-148b-3p could inhibit the tumor growth in nude mice with transplanted irradiated Raji cells.Therefore,in this study,the differentially expressed circRNAs of Raji cells after radiotherapy were detected by circRNA microarray analysis,and the circRNAs that had interaction sites with mi R-148b-3p were screened:hsa?circ?0043415,hsa?circ?0010358 and hsa?circ?0009353.In this study,lentivirus transfection technique,real-time quantitative PCR?qRT-PCR?,CCK-8 and flow cytometry were used to explore the effects of hsa?circ?0043415,hsa?circ?0010358 and hsa?circ?0009353 on the proliferation,cycle and apoptosis of BCL cells,so as to provide a new therapeutic target for the treatment of BCL patients.Methods1.Construction of stable transfected B cell lymphoma cells with up-regulation of circRNAs expressionIn BCL Raji and SU-DHL-10 cells,lentivirus was used to up-regulate circRNA expression,and qRT-PCR was used to detect the expression level of hsa?circ?0043415,hsa?circ?0010358 and hsa?circ?0009353 in Raji/SU-DHL-10 cells,lentivirus control Raji/SU-DHL-10 cells and lentivirus up-regulated circRNA expression group.2.The effect of circRNAs overexpression on the proliferation of B cell lymphoma was detected by CCK-8Raji/SU-DHL-10 cells,lentivirus control Raji/SU-DHL-10 cells and lentivirus up-regulated circRNA expression Raji/SU-DHL-10 cells were seed into a 96-well plate with 2500 cells per well and 3 repeat holes were set.The CCK-8 reagent of 10?l per well was added at 0 h,24 h,48 h and 72 h,respectively,and the cell activity was detected by enzyme labeling instrument after incubation with 37?,5%CO₂ for 2.5 h.3.The effect of circRNAs overexpression on the cell cycle and apoptosis of B cell lymphoma was detected by flow cytometryRaji and SU-DHL-10 cells,lentivirus control Raji and SU-DHL-10 cells and lentivirus up-regulated circRNA expression Raji and SU-DHL-10 cells were digested,terminated,centrifuged,and detected on the computer after adding cycle and apoptosis reagents.4.Statistical processingThe data obtained were expressed by mean plus or minus standard deviation,and the experimental data were statistically analyzed by SPSS25.0 software.The measurement data were measured by paired sample t-test or analysis of variance,and the counting data were compared between groups by chi-square test or Fisher exact probability method.The rank sum test was used for the experimental data which did not conform to the normal distribution,and the significance test was based on P<0.05.The data were plotted by GraphPad Prism 5.0.Results1.Vector identification and lentivirus titerThe sequencing identification of the overexpression circRNAs vector showed that the sequencing peak diagram was normal,there were no heterozygma or overlapping bands,and the sequence comparison was consistent,which indicating that hsa?circ?0043415,hsa?circ?0010358 and hsa?circ?0009353 were successfully inserted into the vector.These results indicated that the construction of the overexpression vector was successful.Besides,the RNA electrophoresis showed clear bands of 28s,18s and 5s,and good RNA integrity.The results of qRT-PCR showed that the expressions of hsa?circ?0043415,hsa?circ?0010358 and hsa?circ?0009353 were all increased in H293T cells with overexpression of circRNAs,which was statistically significant,indicating that the vector was successfully constructed.In addition,lentivirus titer showed that the titer of hsa?circ?0043415 overexpressed lentivirus was 2×10⁷TU/ml,the titer of hsa?circ?0010358 overexpressed lentivirus was 5×10⁶TU/ml,and the titer of hsa?circ?00093535overexpressed lentivirus was 5×10⁶TU/ml.2.Establish a stable transfected B-cell lymphoma cell model with up-regulated circRNAsThe expression of circRNAs was up-regulated by lentivirus particles transfection in Raji and SU-DHL-10 cells to construct stable transfected cell lines.QRT-PCR showed that the expression of circRNAs in the hsa?circ?0043415 overexpression group?circ?0043415?,hsa?circ?0010358 overexpression group?circ?0010358?and hsa?circ?0009353 overexpression group?circ?0009353?was significantly higher than that in Raji/SU-DHL-10 cells group and lentivirus control Raji/SU-DHL-10 cells group?Vector??P<0.001?.3.Up-regulation of hsa?circ?0043415,hsa?circ?0010358 and hsa?circ?0009353expression promoted the proliferation of B-cell lymphoma cellsThe results of CCK-8 showed that with the passage of time,the optical density?OD?value of lentivirus overexpression hsa?circ?0043415,hsa?circ?0010358 and hsa?circ?0009353 Raji and SU-DHL-10 cells group was significantly higher than that of Raji/SU-DHL-10 cells and lentivirus control Raji/SU-DHL-10 cells group?P<0.05?,but there was no significant change of OD value in Raji/SU-DHL-10 cells group and lentivirus control Raji/SU-DHL-10 cells group?P>0.05?.4.Up-regulation of hsa?circ?0043415,hsa?circ?0010358 and hsa?circ?0009353expression promoted the cycle transition of B-cell lymphoma cellsThe results of flow cytometry showed that there were no significant changes in G1phase and S phase in Raji/SU-DHL-10 cells and lentivirus control Raji/SU-DHL-10 cells groups?P>0.05?,but the G1 phase proportion in lentivirus overexpressed hsa?circ?0043415,hsa?circ?0010358 and hsa?circ?0009353 Raji/SU-DHL-10 cells group were lower than those in Raji/SU-DHL-10 cells and lentivirus control Raji/SU-DHL-10cells groups?P<0.05?.The proportion of S phase was higher than that of Raji/SU-DHL-10cells and lentivirus control Raji/SU-DHL-10 cells groups?P<0.05?.5.Overexpression of hsa?circ?0043415,hsa?circ?0010358 and hsa?circ?0009353inhibited apoptosis of B-cell lymphoma cellsThe results of flow cytometry showed that there were no significant changes of apoptosis rate in Raji/SU-DHL-10 cells and lentivirus control Raji/SU-DHL-10 cells groups?P>0.05?,but the apoptosis rate in lentivirus overexpressed hsa?circ?0043415,hsa?circ?0010358 and hsa?circ?0009353 Raji/SU-DHL-10 cells group were lower than those in Raji/SU-DHL-10 cells and lentivirus control Raji/SU-DHL-10 cells groups?P<0.05?.ConclusionHsa?circ?0043415,hsa?circ?0010358 and hsa?circ?0009353 promoted the proliferation of BCL cells,promoted the transition of Raji and SU-DHL-10 cells from G1to S phase,and then reduced the apoptosis of Raji and SU-DHL-10 cells.The role of hsa?circ?0043415,hsa?circ?0010358 and hsa?circ?0009353 as oncogenes in Raji and SU-DHL-10 cells was consistent with previous results,indicating that inhibition of their expression might improve the prognosis of patients with BCL.