Search

Breast cancer is the most common tumor in female, which seriously threatens the health of women. Triple-negative breast cancer is a subtype with the worst prognosis because of its special physiological characteristics and lack of targeted drugs. Therefore, it is urgent to develop new targeted treatments to improve the prognosis and survival rate of the patients. Previous studies have shown that heat shock protein gp96 is expressed on the membrane of a variety of cancer cells but not on the normal cells. Cell membrane gp96 levels are closely related to the poor prognosis of breast cancer, which may serve as a new target for breast cancer treatment. Based on the structure of gp96, we designed an α-helical peptide p37 that specifically targeting the ATP binding region of gp96. To improve the stability and decrease the degradation of the peptide, the N-terminus or C-terminus of p37 was coupled to PEG

AFP is the most widely used biomarker for the diagnosis of hepatocellular carcinoma. However, a substantial proportion of HCC patients have either normal or marginally increased AFP levels in serum, and the underlying mechanisms are not fully understood. In the present study, we provided in vitro as well as in vivo evidence that heat shock protein gp96 promoted AFP expression at the transcriptional level in HCC. NR5A2 was identified as a key transcription factor regulated by AFP and its stability was enhanced by gp96. A further mechanistic study by CO-IP, GST-pull down and molecular docking showed the competitive binding of gp96 and SUMO E3 ligase RanBP2 to NR5A2 at the sites spanning from aa 507 to 539. The binding of gp96 inhibited SUMOylating, ubiquitination, and subsequent degradation of NR5A2. In addition, clinical analysis of HCC patients indicated that gp96 expression was positively correlated to serum AFP levels in tumors. Therefore, our study uncovered the novel regulatory mechanism of gp96 on the stability of its client proteins by directly affecting their SUMOylation and ubiquitination. These findings will help in designing more accurate AFP-based HCC diagnosis and progression monitoring approaches.

The aim of this research is to investigate the relationship between enterprise technological innovation, export product quality, and the upgrading of the manufacturing value chain. The study is based on panel data from Chinese manufacturing enterprises between 2000 and 2013 and uses a multi-dimensional fixed-effect method to analyze the moderating effect of enterprise export product quality on the relationship between technological innovation and the upgrading of the manufacturing value chain. The results show that technological innovation significantly promotes the upgrading of the manufacturing value chain. The regression analysis indicates that a 1% increase in enterprise technological innovation leads to a 0.28% increase in the upgrading of the manufacturing value chain. Similarly, the study demonstrates that improving the quality of exported products significantly enhances the manufacturing industry’s status in the global value chain. The regression analysis reveals that a 1% increase in the quality of exported products leads to a 0.14% increase in the upgrading of the manufacturing value chain. Moreover, the research identifies a threshold value of 0.4438 for the moderating effect of enterprise export product quality on the impact of technological innovation on the upgrading of the manufacturing value chain. When the quality of exported products is below the threshold value, it has a strong positive regulatory effect on technological innovation, promoting the upgrading of the manufacturing value chain. However, once the quality of exported products exceeds the threshold value, its regulatory effect becomes insignificant. The study’s findings have important implications for enterprises looking to overcome the “dilemma of scientific and technological innovation” and promote the intelligent development of the manufacturing industry. The research conclusions have strong reference value for promoting the combination of technology innovation and business model innovation in manufacturing enterprises. This will allow for climbing the high-end links of the global value chain and achieving sustainable development.

Summary Upregulation and stabilization of Foxp3 expression in Tregs are essential for regulating Treg function and immune homeostasis. In this study, gp96 immunization showed obvious therapeutic effects in a Lyn–/– mouse model of systemic lupus erythematosus. Moreover, gp96 alleviated the initiation and progression of MOG-induced experimental autoimmune encephalomyelitis. Immunization of gp96 increased Treg frequency, expansion, and suppressive function. Gene expression profiling identified the NF-κB family member p65 and c-Rel as the key transcription factors for enhanced Foxp3 expression in Treg by gp96. Mutant gp96 within its Toll-like receptor (TLR) binding domain, TLR2 knockout mice, and mice with cell-specific deletion of MyD88, were used to demonstrate that gp96 activated Tregs and induced Foxp3 expression via a TLR2-MyD88-mediated NF-κB signaling pathway. Taken together, these results show that gp96 immunization restricted antibody-induced and Th-induced autoimmune diseases by integrating Treg expansion and activation, indicating its potential clinical usefulness against autoimmune diseases., Graphical abstract, Highlights • SLE symptoms in Lyn–/– mice are ameliorated by gp96 immunization • Tregs expanded by gp96 provide potential in suppressing Th-mediated EAE • Gp96 promotes Treg proliferation, stability, and suppressive function • Gp96 binds to and activates Treg in a TLR2-MyD88-NF-кB-Foxp3 pathway, Immune response, Genomics, Molecular biology

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths and the fifth most common cancer worldwide. Clinically therapeutic options for HCC are very limited, and the overall survival rate of patients is very low. Therefore, early diagnosis and treatment of HCC have important impact on overall survival of patients. At present, alpha-fetoprotein (AFP) is one of the most widely used serological markers for HCC. Many evidences have shown that as a specific onco-protein, AFP has great research value in the occurrence, development, diagnosis and treatment of HCC. Here, we briefly introduce the molecular mechanism of AFP in the regulation of HCC occurrence and development, and its role in tumor escape from immune surveillance. We focus on the application of AFP as an important HCC target or carcino-embryonic antigen (CEA) in HCC clinical diagnosis and treatment.

