Objective:To study the role and possible molecular mechanism of neural precursor cell-expressed developmentally down-regulated gene 4-like ( NEDD4 L) in the replication of hepatitis B virus (HBV). Methods:Small interfering RNA (siRNA) targeting NEDD4 L, plasmid expressing NEDD4 L with hemagglutinin(HA) C-terminal tag (pcDNA3.1- NEDD4 L-HA), plasmid expressing 1.3×HBV genome (pGEM-HBV1.3) and poly (dAT: dAT) were respectively transfected into HepG2 cells using Lipofectamine2000. HepG2.2.15 cells, a cell line that can stably express HBV, were used as control. The mRNA levels of NEDD4 L, interferon (IFN)-α, IFN-β, interferon-stimulated gene 56 ( ISG56), myxovirus resistance protein A ( MxA), oligoadenylate synthetase ( OAS), and the levels of HBV DNA or 3.5 kb HBV RNA were detected by real-time fluorescent quantitative polymerase chain reaction (qRT-PCR). Western blot was used to detect the silence and over-expression of NEDD4 L, and the protein levels of the related signaling molecules. The amount of IFN-β in the cellular supernatant was measured by enzyme linked immunosorbent assay (ELISA). Student t test was used for comparison of continuous data between groups. Results:The levels of NEDD4L mRNA and protein in HepG2.2.15 cells were 10.53±0.47 and 4.17±0.43, respectively, which were both statistically higher than those in HepG2 cells (1.00±0.05, t=3.27, P=0.008 and 1.26±0.25, t=1.68, P=0.030, respectively). In HepG2 cells with knockdown of NEDD4 L, the expression level of HBV DNA in cellular supernatant was 0.32±0.09, which was statistically lower than that in the control (1.00±0.05, t=-0.93, P=0.020), and the expression level of 3.5 kb HBV RNA was 0.49±0.11, which was statistically lower than that in the control (1.00±0.05, t=-0.68, P=0.040), while the mRNA levels of IFN-β and downstream effector molecules ( ISG56, MxA and OAS) were all significantly increased compared with the control ( t=4.66, 9.38, 7.29 and 7.01, respectively, all P<0.01). With poly (dAT: dAT) treatment and vesicular stomatitis virus (VSV) stimulation, the levels of IFN-β in HepG2 cells with knockdown of NEDD4 L were (776.41±115.49) ng/L and (961.21±130.19) ng/L, respectively, which were both statistically higher than those of the control group ((320.15± 56.05) ng/L, t=2.43, P=0.020; (440.17±67.82) ng/L, t=2.85, P=0.030, respectively). With poly (dAT: dAT) treatment and VSV stimulation, the levels of IFN-β in HepG2 cells with overexpression of NEDD4 L were (156.18±26.47) ng/L and (176.67±34.51) ng/L, respectively, which were both statistically lower than those of the control group ((320.38±49.39) ng/L, t=-2.03, P=0.040; (440.59±68.83) ng/L, t=-1.93, P=0.030, respectively). Western blot showed that the replication of HBV reduced the protein level of melanoma differentiation-associated protein 5 (MDA5), a key molecule in upstream of IFN-β, but the down-regulation was not obvious in cells with the knockdown of NEDD4 L. Conclusion:The replication of HBV could promote the up-regulation of NEDD4L protein and subsequently reduce the protein level of MDA5, thereby inhibiting the production of IFN-β, which facilitates HBV to escape the innate immune response.

Objective·To investigate the composition of immune cells in tumor microenvironment of colorectal cancer(CRC),and examine the proportion,phenotype and effector function of natural killer(NK)cells in CRC.Methods·Fresh tumor tissues,paired normal tissues adjacent to tumor,and peripheral blood samples in the same cohort were collected from CRC patients.Tissues were digested and prepared into single cell suspension.The major immune cell lineages were detected by flow cytometry.t-Distributed stochastic neighbor embedding(t-SNE)and statistical analysis were used to analyze the composition of immune cells in tumor microenvironment of CRC.To analyze the phenotype of NK cells,the expression levels of activation markers,including CD16,CD27,CD69,human leukocyte antigen-DR(HLA-DR),and T cell immunoglobulin domain and mucin domain-3(TIM-3),were detected by flow cytometry.NK cell subsets:CD38lowNK cells and CD38highNK cells were also examined by flow cytometry.To assess the effector function of NK cells,they were stimulated with cell stimulation cocktail and the expression levels of interferon-γ(IFN-y),tumor necrosis factor-α(TNF-α),and granulocyte-macrophage colony-stimulating factor(GM-CSF)were measured by flow cytometry.Results·Twenty-five pairs of fresh tumor tissues and normal tissues adjacent to tumor,and 15 peripheral blood samples in the same cohort were collected from CRC patients.The tumor microenvironment of CRC included diverse immune cell types,including T cells,B cells,NK cells and myeloid cells.The proportions of T cells(P=0.000)and myeloid cells(P=0.026)in tumor tissues were significantly higher than those in normal tissues.By contrast,the proportion of NK cells(P=0.007)in tumor was significantly reduced.The proportion of B cells was comparable between tumor and normal tissues.Compared to normal tissues,NK cells in tumor tissues expressed significantly lower CD27(P=0.000)and CD69(P=0.001),while the expression levels of CD16(P=0.008),HLA-DR(P=0.000)and TIM-3(P=0.024)were significantly elevated.The results indicated that NK cells in CRC tumor exhibited a phenotype of late activation and exhaustion.According to the expression level of CD38,NK cells could be divided into two subsets,CD38highNK cells and CD38lowNK cells.The proportion of CD38highNK cells(P=0.003)in tumor tissues was significantly lower than that in normal tissues,while the proportion of CD38lowNK cells was unaffected.Compared to CD38lowNK cells,CD38highNK cells expressed higher CD27,meanwhile significantly less CD16,NKp46,CD57,CD94,HLA-DR and CD158a(P=0.000).These results suggested that CD38highNK cells were at early differentiation state.The secretion of cytokines IFN-y(P=0.032),TNF-α(P=0.042),and GM-CSF(P=0.019)by tumor-infiltrated NK cells was significantly decreased compared to that in normal tissues.The results showed that the function of tumor-infiltrated NK cells was impaired.Conclusion·Together,these data suggest that NK cell compartment is disrupted in tumor tissues of CRC,leading to the impaired anti-tumor immunity mediated by NK cells.