OBJECTIVETo investigate the expression of microRNA-128a (miR-128a) and its role in hepatocellular carcinoma (HCC).

METHODSNineteen pairs of fresh surgical specimens of HCC and adjacent tissues were examined for miR-128a expression using qRT-PCR. A miR-128a mimics or inhibitor was transfected into HCC cells, and the cell viability was analyzed by MTT assay. RND3, one of the potential targets of miR-128a, was predicted by bioinformatics software and demonstrated by dual luciferase reporter assay. The expression of RND3 after transfection was detected using qRT-PCR and Western blotting, and the cell cycle-related proteins were determined with Western blotting.

RESULTSThe expression of miR-128a were significantly up-regulated in HCC tissues as compared to the adjacent tissues (P<0.05). In cultured HCC cells, miR-128a promoted the cell proliferation and resulted in down-regulated RND3 mRNA and protein expressions by targeting RND3' 3'UTR (P<0.05) and also in the down-regulation of cyclin B1, cyclin D1 and CDK4 protein expressions.

CONCLUSIONmiR-128a is up-regulated in HCC and promotes HCC cell proliferation by targeting RND3.

Carcinoma, Hepatocellular ; metabolism ; Cell Proliferation ; Cell Survival ; Down-Regulation ; Humans ; Liver Neoplasms ; metabolism ; MicroRNAs ; genetics ; metabolism ; RNA, Messenger ; Transcriptional Activation ; Tumor Cells, Cultured ; Up-Regulation ; rho GTP-Binding Proteins ; metabolism

NESCA, a newly discovered signaling adapter protein in the NGF-pathway, contains a RUN domain at its N-terminus. Here we report the crystal structure of the NESCA RUN domain determined at 2.0-Å resolution. The overall fold of the NESCA RUN domain comprises nine helices, resembling the RUN domain of RPIPx and the RUN1 domain of Rab6IP1. However, compared to the other RUN domains, the RUN domain of NESCA has significantly different surface electrostatic distributions at the putative GTPase-interacting interface. We demonstrate that the RUN domain of NESCA can bind H-Ras, a downstream signaling molecule of TrkA, with high affinity. Moreover, NESCA RUN can directly interact with TrkA. These results provide new insights into how NESCA participates in the NGF-TrkA signaling pathway.
Adaptor Proteins, Signal Transducing ; chemistry ; genetics ; metabolism ; Amino Acid Sequence ; Binding Sites ; Crystallography, X-Ray ; Gene Expression ; Humans ; Models, Molecular ; Molecular Sequence Data ; Nerve Growth Factor ; chemistry ; genetics ; metabolism ; Oncogene Protein p21(ras) ; chemistry ; genetics ; metabolism ; Protein Binding ; Protein Structure, Tertiary ; Receptor, trkA ; chemistry ; genetics ; metabolism ; Recombinant Proteins ; chemistry ; genetics ; metabolism ; Sequence Homology, Amino Acid ; Signal Transduction ; rab GTP-Binding Proteins ; chemistry

Objective To investigate the effect of Danggui Shaoyao powder on apoptosis of cochlear spiral ganglion cells of age-related hearing loss mice by regulating the inhibitor of kappa B kinase(IKK)/inhibitory subunit of nuclear factor-kappa B alpha(IκBα)/nuclear factor-kappa B(NF-κB)pathway.Methods A total of 48 C57BL/6J mice were randomly divided into age-related hearing loss model group(model group),low-dose Danggui Shaoyao powder group(traditional Chinese medicine-L group,3.2 g/kg crude drug),high-dose Danggui Shaoyao powder group(traditional Chinese medicine-H group,6.4 g/kg crude drug),and high-dose Danggui Shaoyao powder+IKK/IκBα/NF-κB signaling pathway activator resveratrol(RES)group(tradi-tional Chinese medicine-H+RES group,6.4 g/kg crude drug+43.33 mg/kg RES),with 12 mice in each group.Twelve 2-month-old C57BL/6J mice were selected as the control group.Auditory brain stem response(ABR)was used to detect the audi-tory threshold of mice in each group.HE staining was used to observe the pathological changes of spiral ganglions of the mouse cochlea,and the number of spiral ganglion cells in the middle and bottom gyrus of the mouse cochlea was counted.The apoptosis of spiral ganglion cells in mouse cochlea was detected by TUNEL staining.The levels of malondialdehyde(MDA)and superoxide dismutase(SOD)in mouse cochlear tissue were detected by ELISA.The mRNA levels of IL-1β,IL-6 and TNF-α in mouse co-chlear tissues were detected by qRT-PCR.Western blot was used to detect the expression of IKK/IκBα/NF-κB pathway and ap-optosis related proteins in mouse cochlear tissue.Results Compared with the control group,the auditory thresholds at 8 kHz,16 kHz,24 kHz,and 32 kHz,the number of TUNEL positive cells in the cochlear spiral ganglion tissue,the level of MDA,and the mRNA level of IL-1β,IL-6 and TNF-α in the cochlear tissue,the expression of p-IκBα,apoptosis protein(Bax,and cleaved-Caspase-3),and the phosphorylation levels of IKK and NF-κB p65 in the model group were significantly increased(all P＜0.05),the number of cochlear spiral ganglion cells,the level of SOD,and the expression of IκBα protein were significantly re-duced(all P＜0.05),the morphology of cochlear spiral ganglion cells was abnormal,with loose and disordered arrangement,re-sulting in vacuolization and pathological damage.The changes in corresponding indicators of mice in the traditional Chinese med-icine-L group and traditional Chinese medicine-H group were opposite to those in the model group(all P＜0.05).RES alleviated the inhibitory effect of Danggui Shaoyao powder on the apoptosis of cochlear spiral ganglion cells in age-related hearing loss mice.Conclusion Danggui Shaoyao powder may inhibit apoptosis of cochlear spiral ganglion cells of age-related hearing loss mice by downregulating the IKK/IκBα/NF-κB pathway.

