👤 Cuizhi Geng

Endoplasmic reticulum (ER) stress is associated with liver inflammation and hepatocellular carcinoma (HCC). However, how ER stress links inflammation and HCC remains obscure. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an ER stress-inducible secretion protein that inhibits inflammation by interacting with the key subunit of nuclear factor kappa light chain enhancer of activated B cells (NF-κB) p65. We hypothesized that MANF may play a key role in linking ER stress and inflammation in HCC. Here, we found that MANF mRNA and protein levels were lower in HCC tissues versus adjacent noncancer tissues. Patients with high levels of MANF had better relapse-free survival and overall survival rates than those with low levels. MANF levels were also associated with the status of liver cirrhosis, advanced tumor-node-metastasis (TNM) stage, and tumor size. In vitro experiments revealed that MANF suppressed the migration and invasion of hepatoma cells. Hepatocyte-specific deletion of MANF accelerated N-nitrosodiethylamine (DEN)-induced HCC by up-regulating Snail1+2 levels and promoting epithelial-mesenchymal transition (EMT). MANF appeared in the nuclei and was colocalized with p65 in HCC tissues and in tumor necrosis factor alpha (TNF-α)-treated hepatoma cells. The interaction of p65 and MANF was also confirmed by coimmunoprecipitation experiments. Consistently, knockdown of MANF up-regulated NF-κB downstream target genes TNF-α, interleukin (IL)-6 and IL-1α expression in vitro and in vivo. Finally, small ubiquitin-related modifier 1 (SUMO1) promoted MANF nuclear translocation and enhanced the interaction of MANF and p65. Mutation of p65 motifs for SUMOylation abolished the interaction of p65 and MANF. MANF plays an important role in linking ER stress and liver inflammation by inhibiting the NF-κB/Snail signal pathway in EMT and HCC progression. Therefore, MANF may be a cancer suppressor and a potential therapeutic target for HCC. Show less

Background Endometrial adenocarcinoma (EAC) is one of the most commonly diagnosed gynaecological malignancies among female population of the developed countries. DUSP6 is a negative regulator of ERK signaling, which is a molecular switch involved in MAPK signaling during the progress of malignancies. DUSP6 was previously found to inhibit tumorigenesis and EMT-associated properties in several cancers, however, its exact role in EAC remains unclear Methods The level of DUSP6, (E-cad) and (N-cad) in EAC cancerous tissues and respective adjacent non-cancerous tissues were examined by western-blot or immunohistochemistry. The cell growth, invasion and migration abilities were measured in Ishikawa 3H12 endometrial cancer cell lines with overexpressed or knock down DUSP6. Protein levels of EMT-associated markers E-cadherin, N-cadherin and Vimentin were also determined. The impacts of DUSP6 on ERK signaling was assessed by detection of ERK and p-ERK. Results Down-regulation of DUSP6 was observed in EAC compared with the normal controls. The overexpression of DUSP6 significantly attenuated tumor cell growth, invasion, migration abilities and inhibited EMT-associated markers, while knock down of DUSP6 showed opposite trends. Overexpression of DUSP6 also down-regulated p-ERK and the knock down of DUSP6 inversely up-regulated p-ERK level. Conclusions DUSP6 inhibited cell growth, invasion and migration abilities in Ishikawa 3H12 cells as well as attenuating EMT-associated properties. This tumor suppressive effect of DUSP6 in EAC is achieved by inhibiting ERK signaling pathway. Show less

Family with sequence similarity 83, member A (FAM83A), as a potential tumor promoter, was reported to contribute to the progression of several malignant tumors. However, the significance of FAM83A in invasion and metastasis of non-small cell lung cancer (NSCLC) remains largely unknown. In this study, we found that FAM83A expression was significantly increased in NSCLC tissues. High expression of FAM83A was positively associated with tumor metastasis and poor survival of NSCLC patients. Functional experiments revealed that FAM83A knockdown could suppress NSCLC cell migration and invasion both Show less

Anaplastic thyroid carcinoma (ATC) is the most aggressive type of thyroid cancer, with no effective treatment available. Identification of new anti-ATC drugs represents an urgent need. In this study, we find that ATC cells are highly sensitive to THZ531, a potent inhibitor of the transcriptional cyclin-dependent kinase (CDK), CDK12. Cell-based assays demonstrate that CDK12 inhibition significantly impedes cell cycle progression, induces apoptotic cell death, and impairs colony formation in ATC cells. THZ531 causes a loss of elongating RNA polymerase II and suppresses gene expression in ATC cells. An integrative analysis of gene expression profiles and super-enhancer landscape, combining with functional assays, leads to the discovery of two new ATC cancer genes, ZC3H4 and NEMP1. Furthermore, CDK12 inhibition enhances the sensitivity of ATC cells to doxorubicin-mediated chemotherapy. Thus, these findings indicate that CDK12 is a potential therapeutic target for ATC treatment and its inhibition may help to overcome the chemoresistance in patients with ATC. Show less

