Histone demethylase JMJD2D protects against enteric bacterial infection via up-regulating colonic IL-17F to induce ß-defensin expression.

Histone demethylase JMJD2D (also known as KDM4D) can specifically demethylate H3K9me2/3 to activate its target gene expression. Our previous study has demonstrated that JMJD2D can protect intestine from dextran sulfate sodium (DSS)-induced colitis by activating Hedgehog signaling; however, its involvement in host defense against enteric attaching and effacing bacterial infection remains unclear. The present study was aimed to investigate the role of JMJD2D in host defense against enteric bacteria and its underlying mechanisms. The enteric pathogen Citrobacter rodentium (C. rodentium) model was used to mimic clinical colonic infection. The responses of wild-type and JMJD2D-/- mice to oral infection of C. rodentium were investigated. Bone marrow chimeric mice were infected with C. rodentium. JMJD2D expression was knocked down in CMT93 cells by using small hairpin RNAs, and Western blot and real-time PCR assays were performed in these cells. The relationship between JMJD2D and STAT3 was studied by co-immunoprecipitation and chromatin immunoprecipitation. JMJD2D was significantly up-regulated in colonic epithelial cells of mice in response to Citrobacter rodentium infection. JMJD2D-/- mice displayed an impaired clearance of C. rodentium, more body weight loss, and more severe colonic tissue pathology compared with wild-type mice. JMJD2D-/- mice exhibited an impaired expression of IL-17F in the colonic epithelial cells, which restricts C. rodentium infection by inducing the expression of antimicrobial peptides. Accordingly, JMJD2D-/- mice showed a decreased expression of ß-defensin-1, ß-defensin-3, and ß-defensin-4 in the colonic epithelial cells. Mechanistically, JMJD2D activated STAT3 signaling by inducing STAT3 phosphorylation and cooperated with STAT3 to induce IL-17F expression by interacting with STAT3 and been recruited to the IL-17F promoter to demethylate H3K9me3. Our study demonstrates that JMJD2D contributes to host defense against enteric bacteria through up-regulating IL-17F to induce ß-defensin expression.

Prognostic iron-metabolism signature robustly stratifies single-cell characteristics of hepatocellular carcinoma.

Cancer immunotherapy has shown to be a promising method in treating hepatocellular carcinoma (HCC), but suboptimal responses in patients are attributed to cellular and molecular heterogeneity. Iron metabolism-related genes (IRGs) are important in maintaining immune system homeostasis and have the potential to help develop new strategies for HCC treatment. Herein, we constructed and validated the iron-metabolism gene prognostic index (IPX) using univariate Cox proportional hazards regression and LASSO Cox regression analysis, successfully categorizing HCC patients into two groups with distinct survival risks. Then, we performed single-sample gene set enrichment analysis, weighted correlation network analysis, gene ontology enrichment analysis, cellular lineage analysis, and SCENIC analysis to reveal the key determinants underlying the ability of this model based on bulk and single-cell transcriptomic data. We identified several driver transcription factors specifically activated in specific malignant cell sub-populations to contribute to the adverse survival outcomes in the IPX-high subgroup. Within the tumor microenvironment (TME), T cells displayed significant diversity in their cellular characteristics and experienced changes in their developmental paths within distinct clusters identified by IPX. Interestingly, the proportion of Treg cells was increased in the high-risk group compared with the low-risk group. These results suggest that iron-metabolism could be involved in reshaping the TME, thereby disrupting the cell cycle of immune cells. This study utilized IRGs to construct a novel and reliable model, which can be used to assess the prognosis of patients with HCC and further clarify the molecular mechanisms of IRGs in HCC at single-cell resolution.

LINC02875 Upregulation Contributed to Poor Prognosis for the Hepatocellular Carcinoma and Progression for the Cancerous Cells.

The prognostic implications and physiological effect of LINC02875 are unknown in hepatocellular carcinoma (HCC). We sought to examine the prognostic value of LINC02875 in HCC and assessed its role in HCC cellular function. LINC02875 expression was evaluated by RT-qPCR in HCC specimens and cell lines. LINC02875 expression was subjected to assess the correlation with clinical parameters by Chi-squared test and overall survival by Kaplan - Meier curve and Cox regression analysis. The effects of LINC02875 on the biological characteristics of HCC cells were studied by MTS and Transwell assay. LINC02875 was high-expressed in HCC, and this was associated with unfavorable clinical features and poor prognosis of HCC, especially HBV-related HCC. Knockdown of LINC02875 inhibited the proliferation, migration, and invasion of HCC cells. miR-485-5p was a downstream microRNA of LINC02875. LINC02875 affects the prognosis of HCC patients, especially HBV-related ones. LINC02875 represents a suitable therapeutic target for HCC.

