Sirtuin 2 up-regulation suppresses the anti-tumour activity of exhausted natural killer cells in mesenteric lymph nodes in murine colorectal carcinoma.

Natural killer (NK) cells inhibit colorectal carcinoma (CRC) initiation and progression through their tumoricidal activity. However, cumulative evidence suggests that NK cells become functionally exhausted in patients with CRC. To deepen the understanding of the mechanisms underlying CRC-associated NK cell exhaustion, we explored the expression and effect of Sirtuin 2 (Sirt2) in mesenteric lymph node (mLN) NK cells in a murine colitis-associated CRC model. Sirt2 was remarkably up-regulated in mLN NK cells after CRC induction. Particularly, Sirt2 was increased in mLN NK cells expressing high T cell immunoglobulin and mucin domain-3 (TIM3), high lymphocyte activation protein-3 (LAG3), high programmed death-1 (PD-1), high T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT), high NK group 2 member A (NKG2A), but low tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), low interferon-gamma and low granzyme B. In addition, Sirt2 was also increased in NK cells after induction of exhaustion in vitro. Lentivirus-mediated Sirt2 silencing did not affect the acute activation and cytotoxicity of non-exhausted NK cells. However, Sirt2 silencing partially restored the expression of interferon-gamma, granzyme B and CD107a in exhausted NK cells. Meanwhile, Sirt2 silencing down-regulated TIM3, LAG3, TIGIT and NKG2A while up-regulated TRAIL on exhausted NK cells. Consequently, Sirt2 silencing restored the cytotoxicity of exhausted NK cells. Moreover, Sirt2 silencing partially ameliorates the defects in glycolysis and mitochondrial respiration of exhausted NK cells, as evidenced by increases in glycolytic capacity, glycolytic reserve, basal respiration, maximal respiration and spare respiration capacity. Accordingly, Sirt2 negatively regulates the tumoricidal activity of exhausted NK cells in CRC.

Patched 1 and C-C Motif Chemokine Receptor 6 Distinguish Heterogeneous T Helper 17 Subsets in Colitic Lamina Propria.

T helper 17 (Th17) cells contribute to the pathogenesis of inflammatory bowel diseases (IBD). However, their heterogeneity and regulatory mechanisms in IBD are not completely disclosed. A mouse colitis model was established. Th17 cells were enriched from the mesenteric lymph nodes (mLN) and lamina propria (LP). The phenotypes and functions of Th17 subsets were analyzed by flow cytometry, Immunoblotting, and real-time RT-PCR. The contributions of the Th17 subsets to colitis pathogenesis were evaluated by histology, ELISA, and flow cytometry after adoptive transfer. Smoothened (SMO), GLI family zinc finger 1 (Gli1), and GLI family zinc finger 3 (Gli3) were markedly up-regulated while Patched 1 (PTCH1) was down-regulated in LP Th17 cells in colitic lamina propria. Based on the expression of PTCH1 and C-C motif chemokine receptor 6 (CCR6), LP Th17 cells were divided into a PTCH1lowCCR6low Th17 subset and a PTCH1highCCR6high Th17 subset. The former expressed higher T-bet, IFN-γ, TNF-α, IL-1ß, and GM-CSF but lower IL-17A, IL-22, IL-17F, and Gli3 than the latter. The PTCH1highCCR6high Th17 subset was more resistant to polarization towards T helper 1 (Th1) than the PTCH1lowCCR6low Th17 subset. Moreover, the PTCH1highCCR6high Th17 subset was more competent to maintain Th17 identity. The PTCH1highCCR6high Th17 subset induced less severe colitis than the PTCH1lowCCR6low Th17 subset. PTCH1highCCR6high Th17 cells are Th17 cells whereas PTCH1lowCCR6low Th17 cells are Th1-like Th17 cells. Our study deepens the understanding of Th17 heterogeneity and plasticity in colitis.

Zinc finger protein 852 is essential for the proliferation, drug sensitivity, and self-renewal of gastric cancer cells.