This retrospective study investigated the toricity of dual-axis corneal refractive therapy (CRT) orthokeratology lenses and corneal parameters, including flat keratometry (FK), flat eccentricity (e), steep e, corneal astigmatism, and the difference in elevation at 8 mm chord length. We analyzed the right eyes of 143 adolescent patients who underwent ocular examinations, subjective refraction, and corneal topography before CRT lens fitting by trial lens evaluation. After orthokeratology treatment, all patients underwent a topography map with an intact plus power ring and decentration of 0.05). Corneal astigmatism was positively associated with difference in elevation at 8 mm chord length (r = 0.743, p < 0.001, Pearson’s correlation), and corneal astigmatism and the difference in elevation at 8 mm chord length were positively associated with CRT lens toricity (r = 0.657 and r = 0.643, respectively; both p < 0.01, Spearman’s correlation). These results suggest that difference in elevation at 8 mm chord length can be used to conveniently estimate CRT lens toricity in clinical practice, using the equation Y (CRT lens toricity) = 1.02X (difference in elevation at 8 mm chord length) + 20.3.

Kupffer cells (KCs), the largest tissue-resident macrophage population in the body, play a central role in maintaining a delicate balance between immune tolerance and immunity in the liver. However, the underlying molecular mechanism remains elusive. In this study, we show that KCs express high levels of miR-146a, which is under control of the PU.1 transcription factor. miR-146a deficiency promoted KCs differentiation toward a proinflammatory phenotype; conversely, miR-146a overexpression suppressed this phenotypic differentiation. We found that hepatitis B virus (HBV) persistence or HBV surface Ag treatment significantly upregulated miR-146a expression and thereby impaired polarization of KCs toward a proinflammatory phenotype. Furthermore, in an HBV carrier mouse model, KCs depletion by clodronate liposomes dramatically promoted HBV clearance and enhanced an HBV-specific hepatic CD8+ T cell and CD4+ T cell response. Consistent with this finding, miR-146a knockout mice cleared HBV faster and elicited a stronger adaptive antiviral immunity than wild-type mice. In vivo IL-12 blockade promoted HBV persistence and tempered the HBV-specific CTL response in the liver of miR-146a knockout mice. Taken together, our results identified miR-146a as a critical intrinsic regulator of an immunosuppressive phenotype in KCs under inflammatory stimuli, which may be beneficial in maintenance of liver homeostasis under physiological condition. Meanwhile, during HBV infection, miR-146a contributed to viral persistence by inhibiting KCs proinflammatory polarization, highlighting its potential as a therapeutic target in HBV infection.

In hepatitis B virus (HBV) infection, the virus produces redundant hepatitis B surface antigen (HBsAg) that plays a key role in driving T-cell tolerance and viral persistence. However, currently available anti-HBV agents have no direct effect on HBsAg transcription and protein expression. In this study, we designed a heat shock protein gp96 inhibitor p37 with the cell penetrating peptide PTD (protein transduction domain of trans-activator of transcription), which mediated p37 internalization into hepatocytes. PTD-p37 effectively suppressed HBsAg expression and viral replication both in vitro and in vivo. We further provide evidence that PTD-p37 suppressed HBV enhancer/promoter activity via p53 upregulation. Moreover, PTD-p37 had antiviral activity against a lamivudine-resistant HBV strain. Considering that suppression of HBsAg expression is a major goal for treatment of HBV infection, our results provide a basis for developing a new therapeutic approaches targeting host factors against viral expression.

Utilizing regulatory T cells (Treg) is a promising approach for treating autoimmune diseases, including systemic lupus erythematosus (SLE) and rheumatic diseases. Upregulation and stabilization of forkhead box P3 (Foxp3) expression in Tregs are essential for regulating Treg function and immune homeostasis. In this study, we examined the potential effect and mechanism of heat shock protein gp96 in regulating Foxp3 expression and Treg activation in vivo. High-dose gp96 immunization showed obvious therapeutic effects in a Lyn-/- mouse model of SLE, as evidenced by decreased auto-antibody titers, follicular helper T cells, and antibody-production plasma cells. Moreover, immunization of mice with gp96 alleviated the initiation and progression of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (EAE). Immunization of gp96 increased Treg frequency, expansion, and suppressive function with no obvious effector T cell activation. Gene expression profiling was used to identify the NF-κB family member p65 and c-Rel as the key transcription factors for enhanced Foxp3 expression in Treg by gp96. Mutant gp96 within its Toll-like receptor (TLR) binding domain, TLR2 knockout (KO) mice, and mice with cell-specific deletion of MyD88, were used to demonstrate that gp96 activated Tregs and induced Foxp3 expression via a TLR2-MyD88-mediated NF-κB signaling pathway. Taken together, these results show that gp96 immunization restricted antibody- and Th-induced autoimmune diseases by integrating Treg expansion and activation, indicating its potential clinical usefulness against autoimmune diseases.

