Advances and challenges of exosome-derived noncoding RNAs for hepatocellular carcinoma diagnosis and treatment.

Exosomes, also termed extracellular vesicles (EVs), are an important component of the tumor microenvironment (TME) and exert versatile effects on the molecular communications in the TME of hepatocellular carcinoma (HCC). Exosome-mediated intercellular communication is closely associated with the tumorigenesis and development of HCC. Exosomes can be extracted through ultracentrifugation and size exclusion, followed by molecular analysis through sequencing. Increasing studies have confirmed the important roles of exosome-derived ncRNAs in HCC, including tumorigenesis, progression, immune escape, and treatment resistance. Due to the protective membrane structure of exosomes, the ncRNAs carried by exosomes can evade degradation by enzymes in body fluids and maintain good expression stability. Thus, exosome-derived ncRNAs are highly suitable as biomarkers for the diagnosis and prognostic prediction of HCC, such as exosomal miR-21-5p, miR-221-3p and lncRNA-ATB. In addition, substantial studies revealed that the up-or down-regulation of exosome-derived ncRNAs had an important impact on HCC progression and response to treatment. Exosomal biomarkers, such as miR-23a, lncRNA DLX6-AS1, miR-21-5p, lncRNA TUC339, lncRNA HMMR-AS1 and hsa_circ_0004658, can reshape immune microenvironment by regulating M2-type macrophage polarization and then promote HCC development. Therefore, by controlling exosome biogenesis and modulating exosomal ncRNA levels, HCC may be inhibited or eliminated. In this current review, we summarized the recent findings on the role of exosomes in HCC progression and analyzed the relationship between exosome-derived ncRNAs and HCC diagnosis and treatment.

[Study on the relationship between hepatitis B virus genotypes and the effect of polyethylene glycol-interferon-alpha therapy on HBeAg-positive chronic hepatitis B].

OBJECTIVE: To investigate the efficacy of polyethylene glycol (PEG)-interferon α (PEG-IFNα) in treating HBeAg-positive chronic hepatitis B (CHB) and explore the relationship between hepatitis B virus (HBV) genotypes and the effect of interferon α (IFNα) therapy. METHODS: A total of 199 CHB patients with known genotypes were given subcutaneous injection of PEG-IFNα-2a or PEG-IFNα-2b once a week for 48 weeks, with another 24 weeks follow up. The seroconversion of HBeAg influenced by HBV genotypes were analyzed after discontinuation of treatment. RESULTS: In local area, genotype C was the major genotype [64.32% (128/199) ]. Except serum ALT and AST level, the differences in gender, age, liver inflammation, degree of liver fibrosis, HBeAg level and HBV DNA level between genotype B and C were not statistically significant (all P > 0.05). The seroconversion rate of HBeAg in patients with genotype B at early stage of therapy (3 months) was significantly higher than that of patients with genotype C [26.76% (19/71) vs 10.16% (13/128), χ(2) = 9.330, P = 0.002]. While at the end of follow-up, seroconversion rate of HBeAg in patients with genotype B (followed up for 6 months) was higher than that of patients with genotype C [39.44% (28/71) vs 30.47% (39/128)], but the difference was not statistically significant (χ(2) = 1.645, P = 0.200). By univariate analysis based on log-rank test, the time of HBeAg seroconversion in patients with genotype B was much earlier than that of genotype C [(13.99 ± 0.67) months vs (15.47 ± 0.41) months], but the difference was not statistically significant (P = 0.150). CONCLUSIONS: The seroconversion rate of HBeAg in patients with genotype B treated with PEG-IFNα was significantly higher than that of genotype C in early stage of therapy (3 months), while similar at the end of therapy.

[Liver histopathological features influencing HBeAg seroconversion in patients with HBeAg-positive chronic hepatitis B responding to Peg-IFN treatment].