Exploring cellular and molecular mechanisms responsible for gastric cancer growth, survival, self-renewal, and metastasis helps develop efficacious therapeutic strategies. In this study, the expression and function of zinc finger protein 852 (ZNF852) in human gastric cancer cell lines were characterized. ZNF852 was upregulated in gastric cancer cell lines relative to normal gastric epithelial cell line GES-1. When the ZNF852 gene was ablated in gastric cancer cell line MGC-803 using the CRISPR/Cas9-encoding lentivirus, the proliferation of MGC-803 was suppressed. ZNF852 deficiency also resulted in the inhibition of MGC-803 sphere formation, along with decreases in SRY-box 2 (SOX2), octamer-binding transcription factor 4 (OCT4), and Nanog homeobox (NANOG), suggesting that ZNF852 sustains self-renewal of MGC-803 cells. Furthermore, ZNF852 deficiency increased oxaliplatin-induced MGC-803 cell death, implying the role of ZNF852 in drug sensitivity. Subcutaneous infusion of MGC-803 cells into nude mice illustrated the same effects of ZNF852 on the proliferation and self-renewal of gastric cancer cells. Similar effects of ANF852 were also seen in gastric cancer cell line SNU-1. Interestingly, ZNF852 deficiency caused downregulation of epidermal growth factor receptor (EGFR) on gastric cancer cells. In summary, this study uncovers the positive regulatory role of ZNF852 in gastric cancer growth and maintenance. ZNF852 could be a potential therapeutic target for inhibiting gastric cancer initiation or progression.

Primary duodenal papilla lymphoma producing obstructive jaundice: a case report.

BACKGROUND: Obstructive jaundice caused by primary duodenal lymphoma is a rare disease. CASE PRESENTATION: We reported a 59-year-old man who underwent endoscopic ultrasonography for obstructive jaundice and found a duodenal papilla tumor. Light microscopy revealed a non-Hodgkin's lymphoma. Immunohistochemical staining showed that the tumor was aggressive B-cell lymphoma. We carried out molecular targeted therapy combined with CHOP regimen chemotherapy. CONCLUSION: Surgery plays an important role in resolving obstructive jaundice when accurate histological diagnosis cannot be made. After diagnosis, chemotherapy should play a central role in treatment.

Down-regulation of PR/SET domain 10 underlies natural killer cell dysfunction in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the world's leading cause of tumor-related mortalities. Natural killer (NK) cells play a critical role at the first immunological defense line against HCC initiation and progression. NK cell dysfunction is therefore an important mechanism for immune evasion of HCC cells. In the present study using a murine HCC model, we revealed the down-regulation of PR/SET Domain 10 (PRDM10) in hepatic NK cells that were phenotypically and functionally exhausted. PRDM10 silencing diminished the expression of natural killer group 2 member D (NKG2D) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), augmented T cell immunoglobulin and ITIM domain (TIGIT) expression, and decreased the expression of interferon (IFN)-γ, perforin and granzyme B in normal hepatic NK cells in vitro. Consistently, PRDM10-deficient NK cells exhibited impaired cytotoxicity on target cells. In contrast, PRDM10 over-expression promoted NKG2D and Fas ligand (FasL) expression, reduced CD96 expression and enhanced transcripts of IFN-γ, perforin and granzyme B in NK cells in vivo. Moreover, PRDM10 silencing and PRDM10 over-expression down-regulated and up-regulated Eomesodermin (Eomes) expression, respectively. In summary, this study reveals PRDM10 down-regulation as a novel mechanism underlying NK cell dysfunction and identifies PRDM10 as a supporting factor of NK cell function.

NKp46+ lamina propria natural killer cells undergo metabolic reprogramming in a mouse experimental colitis model.

OBJECTIVE: Both innate and adaptive immune system play important roles in the onset and progression of inflammatory bowel diseases (IBDs). However, the significance of natural killer (NK) cells for IBDs remains unclear. To understand the biology of colonic lamina propria natural killer (LPNK) cells in IBDs, we characterized LPNK cell metabolism in a murine acute colitis model. METHODS: C57BL/6J mice were fed with 3% dextran sulfate sodium to establish the acute colitis model. Colonic LPNK cells were isolated from mice through flow cytometry. The expression of metabolic genes in LPNK cells was analyzed by transcriptome sequencing and quantitative RT-PCR. Glucose uptake, Seahorse assay, and ATP assay were conducted to assess the metabolic status of LPNK cells. Phos-flow assay was performed to evaluate cell signaling pathways in LPNK cells. In vitro stimulation and cytotoxicity assay were conducted to measure the function of LPNK cells. RESULTS: In acute colitis, LPNK cells upregulated the expression of genes related to glycolysis and oxidative phosphorylation (oxphos), and enhanced glucose uptake capability. Intracellular ATP production, glycolysis and oxphos in LPNK cells were also promoted in acute colitis. mTORC1 signaling was essential for the metabolic reprogramming in LPNK cells in acute colitis. Although LPNK cells of diseased mice exhibited equivalent cytokine profile to normal LPNK cells upon stimulation with phorbol ester or IL-2, LPNK cells of diseased mice were more cytotoxic to target cells than normal LPNK cells. CONCLUSIONS: LPNK cells undergo metabolic reprogramming which might be a response to upcoming microbial infection in acute colitis.