Toll like receptors (TLRs) are pattern recognition receptors and represent immune receptors in innate immunity. They are very conservative in evolution and extremely important for the survival of organisms. TLRs initiate signal transduction through binding of endogenous or exogenous ligands to activate a series of downstream important gene expression and activation. Studies have shown that regulatory T cells (Tregs) play a central role in maintaining peripheral immune tolerance and preventing transplant rejection. Tregs express certain TLRs, including TLR2, TLR4, TLR5, TLR7, TLR8, and TLR9. Activation of TLRs may directly or indirectly affect (mainly activate) Treg proliferation and immunosuppressive functions, and this regulation is closely related to the occurrence of infection, autoimmune disease and cancer. The heat shock proteins as TLRs ligand molecules play important roles in the regulation of Treg. Therefore, understanding regulatory mechanisms of TLR pathways on Tregs is of great significance for new drug development and targeted therapy. This review introduces how TLR-mediated pathways regulate Tregs' immune function.模式识别受体Toll 样受体 (Toll like receptors，TLRs) 是固有免疫中免疫受体的代表，进化上十分保守，对生物体的生存极为重要。TLRs 通过内源或外源的配体启动信号转导，激活下游一系列重要的基因表达与活化。研究表明调节性T 细胞 (Regulatory T cell，Treg) 在维持机体外周免疫耐受和阻止移植排斥反应等方面发挥核心作用。Treg 细胞表达某些TLRs，包括TLR2、TLR4、TLR5、TLR7、TLR8、TLR9 等。TLRs 的活化可能直接或间接地影响 (主要是活化) Treg 的增殖和免疫抑制功能，这种调节与感染、自身免疫病和癌症的发生密切相关。其中热休克蛋白作为TLRs 配体分子对于Treg 的调节发挥了重要的作用。因此，了解TLRs 通路对研究Treg 免疫调控机制、新药物研发和靶向治疗有重大意义。文中简要介绍了TLRs 通路调节Treg 免疫功能的相关研究进展。.

Liver failure leads to the massive necrosis of hepatocytes, releasing large amounts of intracellular components including damage-associated molecular patterns (DAMPs). We found that extracellular gp96 levels in serum were elevated in patients with chronic hepatitis B infection (CHB) and acute-on-chronic liver failure (ACLF). Meanwhile, the gp96 level positively correlated with hepatic necroinflammation. We employed two mouse liver damage and liver failure models induced by lipopolysaccharide (LPS) plus d-galactosamine (d-Galn), and concanavalin A (ConA) to identify the function of extracellular gp96. As a result, the inhibition of extracellular gp96 by a specific peptide efficiently mitigated both LPS/d-Galn- and ConA-induced liver injury and immune hyperactivation, whereas exogenous gp96 aggravated the symptoms of hepatic injury in mice but not in Kupffer cells-ablated mice. The exposure of Kupffer cells to gp96 induced the secretion of pro-inflammatory cytokines. Collectively, our data demonstrate that gp96 released from necrotic hepatocytes aggravates immune hyperactivation and promotes liver damage and possibly the development of liver failure mainly by activating Kupffer cells.

Programmed death ligand 1 (PD-L1) has been recently shown to be a major obstacle to antiviral immunity by binding to its receptor programmed death 1 (PD-1) on specific IFN-γ producing T cells in chronic hepatitis B. Currently, IFN-α is widely used to treat hepatitis B virus (HBV) infection, but its antiviral effect vary greatly and the mechanism is not totally clear. We found that IFN-α/γ induced a marked increase of PD-L1 expression in hepatocytes. Signal and activators of transcription (Stat1) was then identified as a major transcription factor involved in IFN-α/γ-mediated PD-L1 elevation both in vitro and in mice. Blockage of the PD-L1/PD-1 interaction by a specific mAb greatly enhanced HBV-specific T cell activity by the gp96 adjuvanted therapeutic vaccine, and promoted HBV clearance in HBV transgenic mice. Our results demonstrate the IFN-α/γ-Stat1-PD-L1 axis plays an important role in mediating T cell hyporesponsiveness and inactivating liver-infiltrating T cells in the hepatic microenvironment. These data raise further potential interest in enhancing the anti-HBV efficacy of IFN-α and therapeutic vaccines.

