The CAG repeat in SCA12 functions as a cis element to up-regulate PPP2R2B expression

PPP2R2B, a protein widely expressed in neurons, regulates the protein phosphatase 2A (PP2A) activity for dephosphorylation of tau and other substrates. CAG repeat expansion at the 5′-end of the PPP2R2B gene causes autosomal dominant spinocerebellar ataxia type 12. In the present study, we investigated the roles of CAG repeats and flanking cis elements and the associated proteins in controlling PPP2R2B expression. Deletion/site-directed mutagenesis, in silico searches and cDNA overexpression revealed that CREB1 and SP1 bind to the conserved sequence upstream the CAG repeats to up-regulate PPP2R2B expression, whereas TFAP4 binds to the conserved sequence downstream the CAG repeats to down-regulate PPP2R2B expression. The binding of CREB1, SP1, and TFAP4 to the PPP2R2B promoter was further confirmed by DNA pull-down and ChIP-PCR assays. CAG repeats itself also function as a cis element to up-regulate PPP2R2B expression as AT repeat length has no effect on PPP2R2B expression. Together, our data provide evidence that CREB1, SP1, and TFAP4 play roles in modulating PPP2R2B expression, thus offering a mechanism of regulating PP2A activity as the treatment of neurodegenerative diseases associated with abnormal PP2A activity.     

Expression of GLP-1R protein and its clinical role in intrahepatic cholangiocarcinoma tissues

The study investigates the expression and clinical role of GLP-1R in intrahepatic cholangiocarcinoma (ICC) tissues. ICC tissue, tissue around tumour and normal liver tissue samples from 176 ICC patients were investigated for GLP-1R expression by immunohistochemistry and western blots. Expression levels were correlated to clinical variables and to the postoperative outcome. High GLP-1R expression levels were detected in tumor tissue samples. Kaplan–Meier method was used for survival analysis of patients follow-up data. Results showed that median survival time of patients with high GLP-1R positive expression in ICC tissue were 22 months. Median survival time of patients with low GLP-1R positive expression in ICC tissue were 19.8 months. There wasn’t statistical difference (p = 0.332) between two groups. Immunohistochemistry semi-quantitative analysis showed that tissue differentiation is not prognostic risk factors. In patients with GLP-1R positive expression in ICC tissue, lymph node metastasis was important prognostic factors (p = 0.001). Although statistical analysis showed that GLP-1R can not be judged as a risk prognostic factors, GLP-1 might become a new target for therapy of ICC.     

Genomic organization and mapping of the gene encoding the PP2A B56gamma regulatory subunit

Protein phosphatase 2A (PP2A) is a major serine/threonine phosphatase that regulates a wide variety of cellular processes. The enzymatic activity and intracellular localization of PP2A are determined by three distinct families of cellular regulatory subunits (B, B', and B'). The B' subunit, also known as B56, is the most diverse, consisting of five isoforms (alpha, beta, gamma, delta, and epsilon). The gene encoding B56gamma has been designated as PPP2R5C and encodes three differentially spliced variants: B56gamma1, -gamma2, and -gamma3. However, conflicting chromosomal loci have been reported in human genomic databases. The original cytogenetic mapping placed the gene on chromosome 3p21.3, whereas subsequent studies using radiation hybrid analysis localized PPP2R5C to chromosome 14q. In this study, by radiation hybrid mapping, FISH analysis, BAC clone sequencing, and RT-PCR analysis, we show that the functional gene PPP2R5C exists at 14q32.2 and gives rise to three splicing variants, B56gamma1, -gamma2, and -gamma3, whereas a nonfunctional B56gamma1 pseudogene, PPP2R5CP, is present at 3p21.3. We also report the genomic organization of both the functional gene and the pseudogene.     

