A novel HPV16 splicing enhancer critical for viral oncogene expression and cell immortalization.

High-risk carcinogenic human papillomaviruses (HPVs), e.g. HPV16, express the E6 and E7 oncogenes from two mRNAs that are generated in a mutually exclusive manner by splicing. The HPV16 E7 mRNA, also known as the E6*I/E7 mRNA, is produced by splicing between splice sites SD226 and SA409, while E6 mRNAs retain the intron between these splice sites. We show that splicing between HPV16 splice sites SD226 and SA409 is controlled by a splicing enhancer consisting of a perfect repeat of an adenosine-rich, 11 nucleotide sequence: AAAAGCAAAGA. Two nucleotide substitutions in both 11 nucleotide sequences specifically inhibited production of the spliced E6*I/E7 mRNA. As a result, production of E7 protein was reduced and the ability of HPV16 to immortalize human primary keratinocytes was abolished. The splicing-enhancing effect was mediated by the cellular TRAP150/THRAP3 protein that also enhanced splicing of other high-risk HPV E6*I/E7 mRNAs, but had no effect on low-risk HPV mRNAs. In summary, we have identified a novel splicing enhancer in the E6 coding region that is specific for high-risk HPVs and that is critically linked to HPV16 carcinogenic properties.

A Novel missense mutation of COL2A1 gene in a large family with stickler syndrome type I.

Stickler syndrome type I (STL1, MIM 108300) is characterized by ocular, auditory, skeletal and orofacial manifestations. Nonsyndromic ocular STL1 (MIM 609508) characterized by predominantly ocular features is a subgroup of STL1, and it is inherited in an autosomal dominant manner. In this study, a novel variant c.T100>C (p.Cys34Arg) in COL2A1 related to a large nonsyndromic ocular STL1 family was identified through Exome sequencing (ES). Bioinformatics analysis indicated that the variant site was highly conserved and the pathogenic mechanism of this variant may involve in affected structure of chordin-like cysteine-rich (CR) repeats of ColIIA. Minigene assay indicated that this variant did not change alternative splicing of exon2 of COL2A1. Moreover, the nonsyndromic ocular STL1 family with 16 affected members showed phenotype variability and certain male gender trend. None of the family members had hearing loss. Our findings would expand the knowledge of the COL2A1 mutation spectrum, and phenotype variability associated with nonsyndromic ocular STL1. Search for genetic modifiers and related molecular pathways leading to the phenotype variation warrants further studies.

TIPE1-mediated autophagy suppression promotes nasopharyngeal carcinoma cell proliferation via the AMPK/mTOR signalling pathway.

Recent studies have shown that tumour necrosis factor-α-induced protein 8 like-1(TIPE1) plays distinct roles in different cancers. TIPE1 inhibits tumour proliferation and metastasis in a variety of tumours but acts as an oncogene in cervical cancer. The role of TIPE1 in nasopharyngeal carcinoma (NPC) remains unknown. Interestingly, TIPE1 expression was remarkably increased in NPC tissue samples compared to adjacent normal nasopharyngeal epithelial tissue samples in our study. TIPE1 expression was positively correlated with that of the proliferation marker Ki67 and negatively correlated with patient lifespan. In vitro, TIPE1 inhibited autophagy and induced cell proliferation in TIPE1-overexpressing CNE-1 and CNE-2Z cells. In addition, knocking down TIPE1 expression promoted autophagy and decreased proliferation, whereas overexpressing TIPE1 increased the levels of pmTOR, pS6 and P62 and decreased the level of pAMPK and the LC3B. Furthermore, the decrease in autophagy was remarkably rescued in TIPE1-overexpressing CNE-1 and CNE-2Z cells treated with the AMPK activator AICAR. In addition, TIPE1 promoted tumour growth in BALB/c nude mice. Taken together, results indicate that TIPE1 promotes NPC progression by inhibiting autophagy and inducing cell proliferation via the AMPK/mTOR signalling pathway. Thus, TIPE1 could potentially be used as a valuable diagnostic and prognostic biomarker for NPC.