Background Now, numerous microRNAs (miRNAs) are found to exert effect in retinoblastoma (RB). This research mainly focused on the function of miR-532 in RB, which has not been investigated. Methods RT-qPCR and Western blot analysis were used to measure expressions of miR-532 and genes. Transwell, CCK-8 and luciferase reporter assays were applied to explore functions of miR-532 and MDM4 in RB. Results The expression of miR-532 was reduced in RB. Furthermore, overexpression of miR-532 restrained RB cell survival and metastasis and induced apoptosis. In addition, miR-532 directly targets MDM4. Moreover, downregulation of MDM4 blocked the progression of RB. And upregulation of MDM4 reversed the anti-tumor effect of miR-532 in RB. Conclusion MiR-532 inhibited cell viability and metastasis in RB by targeting MDM4, indicating that miR-532 may be a novel therapeutic target for RB patients.

The reciprocal interaction between influenza virus and host microRNAs (miRNAs) has been implicated in the regulation of viral replication and host tropism. However, the global roles of the cellular miRNA repertoire and the mechanisms of miRNA-mediated antiviral defense await further elucidation. In this study, we systematically screened 297 cellular miRNAs from human and mouse epithelial cells and identified five inhibitory miRNAs that efficiently inhibited influenza virus replication in vitro and in vivo. Among these miRNAs, hsa-mir-127-3p, hsa-mir-486-5p, hsa-mir-593-5p, and mmu-mir-487b-5p were found to target at least one viral gene segment of both the human seasonal influenza H3N2 and the attenuated PR8 (H1N1) virus, whereas hsa-miR-1-3p inhibited viral replication by targeting the supportive host factor ATP6V1A. Moreover, the number of miRNA binding sites in viral RNA segments was positively associated with the activity of host miRNA-induced antiviral defense. Treatment with a combination of the five miRNAs through agomir delivery pronouncedly suppressed viral replication and effectively improved protection against lethal challenge with PR8 in mice. These data suggest that the highly expressed miRNAs in respiratory epithelial cells elicit effective antiviral defenses against influenza A viruses and will be useful for designing miRNA-based therapies against viral infection.

// Xin Li 1, * , Bo Wu 1, * , Lizhao Chen 1 , Ying Ju 1 , Changfei Li 1 and Songdong Meng 1, 2 1 CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences (CAS), Beijing, China 2 College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China * These authors have contributed equally to this work Correspondence to: Songdong Meng, email: mengsd@im.ac.cn Changfei Li, email: lichangfei2006@163.com Keywords: uPAR,miR-17-5p, miR-20a, DR5, apoptosis Received: December 21, 2016 Accepted: July 23, 2017 Published: August 24, 2017 ABSTRACT Dissection and understanding of the molecular pathways driving triple-negative breast cancer (TNBC) are urgently needed to develop efficient tailored therapies. Aside from cell invasion and metastasis, the urokinase-type plasminogen activator receptor (uPAR) has been linked to apoptosis resistance in breast tumors. We explored the mechanism of uPAR-disrupted apoptosis in breast cancer. We found that depletion of uPAR by RNAi increases death receptor 4 (DR4) and death receptor 5 (DR5) expression and triggers TRAIL-induced apoptosis in TNBC cells. The microRNAs miR-17-5p and miR-20a inhibit cell apoptosis via suppression DR4/DR5. We provide evidence that uPAR enhances miR-17-5p/20a expression through upregulation of c-myc. Blocking miR-17-5p/20a with antagomiRNA suppressed the growth of uPAR-overexpressing breast tumor xenografts in mice. These results indicate that uPAR suppresses cell apoptosis by inhibiting the c-myc-miR-17/5p/20a-DR4/DR5 pathway. Therapy directed at uPAR-induced miR-17/20a is a potential option for breast cancer and TNBC.

Persistent inflammation in chronic hepatitis plays a major role in the development of hepatocellular carcinoma (HCC). In this study, the major inflammatory cytokines expressed in chronic hepatitis, IL-6 and TNF-α, induced a marked decrease in microRNA-122 (miR-122) levels, and miR-122 expression was downregulated in the livers of chronic hepatitis B (CHB) patients. The decrease of miR-122 caused upregulation of the proinflammatory chemokine CCL2. IL-6 and TNF-α suppressed miR-122 both by directly downregulating the transcription factor C/EBPα and indirectly upregulating c-myc, which blocks C/EBPα-mediated miR-122 transcription. In addition, IL-6 and TNF-α levels were elevated and miR-122 levels were decreased in mouse and rat models of diethylnitrosamine (DEN)-induced HCC. Restoration of miR-122 levels through delivery of agomir-122 suppressed DEN-induced hepatocarcinogenesis in mice. Our results show that inflammation-induced miR-122 downregulation in hepatitis contributes to carcinogenesis and suggest that increasing miR-122 may be an effective strategy for preventing HCC development in CHB patients.

