[Identification and expression analysis of whole gene family of Isatis indigotica 4-coumarate: CoA ligase].

The 4-coumarate: CoA ligase(4CL) is a key enzyme in the upstream pathway of phenylpropanoids such as flavonoids, soluble phenolic esters, lignans, and lignins in plants. In this study, 13 4CL family members of Arabidopsis thaliana were used as reference sequences to identify the 4CL gene family candidate members of Isatis indigotica from the reported I. indigotica genome. Further bioinformatics analysis and analysis of the expression pattern of 4CL genes and the accumulation pattern of flavonoids were carried out. Thirteen 4CL genes were obtained, named Ii4CL1-Ii4CL13, which were distributed on chromosomes 1, 2, 3, 4, and 6. The analysis of the gene structure and conserved structural domains revealed the intron number of I. indigotica 4CL genes was between 1 and 12 and the protein structural domains were highly conserved. Cis-acting element analysis showed that there were multiple response elements in the promoter sequence of I. indigotica 4CL gene family, and jasmonic acid had the largest number of reaction elements. The collinearity analysis showed that there was a close relationship between the 4CL gene family members of I. indigotica and A. thaliana. As revealed by qPCR results, the expression analysis of the 4CL gene family showed that 10 4CL genes had higher expression levels in the aboveground part of I. indigotica. The content assay of flavonoids in different parts of I. indigotica showed that flavonoids were mainly accumulated in the aboveground part of plants. This study provides a basis for further investigating the roles of the 4CL gene family involved in the biosynthesis of flavonoids in I. indigotica.

Methyltransferase Setd2 prevents T cell-mediated autoimmune diseases via phospholipid remodeling.

Coordinated metabolic reprogramming and epigenetic remodeling are critical for modulating T cell function and differentiation. However, how the epigenetic modification controls Th17/Treg cell balance via metabolic reprogramming remains obscure. Here, we find that Setd2, a histone H3K36 trimethyltransferase, suppresses Th17 development but promotes iTreg cell polarization via phospholipid remodeling. Mechanistically, Setd2 up-regulates transcriptional expression of lysophosphatidylcholine acyltransferase 4 (Lpcat4) via directly catalyzing H3K36me3 of Lpcat4 gene promoter in T cells. Lpcat4-mediated phosphatidylcholine PC(16:0,18:2) generation in turn limits endoplasmic reticulum stress and oxidative stress. These changes decrease HIF-1α transcriptional activity and thus suppress Th17 but enhance Treg development. Consistent with this regulatory paradigm, T cell deficiency of Setd2 aggravates neuroinflammation and demyelination in experimental autoimmune encephalomyelitis due to imbalanced Th17/Treg cell differentiation. Overall, our data reveal that Setd2 acts as an epigenetic brake for T cell-mediated autoimmunity through phospholipid remodeling, suggesting potential targets for treating neuroinflammatory diseases.

Metabolic enzyme Suclg2 maintains tolerogenicity of regulatory dendritic cells diffDCs by suppressing Lactb succinylation.

Metabolic reprogramming plays a pivotal role in the differentiation and function of immune cells including dendritic cells (DCs). Regulatory DCs can be generated in regional tissue niches like splenic stroma and act as an important part of stromal control of immune response for the maintenance of immune tolerance. However, the metabolic alterations during splenic stroma-driven regulatory DCs differentiation and the metabolic enzyme involved in regulatory DCs function remain poorly understood. By combining metabolomic, transcriptomic, and functional investigations of mature DCs (maDCs) and diffDCs (regulatory DCs differentiated from activated mature DCs through coculturing with splenic stroma), here we identified succinate-CoA ligase subunit beta Suclg2 as a key metabolic enzyme that reprograms the proinflammatory status of mature DCs into a tolerogenic phenotype via preventing NF-κB signaling activation. diffDCs downregulate succinic acid levels and increase the Suclg2 expression along with their differentiation from mature DCs. Suclg2-interference impaired the tolerogenic function of diffDCs in inducing T cell apoptosis and enhanced activation of NF-κB signaling and expression of inflammatory genes CD40, Ccl5, and Il12b in diffDCs. Furthermore, we identified Lactb as a new positive regulator of NF-κB signaling in diffDCs whose succinylation at the lysine 288 residue was inhibited by Suclg2. Our study reveals that the metabolic enzyme Suclg2 is required to maintain the immunoregulatory function of diffDCs, adding mechanistic insights into the metabolic regulation of DC-based immunity and tolerance.