Insulin-stimulated phosphorylation of protein phosphatase 1 regulatory subunit 12B revealed by HPLC-ESI-MS/MS

ABSTRACT: BACKGROUND: Protein phosphatase 1 (PP1) is one of the major phosphatases responsible for protein dephosphorylation in eukaryotes. Protein phosphatase 1 regulatory subunit 12B (PPP1R12B), one of the regulatory subunits of PP1, can bind to PP1cdelta, one of the catalytic subunits of PP1, and modulate the specificity and activity of PP1cdelta against its substrates. Phosphorylation of PPP1R12B on threonine 646 by Rho kinase inhibits the activity of the PP1c-PPP1R12B complex. However, it is not currently known whether PPP1R12B phosphorylation at threonine 646 and other sites is regulated by insulin. We set out to identify phosphorylation sites in PPP1R12B and to quantify the effect of insulin on PPP1R12B phosphorylation by using high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. RESULTS: 14 PPP1R12B phosphorylation sites were identified, 7 of which were previously unreported. Potential kinases were predicted for these sites. Furthermore, relative quantification of PPP1R12B phosphorylation sites for basal and insulin-treated samples was obtained by using peak area-based label-free mass spectrometry of fragment ions. The results indicate that insulin stimulates the phosphorylation of PPP1R12B significantly at serine 29 (3.02 +/- 0.94 fold), serine 504 (11.67 +/- 3.33 fold), and serine 645/threonine 646 (2.34 +/- 0.58 fold). CONCLUSION: PPP1R12B was identified as a phosphatase subunit that undergoes insulin-stimulated phosphorylation, suggesting that PPP1R12B might play a role in insulin signaling. This study also identified novel targets for future investigation of the regulation of PPP1R12B not only in insulin signaling in cell models, animal models, and in humans, but also in other signaling pathways.     

Delineation of the frequency and boundary of chromosomal copy number variations in paediatric neuroblastoma

Neuroblastoma, the most common solid tumour in early childhood, is characterized by very frequent chromosomal copy number variations (CNVs). While chromosome 2p amplification, 17q gain, 1p and 11q deletion in human neuroblastoma tissues are well-known, the exact frequencies and boundaries of the chromosomal CNVs have not been delineated. We analysed the publicly available single nucleotide polymorphism (SNP) array data which were originally generated by the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative, defined the frequencies and boundaries of chromosomes 2p11.2 – 2p25.3 amplification, 17q11.1-17q25.3 gain, 1p13.3-1p36.33 deletion and 11q13.3-11q25 deletion in neuroblastoma tissues, and identified chromosome 7q14.1 (Chr7:38254795-38346971) and chromosome 14q11.2 (Chr14:21637401-22024617) deletion in blood and bone marrow samples from neuroblastoma patients, but not in tumour tissues. Kaplan Meier analysis showed that double deletion of Chr7q14.1 and Chr14q11.2 correlated with poor prognosis in MYCN gene amplified neuroblastoma patients. In conclusion, the oncogenes amplified or gained and tumour suppressor genes deleted within the boundaries of chromosomal CNVs in tumour tissues should be studied for their roles in tumourigenesis and as therapeutic targets. Focal deletions of Chr7q14.1 and Chr14q11.2 together in blood and bone marrow samples from neuroblastoma patients can be used as a marker for poorer prognosis and more aggressive therapies.     

A 7-lncRNA signature predict prognosis of Uterine corpus endometrial carcinoma

Current research indicate that long noncoding RNAs (lncRNAs) are associated with the progression of various cancers and can be used as prognostic biomarkers. This study aims to construct a prognostic lncRNA signature for the risk assessment of Uterine corpus endometrial carcinoma (UCEC). The RNA-Seq expression profile and corresponding clinical data of UCEC patients obtained from The Cancer Genome Atlas database. First, some prognosis-related lncRNAs were obtained by univariate Cox analysis. The minimum absolute contraction and selection operator (LASSO) regression and the Cox proportional hazard regression method were used to further identify the lncRNA prognostic model. Finally, seven lncRNAs (AC110491.1, AL451137.1, AC005381.1, AC103563.2, AC007422.2, AC108025.2, and MIR7-3HG) were identified as potential prognostic factors. According to the model constructed by the above analysis, the risk score of each UCEC patient was calculated, and the patients were classified into high and low-risk groups. The low-risk group had significant survival benefits. Moreover, we constructed a nomogram that incorporated independent prognostic factors (age, tumor stage, tumor grade, and risk score). The c-index value for evaluating the predictive nomogram model was 0.801. The area under the curve was 0.797 (3-year survival). The calibration curve also showed that there was a satisfactory agreement between the predicted and observed values in the probability of 1-, 3-, and 5-year overall survival. On the basis of the coexpression relationship, we established a coexpression network of lncRNA-messenger RNA (mRNA) of the 7-lncRNA. The Kyoto Encyclopedia of Genes and Genomes analysis of the coexpressing mRNAs showed that the main pathways related to the 7-lncRNA signature were neuroactive ligand-receptor interaction, serotonergic synapse, and gastric cancer pathway. Therefore, our study revealed that the 7-lncRNA could be used to predict the prognosis of UCEC and for postoperative treatment and follow-up.     