Idiopathic cocoon abdomen with congenital colon malrotation: a case report and review of the literature.

BACKGROUND: Cocoon abdomen is a relatively rare abdominal disease characterized by the total or partial encasement of the small intestinal by a dense fibro-collagenous membrane. CASE PRESENTATION: We reported an unusual case of idiopathic cocoon abdomen with congenital colon malrotation. Laparotomy and sac release were performed on the patient. The patient was no recurrence 6 months after operation. A literature review was also performed. CONCLUSION: Preoperative diagnosis of abdominal cocoon is difficult. A careful history, physical examination and appropriate radiology may be helpful in making a definitive diagnosis. If conservative treatment can't relieve symptoms effectively, surgery is currently considered to be important in the management of this disease.

PPPDE1 promotes hepatocellular carcinoma development by negatively regulate p53 and apoptosis.

We have previously identified that PPPDE1 is a deubiquitinase (DUB) belonging to a cysteine isopeptidase family. Here we sought to explore the biological significance of PPPDE1 in hepatocellular carcinoma and its underlying molecular mechanism. In the present study, we found that amplification and overexpression of PPPDE1 were associated with poor prognosis in hepatocellular carcinoma (HCC). We also demonstrated that knocking down of PPPDE1 could significantly block the clonal growth and tumorigenicity of human HCC cells, which revealed a critical role for PPPDE1 in HCC development. Furthermore, we proved that PPPDE1 is a key modulator of p53 protein level and its down stream apoptosis pathway. Taken together, these results suggested that PPPDE1 is a putative HCC driver gene and extensive studies should be conducted in the future to investigate the role of PPPDE1 in HCC and other tumors.

A Large-scale, multicenter serum metabolite biomarker identification study for the early detection of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the third most lethal cancer worldwide. The lack of effective biomarkers for the early detection of HCC results in unsatisfactory curative treatments. Here, metabolite biomarkers were identified and validated for HCC diagnosis. A total of 1,448 subjects, including healthy controls and patients with chronic hepatitis B virus infection, liver cirrhosis, and HCC, were recruited from multiple centers in China. Liquid chromatography-mass spectrometry-based metabolomics methods were used to characterize the subjects' serum metabolic profiles and to screen and validate the HCC biomarkers. A serum metabolite biomarker panel including phenylalanyl-tryptophan and glycocholate was defined. This panel had a higher diagnostic performance than did α-fetoprotein (AFP) in differentiating HCC from a high-risk population of cirrhosis, such as an area under the receiver-operating characteristic curve of 0.930, 0.892, and 0.807 for the panel versus 0.657, 0.725, and 0.650 for AFP in the discovery set, test set, and cohort 1 of the validation set, respectively. In the nested case-control study, this panel had high sensitivity (range 80.0%-70.3%) to detect preclinical HCC, and its combination with AFP provided better risk prediction of preclinical HCC before clinical diagnosis. Besides, this panel showed a larger area under the receiver-operating characteristic curve than did AFP (0.866 versus 0.682) to distinguish small HCC, and 80.6% of the AFP false-negative patients with HCC were correctly diagnosed using this panel in the test set, which was corroborated by the validation set. The specificity and biological relevance of the identified biomarkers were further evaluated using sera from another two cancers and HCC tissue specimens, respectively. Conclusion: The discovered and validated serum metabolite biomarker panel exhibits good diagnostic performance for the early detection of HCC from at-risk populations. (Hepatology 2018;67:662-675).

Dual-polarized light-field imaging micro-system via a liquid-crystal microlens array for direct three-dimensional observation.