Glycosyltransferase Extl1 promotes CCR7-mediated dendritic cell migration to restrain infection and autoimmunity.

CCR7-triggered DC migration toward draining lymph nodes is critical for the initiation of protective immunity and maintenance of immune tolerance. How to promote CCR7-mediated DC migration to determine T cell responses under inflammatory and homeostatic conditions remains poorly understood. Here we demonstrate that the Extl1 (Exostosin like glycosyltransferase 1) promotes CCR7-triggered DC migration in a heparan sulfate proteoglycans (HSPG)-dependent manner. Mechanistically, Extl1 mediates HSPG production via its glycosyltransferase domain to inhibit C1q expression. Extl1/HSPG axis relieves C1q-mediated restriction of CCR7 surface expression and internalization, and thus enhances CCR7-dependent migratory signaling activation. Consequently, Extl1 is required for DC-mediated Th1 and Th17 responses in immune defense against bacterial infection and for Treg cell development in the prevention of autoimmunity. Our study adds mechanistic insights to the regulation of CCR7-triggered DC migration in immunity and tolerance and provides a potential target for the treatment of infectious and autoimmune diseases.

RNA-binding protein and tumor biotherapy: new opportunities and strategies / 中国肿瘤生物治疗杂志

@#RNA结合蛋白（RBP）由于其独特的生物学功能，目前已经成为肿瘤生物治疗相关靶点筛选的宠儿，很可能为肿瘤生物治疗带来新的机遇。RBP能调控肿瘤细胞及肿瘤微环境免疫细胞和间质细胞的DNA-RNA-蛋白质相互作用网络，进而广泛影响肿瘤发生发展、抗肿瘤免疫应答及肿瘤免疫逃逸过程，目前RBP相关肿瘤生物治疗的研发，主要聚焦在治疗性疫苗、免疫细胞治疗、表观调控治疗等方面，部分研发成果已处于临床试验阶段。随着新理论、新技术的发展以及研究模式的创新，靶向RBP的治疗逐渐摆脱了既往靶向难、疗效欠佳的困局，迎来了新的机遇，通过改良精准靶向和优化组合用药等新策略，为肿瘤生物治疗注入了新的活力，对精准个体化医疗的发展具有重要意义。

lncRNA-GM targets Foxo1 to promote T cell-mediated autoimmunity.

RNA-RBP interaction is important in immune regulation and implicated in various immune disorders. The differentiation of proinflammatory T cell subset TH17 and its balance with regulatory T cell (Treg) generation is closely related to autoimmune pathogenesis. The roles of RNA-RBP interaction in regulation of TH17/Treg differentiation and autoinflammation remain in need of further investigation. Here we report that lncRNA-GM polarizes TH17 differentiation but inhibits iTreg differentiation by reducing activity of Foxo1, a transcriptional factor that is important in inhibiting TH17 differentiation but promoting Treg generation. lncRNA-GM-deficient mice were protected from experimental autoimmune encephalomyelitis. Mechanistically, lncRNA-GM directly binds to cytoplasmic Foxo1, thus inhibiting its activity through blocking dephosphorylation of Foxo1 by phosphatase PP2A to promote Il23r transcription. The human homolog of lncRNA-GM (AK026392.1) also polarizes human TH17 differentiation. Our study provides mechanistic insight into the interaction of lncRNA and transcriptional factor in determining T cell subset differentiation during T cell-mediated autoimmune diseases.

RNA-binding protein ZCCHC4 promotes human cancer chemoresistance by disrupting DNA-damage-induced apoptosis.

RNA-binding proteins (RBPs) play important roles in cancer development and treatment. However, the tumor-promoting RBPs and their partners, which may potentially serve as the cancer therapeutic targets, need to be further identified. Here, we report that zinc finger CCHC domain-containing protein 4 (ZCCHC4) is of aberrantly high expression in multiple human cancer tissues and is associated with poor prognosis and chemoresistance in patients of hepatocellular carcinoma (HCC), pancreatic cancer and colon cancer. ZCCHC4 promotes chemoresistance of HCC cells to DNA-damage agent (DDA) both in vitro and in vivo. HCC cell deficiency of ZCCHC4 reduces tumor growth in vivo and intratumoral interference of ZCCHC4 expression obviously enhances the DDA-induced antitumor effect. Mechanistically, ZCCHC4 inhibits DNA-damage-induced apoptosis in HCC cells by interacting with a new long noncoding RNA (lncRNA) AL133467.2 to hamper its pro-apoptotic function. Also, ZCCHC4 blocks the interaction between AL133467.2 and γH2AX upon DDA treatment to inhibit apoptotic signaling and promote chemoresistance to DDAs. Knockout of ZCCHC4 promotes AL133467.2 and γH2AX interaction for enhancing chemosensitivity in HCC cells. Together, our study identifies ZCCHC4 as a new predictor of cancer poor prognosis and a potential target for improving chemotherapy effects, providing mechanistic insights to the roles of RBPs and their partners in cancer progression and chemoresistance.