The landscape and prognostic value of immune characteristics in uterine corpus endometrial cancer

In the present study, we explored the clinical and immunological characteristics of 575 uterine corpus endometrial carcinoma (UCEC) samples obtained from The Cancer Genome Atlas (TCGA) using the ESTIMATE and CIBERSORT algorithms. First, Kaplan–Meier and univariate Cox regression analyses indicated that the immune cell score was a prognostic factor for overall survival (OS) and recurrence-free survival (RFS). Multivariate Cox regression analysis further revealed that the immune cell score was an independent prognostic factor for UCEC patients. Second, we investigated the correlation between the infiltration levels of 22 types of immune cells and the immune score. Survival analysis based on the 22 immune cell types showed that higher levels of regulatory T cell, activated NK cell, and follicular helper T-cell infiltration were associated with longer OS, while higher levels of CD8+ T cell and naive B-cell infiltration were associated with longer RFS. Next, we performed differential expression and prognosis analyses on 1534 immune-related genes and selected five from 14 candidate genes to construct a prognostic prediction model. The area under the receiver-operating characteristic (ROC) curve (AUC) for 3- and 5-year survival were 0.711 and 0.728, respectively. Further validation using a stage I–II subgroup showed similar results, presenting AUC values for 3- and five-year survival of 0.677 and 0.692, respectively. Taken together, the present study provides not only a deeper understanding of the relationship between UCEC and the immune landscape but also guidance for the future development of UCEC immunotherapy.     

Matrix Metalloproteinase-14 Expression and Its Prognostic Value in Cervical Carcinoma

The objective of the study was to evaluate the expression of matrix metalloproteinase-14 (MMP-14) in cervical carcinoma and correlate its expression with clinicopathological parameters, recurrence, and survival of the patients. The expressions of MMP-14 in normal cervical mucosa and cervical carcinoma tissue were detected with immunohistochemistry. Survival analysis by the Kaplan–Meier method was performed to assess prognostic significance. The positive expression rate of MMP-14 in cervical carcinoma tissue was 81.6 %(111/136), and there was significant difference on their positive expression rates between in cervical carcinoma tissue and in normal cervical mucosa(22.4 %)(13/58)(P < 0.05);The positive expression rates of MMP-14 in patients with poor histologic differentiation, lymph node metastasis, and recurrence group were heightened. Using Kaplan–Meier analysis, a comparison of survival curves of low versus high expressions of MMP-14 revealed a highly significant difference in human cervical carcinoma tissue (P < 0.05), which suggests that overexpression of MMP-14 is associated with a worse prognosis. The MMP-14 which promotes angiogenes is associated with lymph node metastasis, vascular invasion, and poor prognosis of cervical carcinoma. The current study shows that MMP-14 may be an independent prognostic factor for cervical carcinoma patients.     

PHF6 promotes the progression of endometrial carcinoma by increasing cancer cells growth and decreasing T-cell infiltration

Uterine corpus endometrial carcinoma (UCEC) is the most common cancer of the female reproductive tract. The overall survival of advanced and recurrent UCEC patients is still unfavourable nowadays. It is urgent to find a predictive biomarker and block tumorgenesis at an early stage. Plant homeodomain finger protein 6 (PHF6) is a key player in epigenetic regulation, and its alterations lead to various diseases, including tumours. Here, we found that PHF6 expression was upregulated in UCEC tissues compared with normal tissues. The UCEC patients with high PHF6 expression had poor survival than UCEC patients with low PHF6 expression. PHF6 mutation occurred in 12% of UCEC patients, and PHF6 mutation predicted favourable clinical outcome in UCEC patients. Depletion of PHF6 effectively inhibited HEC-1-A and KLE cell proliferation in vitro and decreased HEC-1-A cell growth in vivo. Furthermore, high PHF6 level indicated a subtype of UCECs characterized by low immune infiltration, such as CD3⁺ T-cell infiltration. While knockdown of PHF6 in endometrial carcinoma cells increased T-cell migration by promoting IL32 production and secretion. Taken together, our findings suggested that PHF6 might play an oncogenic role in UCEC patients. Thus, PHF6 could be a potential biomarker in predicting the prognosis of UCEC patients. Depletion of PHF6 may be a novel therapeutic strategy for UCEC patients.     