Light-field imaging is a crucial and straightforward way of measuring and analyzing surrounding light worlds. In this paper, a dual-polarized light-field imaging micro-system based on a twisted nematic liquid-crystal microlens array (TN-LCMLA) for direct three-dimensional (3D) observation is fabricated and demonstrated. The prototyped camera has been constructed by integrating a TN-LCMLA with a common CMOS sensor array. By switching the working state of the TN-LCMLA, two orthogonally polarized light-field images can be remapped through the functioned imaging sensors. The imaging micro-system in conjunction with the electric-optical microstructure can be used to perform polarization and light-field imaging, simultaneously. Compared with conventional plenoptic cameras using liquid-crystal microlens array, the polarization-independent light-field images with a high image quality can be obtained in the arbitrary polarization state selected. We experimentally demonstrate characters including a relatively wide operation range in the manipulation of incident beams and the multiple imaging modes, such as conventional two-dimensional imaging, light-field imaging, and polarization imaging. Considering the obvious features of the TN-LCMLA, such as very low power consumption, providing multiple imaging modes mentioned, simple and low-cost manufacturing, the imaging micro-system integrated with this kind of liquid-crystal microstructure driven electrically presents the potential capability of directly observing a 3D object in typical scattering media.

PPPDE1 is a novel deubiquitinase belonging to a cysteine isopeptidase family.

Ubiquitinlation of proteins is prevalent and important in both normal and pathological cellular processes. Deubiquitinating enzymes (DUBs) can remove the ubiquitin tags on substrate proteins and dynamically regulate the ubiquitination process. The PPPDE family proteins were predicted to be a novel class of deubiquitinating peptidase, but this has not yet been experimentally proved. Here we validated the deubiquitinating activity of PPPDE1 and revealed its isopeptidase activity against ubiquitin conjugated through Lys 48 and Lys 63. We also identified ribosomal protein S7, RPS7, as a substrate protein of PPPDE1. Moreover, PPPDE1 could mediate the ubiquitin chain editing of RPS7, deubiquitinating Lys 48-linked ubiquitination, and finally stabilize RPS7 proteins. Taken together, we report that PPPDE1 is a novel deubiquitinase that belongs to a cysteine isopeptidase family.

Down-regulation of zinc finger protein 335 undermines natural killer cell function in mouse colitis-associated colorectal carcinoma.

Natural killer (NK) cells constitute an active and potent anti-tumor effector population against multiple malignancies. NK cells exploit tumoricidal machinery to restrain colorectal carcinoma (CRC) expansion and invasion. Nonetheless, it is becoming increasingly evident that functional exhaustion considerably compromises the potency of NK cells in patients with CRC. To elucidate the factors that impair NK cell function in the context of CRC, we determined the role of zinc finger protein 335 (ZFP335) in modulating NK cell activity in mouse CRC induced by azoxymethane and dextran sulfate sodium. ZFP335 was profoundly decreased in NK cells in mesenteric lymph nodes of CRC-bearing mice. ZFP335 was especially diminished in NK cells that were both phenotypically and functionally exhausted. Besides, effective ZFP335 knockdown markedly undermined NK cell proliferation, tumoricidal protein production, degranulation, and cytotoxic efficacy on malignant cells, strongly suggesting that ZFP335 reinforces NK cell function. Importantly, ZFP335 knockdown lowered the expression of Janus kinase 1 (JAK1) and Janus kinase 3 (JAK3), both of which play crucial roles in NK cell homeostasis and activation. Collectively, ZFP335 down-regulation is essential for NK cell exhaustion in mesenteric lymph nodes of mice with CRC. We discovered a new ZFP335-JAK1/3 signaling pathway that modulates NK cell exhaustion.

GTP binding protein 2 maintains the quiescence, self-renewal, and chemoresistance of mouse colorectal cancer stem cells via promoting Wnt signaling activation.