TIPE1 promotes cervical cancer progression by repression of p53 acetylation and is associated with poor cervical cancer outcome.

Previous studies have shown that TIPE1 inhibits tumor proliferation and metastasis in certain cancers; however, increased expression of TIPE1 is observed in cervical cancer cell lines and tissues, indicating it might exert a distinctive role in cervical cancer. Cell and xenograft tumorigenicity assays showed that TIPE1 facilitates cervical cancer progression in this study. Further investigation demonstrated that TIPE1 binds to p53 and impairs its activity via inhibition of its acetylation. In addition, TIPE1 promoted cell proliferation and suppressed cisplatin susceptibility in a p53-dependent manner, indicating that TIPE1 facilitates cervical cancer progression primarily through the p53 pathway. TIPE1 expression in clinical samples also demonstrated that its upregulation predicts poor prognosis in patients with cervical cancer. Taken together, the results of this study showed that TIPE1 serves as an oncogene by restricting p53 activity in the development of cervical cancer, suggesting that TIPE1 will provide a new potential target for cervical cancer therapy and can be used as a biomarker to predict patient prognosis.

Upregulation of Tumor Necrosis Factor-α-Induced Protein 8-Like 2 mRNA Is Negatively Correlated with Serum Concentrations of Tumor Necrosis Factor-α and Interleukin 6 in Type 2 Diabetes Mellitus.

BACKGROUND: Tumor necrosis factor-α-induced protein 8-like 2 (TIPE2 or TNFAIP8L2) is a negative regulator of natural and adaptive immunity. The role of TIPE2 in type 2 diabetes mellitus (T2DM) remains unknown, although TIPE2 plays key roles in preserving inflammatory homeostasis. METHODS: TIPE2 expression was measured by Western blotting and real-time polymerase chain reaction (RT-PCR) in peripheral blood mononuclear cells (PBMCs) isolated from T2DM patients and healthy controls, and tumor necrosis factor-α (TNF-α), high-sensitivity C-reactive protein (hsCRP), interleukin 6 (IL-6), and other related biometabolic parameters were detected using a nephelometer or by ELISA. Differentiated THP-1 cells were exposed to siTIPE2 and TIPE2 adenovirus. RESULTS: TIPE2 was significantly increased in PBMCs from T2DM patients compared with those from healthy controls and was negatively correlated with serum TNF-α, IL-6, and hsCRP concentrations but positively correlated with HbA1c and LDL-C in T2DM patients. High glucose treatment (50 mmol/L) can upregulate the expression of TIPE2 and cytokine secretion in differentiated THP-1 cells. siTIPE2 infection exacerbated the increased TNF-α and IL-6 concentrations in differentiated THP-1 cells under high glucose conditions (50 mmol/L), while infection with TIPE2 adenovirus reversed the increased TNF-α concentration. CONCLUSIONS: The present study indicates that TIPE2 may participate in T2DM by regulating TNF-α production.

Role of citrullination modification catalyzed by peptidylarginine deiminase 4 in gene transcriptional regulation.

Peptidylarginine deiminase 4 (PADI4), a new histone modification enzyme, which converts both arginine and monomethyl-arginine to citrulline, has gained massive attention in recent years as a potential regulator of gene transcription. Recent studies have shown that arginine residues R2, R8, R17, and R26 in the H3 tail and R3 in the H4 tail can be deiminated by PADI4. This kind of histone post-translational modification has the potential to antagonize histone methylation and coordinate with histone deacetylation to regulate gene transcription. PADI4 also deiminates non-histone proteins, such as p300, NPM1, ING4, RPS2, and DNMT3A. PADI4 has been shown to involve in cell apoptosis and differentiation. Moreover, PADI4 can interact with tumor suppressor p53 and regulate the transcriptional activity of p53. Dysregulation of PADI4 is implicated in a variety of diseases, including rheumatoid arthritis, tumor development, and multiple sclerosis. A wide variety of PADI4 inhibitors have been identified. Further understanding of PADI4 functions may lead to novel diagnostic and therapeutic approaches in these diseases. This review summarizes the recent progress in the study of the regulation mechanism of PADI4 on gene transcription and the major physiological functions of PADI4 in human diseases.