子宫内膜癌中的lncRNA-miRNA-mRNA ceRNA网络的整合分析

子宫内膜癌(uterine corpus endometrial carcinoma,UCEC)是危害女性健康的癌症之一,但其发生发展机制尚不完全清楚.基因的异常表达在细胞癌变过程中发挥着重要的作用.本研究利用生物信息学方法对UCEC中异常表达的基因进行网络调控分析,为UCEC的机制研究及预后治疗提供理论依据.首先,利...     

Over expression of PPP2R2C inhibits human glioma cells growth through the suppression of mTOR pathway

PPP2R2C encodes a gamma isoform of the subunit B55 subfamily, which is a regulatory subunit of Protein phosphatase type 2A (PP2A). Our study shows that PPP2R2C is downregulated in glioma cells and human brain cancer patient samples. Overexpression of PPP2R2C inhibited cancer cell proliferation both in vitro and in vivo through the suppression of the activity of S6K in the mTOR pathway. Moreover, exogenous expression of PPP2R2C promoted the formation of a complex with the PP2A-C subunit to further enhance the binding of PP2A-C with S6K. Our results suggest that PPP2R2C is a potential tumor suppressor gene in human brain cancers. This study will provide novel insight into the development of therapeutic strategies in the treatment of human brain tumors.     

Novel gene signatures for prognosis prediction in ovarian cancer

Ovarian cancer (OV) is one of the leading causes of cancer deaths in women worldwide. Late diagnosis and heterogeneous treatment result to poor survival outcomes for patients with OV. Therefore, we aimed to develop novel biomarkers for prognosis prediction from the potential molecular mechanism of tumorigenesis. Eight eligible data sets related to OV in GEO database were integrated to identify differential expression genes (DEGs) between tumour tissues and normal. Enrichment analyses discovered DEGs were most significantly enriched in G2/M checkpoint signalling pathway. Subsequently, we constructed a multi‐gene signature based on the LASSO Cox regression model in the TCGA database and time‐dependent ROC curves showed good predictive accuracy for 1‐, 3‐ and 5‐year overall survival. Utility in various types of OV was validated through subgroup survival analysis. Risk scores formulated by the multi‐gene signature stratified patients into high‐risk and low‐risk, and the former inclined worse overall survival than the latter. By incorporating this signature with age and pathological tumour stage, a visual predictive nomogram was established, which was useful for clinicians to predict survival outcome of patients. Furthermore, SNRPD1 and EFNA5 were selected from the multi‐gene signature as simplified prognostic indicators. Higher EFNA5 expression or lower SNRPD1 indicated poorer outcome. The correlation between signature gene expression and clinical characteristics was observed through WGCNA. Drug‐gene interaction was used to identify 16 potentially targeted drugs for OV treatment. In conclusion, we established novel gene signatures as independent prognostic factors to stratify the risk of OV patients and facilitate the implementation of personalized therapies.     