Deficiency of GFRα1 promotes hepatocellular carcinoma progression but enhances oxaliplatin-mediated anti-tumor efficacy.

Neurotrophic factors and their receptors have been identified to promote tumor progression. GFRα1, the receptor for glial cell line-derived neurotrophic factor (GDNF), has been demonstrated to be predominantly expressed in adult liver tissue. Our preliminary data showed that GFRα1 is significantly downregulated in hepatocellular carcinoma (HCC) tissue, compared to the matched non-neoplastic tissue. However, the role of GFRα1 in HCC progression remains unknown. Here we found that the expression of GFRα1 in HCC tissue is inversely correlated with the poorer prognosis of HCC patients. Silencing of GFRα1 expression markedly enhances HCC cell growth, tumor metastasis, as well as shortens the survival of HCC tumor-bearing mice. Forced expression of GFRα1 in HCC cells significantly reverses the tumor-promoting effects of GFRα1 silencing, and AAV8-mediated GFRα1 transfection in HCC tumor tissues significantly impedes tumor growth and prolongs the survival of HCC tumor-bearing mice. These results are also verified in vivo in GFRα1 knock-out mice model, with increased DEN-induced HCC carcinogenesis. Mechanistically, GFRα1 could inhibit epithelial-to-mesenchymal transition (EMT) of HCC cells, by upregulating expression of Claudin-1 and ZO-1. Of note, silencing of GFRα1 expression promotes oxaliplatin-mediated HCC cell apoptosis resulting in prolonged survival of HCC-bearing mice, and forced expression of GFRα1 markedly increased oxaliplatin resistance of HCC cells. These results demonstrate that deficiency of GFRα1 promotes HCC progression but enhances chemotherapeutic anti-tumor efficacy, suggesting that GFRα1 may be a candidate prognostic biomarker and a potential therapeutic target in HCC.

Do Elderly Patients With Stage I-II Hepatocellular Carcinoma Benefit From More Radical Surgeries? A Population-Based Analysis.

Background and Aims: The best treatment modalities for elderly patients with stage I-II HCC (hepatocellular carcinoma) remain controversial in an era of a shortage of liver donors. Methods: From the SEER database (Surveillance, Epidemiology, and End Results program), 2,371 elderly patients were sampled as Cohort 1. OS (Overall Survival) and CSS (Cancer-Specific Survival) were compared between the Non-surgery and Surgery groups. A stratification analysis in a CSS Cox model was also conducted among sub-groups, and propensity score matching was performed to generate Cohort 2 (746 pairs), reducing the influences of confounders. Results: For Cohort 1, the median follow-up times of the Non-surgery and Surgery groups were 11 months (95% CI, confidence interval: 9.74-12.26) vs. 49 months (44.80-53.21) in OS, and 14 months (12.33-15.67) vs. 74 months (64.74-83.26) in CSS, respectively. In the stratification analysis, for the elderly patients (age >= 70 years), Larger Resection was associated with a higher HR (hazard ratio) than Segmental Resection: 0.30 (95% CI, confidence interval: 0.22-0.41) vs. 0.29 (0.21-0.38) in 70-74 year-olds; 0.26 (0.18-0.38) vs. 0.23 (0.16-0.32) in 75-79 year-olds; 0.32 (0.21-0.49) vs. 0.21 (0.13-0.32) in those 80+ years old. For Cohort 2, a similar result could be seen in the CSS Cox forest plot. The HRs of Larger Resection and Segmental Resection were 0.27 (0.21-0.33) and 0.25 (0.20-0.31), respectively. Conclusions: It is cautiously recommended that, when liver transplantation is not available, segmental or wedge liver resection is the better treatment choice for elderly patients with stage I-II HCC (AJCC edition 6), especially those over 70 years old, compared with other surgeries, based on the SEER data.

CCR7 Chemokine Receptor-Inducible lnc-Dpf3 Restrains Dendritic Cell Migration by Inhibiting HIF-1α-Mediated Glycolysis.