Endothelial barrier function in the onset and Tongxinluo (TXL) protection of myocardial ischemia/reperfusion (I/R) injury, and TXL can induce the secretion of Angiopoietin-like 4 (Angptl4) in human cardiac microvascular endothelial cells during hypoxia/reoxygenation. We intend to demonstrate whether TXL can attenuate myocardial I/R injury in diabetes, characterized with microvascular endothelial barrier disruption, by induction of Angptl4-mediated protection of endothelial barrier integrity. I/R injury was created by coronary ligation in ZDF diabetic and non-diabetic control rats. The animals were anesthetized and randomized to sham operation or I/R injury with or without the exposure to insulin, rhAngptl4, TXL, Angptl4 siRNA, and the PPAR-α inhibitor MK886. Tongxinluo, insulin and rhAngptl4 have the similar protective effect on diabetic hearts against I/R injury. In I/R-injured diabetic hearts, TXL treatment remarkably reduced the infarct size, and protected endothelial barrier integrity demonstrated by decreased endothelial cells apoptosis, microvascular permeability, and myocardial hemorrhage, fortified tight junction, and upregulated expression of JAM-A, integrin-α5, and VE-cadherin, and these effects of TXL were as effective as insulin and rhAngptl4. However, Angptl4 knock-down with siRNA interference and inhibition of PPAR-α with MK886 partially diminished these beneficial effects of TXL and rhAngptl4. TXL induced the expression of Angptl4 in I/R-injured diabetic hearts, and was canceled by Angptl4 siRNA and MK886. TXL treatment increased myocardial PPAR-α activity, and was abolished by MK886 but not by Angptl4 siRNA. TXL protects diabetic hearts against I/R injury by activating Angptl4-mediated restoration of endothelial barrier integrity via the PPAR-α pathway. Show less

Protein kinase N2 (PKN2) is a PKC-related serine/threonine-protein kinase. PKN2 is required for tumor cell migration, invasion and apoptosis. However, the functional role of PKN2 in regulating tumor associated macrophages (TAMs) polarization in colon cancer has never been reported. PKN2 expression in human colon cancer tissues was examined with immunohistochemistry (IHC). M1/M2 macrophage signatures were evaluated by RT-PCR, IHC and flow cytometry. The effects of PKN2 on tumor growth and TAM polarization were investigated both in vitro and in vivo. PKN2 targeted cytokines/pathway were analyzed by gene expression analysis and further confirmed by PCR, luciferase assay or western blot. Correlations between PKN2 and transcriptional factors for IL4 and IL10 were confirmed by ChIP-qPCR. The catalytic activities of PKN2 and DUSP6 were determined by kinase activity assay. Interactions between PKN2 and DUSP6 were confirmed by Co-IP. The expression of PKN2 in colon cancer cells predicted a favorable prognosis and was associated with low M2 macrophage content in human colon cancer tissues. PKN2 inhibited tumor growth in mice xenograft model and inhibited M2 phenotype polarization both in vitro and in vivo. Mechanistically, PKN2 suppresses the expression of IL4 and IL10 from colon cancer cells by inhibiting Erk1/2 phosphorylation, which is required for phosphorylation and binding of CREB and Elk-1 to the promoters of IL4 and IL10. DUSP6, which is phosphorylated and activated through direct association with PKN2, suppresses Erk1/2 activation. The expression of PKN2 in colon cancer cells suppresses tumor associated M2 macrophage polarization and tumor growth. Targeting PKN2 signaling pathway may provide a potential therapeutic strategy for colon cancer. Show less

A large number of drugs can induce prolongation of cardiac repolarization and life-threatening cardiac arrhythmias. The prediction of this side effect is however challenging as it usually develops in some genetically predisposed individuals with normal cardiac repolarization at baseline. Here, we describe a platform based on a genetically diverse panel of induced pluripotent stem cells (iPSCs) that reproduces susceptibility to develop a cardiotoxic drug response. We generated iPSC-derived cardiomyocytes from patients presenting in vivo with extremely low or high changes in cardiac repolarization in response to a pharmacological challenge with sotalol. In vitro, the responses to sotalol were highly variable but strongly correlated to the inter-individual differences observed in vivo. Transcriptomic profiling identified dysregulation of genes ( Show less