Objective To investigate the effect of overexpression of ubiquitin-conjugating enzyme 2T(UBE2T)on radiosensitivity of hepatocellular carcinoma(HCC).Methods Hepa1-6 cells were transfected with a UBE2T-overexpressing or a control lentiviral vector,and the changes in their radiotherapy sensitivity and concentrations of glucose and lactate in the supernatant were assessed using colony-forming assay and colorimetric assay.The transfected cells were inoculated subcutaneously in nude mice or C57BL/6 mice,and tumor growth following irradiation were recorded.The xenografts were collected for analyzing infiltration of CD4+T cells and regulatory T cells(Tregs)using flow cytometry and detecting expressions of HK1 and LDHA using Western blotting.The correlations of UBE2T expression with immune cell infiltration,glycolysis and Tregs in HCC were analyzed using CIBERSORT algorithm and TCGA database,and the results were verified in a co-culture system of Hepa1-6 cells and Tregs.Results UBE2T overexpression caused radiotherapy resistance in both cultured Hepa1-6 cells and xenografts in the tumor-bearing mouse models(especially in C57BL/6 mice).CIBERSORT analysis suggested that a high expression of UBE2T was associated with increased percentages of dendritic cells,T follicular helper cells,M2 macrophages,monocytes,lymphocytes and Tregs in HCC.The UBE2T-overexpressing xenografts showed an increased percentage of Tregs and enhanced expressions of HK1 and LDHA,and irradiation increased infiltration of CD4+T cells and Tregs in the tumor microenvironment.Hepa1-6 cells overexpressing UBE2T showed a decreased glucose concentration and an increased lactate concentration.GSEA analysis suggested that a high UBE2T expression was positively correlated with increased glycolysis and Tregs infiltration in HCC.In the cell co-culture system,UBE2T overexpression significantly enhanced lactate production,proliferation and immunosuppressive functions of Tregs.Conclusion A high UBE2T expression results in radiotherapy resistance of HCC possibly by enhancing glycolysis and cause enrichment of Tregs in the tumor microenvironment.

Objective To investigate the effect of overexpression of ubiquitin-conjugating enzyme 2T(UBE2T)on radiosensitivity of hepatocellular carcinoma(HCC).Methods Hepa1-6 cells were transfected with a UBE2T-overexpressing or a control lentiviral vector,and the changes in their radiotherapy sensitivity and concentrations of glucose and lactate in the supernatant were assessed using colony-forming assay and colorimetric assay.The transfected cells were inoculated subcutaneously in nude mice or C57BL/6 mice,and tumor growth following irradiation were recorded.The xenografts were collected for analyzing infiltration of CD4+T cells and regulatory T cells(Tregs)using flow cytometry and detecting expressions of HK1 and LDHA using Western blotting.The correlations of UBE2T expression with immune cell infiltration,glycolysis and Tregs in HCC were analyzed using CIBERSORT algorithm and TCGA database,and the results were verified in a co-culture system of Hepa1-6 cells and Tregs.Results UBE2T overexpression caused radiotherapy resistance in both cultured Hepa1-6 cells and xenografts in the tumor-bearing mouse models(especially in C57BL/6 mice).CIBERSORT analysis suggested that a high expression of UBE2T was associated with increased percentages of dendritic cells,T follicular helper cells,M2 macrophages,monocytes,lymphocytes and Tregs in HCC.The UBE2T-overexpressing xenografts showed an increased percentage of Tregs and enhanced expressions of HK1 and LDHA,and irradiation increased infiltration of CD4+T cells and Tregs in the tumor microenvironment.Hepa1-6 cells overexpressing UBE2T showed a decreased glucose concentration and an increased lactate concentration.GSEA analysis suggested that a high UBE2T expression was positively correlated with increased glycolysis and Tregs infiltration in HCC.In the cell co-culture system,UBE2T overexpression significantly enhanced lactate production,proliferation and immunosuppressive functions of Tregs.Conclusion A high UBE2T expression results in radiotherapy resistance of HCC possibly by enhancing glycolysis and cause enrichment of Tregs in the tumor microenvironment.