CCR7 chemokine receptor stimulation induces rapid but transient dendritic cell (DC) migration toward draining lymph nodes, which is critical for the initiation of protective immunity and maintenance of immune homeostasis. The mechanisms for terminating CCR7-mediated DC migration remain incompletely understood. Here we have identified a long non-coding RNA lnc-Dpf3 whose feedback restrained CCR7-mediated DC migration. CCR7 stimulation upregulated lnc-Dpf3 via removing N6-methyladenosine (m6A) modification to prevent RNA degradation. DC-specific lnc-Dpf3 deficiency increased CCR7-mediated DC migration, leading to exaggerated adaptive immune responses and inflammatory injuries. Mechanistically, CCR7 stimulation activated the HIF-1α transcription factor pathway in DCs, leading to metabolic reprogramming toward glycolysis for DC migration. lnc-Dpf3 directly bound to HIF-1α and suppressed HIF-1α-dependent transcription of the glycolytic gene Ldha, thus inhibiting DC glycolytic metabolism and migratory capacity. We demonstrate a critical role for CCR7-inducible lnc-Dpf3 in coupling epigenetic and metabolic pathways to feedback-control DC migration and inflammatory responses.

Tumor-Induced Generation of Splenic Erythroblast-like Ter-Cells Promotes Tumor Progression.

Identifying tumor-induced leukocyte subsets and their derived circulating factors has been instrumental in understanding cancer as a systemic disease. Nevertheless, how primary tumor-induced non-leukocyte populations in distal organs contribute to systemic spread remains poorly defined. Here, we report one population of tumor-inducible, erythroblast-like cells (Ter-cells) deriving from megakaryocyte-erythroid progenitor cells with a unique Ter-119+CD45-CD71+ phenotype. Ter-cells are enriched in the enlarged spleen of hosts bearing advanced tumors and facilitate tumor progression by secreting neurotrophic factor artemin into the blood. Transforming growth factor ß (TGF-ß) and Smad3 activation are important in Ter-cell generation. In vivo blockade of Ter-cell-derived artemin inhibits hepatocellular carcinoma (HCC) growth, and artemin deficiency abolishes Ter-cells' tumor-promoting ability. We confirm the presence of splenic artemin-positive Ter-cells in human HCC patients and show that significantly elevated serum artemin correlates with poor prognosis. We propose that Ter-cells and the secreted artemin play important roles in cancer progression with prognostic and therapeutic implications.

Small GTPase RBJ promotes cancer progression by mobilizing MDSCs via IL-6.

RBJ has been identified to be dysregulated in gastrointestinal cancer and promotes tumorigenesis and progression by mediating nuclear accumulation of active MEK1/2 and sustained activation of ERK1/2. Considering that nuclear accumulation and constitutive activation of MEK/ERK not only promotes tumor progression directly, but also induces chronic inflammation, we wonder whether and how RBJ impairs host immune-surveillance via chronic inflammation and consequently supports tumor progression. Here, we report that higher expression of RBJ in human breast cancer tissue has been significantly correlated with poorer prognosis in breast cancer patients. The forced expression of RBJ promotes tumor growth and metastasis both in vitro and in vivo. In addition, more accumulation of immune suppressive cells but less antitumor immune cell subpopulations were found in spleen and tumor tissue derived from RBJ force-expressed tumor-bearing mice. Furthermore, forced RBJ expression significantly promotes tumor cell production of pro-inflammatory cytokine IL-6 by constitutive activating MEK/ERK signaling pathway. Accordingly, RBJ knockdown significantly decreases tumor growth and metastasis in vitro and in vivo, with markedly reduced production of IL-6. Administration of anti-IL-6 neutralizing antibody could reduce MDSCs accumulation in tumor tissue in vivo. Therefore, our results demonstrate that RBJ-mediated nuclear constitutive activation of ERK1/2 leads to persistent production of IL-6 and increase of MDSCs recruitment, contributing to promotion of tumor growth and metastasis. These results suggest that RBJ contributes to tumor immune escape, maybe serving a potential target for design of antitumor drug.

CXCR2+ MDSCs promote breast cancer progression by inducing EMT and activated T cell exhaustion.