OBJECTIVE: To investigate the therapeutic efficiency of antiviral treatment with pegylated-interferon (Peg-IFN) for hepatitis B e antigen (HBeAg)-positive chronic hepatitis B (CHB) and to explore whether liver histopathological features or other factors influence the HBeAg seroconversion treatment response. METHODS: Eighty HBeAg-positive CHB patients with diagnosis confirmed by liver puncture were treated with Peg-IFN(2a or 2b)body weight dose, once weekly). At treatment week 48, the rate of HBeAg seroconversion was determined and used to analyze the influence of liver histopathological features (liver biopsy assessment of: inflammation, graded G0 to G4; fibrosis stage, graded S0 to S4), sex, age, differential levels (pre-treatment baseline vs. week 48 post-treatment) of serum alanine transferase (ALT), and HBV DNA, by binary logistic analysis. RESULTS: At week 48, the overall rate of HBeAg seroconversion was 30.0%. The rate of HBeAg seroconversion gradually advanced with increased liver inflammation (X2 = 8.435, P = 0.015): 9.09% of the 22 patients with G1; 31.58% of the 38 patients with G2; 47.30% of the 19 patients with G3; the one patient with G4. In contrast, the rate of HBeAg seroconversion showed a much weaker association with liver fibrosis (X2 = 5.917, P = 0.116). Only baseline HBeAg level, and no other baseline index, was significantly different between the patients who achieved HBeAg seroconversion and those who did not. Liver inflammation and baseline HBeAg level were identified as influencing factors of HbeAg seroconversion in response to Peg-IFN treatment. CONCLUSION: Peg-IFN therapy induces a higher rate of HBeAg seroconversion in HBeAg-positive CHB patients with severe liver inflammation; histological analysis of pre-treatment liver biopsies may help to identify patients most likely to benefit from the antiviral regimen.

HBV inhibits apoB production via the suppression of MTP expression.

BACKGROUND: Liver dominates the production and secretion of apolipoprotein B (apoB) and evidence shows that liver malfunction induced by hepatitis B virus (HBV) infection could lead to apolipoprotein metabolism disorders. The present study was undertaken to assess the effects of HBV on apoB expression. METHODS: Clinical examination: serum apoB levels in patients with chronic HBV infection and in healthy individuals were measured by immunoturbidimetry using biochemical analyzer Olympus 5400. Cell study: mRNA and protein expression levels of apoB in HepG2 and HepG2.2.15 cells were measured by RT-PCR and Western blot. Alternatively, HBV infectious clone pHBV1.3 or control plasmid pBlue-ks were tranfected into HepG2 cells, and mRNA and protein expression levels of apoB, as well as the microsomal triglyceride transfer protein (MTP) in tranfected HepG2 cells were also measured by RT-PCR and western blot. RESULTS: Serum apoB level was much lower in chronic HBV patients as compared to healthy individuals (P < 0.05). Expression of apoB mRNA and protein was lower in HepG2.2.15 cells than in HepG2 cells. Similarly, expression of apoB mRNA and protein was lower in pHBV1.3 transfected HepG2 cells than in pBlue-ks transfected HepG2 cells. Expression of MTP mRNA and protein in pHBV1.3 transfected HepG2 cells was reduced in a dose-dependent fashion. CONCLUSION: HBV infection plays an inhibitory effect on apoB expression.

[Study of the effect and mechanism of spastic paraplegia 21 protein on the replication of hepatitis B virus].

OBJECTIVE: To study the effect of human spastic paraplegia 21 protein (SPG21) on the replication of hepatitis B virus(HBV) and its regulatory mechanism. METHODS: HBV infectious clone pHBV1.3 and its promoter pHBV-Luc were transfected respectively into HepG2 cells with SPG21 of different concentrations, HBsAg and HBeAg in the supernatants were measured by enzyme linked immunosorbent assay (ELISA), expression of HBV core mRNA and protein were detected by RT-PCR and western blot, covalently closed circular DNA(ccc DNA) levels were measured by real-time PCR, and HBV promoter activity was measured by luminometer fluorescence detector. RESULTS: Expression of HBsAg, HBeAg, HBV core protein and cccDNA were upregulated by SPG21 as well as HBV promoter activity in a dose-dependent approach. The activity of HBV promoter increased to 1.63, 3.09 and 4.66 times in HepG2 cells treated with 50mug/ml, 100mug/ml and 200mug/ml SPG21 respectively during 48 hour-treated ( P less than 0.05), as compared to the control group. CONCLUSIONS: SPG21 can enhance the replication of HBV in HepG2 cells.

Combination of oridonin and TRAIL induces apoptosis in uveal melanoma cells by upregulating DR5.