Increased expression of TIPE2 mRNA in PBMCs of patients with ankylosing spondylitis is negatively associated with the disease severity.

Ankylosing spondylitis (AS) is an autoimmune disease and characterized by chronic inflammatory arthritis. Tumor necrosis factor α induced protein-8 like-2 (TIPE2) is responsible for maintaining immune homeostasis by inhibiting the secretion of inflammatory cytokines in the condition of inflammation. However, whether TIPE2 participates in the development of AS remains unknown. In this study, we measured the mRNA expression of TIPE2 and TIPE1 in peripheral blood mononuclear cells (PBMCs) from 45 AS patients and 40 healthy controls by qRT-PCR. The results showed TIPE2 expression was significantly increased in AS patients compared with controls (P=0.0066), while there was no significant difference for TIPE1 between two groups (P=0.2302). Moreover, the expression of TIPE2 mRNA in AS patients were decreased after treatment with TNF-α blocker (P<0.001). In addition, we found that TNF-α or plasma from AS patients induced TIPE2 expression in THP-1 cells in vitro. More importantly, the TIPE2 mRNA expression levels were negatively correlated with TNF-α, hsCRP and bath ankylosing spondylitis disease activity index (BASDAI) (r=-0.3574, P=0.0159; r=-0.3174, P=0.0336; r=-0.6000, P<0.0001; respectively) in the AS patients. These results indicated that TIPE2 contributes to the pathogenesis of AS.

A transcriptional target of androgen receptor, miR-421 regulates proliferation and metabolism of prostate cancer cells.

Prostate cancer is one of the most common malignancies, and microRNAs have been recognized to be involved in tumorigenesis of various kinds of cancer including prostate cancer (PCa). Androgen receptor (AR) plays a core role in prostate cancer progression and is responsible for regulation of numerous downstream targets including microRNAs. This study identified an AR-repressed microRNA, miR-421, in prostate cancer. Expression of miR-421 was significantly suppressed by androgen treatment, and correlated to AR expression in different prostate cancer cell lines. Furthermore, androgen-activated AR could directly bind to androgen responsive element (ARE) of miR-421, as predicted by bioinformatics resources and demonstrated by ChIP and luciferase reporter assays. In addition, over-expression of miR-421 markedly supressed cell viability, delayed cell cycle, reduced glycolysis and inhibited migration in prostate cancer cells. According to the result of miR-421 target genes searching, we focused on 4 genes NRAS, PRAME, CUL4B and PFKFB2 based on their involvement in cell proliferation, cell cycle progression and metabolism. The expression of these 4 downstream targets were significantly repressed by miR-421, and the binding sites were verified by luciferase assay. Additionally, we explored the expression of miR-421 and its target genes in human prostate cancer tissues, both in shared microarray data and in our own cohort. Significant differential expression and inverse correlation were found in PCa patients.

A Novel Insertion Variant of CRYGD Is Associated with Congenital Nuclear Cataract in a Chinese Family.

OBJECTIVE: To investigate a novel insertion variant of CRYGD identified in a Chinese family with nuclear congenital cataract. METHODS: A Chinese family with congenital nuclear cataract was recruited for the mutational screening of candidate genes by direct sequencing. Recombinant N-terminal Myc tagged wildtype or mutant CRYGD was expressed in HEK293T cells. The expression pattern, protein solubility and subcellular distribution were analyzed by western blotting and immunofluorescence. PRINCIPAL FINDINGS: A novel insertion variant, c.451_452insGACT, in CRYGD was identified in the patients. It causes a frameshift and a premature termination of the polypeptide to become Y151*. A significantly reduced solubility was observed for this mutant. Unlike wildtype CRYGD, which existed mainly in the cytoplasm, Y151* was mis-located in the nucleus. CONCLUSIONS: We have identified a novel mutation, c.451_452insGACT, in CRYGD, which is associated with nuclear cataract. This is the first insertion mutation of CRYGD found to cause autosomal dominant congenital cataract. The mutant protein, with loss of solubility and localization to the nucleus, is hypothesized to be the major cause of cataract in these patients.