Although myeloid-derived suppressor cells (MDSCs) have been demonstrated to contribute to tumor initiation, progression and metastasis, however, which MDSC subsets are preferentially expanded and activated, and what's the key molecular mechanism responsible for specific MDSC subsets in promoting tumor progression need to be fully addressed. Here we identify that Ly6GmiLy6CloCD11b+CXCR2+ subpopulation (named CXCR2+ MDSCs) are predominately expanded and recruited in systemic and local tumor microenvironment during breast cancer progression and metastasis. The proportion of CXCR2+ MDSCs is inversely correlated with the infiltration of CD4+ or CD8+ T cells. Besides, CXCR2+ MDSCs promote breast cancer growth and metastasis to lung and/or lymph node in vivo. Furthermore, CXCR2+ MDSCs induce epithelial mesenchymal transition (EMT) of breast cancer cells via IL-6. Moreover, CXCR2+ MDSCs upregulate the expression of immunosuppressive molecules programmed cell death protein 1(PD1), PD1 ligand 1 (PDL1), lymphocyte activation gene 3 protein (LAG3), cytotoxic T lymphocyte antigen 4 (CTLA4), and T cell immunoglobulin domain and mucin domain protein 3 (TIM3) on CD4+ or CD8+ T cells, and induce exhaustion of the activated T cells partially via IFN-γ. These results demonstrate that CXCR2+ MDSCs accelerate breast cancer progression via directly inducing cancer cell EMT and indirectly promoting T cell exhaustion, suggesting that CXCR2+ MDSCs may be a potential therapeutic target of breast cancer.

Tumor-Derived CXCL1 Promotes Lung Cancer Growth via Recruitment of Tumor-Associated Neutrophils.

Neutrophils have a traditional role in inflammatory process and act as the first line of defense against infections. Although their contribution to tumorigenesis and progression is still controversial, accumulating evidence recently has demonstrated that tumor-associated neutrophils (TANs) play a key role in multiple aspects of cancer biology. Here, we detected that chemokine CXCL1 was dramatically elevated in serum from 3LL tumor-bearing mice. In vitro, 3LL cells constitutively expressed and secreted higher level of CXCL1. Furthermore, knocking down CXCL1 expression in 3LL cells significantly hindered tumor growth by inhibiting recruitment of neutrophils from peripheral blood into tumor tissues. Additionally, tumor-infiltrated neutrophils expressed higher levels of MPO and Fas/FasL, which may be involved in TAN-mediated inhibition of CD4(+) and CD8(+) T cells. These results demonstrate that tumor-derived CXCL1 contributes to TANs infiltration in lung cancer which promotes tumor growth.

Endometrial stromal cells and decidualized stromal cells: origins, transformation and functions.

Decidualization of endometrium, which is characterized by endometrial stromal cell (ESC) decidualization, vascular reconstruction, immune cell recruitment, and plentiful molecule production, is a crucial step for uterus to become receptive for embryo. When implantation takes place, ESCs surround and directly interact with embryo. Decidualized stromal cells (DSCs) are of great importance in endometrial decidualization, having a broad function in regulating immune activity and vascular remodeling of uterus. DSCs are shown to have a higher metabolic level and looser cytoskeleton than ESCs. What's the origin of ESCs and how ESCs successfully transform into DSCs had puzzled scientists in the last decades. Breakthrough had been achieved recently, and many studies had elucidated some of the characters and functions of DSCs. However, several questions still remain unclear. This paper reviews current understanding of where ESCs come from and how ESCs differentiate into DSCs, summarizes some characters and functions of DSCs, analyzes current studies and their limitations and points out research areas that need further investigation.

[Molecular genetics study of ED1 gene for two X-linked hypohidrotic ectodermal dysplasia families].

OBJECTIVE: To provide genetic diagnosis and counseling for patients from two families affected with X-linked hypohidrotic ectodermal dysplasia. METHODS: Potential mutation of the ED1 gene was screened by DNA sequencing. For family 1, multiplex ligation-dependent probe amplification (MLPA) analysis and haplotyping of ED1 gene were also carried out for prenatal diagnosis. RESULTS: For the patient from family 1, deletion of the exon 1 of the ED1 gene and 2 short tandem repeat(STR) sites (DXS8269 and DXS1422) were detected. His daughter was carrier of the deletion. Upon prenatal diagnosis, the fetus was confirmed to be a normal male, for whom the haplotype of ED1 gene has differed from that of the proband. In family 2, a c.463C>T mutation in exon 3 of the ED1 gene was detected in the proband, whose mother was heterozygous for the same mutation. CONCLUSION: The deletion (exon 1) and missense (R155C) mutation in ED1 gene have probably underlied the disease in the two families. During prenatal diagnosis, it may be necessary to obtain precise results through combining mutation detection and haplotype analysis of the ED1 gene.