AIM: To investigate the inhibitory effect of the combined use of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and oridonin on choroidal melanoma cell lines, and to explore its underlying mechanism. METHODS: MUM-2B and C918 cells were treated with different concentrations of TRAIL and oridonin, and MTT assay used to evaluate the inhibition rate of the two compounds on cells. Then, the cell cycle distribution and apoptosis were detected by flow cytometry, and changes in apoptosis-related proteins such as death receptor 5 (DR5), a-caspase-3, and x-linked inhibitor of apoptosis protein (XIAP) were detected by Western blot. MUM-2B cells were transfected with si-DR5, which interfered with the expression of the DR5 gene. MTT and Western blot assay were used to detect cell activity and apoptosis-related proteins. RESULTS: When TRAIL and oridonin were simultaneously administered to the MUM-2B cells, the apoptosis rate was significantly higher than that by the two drugs individually. However, the effect of combined use of TRAIL and oridonin on C918 cells was not significantly different from that used alone. Cell cycle analysis showed that TRAIL and oridonin could induce G2/M arrest in MUM-2B cells. The Western blot results showed that the protein expression levels of the DR5, a-caspase-3, and BAX increased, while the expression levels of the anti-apoptosis-related proteins XIAP and BCL-2 were suppressed when TRAIL and oridonin simultaneously administered to MUM-2B cells. Interfering the expression of DR5 gene in MUM-2B cells could reverse the inhibitory effect of oridonin and TRAIL on the proliferation and apoptosis induction of MUM-2B cells. CONCLUSION: The inhibitory effects of oridonin and TRAIL on MUM-2B cells are significantly enhanced when they were administered as a combined treatment, which may ascribe to up-regulation of DR5.

Interleukin-28B Polymorphisms Predict the Efficacy of Peginterferon Alpha in Patients With Chronic Hepatitis B: A Meta-Analysis.

Background: Polyethylene glycol interferon alpha (PEG-IFN-α) is the most frequently used pharmacotherapeutic approach in patients infected with hepatitis B virus (HBV). Numerous studies have reported that interleukin-28B (IL-28B) genetic polymorphisms are related to the therapeutic efficacy of PEG-IFN-α, but the results are inconsistent. The present meta-analysis aimed to analyze the association between IL-28B genetic polymorphisms and the prognosis of patients with chronic hepatitis B (CHB) treated with PEG-IFN-α to inform clinical practice. Methods: PubMed, EBSCO, and Scopus databases were searched for relevant literature published before February 30, 2021. We calculated the crude odds ratios (ORs) with 95% confidence intervals (CIs) of the cited articles. A total of 2510 patients with CHB treated with PEG-IFN-α in 13 clinical cohort studies were analyzed. Results: The overall analysis demonstrated a potential association between IL-28B genetic polymorphisms and response to PEG-IFN-α; however, the association was not statistically significant. Furthermore, the subgroup analysis revealed that among patients with HBeAg-negative CHB, the rs12979860 CC genotype and rs8099917 TT genotype were associated with more significant treatment response to PEG-IFN-α (CC vs. non-CC: OR 2.78, 95% CI 1.00-7.76, I 2 = 83%; TT vs. non-TT: OR 2.16, 95% CI 1.35-3.48, I 2 = 0%). Among Asian patients with CHB, the rs12979860 CC genotype was associated with a more significant treatment response to PEG-IFN (CC vs. non-CC: OR 1.88, 95% CI 1.18-2.99, I 2 = 0%). Conclusion: This meta-analysis revealed that the IL-28B rs12979860 CC genotype and rs8099917 TT genotype indicated a better treatment response than non-CC and non-TT genotypes for PEG-IFN-α in patients with CHB.

[Effect of hepatitis B virus X gene on the expression of spastic paraplegia 21].

OBJECTIVE: To investigate the effect of hepatitis B virus(HBV) X gene on the expression of SPG21. METHODS: The expressions of SPG21 mRNA and protein in HepG2 and HepG2.2.15 cells were tested by RT-PCR and western blot. HepG2 cells were co-transfected with reporter plasmid pGL3-SPG21 and plasmids carrying individual genes of HBV, the luciferase activity was measured and the expressions of SPG21 were detected by RT-PCR and western blot. RESULTS: The expressions of SPG21 mRNA and protein were higher in HepG2.2.15 cells than in HepG2 cells (0.36+/-0.06 vs 0.21+/-0.05, P value is less than 0.05). The activity of SPG21 in HepG2 cells transfected with pCMV-X was higher (875+/-27 vs 67+/-12, P value is less than 0.01) as compared to blank control group (transfected with pCMV-tag2B). HBV X gene enhanced SPG21 gene promoter activity, SPG21 mRNA expression and SPG21 protein production in HepG2 cells in a dose-dependent manner. CONCLUSION: HBV X gene can specially activate SPG21 expression.

miR-200a overexpression in advanced ovarian carcinomas as a prognostic indicator.