Colorectal cancer (CRC) is one of the most common cancers and the second most deadly cancer across the globe. Colorectal cancer stem cells (CCSCs) fuel CRC growth, metastasis, relapse, and chemoresistance. A complete understanding of the modulatory mechanisms of CCSC biology is essential for developing efficacious CRC treatment. In the current study, we characterized the expression and function of GTP binding protein 2 (GTPBP2) in a chemical-induced mouse CRC model. We found that GTPBP2 was expressed at a higher level in CD133+CD44+ CCSCs compared with other CRC cells. Using a lentivirus-based Cas9/sgRNA system, GTPBP2 expression was ablated in CRC cells in vitro. GTPBP2 deficiency caused the following effects on CCSCs: 1) Significantly accelerating proliferation and increasing the proportions of cells at G1, S, and G2/M phase; 2) Impairing resistance to 5-Fluorouracil; 3) Weakening self-renewal but not impacting cell migration. In addition, GTPBP2 deficiency remarkably decreased ß-catenin expression while increasing ß-catenin phosphorylation in CCSCs. These effects of GTPBP2 were present in CCSCs but not in other CRC cell populations. The Wnt agonist SKL2001 completely abolished these changes in GTPBP2-deficient CCSCs. When GTPBP2-deficient CCSCs were implanted in nude mice, they exhibited consistent changes compared with GTPBP2-expressing CCSCs. Collectively, this study indicates that GTPBP2 positively modulates Wnt signaling to reinforce the quiescence, self-renewal, and chemoresistance of mouse CCSCs. Therefore, we disclose a novel mechanism underlying CCSC biology and GTPBP2 could be a therapeutic target in future CRC treatment.

Toward a comprehensive solution for treating solid tumors using T-cell receptor therapy: A review.

T-cell receptor therapy (TCR-T) has demonstrated efficacy, durability, and safety advantages in certain solid tumors (such as human papillomavirus-related tumors, synovial sarcoma, and melanoma). This study aimed to provide careful considerations for developing TCR-T for solid tumors. Therefore, in this review, we have summarized the current clinical application, advantage of TCR-T modalities and explored efficacy/safety-related parameters, particularly avidity, pharmacokinetics/pharmacodynamics, and indications, for solid tumors. Furthermore, we have investigated critical factors related to avidity, including antigen selection, T-cell receptor acquisition, optimization, and co-receptor engagement. Moreover, we have re-examined the expression of tumor antigens for a potentially higher coverage rate of solid tumors based on the current RNA-seq datasets. Finally, we have discussed the current limitations and future directions of TCR-Ts.

Dominant neoantigen verification in hepatocellular carcinoma by a single-plasmid system coexpressing patient HLA and antigen.

BACKGROUND: Previous studies confirmed that most neoantigens predicted by algorithms do not work in clinical practice, and experimental validations remain indispensable for confirming immunogenic neoantigens. In this study, we identified the potential neoantigens with tetramer staining, and established the Co-HA system, a single-plasmid system coexpressing patient human leukocyte antigen (HLA) and antigen, to detect the immunogenicity of neoantigens and verify new dominant hepatocellular carcinoma (HCC) neoantigens. METHODS: First, we enrolled 14 patients with HCC for next-generation sequencing for variation calling and predicting potential neoantigens. Then, the Co-HA system was established. To test the feasibility of the system, we constructed target cells coexpressing HLA-A*11:01 and the reported KRAS G12D neoantigen as well as specific T-cell receptor (TCR)-T cells. The specific cytotoxicity generated by this neoantigen was shown using the Co-HA system. Moreover, potential HCC-dominant neoantigens were screened out by tetramer staining and validated by the Co-HA system using methods including flow cytometry, enzyme-linked immunospot assay and ELISA. Finally, antitumor test in mouse mode and TCR sequencing were performed to further evaluate the dominant neoantigen. RESULTS: First, 2875 somatic mutations in 14 patients with HCC were identified. The main base substitutions were C>T/G>A transitions, and the main mutational signatures were 4, 1 and 16. The high-frequency mutated genes included HMCN1, TTN and TP53. Then, 541 potential neoantigens were predicted. Importantly, 19 of the 23 potential neoantigens in tumor tissues also existed in portal vein tumor thrombi. Moreover, 37 predicted neoantigens restricted by HLA-A*11:01, HLA-A*24:02 or HLA-A*02:01 were performed by tetramer staining to screen out potential HCC-dominant neoantigens. HLA-A*24:02-restricted epitope 5'-FYAFSCYYDL-3' and HLA-A*02:01-restricted epitope 5'-WVWCMSPTI-3' demonstrated strong immunogenicity in HCC, as verified by the Co-HA system. Finally, the antitumor efficacy of 5'-FYAFSCYYDL-3'-specific T cells was verified in the B-NDG-B2mtm1Fcrntm1(mB2m) mouse and their specific TCRs were successfully identified. CONCLUSION: We found the dominant neoantigens with high immunogenicity in HCC, which were verified with the Co-HA system.

[Correlation between interleukin-28B genetic polymorphisms and primary hepatocellular carcinoma].

