RESUMO
OBJECTIVE@#To explore the role of small nuclear noncoding RNA 7SK in embryonic stem cell (ESCs) proliferation and the value of 7SK as a target for early diagnosis and treatment for primordial dwarfism (PD).@*METHODS@#ESC line R1 was transfected with the CRISPR/Cas9 system, and sequencing of the PCR product and glycerol gradient analysis were performed to identify novel 7SK deletion mutations. A lentivirus system was used to knock down cyclin-dependent kinase 9 (CDK9) in clones with 7SK deletion mutations, and the effect of CDK9 knockdown on the protein level of cell division cycle 6 (CDC6) was analyzed with Western blotting.@*RESULTS@#We identified a novel deletion mutation of 7SK at 128-179 nt in the ESCs, which resulted in deficiency of cell proliferation. 7SK truncation at 128-179 nt significantly reduced the protein expressions of La-related protein 7 (LARP7) and CDC6.@*CONCLUSIONS@#7SK truncation at 128-179 nt can significantly impair proliferation of ESCs by downregulating CDC6. 7SK is a key regulator of proliferation and mediates the growth of ESCs through a mechanism dependent on CDK9 activity, suggesting the value of 7SK truncation at 128-179 nt as a potential target for early diagnosis and treatment of PD.
Assuntos
Humanos , Proteínas de Ciclo Celular , Proliferação de Células , Células-Tronco Embrionárias/metabolismo , Células HeLa , Proteínas Nucleares , Fator B de Elongação Transcricional Positiva/metabolismo , RNA Longo não Codificante/genética , Proteínas de Ligação a RNA , Ribonucleoproteínas , Fatores de TranscriçãoRESUMO
Objective To investigate the role of cyclin-dependent kinase 9 ( CDK9) in regulating lytic replication of Kaposi′s sarcoma-associated herpesvirus ( KSHV) .Methods Percentages of red fluores-cent protein (RFP) positive cells were counted after treating and stimulating iSLK .219 cells with FIT-039, a CDK9 specific inhibitor , and doxycycline ( Dox ) , respectively , or transfecting and stimulating them with CDK9 siRNA (si-CDK9) and Dox, respectively.Quantitative real-time PCR was used to detect the expres-sion of KSHV genes at mRNA level in TREx-K-Rta BCBL-1 cells treated with FIT-039 or transfected with si-CKD9 in the presence of Dox.Chromatin immunoprecipitation (ChIP) assay was used to examine the enrich-ment of RNA polymerase Ⅱ( RNA PolⅡ) and phosphorylated CTD-S2 of RNA PolⅡon the PAN promot-er in TREx-K-Rta BCBL-1 cells treated with FIT-039.Results Both FIT-039 intervention and CDK9 knockdown dramatically deceased the percentage of RFP-positive iSLK.219 cells and suppressed the expres-sion of ORF50, PAN and K2 in TREx-K-Rta BCBL-1 cells at mRNA level.Furthermore, FIT-039 signifi-cantly inhibited the enrichment of RNA Pol Ⅱand phosphorylated CTD-S2 of RNA Pol Ⅱon the PAN pro-moter in TREx-K-Rta BCBL-1 cells.Conclusion CDK9 is involved in regulating the lytic replication of KSHV through promoting the phosphorylation of CTD-S2 of RNA Pol Ⅱ.
RESUMO
AIM To investigate cyclin dependent kinase 9(Cdk9) and cyclin T1 protein expressions in diabetic rat kidney and the effect of berberine on them. METHODS Type 2 diabetes mellitus (T2DM) rats were induced by injection (ip) with diet for 16 weeks. From week 17 to 32, diabetic rats were given berberine 75, respectively. The kidney tissue structure was observed with hematoxylin/eosin (HE) staining, kidney to body weight ratio was calculated, and Cdk9 and cyclin T1 expressions were examined by immunohistochemistry. RESULTS Compared with control rats, the volume of diabetic model rat glomerulus accreted, some intercapillary cells proliferated and mesangial region expanded, both glomerular basement membrane and renal tubular basement membrane thickened. Treatment with diabetic nephropathy symptom. The diabetic kidney to body weight ratio protein expressions in diabetic kidney to near control level. CONCLUSION Berberine regulates Cdk9 and cyclin T1 protein expressions in diabetic kidney which may partly contribute to ameliorate nephropathy complication induced by STZ and the high-carbohydrate/high-fat diet.