BACKGROUND: miR-200a expression is frequently altered in numerous cancers. The aim of the present study was to determine the role of microRNA-200a in advanced ovarian carcinomas. MATERIALS AND METHODS: We measured miR-200a expression in 72 matched normal ovarian tissues and advanced ovarian carcinomas, and also two ovarian carcinoma cell lines (SKOV3 and SKOV3.ip1--the latter being more invasive and metastatic than the parental SKOV3) by stem-loop real-time RT-PCR based on TaqMan microRNA assay using U6 as a reference. Levels of miR-200a expression were compared by disease stage, tumor grade, histology, and lymph node involvement. To evaluate the role of microRNA-200a, cell proliferation and invasion of SKOV-3 and SKOV-3.ip1 were analyzed with miR-200a inhibitor/mimic transfected cells. RESULTS: Of 72 paired samples, 65 cancer tissues overexpressed microRNA-200a greater than two fold in comparison with matched normal epithelium. Specifically, patients with lymph node metastasis showed significant elevation. The level correlated with clinicopathological features, including high tumor grade, late disease stage, most notably with lymph node metastasis, but not with tumor histology. In addition, SKOV-3.ip1 cells also overexpressed miR-200a compared with SKOV-3, and miR-200a inhibitor transfected SKOV-3.ip1 cells showed significant reduction in cellular proliferation and invasion, while a miR-200a mimic stimulated the opposite behavior. CONCLUSIONS: We provide definitive evidence that miR-200a is up-regulated in a significant proportion of advanced ovarian carcinomas, and that elevated miR-200a expression facilitates tumor progression. Our findings support the notion that miR- 200a is an onco-microRNA for ovarian cancer, and elevation is a useful potential diagnostic indicator. This study also provides a solid basis for further functional analysis of miR-200a in advanced ovarian cancer.

Cryptotanshinone induces inhibition of breast tumor growth by cytotoxic CD4+ T cells through the JAK2/STAT4/ perforin pathway.

Cryptotanshinone (CPT), is a quinoid diterpene isolated from the root of the Asian medicinal plant, Salvia miotiorrhiza bunge. Numerous researchers have found that it could work as a potent antitumor agent to inhibit tumor growth in vitro, buith there has been much less emphasis on its in vivo role against breast tumors. Using a mouse tumor model of MCF7 cells, we showed that CPT strongly inhibited MCF7 cell growth in vivo with polarization of immune reactions toward Th1-type responses, stimulation of naive CD4+ T cell proliferation, and also increased IFN-γ and perforin production of CD4+ T cells in response to tumor-activated splenocytes. Furthermore, data revealed that the cytotoxic activity of CD4+ T cells induced by CPT was markedly abrogated by concanamycin A(CMA), a perforin inhibitor, but not IFN-γ Ab. On the other hand, after depletion of CD4+ T cells or blocked perforin with CMA in a tumor-bearing model, CPT could not effectively suppress tumor growth, but this phenomenon could be reversed by injecting naive CD4+ T cells. Thus, our results suggested that CPT mainly inhibited breast tumor growth through inducing cytotoxic CD4+ T cells to secrete perforin. We further found that CPT enhanced perforin production of CD4+ T cells by up-regulating JAK2 and STAT4 phosphorylation. These findings suggest a novel potential therapeutic role for CPT in tumor therapy, and demonstrate that CPT performs its antitumor functions through cytotoxic CD4+ T cells.

Resistant mutants induced by adefovir dipivoxil in hepatitis B virus isolates.