Background HER2 gene amplification generates an enormous number of HER2 transcripts, but the global effects on endogenous miRNA targets including HER family members in breast cancer are unexplored. Methods We generated a HER2–3’UTR expressing vector to test the tumor-promoting properties in HER2 low expressing T47D and MCF7 cells. Through microarray analysis and real-time PCR analysis we identified genes that were regulated by HER2–3’UTR. Positive and negative manipulation of miRNA expression, response element mutational studies and transcript reporter assays were performed to explore the mechanism of competitive sequestration of miR125a/miRNA125b by HER2 3’UTR. To investigate if trastuzumab-induced upregulation of HER3 is also mediated through miRNA de-repression, we used the CRISPR/cas9 to mutate the endogenous HER2 mRNA in HER2 over-expressing Au565 cells. Finally, we looked at cohorts of breast cancer samples of our own and the TCGA to show if HER2 and HER3 mRNAs correlate with each other. Results The HER2 3’UTR pronouncedly promoted cell proliferation, colony formation, and breast tumor growth. High-throughput sequencing revealed a significant increase in HER3 mRNA and protein levels by the HER2 3’untranslated region (3’UTR). The HER2 3’UTR harboring a shared miR-125a/b response element induced miR-125a/b sequestration and thus resulted in HER3 mRNA derepression. Trastuzumab treatment upregulated HER3 via elevated HER2 mRNA expression, leading to trastuzumab resistance. Depletion of miR-125a/b enhanced the antitumor activity of trastuzumab. Microarray data from HER2-overexpressing primary breast cancer showed significant elevation of mRNAs for predicted miR-125a/b targets compared to non-targets. Conclusions These results suggest that HER2 3’UTR-mediated HER3 upregulation is involved in breast cell transformation, increased tumor growth, and resistance to anti-HER2 therapy. The combinatorial targeting of HER3 mRNA or miR-125a/b may offer an effective tool for breast cancer therapy. Electronic supplementary material The online version of this article (10.1186/s12943-018-0862-5) contains supplementary material, which is available to authorized users.

Background: Cytokeratin-18 Neoepitope M30 (CK-18 M30) has been reported to be associated with chronic HBV infection and severity of liver injury; however, the results of these studies are inconsistent. Objectives: We sought to investigate the association between serum CK-18 M30 levels and the severity of liver injury in chronic hepatitis B (CHB) patients. Methods: A systematic literature search was performed in PubMed, Embase, and Cochrane Library databases for relevant studies published in English up to August 2017. Heterogeneity among individual studies was investigated for summarizing all the studies. The standard mean difference (SMD) and 95% confidence interval (CI) were calculated using a random-effects model or fixed-effects model. Finally, the sensitivity analysis and publication bias were performed to evaluate the accuracy of this meta-analysis. Statistical analysis was conducted using Review Manager 5.3 and Stata 12.0. Results: Five case-control studies were included in the ultimate analysis, recruiting 488 CHB patients, 276 inactive carriers, and 193 healthy controls. The major results of the meta-analysis revealed significantly elevated serum CK-18 M30 levels in chronic HBV infected patients including CHB patients with severe liver injury and inactive HBV carriers when compared to healthy controls (SMD = 1.13, 95% CI: 0.75 - 1.50, P < 0.001; SMD = 0.63, 95% CI: 0.38 - 0.89, P < 0.001, respectively). Furthermore, the serum CK-18 M30 levels were significantly higher in CHB patients with severe liver injury than in inactive carriers (SMD =1.29, 95% CI: 0.60 - 1.98, P < 0.001). The sensitivity and publication bias analysis verified the stability and reliability of our analysis. Conclusions: The elevated serum CK-18 M30 levels could be regarded as a useful non-invasive biomarker for the diagnosis of chronic HBV infection, and were associated with the severity of liver injury in chronic Hepatitis B patients. The serum CK-18 M30 levels could reflect the liver inflammation in inactive carriers, representing the early stage of chronic HBV infection.

Figure S5. Reciprocal ceRNA activity between HER2 and HER3 3â UTR. HER2 3â UTR-luciferase reporter assay in T47D cells transfected with the HER3 3â UTR or control vector. (PDF 150 kb)

Figure S3. Combinatory treatment with HER3 siRNA and trastuzumab is useful for overcoming trastuzumab resistance. (A-C) Real-time PCR (A), western blotting (B), and FACS (C) analysis of HER2 and HER3 expression in AU565 parental and trastuzumab-resistant (TtzmR) cell lines. (D) Proliferation of AU565 parental and TtzmR cells treated with 10 μg/ml trastuzumab or control IgG, along with a cholesterol-conjugated siRNA targeting HER3 or a randomized oligonucleotide (control). All error bars represent the standard deviation. All quantitative data were generated from a minimum of three replicates. (E, F) AU565 TtzmR cells were s.c. injected into female BALB/c-nude mice. Mice were treated with cholesterol-conjugated HER3 siRNA or trastuzumab at days 0, 7, and 14. Representative in vivo luciferase images of mice at days 0, 10, and 21(E). The results are presented as means ± SD from five mice. Immunostaining of HER3 in xenograft tumor sections (F). Red, HER3; Blue, DAPI. Scale bar, 40 μm. (PDF 3149 kb)