Identification and Validation of an Immune Evasion Molecular Subgroup of Patients With Colon Cancer for Implications of Immunotherapy.

Immune evasion (IEV) plays a critical role in the development and progression of colon cancer. However, studies to predict the prognosis of colon cancer via IEV-related genes are limited. Therefore, based on the 182 IEV-related genes, we used the univariate and Lasso Cox regression model to construct the IEV-related genes signature (IEVSig) of 16 prognostic IEV-related genes using the Gene Expression Omnibus and The Cancer Genome Atlas online databases. We found that IEVSig was an independent prognostic factor, and patients with high IEVSig had higher TNM stage and shorter recurrence-free survival than their counterparts. Kyoto Encyclopedia of Genes and Genomes and gene set enrichment analyses revealed that patients with high and low IEVSig had significantly different enrichment pathways. Immune cell infiltration analysis showed that nine immune cells obviously increased in the high-IEVSig group, whereas five immune cells increased in the low-IEVSig group. Immunotherapy cohort analysis revealed that patients with high IEVSig had a higher proportion of progressive disease or stable disease after receiving immunotherapy than patients with low IEVSig. Furthermore, patients with low IEVSig had higher tumor mutation load and neoantigen burden, which indicated an improved response to immunotherapy, than patients with high IEVSig. Thus, an IEV-related prognostic signature was established to predict the prognosis of patients with colon cancer and derive a prediction marker to offer insights into therapeutic strategies.

Role of betaine in inhibiting the induction of RNA Pol III gene transcription and cell growth caused by alcohol.

Alcohol has been classified as carcinogenic to humans by the International Agency for Research on Cancer (IARC). Studies have demonstrated that alcohol intake increases the risk of breast cancer, and alcohol also stimulates breast cancer cell growth. Deregulation of Pol III genes is tightly associated with tumour development. Transcription factor II-B (TFIIB)-related factor 1 (Brf1) is a transcription factor that specifically regulates Pol III gene transcription. Our in vivo and in vitro studies have indicated that alcohol enhances the transcription of Pol III genes to cause an alteration of cellular phenotypes, which is closely related with human breast cancer. Betaine is a vegetable alkaloid and has antitumor functions. Most reports about betaine show that the consumption level of betaine is inversely associated with a risk of breast cancer. Although different mechanisms of betaine against tumour have been investigated, nothing has been reported on the effect of betaine on the deregulation of Brf1 and Pol III genes. In this study, we determine the role of betaine in breast cancer cell growth and colony formation and explore its mechanism. Our results indicate that alcohol increases the rates of growth and colony formation of breast cancer cells, whereas betaine is able to significantly inhibit the effects of alcohol on these cell phenotypes. Betaine decreases the induction of Brf1 expression and Pol III gene transcription caused by ethanol to reduce the rates of cell growth and colony formation. Together, these studies provide novel insights into the role of betaine in alcohol-caused breast cancer cell growth and deregulation of Brf1 and Pol III genes. These results suggest that betaine consumption is able to prevent alcohol-associated human cancer development.

The significance of Runx2 mediating alcohol-induced Brf1 expression and RNA Pol III gene transcription.

Runx2 (Runt-related transcription factor 2) is a key transcription factor which is associated with osteoblast differentiation and expressed in ER+ (estrogen receptor positive) human breast cancer cell lines. Runx2 also participates in mammary gland development. Deregulation of RNA Pol III genes (polymerase III-dependent genes) is tightly linked to tumor development, while Brf1 (TFIIB-related factor 1) specifically regulates these gene transcription. However, nothing is known about the effect of Runx2 on Brf1 expression and Pol III gene transcription. Expression of Runx2, Brf1 and Pol III genes from the samples of human breast cancer and cell culture model were determined by the assays of RT-qPCR, immunoblot, luciferase reporter activity, immunohistochemistry, chromatin immunoprecipitation and Immunofluorescence. High expression of Runx2 is observed in the cases of breast cancer. The patients of high Runx2 expression at early stages display longer survival period, whereas the cases of high Runx2 at advanced stages reveal faster recurrence. The identification of signaling pathway indicates that JNK1 and c-Jun mediate Runx2 transcription. Repression of Runx2 reduces Brf1 expression and Pol III gene transcription. Further analysis indicates that Runx2 is colocalized with Brf1 in nucleus of breast cancer tissue. Both Runx2 and Brf1 synergistically modulate Pol III gene transcription. These studies indicate that Brf1 overexpression is able to be used as an early diagnosis biomarker of breast cancer, while high Runx2 expression indicates long survival period and faster recurrence. Runx2 mediates the deregulation of Brf1 and Pol III genes and its abnormal expression predicts the worse prognosis of breast cancer.