Dual-specificity MAP kinase (MAPK) phosphatases (DUSPs) are well-established negative modulators in regulating MAPK signaling in mammalian cells and tissues. Our previous studies have shown the involvement of DUSP6 in regulating innate immunity in Japanese flounder Paralichthys olivaceus. In order to gain a better understanding of the role of DUSPs in fish innate immunity, in the present study we identified and characterized three additional DUSP genes including DUSP1, 2 and 5 in P. olivaceus. The three Japanese flounder DUSP proteins share common domain structures composed of a conserved N-terminal Rhodanase/CDC25 domain and a C-terminal catalytic phosphatase domain, while they show only less than 26% sequence identities, indicating that they may have different substrate selectivity. In addition, mRNA transcripts of all the three DUSP genes are detected in all examined Japanese flounder tissues; however, DUSP1 is dominantly expressed in spleen while DUSP2 and 5 are primarily expressed in skin. Furthermore, all the three DUSP genes are constitutively expressed in the Japanese flounder head kidney macrophages (HKMs) and peripheral blood leucocytes (PBLs) with unequal distribution patterns. Moreover, all the three DUSPs gene expression was induced differently in response to the LPS and double-stranded RNA mimic poly(I:C) stimulations both in the Japanese flounder HKMs and PBLs, suggesting an association of DUSPs with TLR signaling in fish. Taken together, the co-expression of various DUSPs members together with their different responses to the immune challenges indicate that the DUSP members may operate coordinately in regulating the MAPK-dependent immune responses in the Japanese flounder. Show less

Dual-specificity phosphatase 6 (Dusp6) is a member of mitogen-activated protein kinase (MAPK) phosphatases that play crucial roles in regulating MAPK signaling and immune response. The immunological relevance of Dusp6 in fish, however, remains largely uncharacterized. In the present study, a full-length Japanese flounder dusp6 cDNA ortholog, termed PoDusp6, was identified and characterized from Paralichthys olivaceus. The deduced PoDusp6 protein is comprised of 383 amino acids with a conserved N-terminal regulatory rhodanese homology domain and a C-terminal catalytic domain. Immunofluorescence microscopy revealed that PoDusp6 protein is mainly localized in cytoplasm. Sequence analysis indicates that PoDusp6 is highly conserved (>70% identity) throughout the evolution from teleost to mammals. In unstimulated conditions, PoDusp6 mRNA was present in all examined tissues and showed the highest expression in Japanese flounder head kidney macrophages (HKMs). Immune challenge experiments revealed that the expression of PoDusp6 was down-regulated at the early stage after LPS and poly(I:C) stimulations but significantly up-regulated at the later stage in the HKMs. The similar expression pattern was also observed in the Japanese flounder immune-related tissues including head kidney, gill and spleen upon bacterial challenge with Edwardsiella tarda. Overexpression of PoDusp6 in Japanese flounder FG-9307 cells led to a significant down-regulation of proinflammatory cytokine genes IL-1beta, TNF-alpha and IFN-gamma, and antiviral gene Mx. Interestingly, inhibition of Dusp6 activity also down-regulated the LPS-induced IL-beta gene expression but did not affected on the LPS-induced IFN-gamma and TNF-alpha expression in the HKMs. Our findings suggest that the expression of PoDusp6 is modulated by immune stimuli and PoDusp6 may act as an essential modulator in fish inflammatory response. Show less

Global prevalence of non-alcoholic fatty liver disease (NAFLD) constitutes a threat to human health. Goose is a unique model of NAFLD for discovering therapeutic targets as its liver can develop severe steatosis without overt injury. Fatty acid desaturase (Fads) is a potential therapeutic target as Fads expression and mutations are associated with liver fat. Here, we hypothesized that Fads was promoted to provide a protection for goose fatty liver. To test this, goose Fads1 and Fads2 were sequenced. Fads1/2/6 expression was determined in goose liver and primary hepatocytes by quantitative PCR. Liver fatty acid composition was also analyzed by gas chromatography. Data indicated that hepatic Fads1/2/6 expression was gradually increased with the time of overfeeding. In contrast, trans-C18:1n9 fatty acid (Fads inhibitor) was reduced. However, enhanced Fads capacity for long-chain polyunsaturated fatty acid (LC-PUFA) synthesis was not sufficient to compensate for the depleted LC-PUFAs in goose fatty liver. Moreover, cell studies showed that Fads1/2/6 expression was regulated by fatty liver-associated factors. Together, these findings suggest Fads1/2 as protective components are promoted to meet instant need for LC-PUFAs in goose fatty liver, and we propose this is required for severe hepatic steatosis without liver injury. Show less