Figure S2. Effect of trastuzumab on HER2 and HER3 levels. FACS analysis of HER2 and HER3 levels in AU565 cells treated with 10Â Îźg/ml trastuzumab at the indicated times or the indicated concentrations of trastuzumab. (PDF 1027 kb)

Figure S1. Effect of trastuzumab on miR-125a/b levels. Real-time PCR analysis of miR-125a and miR-125b levels in AU565 cells treated with 10Â Îźg/ml trastuzumab at the indicated times. Data were generated from three replicates. (PDF 295 kb)

Figure S4. Correlation between miR-125a/b and EGFR family proteins in HER2 positive breast cancer patients. Correlation between miR-125a and miR-125b and EGFR family proteins (EGFR, HER2 and HER3) in HER2 positive breast cancer patients. (PDF 1450 kb)

The resistance to apoptosis displayed by liver cancer plays a key role in hepatocarcinogenesis, tumor progression, and resistance to chemo- or radio-therapy. In this study, we uncovered the potential role and mechanism of heat shock protein gp96 in regulating liver tumor cell growth and apoptosis. P53 protein was identified as a gp96 client protein by profiling apoptosis-related proteins in gp96-knockdown liver cancer cells. Overexpression and knockdown studies both demonstrated that gp96 decreases p53 protein levels, and gp96 regulated cell apoptosis in a p53-dependent manner. We further provide evidence that gp96 interacts with both p53 and Mdm2 to enhance Mdm2-mediated p53 ubiquitination and degradation. Moreover, targeting gp96 with siRNA induced cell apoptosis and led to the suppression of liver tumor growth in vivo. In conclusion, we elucidated an underlying mechanism by which gp96 promotes p53 degradation via increasing Mdm2 E3 ligase activity and provided a new therapeutic strategy to target the gp96-mediated anti-apoptotic characteristic of hepatocellular carcinoma.

Cancer stem cells are currently under intensive investigation due to their capabilities for tumor initiation, self-renewal, and resistance to chemotherapy. CD133 is implicated in stemness and the malignancy of tumor cells. Here, we explored heat shock protein gp96 adjuvanted CD133 epitope vaccine against leukemia. We screened and identified three H2-Kd-restricted cytotoxic T lymphocyte (CTL) epitopes derived from CD133, CD133₄₁₉₋₄₂₈, CD133₇₀₂₋₇₁₀ and CD133₇₆₀₋₇₆₉. The immunogenicity and antitumor activity of the epitope vaccine using heat shock protein gp96 as adjuvant were further determined in CD133⁺ leukemia xenograft mice. Finally, we demonstrate that adoptive transfer of epitope-specific CTLs led to suppression of leukemia growth. Our data therefore provide the basis for designing a CD133 epitope vaccine to activate specific CTLs against CD133⁺ leukemia and other cancers.肿瘤干细胞具有肿瘤形成、自我更新和抗化疗的特性，因而受到广泛关注和研究。CD133作为重要的肿瘤干细胞标志物与肿瘤细胞的干性与恶性密切相关。本研究筛选并且鉴定了CD133的3个H2-Kd限制性的细胞毒性T细胞(CTL)的表位，分别是CD133₄₁₉₋₄₂₈、CD133₇₀₂₋₇₁₀和CD133₇₆₀₋₇₆₉。将重组热休克蛋白gp96为佐剂联合CD133表位制备表位疫苗，将疫苗免疫CD133⁺白血病移植的小鼠后引发抗肿瘤的特异性T细胞免疫应答。转输CD133表位特异的T细胞同样可以抑制小鼠淋巴瘤的生长。该研究为设计抗CD133⁺的白血病和其他肿瘤的表位疫苗提供了依据。.

Background Some studties reported that the polymorphism of TM6SF2 gene E167K affects the occurrence and the progression of hepatocytes carcinoma (hepatocellular, HCC). In oeder to investigate the effects of the polymorphism of TM6SF2 gene E167K in the pathogenesis of HCC, we explored its influence on the cell cycle in hepatocellular carcinoma cell HEPA1-6. Methods HEPA 1–6 cells which could respectively overexpress TM6SF2 wild type and E167K variant were cultured and HEPA 1–6 cells with zero load plasmids were used as matched control. Flow cytometry was used to detect the cell cycles of these 3 type of HEPA 1–6 cells. Realtime fluores-cence quantitative PCR and western blot were used to analyzed the expression of regulatory factors (Cyclin D1、p53、P16、P27、P21 and Rb) of cell cycle. T-test was used in statistical analysis. Results Cell cycle phase distribution was presented by the proportion of cells in each phases (%). Compared with the control group, the cell cycle phase distribution (G1 phase 57.36 ± 0.21%, G2/M phase 25.61 ± 0.36%,S phases 19.31 ± 0.25%) had no differences in wild type group (G1 phase 57.63 ± 0.28%, G2/M phase 25.77 ± 0.51%, S phases 19.54 ± 0.25%; P 0.05). P16 and P21 expression showed no statistical sigtfificance in any of these three groups (P > 0.05). Conclusion E167K polymorphism of TM6SF2 gene affects cell cycles of HEPA1–6 cells via up-regulating CyclinD1、P53 and Rb and down-regulating P27.