Upregulation of DPY30 promotes cell proliferation and predicts a poor prognosis in cholangiocarcinoma.

OBJECTIVES: Modification of lysine 4 on histone H3 methylation by SET1 and MLL family methyltransferase complexes is tightly linked to cancer progression. DPY30 is an important subunit of SET1 and MLL complexes, however, its expression and roles in cancer progression was little known, especially in cholangiocarcinoma (CCA). MATERIALS AND METHODS: The Q-PCR and IHC were performed to detect the levels of DPY30 mRNA and protein in CCA tissues. Effect of DPY30 knockdown on the proliferation of CCA cells was detected by MTS and colony formation, and cell cycle distribution was analyzed by flow cytometer. The glucose uptake, lactate release and ATP production assays were performed to detect the glycolysis of CCA cells. RESULTS: The level of DPY30 mRNA and protein in CCA tissues were all significantly higher than that of pericancer tissues, and its upregulation was closely associated with pathological differentiation, tumor size, and TNM stage. In addition, Kaplan-Meier analysis of overall survival revealed that DPY30 upregulation was significantly associated with poor survival, and univariate and multivariate analysis indicated that it was an independently prognosis factor in CCA patients. Moreover, DPY30 knockdown inhibited in-vitro growth and induced cell cycle arrest at G2/M and decreased glycolysis in CCA cells. CONCLUSIONS: DPY30 upregulation may promote the development of CCA and was associated with the aggressive malignant behavior and poor survival outcome of CCA patients. DPY30 might serve as a potential novel target for treatment of CCA patients.

The influences of red wine in phenotypes of human cancer cells.

Alcohol intake increases the risk of cancer development. Approximately 3.6% human cancers worldwide derive from chronic alcohol drinking, including oral, liver, breast and other organs. Our studies in vivo and in vitro have demonstrated that diluted ethanol increase RNA Pol III gene transcription and promotes cell proliferation and transformation, as well as tumor formation. However, it is unclear about the effect of red wines on the human cancer cells. In present study, we investigated the roles of red wine in human cancer cell growth, colony formation and RNA Pol III gene transcription. Low concentration (12.5 mM to 25 mM) of ethanol enhances cell proliferation of breast and esophageal cancer lines, whereas its higher concentration (100 mM to 200 mM) slightly decreases the rates. In contrast, red wines significantly repress cell proliferation of different human cancer lines from low dose to high dose. The results reveal that the red wine also inhibits colony formation of human breast cancer and esophageal carcinoma cells. The effects of repression on different human cancer lines are in a dose-dependent manner. Further analysis indicates that ethanol increases RNA Pol III gene transcription, whereas the red wines significantly reduce transcription of the genes. Interestingly, the effects of mature wine (brick red) on cancer cell phenotypes are much stronger than young wine (intense violet). Together, these new findings suggest that red wines may contain some bioactive components, which are able to inhibit human cancer cell growth and colony formation.

Natural killer cells inhibit pulmonary metastasis of hepatocellular carcinoma in nude mice.

Natural killer (NK) cells have been demonstrated to inhibit tumor growth. However, the role of NK cells in the inhibition of hepatocellular carcinoma metastasis is not well understood. The present study aimed to investigate the roles that NK cells may serve in inhibiting hepatocellular carcinoma metastasis. The role of isolated NK cells in the inhibition, proliferation, migration and invasion of the hepatoma cell line, MHCC97-H, was examined in vitro. Additionally, the survival rate of NK cells labeled with carboxyfluorescein diacetate-succinimidyl ester was assessed in vivo. An orthotopic implantation model was used to evaluate the role of NK cells in suppressing MHCC97-H cells in vivo. The effect of interleukin (IL)-2 stimulation on the tumor-inhibitory role of the NK cells was measured indirectly by analyzing the expression of various NK cell receptors and activated NK cell markers. It was observed that the NK cells inhibited the proliferation, migration and invasion of the MHCC97-H cells in vitro. Furthermore, the NK cells demonstrated long-term survival in the livers of the nude mice, and inhibited lung metastasis of hepatocellular carcinoma in vivo. However, liver tumor growth was not inhibited by the NK cells. IL-2 was identified to enhance the tumor-inhibitory effect of NK cells. The present study concludes that IL-2 may enhance the antitumor activity of the NK cells, and thereby inhibit the metastases of hepatocellular carcinoma in mice.

Activated hepatic stellate cells promote liver cancer by induction of myeloid-derived suppressor cells through cyclooxygenase-2.