OBJECTIVE: To explore the correlation between single nucleotide polymorphisms (SNPs) of interleukin-28B (IL-28B) gene and the susceptibility to primary hepatocellular carcinoma (HCC). METHODS: A total of 300 histologically confirmed HCC cases (from November 2001 to April 2010) and 310 healthy controls with no history of chronic hepatitis B or hepatocellular carcinoma (2009-2010) were selected from a hospital in Guilin and a hospital in Beijing for this case-control study.139 HCC patients in the case group had complete clinical tracking data. All the subjects were Han Chinese, with no age or gender restrictions.2 ml peripheral blood samples were drawn from each subject with informed consent. SNP of rs12972991, rs4803223, rs8099917 and rs12979860 four loci in IL-28B gene were analyzed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF). RESULTS: The frequencies of C allele at rs12972991, G allele at rs8099917 and G allele at rs4803223 were 6.7% (40/598), 7.9% (47/598) and 10.0% (59/588) respectively in case group; all higher than the corresponding frequencies in control group, separately 2.9% (18/618), 4.1% (25/616) and 3.6% (21/608). The differences were statistically significant (χ2=9.542, 7.858, 20.736, P values all<0.05). The above alleles could increase the risk of HCC, and the OR (95%CI) values were separately 1.67 (1.13-2.46), 1.49 (1.08-2.06) and 2.91 (1.79-4.72). The genotype frequencies of AC+CC at rs12972991, GT+GG at rs8099917, GA+GG at rs4803223 were 13.0% (39/299), 14.7% (44/299) and 19.0% (56/296) respectively in case group; while the frequencies were lower in control group, separately 5.8% (18/309), 8.1% (25/308) and 6.6% (20/304). The differences were statistically significant (χ2=9.319, 6.557, 20.948, P values all<0.05). These genotypes may increase the risk of HCC, and the adjusted OR (95%CI) values were 2.24 (1.31-3.83), 1.81 (1.14-2.88) and 2.90 (1.78-4.70), respectively. The stratified analysis of the clinical data indicated that the frequency of genotype GA+GG at rs4803223 was 50.0% (13/26) in patients of tumor thrombosis in portal vein (TTPV), higher than the frequency of genotype AA (21.1%, 23/109). The difference was statistically significant (χ2=8.965, P=0.003). CONCLUSION: The results suggested that IL-28B gene polymorphisms was correlated to the susceptibility to HCC in Chinese Han ethnic population. Among them, GA + GG genotype at rs4803223 could increase the risk of TTPV in HCC patients.

FOXP3 expression in FOXP3+ tumor cells promotes hepatocellular cells metastasis.

BACKGROUND: Forkhead transcription factor 3 (FOXP3) is a key molecule for the development of regulatory T cell. Recent studies showed that FOXP3 was also expressed in tumor cells. This study is designed to identify the expression of FOXP3 and its pathogenesis in hepatocellular carcinoma (HCC). METHODS: Through immunohistochemistry, RNA extraction and Real-Time Quantitative PCR, western blot analysis, transwell cell migration assay and invasion assays, and in vivo experiment, we detected FOXP3 expression in HCC and analyzed the expression of tumor metastasis-related genes in HCC cells using female BALB/c-Nude mice. RESULTS: The results showed that FOXP3 was expressed in partial HCC tissues samples and cell lines. The distant metastasis rate was remarkably higher in the FOXP3 positive HCCs than that in the negative group. The positive rate of FOXP3 expression in the metastatic HCC was higher than that in primary HCC, and the expression level of FOXP3 was found to increase as the enhancement in the metastatic potential of the cell lines. Furthermore, in HCC cell lines, FOXP3 overexpression can promote the cell metastasis and invasion by regulating MMP-1. The in vivo experiment showed that the proliferation ability of HepG2 cells in nude mice increased significantly after FOXP3 was overexpressed, and the incidence rate of lung metastasis in MHCC97L cells was significantly deceased after knocking down the FOXP3 expression. CONCLUSIONS: Our findings support that partial HCC tissues and cell lines expressing FOXP3 can promote the metastasis by regulating MMP-1.

RFX5 promotes the progression of hepatocellular carcinoma through transcriptional activation of KDM4A.