During virus infection, viral RNAs and mRNAs function as blueprints for viral protein synthesis and possibly as pathogen-associated molecular patterns (PAMPs) in innate immunity. Here, considering recent research progress in microRNAs (miRNAs) and competitive endogenous RNAs (ceRNAs), we speculate that viral RNAs act as sponges and can sequester endogenous miRNAs within infected cells, thus cross-regulating the stability and translational efficiency of host mRNAs with shared miRNA response elements. This cross-talk and these reciprocal interactions between viral RNAs and host mRNAs are termed “competitive viral and host RNAs” (cvhRNAs). We further provide recent experimental evidence for the existence of cvhRNAs networks in hepatitis B virus (HBV), as well as Herpesvirus saimiri (HVS), lytic murine cytomegalovirus (MCMV) and human cytomegalovirus (HCMV) infections. In addition, the cvhRNA hypothesis also predicts possible cross-regulation between host and other viruses, such as hepatitis C virus (HCV), HIV, influenza virus, human papillomaviruses (HPV). Since the interaction between miRNAs and viral RNAs also inevitably leads to repression of viral RNA function, we speculate that virus may evolve either to employ cvhRNA networks or to avoid miRNA targeting for optimal fitness within the host. CvhRNA networks may therefore play a fundamental role in the regulation of viral replication, infection establishment, and viral pathogenesis.

The heat shock protein gp96 is an adjuvant that can elicit T cell responses against cancer and infectious diseases, via antigen presentation, in both rodent models and clinical trials. Its uptake and internalization into antigen presenting cells (APCs) is a critical step in gp96-mediated immune responses. This study examined strategies to improve the cell internalization and T cell activation of gp96. It was found that recombinant fusion with the cell-penetrating peptide TAT (trans-activator of transcription) slightly decreased the aggregation level of gp96 and significantly increased its internalization into macrophages. Furthermore, immunization with the TAT-gp96 fusion dramatically enhanced gp96-mediated hepatitis B virus (HBV)-specific T cell responses and its antiviral efficiency in HBV transgenic mice compared to rgp96. In addition, the inclusion of TAT significantly improved the antitumor T cell immune response to a gp96 vaccine in the B16 melanoma model. These results provide evidence that the efficient transduction of gp96 into APCs can significantly enhance the outcome of gp96-based immunotherapy, and therefore provide a basis for more efficient approaches to improving the immunoregulatory and adjuvant functions of this unique T cell adjuvant.

Hepatitis B virus (HBV) infection induces hepatocarcinogenesis and malignant progression, yet global effects of the redundant viral mRNAs produced during infection are unexplored. Here, microRNA (miRNA) target prediction and whole genome expression analysis revealed that HBV pre-C/C mRNA leads to upregulation of multiple let-7a targeted genes. A let-7a complementary region from nt 86 to 108 in the HBV genome was then identified in HBV pre-C/C, pre-S, and S mRNAs. The let-7a sequestration effect by HBV mRNAs was observed under transfection and virus infection, which is dependent on the let-7a response sequence. Moreover, we found reduced AGO2 binding, as well as functional mRNA and protein de-repression of let-7a targets (e.g., c-myc, K-RAS, and CCR7), upon viral mRNA expression. Let-7a levels in the liver were significantly decreased in hepatocellular carcinoma (HCC) patients with HBV infection and were negatively correlated with intrahepatic pre-S2 mRNA levels. Finally, both in vitro and in vivo studies demonstrated that let-7a inhibition by HBV mRNAs resulted in enhanced HCC cell colony formation and tumor growth, providing evidence of the oncogenic potential of HBV mRNAs.

To study the effect of pituitary tumor-transforming gene 1 (PTTG1) on Hepatitis B Virus (HBV) replication.The effect and mechanism of PTTG1 on HBV replication were examined by enzyme-linked immunosorbent assay (ELISA), immunoblot analysis, real-time PCR, dual-luciferase reporter assays and immunoblot analysis.PTTG1 impairs the repression of P53 on HBV enhancer I and enhancer II through decreasing the protein level of P53, resulting in promotion of HBV replication.PTTG1 enhances HBV replication through suppression of P53.