Hepatic stellate cells (HSCs) are critical mediators of immunosuppression and the pathogenesis of hepatocellular carcinoma (HCC). Our previous work indicates that HSCs promote HCC progression by enhancing immunosuppressive cell populations including myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs). MDSCs are induced by inflammatory cytokines (e.g., prostaglandins) and are important in immune suppression. However, how HSCs mediate expansion of MDSCs is uncertain. Thus, we studied activated HSCs that could induce MDSCs from bone marrow cells and noted that HSC-induced MDSCs up-regulated immunosuppressive activity via iNOS, Arg-1, and IL-4Rα. After treating cells with a COX-2 inhibitor or an EP4 antagonist, we established that HSC-induced MDSC accumulation was mediated by the COX2-PGE2-EP4 signaling. Furthermore, in vivo animal studies confirmed that inhibition of HSC-derived PGE2 could inhibit HSC-induced MDSC accumulation and HCC growth. Thus, our data show that HSCs are required for MDSC accumulation mediated by the COX2-PGE2-EP4 pathway, and these data are the first to link HSC and MDSC subsets in HCC immune microenvironment and provide a rationale for targeting PGE2 signaling for HCC therapy.

Capsaicin Enhances the Drug Sensitivity of Cholangiocarcinoma through the Inhibition of Chemotherapeutic-Induced Autophagy.

Cholangiocarcinoma (CCA), a devastating cancer with a poor prognosis, is resistant to the currently available chemotherapeutic agents. Capsaicin, the major pungent ingredient found in hot red chili peppers of the genus Capsicum, suppresses the growth of several malignant cell lines. Our aims were to investigate the role and mechanism of capsaicin with respect to the sensitivity of CCA cells to chemotherapeutic agents. The effect of capsaicin on CCA tumor sensitivity to 5-fluorouracil (5-FU) was assessed in vitro in CCA cells and in vivo in a xenograft model. The drug sensitivity of QBC939 to 5-FU was significantly enhanced by capsaicin compared with either agent alone. In addition, the combination of capsaicin with 5-FU was synergistic, with a combination index (CI) < 1, and the combined treatment also suppressed tumor growth in the CCA xenograft to a greater extent than 5-FU alone. Further investigation revealed that the autophagy induced by 5-FU was inhibited by capsaicin. Moreover, the decrease in AKT and S6 phosphorylation induced by 5-FU was effectively reversed by capsaicin, indicating that capsaicin inhibits 5-FU-induced autophagy by activating the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway in CCA cells. Taken together, these results demonstrate that capsaicin may be a useful adjunct therapy to improve chemosensitivity in CCA. This effect likely occurs via PI3K/AKT/mTOR pathway activation, suggesting a promising strategy for the development of combination drugs for CCA.

[Immunocharacteristics of bone marrow mesenchymal stem cell].

OBJECTIVE: To study the immunocharacteristics of bone marrow mesenchymal stem cell (MSC) and provide experimental evidence for the potential therapeutic application. METHODS: MSCs were isolated from rat bone marrow and confirmed by immunophenotype, and the growth dynamic and cell cycle were analyzed. MSCs were cultured with or without 200 U/ml interferon gamma (IFNg) , the expression of PDL-1, CD54, CD40, CD80, CD86, MHC-I, and MHC-II was detected by flow cytometry. MSCs were used as regulatory cells in mixed lymphocyte reaction (MLR), the PDL-1 and CD54 molecules on MSCs were blocked to explore their roles in MLR. The IFN, IL-2, IL-4 and IL-10 molecules in culture supernatant were quantified by ELISA. The homing of MSCs to liver and induction of microchimerism were analyzed after MSCs transplantation. RESULTS: The purity of MSCs was high. The growth curve showed that the first two days were the lag phase; the third, fourth, fifth days were the log phase; the sixth and seventh days were the stationary phase. Flow cytometry indicated that 76.0%+/-2.0% of the MSCs were in G1/G0 phase, 13.0%+/-2.0% in S phase, 10.0%+/-1.7% in G2 and M phase. IFNg treatment led to up-regulation of CD54, PDL-1, MHC-I and MHC-II, however, CD40, CD80 and CD86 were not expressed on MSCs even after IFNg treatment. MSCs inhibited MLR, IFNg treatment enhanced the inhibitory effect of MSCs on MLR. Blocking of PDL-1 or CD54 on MSCs partially alleviated the inhibition effect. There were high levels of IFNg and IL-10, and low level of IL-4 in the culture supernatant of MLR, however, IL-2 was not detected. MSCs can home to the liver and induce formation of microchimerism after transplantation. CONCLUSION: IFNg treatment enhances the inhibitory effect of MSCs on MLR, PDL-1 and CD54 are key molecules mediating this inhibitory effect. MSC can home to the liver and induce formation of microchimerism after transplantation.