Studying mechanisms of malignant transformation of human pre-B cells, we found that acute activation of oncogenes induced immediate cell death in the vast majority of cells. Few surviving pre-B cell clones had acquired permissiveness to oncogenic signaling by strong activation of negative feedback regulation of Erk signaling. Studying negative feedback regulation of Erk in genetic experiments at three different levels, we found that Spry2, Dusp6, and Etv5 were essential for oncogenic transformation in mouse models for pre-B acute lymphoblastic leukemia (ALL). Interestingly, a small molecule inhibitor of DUSP6 selectively induced cell death in patient-derived pre-B ALL cells and overcame conventional mechanisms of drug-resistance. Show less

The excessive proliferation of vascular smooth muscle cells was key factor in the restenosis of vein graft. And the Notch signaling was demonstrated to regulate vSMC proliferation and differentiation. Soluble Jagged-1 (sJag1) can inhibit Notch signaling in vitro and in vivo; however, its capacity to suppress restenosis of vein graft remains unknown. Under the microscope, the left jugular vein of these rats was interposed into the left common carotid artery, followed without any treatment (control), or with Ad-Jag1 (treatment) or placebo (DMSO) post operation. We showed that Ad-Jag1 can attenuate restenosis of vein graft by inducing decreased proliferation and increased apoptosis in vivo. Notch1-Hey2 signaling is critical for the development of intima thickening by controlling vSMC-fate determination. By blocking Notch signaling, Ad-Jag1 can significantly inhibit intima thickening. These studies identify that Ad-Jag1 can restore the vSMC phenotype and inhibit the vSMC proliferation by suppression of Notch1 signaling, and thus open a new avenue for the treatment of restenosis in vein graft. Show less

Existing evidence suggests that adverse pregnancy outcomes are closely related with dietary factors. Previous studies in mice have focused on the harm of folate deficiency (FD) on development of embryo, while the effect of low maternal folate levels on maternal intrauterine environment during early pregnancy remains unclear. Since our previous study found that FD treatment of mice causes no apparent defects in embryo implantation but is accompanied by female subfertility, we next chose to investigate a potential role of FD on molecular events after implantation. We observed that the decidual bulges began to be stunted on pregnancy day 6. The results of functional experiments in vivo and in vitro showed that FD inhibited the process of endometrial decidualization. It has been confirmed that DNA methylation participates in decidualization, and folate as a methyl donor could change the methylation patterns of genes. Thus, we hypothesized that FD impairs maternal endometrial decidualization by altering the methylation profiles of related genes. Reduced representation bisulphite sequencing was carried out to detect the methylation profiles of endometrium on pregnancy day 6-8, which is equivalent to the decidualization period in mice. The results confirmed that FD changes the methylation patterns of genome, and GO analysis of the differentially methylated regions revealed that the associated genes mainly participate in biological adhesion, biological regulation, cell proliferation, development, metabolism and signalling. In addition, we found some candidates for regulators of decidual transformation, such as Nr1h3 and Nr5a1. The data indicate that FD inhibits decidualization, possibly by altering methylation patterns of the genome in mice. Show less

To investigate the association between two polymorphisms of the APOC3 gene (T-455C and C-482T) and hereditary risk of non-alcoholic fatty liver disease (NAFLD). A total of 287 patients with NAFLD and 310 control subjects were genotyped by PCR and direct sequencing. Serum lipid profiles were also detected by standard biochemical One-hundred-and-eighty of the study participants were used to measure the APOC3 content by enzyme-linked immunosorbent assay. Inter-group differences and associations were assessed statistically using Chi square and t tests and logistic and linear regression analyses. The frequencies of neither the genotypes or alleles were significantly different between the NAFLD cases and the controls. Compared with the most common genotypes-455TT or-482CC, none of the variants showed a significant increase in risk of NAFLD or for the clinical and biochemical parameters. The adjusted odds ratios (with 95% confidence intervals) of NAFLD were 1.25 (0.79-1.96) and 1.20 (0.76-1.89) for carriers of the APOC3-455C and-482 T variants respectively (P more than 0.05). The T-455C and C-482T polymorphisms of the APOC3 gene are not associated with risk of NAFLD, pathogenic changes in lipid profiles, or insulin resistance in Han Chinese. Show less