Hepatitis B virus (HBV) chronically infects approximately 350 million people worldwide. The replication of HBV which genome is only 3.2 kb long relies heavily on host factors. Previous studies demonstrated that a highly expressed liver-specific microRNA (miRNA) miR-122 suppresses HBV expression and replication in multiple ways. In this study, we found that the miR-122 response elements in viral genome facilitate HBV expression and replication in miR-122 highly-expressed hepatocytes. Moreover, mutations in miR-122 response elements are correlated with viral loads and disease progression in HBV-infected patients. We next found that HBV mRNA with miR-122 response elements alone could lead to altered expression of multiple host genes by whole genome expression analysis. HBV mRNA-mediated miR-122 down-regulation plays a major role in HBV mRNA-induced differential gene expression. HBV mRNA could enhance viral replication via miR-122 degradation and the up-regulation of its target cyclin G1. Our study thereby reveals that under the unique condition of high abundance of miR-122 and viral mRNAs and much lower level of miR-122 target in HBV infection, HBV may have evolved to employ the miRNA-mediated virus and host mRNAs network for optimal fitness within hepatocytes.

In Hepatitis B virus (HBV) infection, the virus generates numerous viral mRNAs/proteins and viral loads, which plays a major role in driving T cell tolerance, viral persistence, and hepatocellular carcinoma. However, currently available anti-HBV agents have no direct effect on viral mRNA transcription and protein expression. In this study, we designed a recombinant fusion of p53 protein with the cell-penetrating peptide PTD (protein transduction domain of trans-activator of transcription), which mediated p53 internalization into hepatocytes. PTD-p53 effectively suppressed HBV transcription and antigen expression by interaction with viral enhancers. We further provide evidence that PTD-p53 counteracts the viral transcription feedback loop and effectively suppressed HBV production of viral mRNAs, as well as HBsAg, HBeAg, and HBcAg, both in vitro and in vivo. Our results thereby provide a basis for developing a new therapeutic approach against HBV infection.

To investigate the mechanism of gp96 raised during hepatitis B virus (HBV) infection and the pathological mechanism.The mechanism of NF-KB activating gp96 expression was determined by bioinformatics analysis, luciferase reporter assay, real-time PCR and Western blot. The effect of over-expression and knockdown gp96 expression by transfection or RNA interference on hepatocyte proliferation, apoptosis and cell cycle was examined by CCK-8 and flow cytometry. The role of gp96 for HCC development was determined by epithelial-mesenchymal transition (EMT) and colony formation assay.NF-kB significantly increased the gp96 expression by binding to the NF-kappaB binding site. Over-expression and knockdown studies both show that gp96 promoted hepatocyte proliferation, inhibited apoptosis, and induced G0/G1 to S phase cell cycle progression. Moreover, gp96 induced epithelial-mesenchymal transition and increased colony formation ability of hepatocytes.Our results therefore provide insights in chronic HBV infection-induced gp96 expression, and indicate that elevated gp96 may contribute to HCC development during chronic inflammation.

Targeted therapy is currently under intensive investigation due to the resistance of liver cancer to cytotoxic chemotherapies. Dissecting the molecular events that drive the progression of liver cancer and defining specific targets are urgently needed to develop efficient tailored therapies. Cell membrane gp96 (mgp96) has been implicated in tumor growth and malignancy. Here, we explored the functional and clinical relevance of mgp96 in liver cancer. We found that elevated mgp96 abundance was associated with tumor metastasis and recurrence in patients with primary liver tumors. Decreased KDELR1 levels in hepatoma cells contribute to cell membrane translocation of the normally ER-resident gp96. Urokinase-type plasminogen activator receptor (uPAR) was identified as a mgp96 client protein, and mgp96 stabilized uPAR protein. Our clinical results proved that elevated mgp96 abundance is positively correlated with uPAR expression levels in liver tumors. We further provided evidence that targeting mgp96 with siRNA or a specific mAb that blocked the mgp96-uPAR interaction led to inhibited cell growth, survival, and invasion in vitro, as well as the suppression of liver tumor growth and metastasis in vivo. mgp96 promotes liver cancer progression through increasing the protein stability and signaling of uPAR, and may be a new promising target for suppressing uPAR-mediated tumor growth and metastasis in liver cancer.

Secretory anti-gp96 scFv fragment was expressed in Pichia pastoris to obtain a small molecule antibody that specifically recognizes heat shock protein gp96. The gp96-scFv fragment gene was synthesized and cloned to Pichia pastoris expression plasmid pPICZa-A. Pichia pastoris X33 was electroporated with the linearized recombinant expression vector, and expression of gp96-scFv fragment was induced by methanol. The His-tagged recombinant protein was then purified by affinity chromatography and analyzed with SDS-PAGE and Western blotting assays. The biological activities of recombinant gp96-scFv fragment were determined by Western blotting, Immunofluorescence, ELISA and FACS assays. The gp96-scFv fragment was expressed successfully in Pichia pastoris. About 50 mg of recombinant protein could be purified from 1 liter of the Pichia pastoris culture supernatant. Its molecular weight was about 15 kDa. The gp96-scFv fragment could specifically bind to gp96 protein by Western blotting, immunofluorescence, ELISA and FACS analyses. Pichia pastoris-expressed gp96-scFv fragment specifically recognizes gp96 protein, which could be used for Western blotting, Immunofluorescence, ELISA and FACS analyses.