ABSTRACT
Objective To investigate the effect of cyclin D1 (with CCND1 as the gene name) on HBV replication and its potential mechanism. Methods With reference to GSE84044 dataset, the Spearman's rank correlation analysis was used to investigate the correlation between the expression levels of genes in liver tissue and serum HBV DNA load in patients with HBV-related liver fibrosis. Cyclin D1 and cyclin D1 T286A mutant were transiently expressed in the HBV cell replication model, and time-resolved immunofluorescence and quantitative real-time PCR were used to measure the levels of HBsAg/HBeAg and HBV DNA in cell culture supernatant; Western blot was used to measure the level of HBV core protein in cells; reverse-transcription quantitative real-time PCR was used to measure the level of HBV RNA in cells; dual-luciferase reporter assay was used to observe the effect of cyclin D1 on the activity of HBV basic core promoter (BCP). GSE83148 dataset was used to investigate the correlation between CCND1 and HBV-related regulatory factors. The independent samples t -test was used for comparison of normally distributed continuous data between two groups, and the Mann-Whitney U test was used for comparison of non-normally distributed continuous data between two groups. Results The analysis of GSE84044 data showed that 7 cell cycle genes were significantly negatively correlated with HBV DNA load in liver tissue of the patients with HBV-related liver fibrosis (all r < -0.3, all P < 0.05), which included the CCND1 gene ( r =-0.474, P < 0.001). Exogenous expression of cyclin D1 and cyclin D1 T286A mutant reduced the levels of HBsAg, HBeAg, and HBV DNA in culture supernatant of the HBV replication cell model, as well as the levels of HBV core protein and HBV RNA in cells. Exogenous expression of cyclin D1 significantly inhibited the transcriptional activity of HBV BCP. The expression level of CCND1 in liver tissue of chronic hepatitis B patients was significantly positively correlated with the expression of APOBEC3G ( r =0.575, P < 0.001), SMC5 ( r =0.341, P < 0.001), and FOXM1 ( r =0.333, P < 0.001) which inhibited HBV replication, while it was significantly negatively correlated with the expression of the HBV entry receptor NTCP ( r =-0.511, P < 0.001) and HNF1α as the transcription factor for positive regulation of HBV replication ( r =-0.430, P < 0.001). Overexpression of cyclin D1 in HepG2 cells reduced the transcriptional levels of HNF1α and NTCP. Conclusion Cyclin D1 inhibits HBV transcription and replication possibly by downregulating the expression of HNF1α and NTCP.
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Objective@#To explore and analyze the possible mechanism of liver injury in patients with coronavirus disease 2019 (novel coronavirus pneumonia, NCP).@*Methods@#The correlation between ALT, AST and other liver enzyme changes condition and NCP patients’ disease status reported in the literature was comprehensively analyzed. ACE2 expression in liver tissue for novel coronavirus was analyzed based on single cell sequencing (GSE115469) data. RNA-Seq method was used to analyze Ace2 expression and transcription factors related to its expression in liver tissues at various time-points after hepatectomy in mouse model of acute liver injury with partial hepatectomy. t-test or Spearman rank correlation analysis was used for statistical analysis.@*Results@#ALT and AST were abnormally elevated in some patients with novel coronavirus infection, and the rate and extent of ALT and AST elevation in severe NCP patients were higher than those in non-severe patients. Liver tissue results of single cell sequencing and immunohistochemistry showed that ACE2 was only expressed in bile duct epithelial cells of normal liver tissues, and very low in hepatocytes. In a mouse model of acute liver injury with partial hepatectomy, Ace2 expression was down-regulated on the first day, but it was elevated up to twice of the normal level on the third day, and returned to normal level on seventh day when the liver recovered and hepatocyte proliferation stopped. Whether this phenomenon suggests that the bile duct epithelial cells with positive expression of Ace2 participate in the process of liver regeneration after partial hepatectomy deserves further study. In RNA-Seq data, 77 transcription factors were positively correlated with the expression of ACE2 (r > 0.2, FDR < 0.05), which were mainly enriched in the development, differentiation, morphogenesis and cell proliferation of glandular epithelial cells.@*Conclusion@#We assumed that in addition to the over activated inflammatory response in patients with NCP, the up-regulation of ACE2 expression in liver tissue caused by compensatory proliferation of hepatocytes derived from bile duct epithelial cells may also be the possible mechanism of liver tissue injury caused by 2019 novel coronavirus infection.
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The protocorm-suspension-system of Cremastra appendiculata was founded by liquid-suspension culture. The factors to effect germination rate and seedling conversion rate of C. appendiculata synthetic seeds, such as synthetic coating materials, synthetic endosperm components, storing conditions and germination materials, etc. were studied. The result showed that the germination rate and seedling conversion rate of synthetic seeds were the highest on the MS solid-medium while using 4% sodium alginate + 2% CaCl2 + 2% chitosan as synthetic coating materials, with 1/2 MS liquid-medium + 0.2 mg x L(-1) NAA + 0.1 mg x L(-1) GA3 + 0.5 mg L(-1) BA + 0.4 mg x L(-1) penicillin + 10.0 mg x L(-1) endophyte extract +0.3% carbendazim powder + 0.2% sodium benzoate + 1.0% sucrose as synthetic endosperm. And the germination rate and seedling conversion rate of synthetic seeds could attain to 68% and 65% after 20 days storing at 4 degrees C. The germination rate and seedling conversion rate of synthetic seeds decreased to a great extent with increasing the storing temperature and prolonging storing time.