The precise morphology of the mechanosensitive hair bundle requires seamless integration of actin and microtubule networks. Here, we identify Acf7a (actin crosslinking family protein 7a) as a protein positioned to bridge these distinct cytoskeletal networks in hair cells. By imaging Acf7a-Citrine fusion protein in zebrafish and immunolabeling of vestibular and cochlear mouse hair cells, we show that Acf7a and ACF7 circumscribe, underlie, and are interwoven into the cuticular plate (CP), and they also encircle the basal body of the kinocilium. In cochlear hair cells, ACF7 localization is graded, with the highest concentration near each fonticulus--an area free of F-actin in the region of the CP that contains the basal body. During hair-cell development and regeneration, Acf7a precedes formation of the hair bundle and CP. Finally, electron tomography demonstrates that the ends of microtubules insert into the CP and are decorated with filamentous linkers connecting microtubules to the CP. These observations are consistent with ACF7 being a linker protein, which may shape the cytoskeleton of the hair cell early during hair-bundle genesis. Show less

Duodenal signaling affects esophageal motility and perception, both pathophysiological factors in gastroesophageal reflux disease (GERD). Duodenal gene expression abnormalities, contributing to altered esophageal sensorimotor function, have not been reported to date. To identify differentially expressed genes in GERD patients' duodenum. Twenty GERD patients (total 24-h acid exposure 6-12%, SAP ≥95%) and ten healthy controls (HC) were included. Two weeks prior to duodenal biopsy collection, ten patients discontinued proton pump inhibitor (PPI) treatment and ten took maximum dose PPI. RNA was profiled on an Affymetrix Human Genome U133 Plus 2.0 array (Affymetrix, Santa Clara, CA, USA). Genes exhibiting a fold change ≥ 1.4 (t test p value <1E-4) were considered differentially expressed. A subset of 21 differentially expressed genes was selected for confirmatory TaqMan low-density array RT-PCR. Mucosal apolipoprotein A-IV (apoA-IV) and cholecystokinin (CCK) concentrations were determined by ELISA and RIA, respectively. In GERD patients off PPI, 23 up- and 23 down-regulated genes relative to HC were found. In GERD patients on PPI, 33 and five genes were higher, respectively, lower expressed. The majority of up-regulated genes were associated with lipid absorption, particularly triglyceride resynthesis and intracellular vesicular transport, rate-limiting processes for chylomicron production and secretion. Differential expression of 11 genes was confirmed by RT-PCR. Mucosal apoA-IV and CCK concentrations (signaling proteins released upon chylomicron secretion) were similar in GERD patients and HC. The identified mRNA expression differences suggest that in GERD patients' duodenum, the chylomicron production and secretion potential is elevated, and may underlie a mechanism by which postprandial duodenal signaling contributes to GERD symptom generation. Show less

The objective of this study was to compare the expression of Col1a1, Col1a2, and procollagen I in the seminiferous tubules of immature and adult mice and to characterize the cellular expression pattern of procollagen I in germ cells during spermatogenesis in order to provide necessary groundwork for further functional studies in the process of spermatogenesis. Microarray analysis demonstrated that Col1a1 and Col1a2 were abundantly expressed in the seminiferous tubules of 6-day-old mice compared with 60-day-old mice, and the expression levels of Col1a1 and Col1a2 mRNA were validated using a semi-quantitative RT-PCR assay. Western blot analysis further confirmed that procollagen I was expressed at a higher level in the seminiferous tubules of 6-day-old mice compared with 60-day-old mice. Immunohistochemical analysis revealed that type A spermatogonia were positive for procollagen I in the testis of 6-day-old mice, whereas Sertoli cells were negative for this protein. The in vivo procollagen I staining in type A spermatogonia was corroborated in spermatogonia exhibiting a high potential for proliferation and the ability to form germ cell colonies in in vitro culture. Moreover, procollagen I was also detected in type A spermatogonia, intermediate spermatogonia, type B spermatogonia, and preleptotene spermatocytes in the adult mouse testes, but positive staining disappeared in more differentiated germ cell lineages detaching from the basement membrane, including leptotene spermatocytes, pachytene spermatocytes, round spermatids and elongated spermatids. These data suggest that Col1a1, Col1a2 and procollagen I are associated with type A spermatogonia and play a potential role in mediating the detachment and migration of germ cells during spermatogenesis. Show less