Regulatory factor X-5 (RFX5) represents a key transcription regulator of MHCII gene expression in the immune system. This study aims to explore the molecular mechanisms and biological significance of RFX5. Firstly, by analyzing ENCODE chromatin immunoprecipitation (ChIP)-seq in HepG2 and TCGA RNA-seq data, we discovered lysine-specific demethylase 4A (KDM4A), also named JMJD2A, to be a major downstream target gene of RFX5. Moreover, RFX5 was verified to bind directly to the KDM4A's promoter region and sequentially promoted its transcription determined by the ChIP-PCR assay and luciferase assay. In addition, RFX5-dependent regulation of KDM4A was demonstrated in HCC. Compared with adjacent non-tumor tissues, the expression levels of KDM4A were significantly raised in HCC tumor tissues. Notably, elevated levels of KDM4A were strongly correlated with HCC patient prognosis. Functionally, KDM4A overexpression largely rescued the growth inhibitory effects of RFX5 deletion, highlighting KDM4A as a downstream effector of RFX5. Mechanistically, the RFX5-KDM4A pathway promoted the progression of the cell cycle from G0/G1 to S phase and was protective against cell apoptosis through regulation of p53 and its downstream genes in HCC. In conclusion, RFX5 could promote HCC progression via transcriptionally activating KDM4A expression.

Expression of CIITA-related MHCII molecules in tumors linked to prognosis in hepatocellular carcinoma.

The prognosis of hepatocellular carcinoma (HCC) after surgery is poor due to its high recurrence rate. In order to unfold the mechanism of different recurrent-free survival (RFS) times following resection, expression profiling of tumor tissues from 32 HCC patients with different RFS time were used to identify differential expression of individual genes and signaling pathway components correlated with RFS time. Quantitative RT-PCR, Western blotting, and immunohistochemistry were used to validate the expression of selected genes. Up-regulation of several immune related genes and pathways, especially HLA II-related antigen presenting pathways, significantly correlated with longer RFS time. The expression of MHCII molecules were found to be mainly located in either CD68+ cells or CD45+ cells, and their expression significantly correlated with the expression of CIITA (HLA II genes transactivator) in the tumor. The results suggest that the high expression level of CIITA and MHCII molecules in hepatocellular carcinoma tissue is an effective prognostic marker for longer RFS time in HCC.

[Specific suppression in regulatory T cells by Foxp3 siRNA contributes to enhance the in vitro anti-tumor immune response in hepatocellular carcinoma patients].

OBJECTIVE: To design the suitable sequence siRNA of Foxp3 to interfere the function of regulatory T cells(Treg), and to evaluate whether the suppression of Treg could enhance the anti-tumor immune response in hepatocellular carcinoma patients or not. METHODS: Foxp3-specific siRNAs by chemical synthesis were delivered into regulatory T cells. The inhibition efficiencies of Foxp3-specific siRNAs were evaluated by real-time PCR and fluorescently stained for intracellular Foxp3 and analyzed using multiparameter FCM. The Foxp3(+) Treg subpopulation was selectively analyzed for surface expression levels of CD127, CTLA-4 and GITR. The suppression of Treg to CD4(+)CD25(-) T cells or anti-tumor specific CD8(+) T-cell responses induced by tumor specific antigen (NY-ESO-1b) was evaluated by CFSE, Elispot and Pentamer analysis. RESULTS: A subpopulation of Tregs with reduced levels of Foxp3 mRNA and protein mediated by siRNA was CD127 up-regulation and CTLA-4 or GITR down-regulation compared with those in Foxp3(high) Tregs. Knockdown of intracellular Foxp3 in Treg reduced suppression of Foxp3+ Treg to proliferative capacity of CD4(+)CD25(-) T cells. IFN-gamma released of NY-ESO-1-specific CD8(+) T cells from the group of Tregs with Foxp3 specific siRNA transfected was increased as compared with the group of Treg with non-specific control siRNA transfected(132+/-55 vs 27+/-11, P<0.05). Frequency of NY-ESO-1b-specific CD8(+) T cells by Pentamer analysis from the group of Tregs with Foxp3 specific siRNA transfected was increased as compared with the group of Treg with non-specific control siRNA transfected (0.21%+/-0.17% vs 0.57%+/-0.39%, P<0.05). CONCLUSION: Knockdown of intracellular Foxp3 in Treg mediated by siRNAs can inhibit the suppression of Foxp3+ Treg and enhance the anti-tumor immune response in hepatocellular carcinoma patients in vitro.