Objective To explore the molecular mechanism of circular RNA(circ)VAPA promoting the Hippo pathway to inhibit liver regeneration through the miR-101a-3p/TEAD3 axis.Methods A mouse model of 70％partial hepatectomy-induced liver regeneration was constructed,and the expressions of circVA-PA,miR-101a-3p,and TEAD3 were analyzed.Mouse embryonic hepatocyte BNL CL.2 cells were transfected with siRNA or overexpression plasmids and divided into the VAPA-NC group,the VAPA-NC+miR-101a-3p mimic group,the VAPA-NC+miR-101a-3p mimic-NC group,the VAPA-OE group,the VAPA-OE+miR-101a-3p mimic group and the VAPA-OE+miR-101a-3p mimic-NC group.CCK-8 method and flow cytometry were used to analyze the proliferation,apoptosis,and cell cycle changes of hepatocytes.Immunofluorescence staining was used to analyze the nuclear translocation of YAP1.Quantitative Reverse Transcription Real-Time PCR(qRT-PCR)was used to analyze the expressions of key genes in the Hippo pathway.The dual-lu-ciferase reporter gene assay was used to verify the targeting relationships between circVAPA and miR-101a-3p,as well as between miR-101a-3p and TEAD3.Results The level of circVAPA gradually increased during liver regeneration(P＜0.05),while the level of miR-101a-3p first increased and then decreased(P＜0.05).When miR-101a-3p was overexpressed alone,the cell proliferation rate was the highest(P＜0.05),but it had no effect on the cell apoptosis rate(P＞0.05).When circVAPA was overexpressed alone,it had no effect both on the cell proliferation rate and apoptosis(P＞0.05).After co-overexpression of circVAPA and miR-101a-3p,the cell proliferation rate significantly decreased,and the cell apoptosis significantly increased(P＜0.05).When miR-101a-3p was overexpressed alone,a large number of cells entered into the S phase.After co-overex-pression of circVAPA and miR-101a-3p,a large number of cells were blocked in the G2/M phase.The phos-phorylation level of the Hippo upstream gene YAP1 significantly increased at 6 hours after liver regeneration(P＜0.05)and then rapidly decreased.However,co-overexpression of circVAPA and miR-101a-3p did not af-fect the level of p-YAP1 and the nuclear translocation of YAP1(P＞0.05).The expression levels of the Hip-po downstream gene CTGF and the transcription factor TEAD3 first increased and then decreased during liver regeneration(P＜0.05),and there was no significant change in CYR61(P＞0.05).After co-overexpression of circVAPA and miR-101a-3p,the expression level of CTGF increased(P＜0.05).Knocking down or overex-pressing circVAPA did not affect the expression of TEAD3(P＞0.05),while overexpressing miR-101a-3p could significantly inhibit the expression level of TEAD3(P＜0.05).The dual-luciferase reporter gene assay confirmed the targeting relationships between circVAPA and miR-101a-3p,as well as between miR-101a-3p and TEAD3.Conclusion circVAPA promotes the Hippo pathway to inhibit liver regeneration through the miR-101a-3p/TEAD3 axis.

Objective:To explore the predictive value of peripheral blood cytokine models on organ functional impairment after chimeric antigen receptor T(CAR-T) cell therapy in children with B-lineage lymphocytic leukemia.Methods:The clinical data of 44 children with acute B-lineage lymphoblastic leukemia who received CAR-T cell therapy at Children′s Hospital of Soochow University from September 2018 to October 2020 were retrospectively analyzed.Peripheral blood cytokines, including interleukin(IL)-2, IL-4, IL-6, IL-10, tumor necrosis factor-α, interferon(IFN)-γ and IL-17A, were measured daily for 14 days after receiving CAR-T cell therapy.The trend of peripheral blood cytokine levels was analyzed at the endpoint of organ function recovery or death within 14 days after CAR-T cell treatment.Receiver operating characteristic curve was used to establish a mathematical prediction model to predict the occurrence of organ damage in the children.Results:Of the 44 children, 31 cases were boys and 13 cases were girls, with a median age of 7.96 (5.19, 11.48)years.Cytokine release syndrome(CRS) response occurred in 95.5% (42/44) children, with 88.1% (37/42) had a grade 1-3 CRS response, and 16.7% (7/42) had a severe grade 4-5 CRS response.Using IL-6>3 892.95 pg/mL as cut-off value, the area under the curve(AUC) for predicting acute respiratory failure was 0.818, with a sensitivity of 0.8 and a specificity of 0.735, while combining IFN-γ>414.4 pg/mL, IL-6>3 892.95 pg/mL and IL-2>27.05 pg/mL were the three cut-off values, with an AUC of 0.741, sensitivity of 0.6 and specificity of 0.912 for predicting acute respiratory failure. Using IFN-γ>1 699.5 pg/mL as cut-off value, the AUC for predicting shock was 0.908, with a sensitivity of 0.722 and a specificity of 1.With IL-6>4 607.3 pg/mL as cut-off value, the AUC for predicting liver injury was 0.964, with a sensitivity of 1 and a specificity of 0.906, while combining both IL-6>4 607.3 pg/mL and IFN-γ>1 446.2 pg/mL as cut-off values, the AUC for predicting liver injury was 0.977, with a sensitivity of 1 and a specificity of 0.906.Combining both IL-6>6 972.2 pg/mL and IFN-γ>3 981.5 pg/mL predicted a positive predictive value of 62.5% and a negative predictive value of 94.4% for grade 4-5 CRS response, with an AUC of 0.846, a predictive sensitivity of 0.714 and a specificity of 0.838, and all children had a combination of two or more organ function injuries.Conclusion:The combination of IL-6 and IFN-γ can effectively predict the incidence of liver injury and cytokine release syndrome.The combination of peripheral blood cytokines IFN-γ, IL-6 and IL-2 can be used to predict the incidence of acute respiratory failure after the treatment of CAR-T cells in children with acute B-lineage lymphoblastic leukaemia.IFN-γ single index can be used to predict the incidence of shock.The combination of IL-6 and IFN-γ can be used to predict the incidence of liver injury and the severity of CRS.