Characterization of gene mutations and phenotypes of cystic fibrosis in Chinese patients.

BACKGROUND AND OBJECTIVE: Cystic fibrosis (CF) is a relatively common autosomal recessive disorder in Caucasians. CF is considered a very rare disease in Asians, and fewer than 30 Chinese CF patients are reported in the literature. We enrolled seven patients of Chinese Han origin diagnosed with CF at the Peking Union Medical College Hospital, to characterize gene mutations and phenotypes of CF in Chinese patients. METHODS: We analysed the clinical presentation and screened the coding region of the CFTR gene for each patient. RESULTS: Patients were 0-6 years old at onset of symptoms and were 10-28 years old at the time of diagnosis with CF. None of the seven patients had a family history of CF, and only one patient had parents who were consanguineous. Two patients had gastrointestinal symptoms but stool Sudan III results were normal. Four of the seven CF patients also had allergic bronchopulmonary aspergillosis. The concentration of chloride in patients' sweat ranged from 66 mmol/l to 154 mmol/l. In total, we identified 11 different mutations in seven CF patients, including one novel mutation (△E7-E11). Only one of these mutations (R553X) is present in the Caucasian CFTR common mutation-screening panel; and none of the 11 mutations are common in Caucasian CF patients. CONCLUSIONS: CF in China is difficult to diagnose because of a combination of low awareness, atypical clinical symptoms, and a lack of sweat and genetic testing facilities in most hospitals. The mutations identified in Chinese CF patients are different from the common Caucasian gene mutations.

Human cataract mutations in EPHA2 SAM domain alter receptor stability and function.

The cellular and molecular mechanisms underlying the pathogenesis of cataracts leading to visual impairment remain poorly understood. In recent studies, several mutations in the cytoplasmic sterile-α-motif (SAM) domain of human EPHA2 on chromosome 1p36 have been associated with hereditary cataracts in several families. Here, we have investigated how these SAM domain mutations affect EPHA2 activity. We showed that the SAM domain mutations dramatically destabilized the EPHA2 protein in a proteasome-dependent pathway, as evidenced by the increase of EPHA2 receptor levels in the presence of the proteasome inhibitor MG132. In addition, the expression of wild-type EPHA2 promoted the migration of the mouse lens epithelial αTN4-1 cells in the absence of ligand stimulation, whereas the mutants exhibited significantly reduced activity. In contrast, stimulation of EPHA2 with its ligand ephrin-A5 eradicates the enhancement of cell migration accompanied by Akt activation. Taken together, our studies suggest that the SAM domain of the EPHA2 protein plays critical roles in enhancing the stability of EPHA2 by modulating the proteasome-dependent process. Furthermore, activation of Akt switches EPHA2 from promoting to inhibiting cell migration upon ephrin-A5 binding. Our results provide the first report of multiple EPHA2 cataract mutations contributing to the destabilization of the receptor and causing the loss of cell migration activity.

Mutations in SLC20A2 link familial idiopathic basal ganglia calcification with phosphate homeostasis.

Familial idiopathic basal ganglia calcification (IBGC) is a genetic condition with a wide spectrum of neuropsychiatric symptoms, including parkinsonism and dementia. Here, we identified mutations in SLC20A2, encoding the type III sodium-dependent phosphate transporter 2 (PiT2), in IBGC-affected families of varied ancestry, and we observed significantly impaired phosphate transport activity for all assayed PiT2 mutants in Xenopus laevis oocytes. Our results implicate altered phosphate homeostasis in the etiology of IBGC.