上皮性卵巢癌组织miR-338-3p、miR-1294的表达及临床意义

摘要 目的：探讨上皮性卵巢癌组织中微小RNA（miR）-338-3p、miR-1294表达与患者临床病理参数的关系,并分析其表达对上皮性卵巢癌预后的影响。方法：收集2010年6月～2015年6月我院行手术治疗的上皮性卵巢癌患者的石蜡组织样本（癌组织和癌旁正常组织）,实时荧光定量PCR（RT-PCR）技术检测组织样本中miR-338-3p、miR-1294的表达情况;分析癌组织中miR-338-3p、miR-1294表达与患者临床病理参数的关系;Kaplan-Meier生存曲线分析miR-338-3p、miR-1294表达与患者预后的关系;Cox回归模型分析上皮性卵巢癌患者的预后影响因素。结果：与癌旁正常组织相比,上皮性卵巢癌组织中miR-338-3p、miR-1294表达均降低（均P<0.05）;miR-338-3p表达与组织学分级、淋巴结转移、FIGO分期相关（均P<0.05）;miR-1294表达与肿瘤直径、淋巴结转移、FIGO分期相关（均P<0.05）。Kaplan-Meier生存曲线结果显示,miR-338-3p低表达组患者、miR-1294低表达组患者的预后较差（均P<0.05）。Cox回归模型分析结果显示,淋巴结转移、较高FIGO分期、miR-338-3p低表达、miR-1294低表达是上皮性卵巢癌预后的危险因素（均P<0.05）。结论：上皮性卵巢癌组织中miR-338-3p、miR-1294均低表达,且均与较差临床病理参数、预后不良相关;miR-338-3p、miR-1294可能是上皮性卵巢癌患者预后预测的潜在指标。     

The spinocerebellar ataxia 12 gene product and protein phosphatase 2A regulatory subunit Bbeta2 antagonizes neuronal survival by promoting mitochondrial fission

The neurodegenerative disorder spinocerebellar ataxia 12 (SCA12) is caused by CAG repeat expansion in the non-coding region of the PPP2R2B gene. PPP2R2B encodes Bbeta1 and Bbeta2, alternatively spliced and neuron-specific regulatory subunits of the protein phosphatase 2A (PP2A) holoenzyme. We show here that in PC12 cells and hippocampal neurons, cell stressors induced a rapid translocation of PP2A/Bbeta2 to mitochondria to promote apoptosis. Conversely, silencing of PP2A/Bbeta2 protected hippocampal neurons against free radical-mediated, excitotoxic, and ischemic insults. Evidence is accumulating that the mitochondrial fission/fusion equilibrium is an important determinant of cell survival. Accordingly, we found that Bbeta2 expression induces mitochondrial fragmentation, whereas Bbeta2 silencing or inhibition resulted in mitochondrial elongation. Based on epistasis experiments involving Bcl2 and core components of the mitochondrial fission machinery (Fis1 and dynamin-related protein 1), mitochondrial fragmentation occurs upstream of apoptosis and is both necessary and sufficient for hippocampal neuron death. Our data provide the first example of a proapoptotic phosphatase that predisposes to neuronal death by promoting mitochondrial division and point to a possible imbalance of the mitochondrial morphogenetic equilibrium in the pathogenesis of SCA12.     

Systematic profiling of invasion-related gene signature predicts prognostic features of lung adenocarcinoma

Due to the high heterogeneity of lung adenocarcinoma (LUAD), molecular subtype based on gene expression profiles is of great significance for diagnosis and prognosis prediction in patients with LUAD. Invasion-related genes were obtained from the CancerSEA database, and LUAD expression profiles were downloaded from The Cancer Genome Atlas. The ConsensusClusterPlus was used to obtain molecular subtypes based on invasion-related genes. The limma software package was used to identify differentially expressed genes (DEGs). A multi-gene risk model was constructed by Lasso-Cox analysis. A nomogram was also constructed based on risk scores and meaningful clinical features. 3 subtypes (C1, C2 and C3) based on the expression of 97 invasion-related genes were obtained. C3 had the worst prognosis. A total of 669 DEGs were identified among the subtypes. Pathway enrichment analysis results showed that the DEGs were mainly enriched in the cell cycle, DNA replication, the p53 signalling pathway and other tumour-related pathways. A 5-gene signature (KRT6A, MELTF, IRX5, MS4A1 and CRTAC1) was identified by using Lasso-Cox analysis. The training, validation and external independent cohorts proved that the model was robust and had better prediction ability than other lung cancer models. The gene expression results showed that the expression levels of MS4A1 and KRT6A in tumour tissues were higher than in normal tissues, while CRTAC1 expression in tumour tissues was lower than in normal tissues. The 5-gene signature prognostic stratification system based on invasion-related genes could be used to assess prognostic risk in patients with LUAD.