AIM: To investigate the loci of adefovir dipivoxil (ADV)-induced resistance in hepatitis B virus (HBV) isolates and optimize the management of ADV-treated patients. METHODS: Between June 2008 and August 2010, a cross-sectional control study was conducted comprising 79 patients with chronic HBV infection-related liver disease who had been administered ADV monotherapy. Patients underwent liver imaging. Serum DNA extracts were analyzed for HBV DNA levels, genotypes, and serology markers, and deep sequencing of the HBV P gene was performed. RESULTS: ADV-resistant patients were found either with a single mutated locus, or with coexisting mutated loci. The most prevalent mutations were rtA181T, rtV214A, and rtN236T. Twenty-six patients had more than two mutated loci. The mutants were distributed among the patients without any significant affinity for gender, age, end-stage of liver disease, complications of non-alcoholic fatty liver disease, or HBV DNA levels. Patients with the rtA181T mutant were primarily infected with genotype C and e-antigen negative HBV, while patients with the rtN236T mutant were primarily infected by genotype B HBV (χ(2) = 6.004, 7.159; P = 0.023, 0.007). The duration of treatment with ADV was shorter in the single mutant group compared with the multi-mutant group (t = 2.426, P = 0.018). CONCLUSION: Drug-resistant HBV mutants are complex and diverse. Patients should receive the standard and first-line antiviral treatment, strictly comply with medication dosage, and avoid short-term withdrawal.

[HBV genotype and liver histology effect of peginterferon alpha treatment of HBeAg-position chronic hepatitis B].

OBJECTIVE: To investigate the efficacy of PEG-interferon alpha (PEG-IFN alpha) treatment of HBeAg-positive chronic hepatitis B and HBV genotypes and liver tissues effect of HBeAg seroconversion. METHODS: 54 cases confirmed by liver biopsy, genotype clear HBeAg positive chronic hepatitis B (CHB) patients according to body weight, respectively, subcutaneous injection of PEG-IFN-alpha2a 135 microg or 180 microg, or PEG-IFN-alpha2b 50 microg, 80 microg or 100 microg once weekly treatment for 48 weeks and followed for 24 weeks after discontinuation. Statistics of HBeAg seroconvertion, HBV genoty pes and liver histology e antigen seroconversion after the end of treatment. RESULTS: 54 patients were followed up at the end of HBeAg seroconversion rate was 29.63% (16/54). Genotype B patients with HBeAg seroconversion rate was 35.29%, 27.03% higher than the C-type patients, but the difference was not statistically significant (chi2 = 0.382, P = 0.537). Inflammation of the liver activity highter ( > G2) , the degree of fibrosis heavier ( > S1) HBeAg seroconversion rate (50.00% vs. 25.00%, 40.90% vs. 21.88%), but were not statistically significant (chi2 = 1.391, 1.444, P = 0.238, 0.229). Activity of HBV genotype, liver inflammation, liver fibrosis and other factors by multivariate Logistic regression analysis, only liver inflammation activity of the important factors of HBeAg seroconversion. CONCLUSION: Important factors, liver inflammation activity of PEG-interferon alpha treatment of HBeAg-position chronic hepatitis B patients and HBV genotypes and liver fibrosis may be of little significance.

[Association of liver histopathology and clinical features among patients with chronic hepatitis B virus infection].

OBJECTIVE: To explore the histopathological features of chronic hepatitis B virus (HBV) carriers and chronic hepatitis B (CHB) patients with mildly elevated serum alanine aminotransferase (ALT). METHODS: 105 patients were divided into three groups according to serum ALT levels: Group A [ALT level < or = 0.5 x upper limits of normal (ULN)], Group B (0.5 x ULN < ALT level < or = 1 x ULN) and Group C(1 x ULN < ALT level < 2 x ULN). Grade of liver inflammation and stage of liver fibrosis in the three groups were compared. The changes in clinical parameters were then observed in patients who had liver histopathological changes. RESULTS: Among 40.95% of the patients, hepatitis degree went to G2 or even worse; and among 30.43% of the patients whose ALT level were normal, the hepatitis degree reached G2 or even worse. In 26.67% of the patients, degree of fibrosis went to S2 or even worse, and for the 17.39% patients whose ALT level were normal, degree of fibrosis went to S2 or even worse. The aggravation of liver inflammation and fibrosis was correlated with ALT and hyaluronic acid increasing (all P < 0.05). CONCLUSIONS: Frequent monitoring of serum ALT and hyaluronic acid may help to understand histopathological changes in the liver. Liver biopsy applied to CHB should be regarded as a main basis if antiviral therapy should be conducted.