Exploration of the relationship between tumor mutation burden and immune infiltrates in colon adenocarcinoma

Background: Tumor mutation burden (TMB) was correlated with the immunotherapeutic response in various malignancies. We aimed to evaluate the TMB immune signature in colon adenocarcinoma (COAD).Methods: Gene expression profile, mutation and clinical data of COAD patients were obtained from The Cancer Genome Atlas (TCGA) database. The samples were divided into high and low TMB level groups to identify differentially expressed genes (DEGs). Functional enrichments analyzes were performed to identify the biological functions of the DEGs. Then, immune cell infiltration signatures were calculated by the CIBERSORT algorithm. Finally, Cox proportional hazard model was constructed to estimate the prognostic value of the identified immune-related genes.Results: Gene set enrichment analysis in the high-TMB level group showed that DEGS were enriched in immune-related pathways, such as antigen processing and presentation, Toll-like receptor signaling and natural killer cell-mediated cytotoxicity. A higher infiltration level of CD8+ T cells, CD4+ T cells, activated NK cells , M1 Macrophages and T follicular helper cells was observed in the high-TMB level group. Furthermore, a Cox regression model combined with survival analysis based on the expression level of four identified prognostic genes was constructed, validated anf revealed that higher risk-score levels conferred poor survival outcomes in COAD patients.Conclusions: Our data demonstrate that the high TMB levels are associated with an immune signature in COAD and deepen the molecular understanding of TMB function in tumor immunotherapy.     

Heat shock factor 5 correlated with immune infiltration serves as a prognostic biomarker in lung adenocarcinoma

Lung adenocarcinoma (LUAD) is the predominant subtype of lung cancer with a relatively poor prognosis. The dramatic improvements of new immunotherapy strategies have shown promising results in lung cancer patients. This study aimed to elucidate the functions of immune-associated genes in LUAD prognosis and pathogenesis by analyzing public databases. We obtained expression profiles of LUAD patients from The Cancer Genome Atlas (TCGA) database and applied the ESTIMATE algorithm to calculate immune scores and stromal scores. A series of microenvironment-related genes with prognostic value was then identified. Of note, heat shock factor 5 (HSF5) was found to be decreased in LUAD patients and positively correlated with overall survival, which was further confirmed in the Gene Expression Omnibus (GEO) database. Moreover, Gene Ontology (GO) analysis based on the correlated genes of HSF5 demonstrated that HSF5 expression was significantly associated with the immune response and inflammatory activities. Based on the Tumor IMmune Estimation Resource (TIMER) and Gene Expression Profiling Interactive Analysis (GEPIA) datasets, HSF5 expression showed strong correlations with various immune cell infiltration and diverse immune marker sets. These findings suggest that HSF5 can be used as a promising biomarker for determining prognosis and immune infiltration in LUAD patients.     

Co-expression of XIAP and cyclin D1 complex correlates with a poor prognosis in patients with hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. Despite improved diagnosis and treatment, the prognosis for HCC patients remains poor. The goal of this study was to identify key regulatory proteins and signaling pathways important for cell apoptosis and proliferation as biomarkers for prognostication and targeted therapy. Protein Pathway Array was applied to screen 38 signaling proteins and phosphoproteins in 12 paired HCC tumors and surrounding benign tissues and found that 20 of them, including XIAP, CDK4, CDK6, and Cyclin D1, were overexpressed in HCC tissues. Immunostaining results of XIAP, CDK4, and Cyclin D1 in an additional 59 HCC tissues showed that the expression of XIAP correlated with the expression of CDK4/Cyclin D1, and that the increased expression of these proteins correlated with poor overall survival in these patients. Further studies using the HCC Huh7 cell line transfected with XIAP siRNA or expression vector demonstrated that XIAP regulated the expression of CDK4, CDK6, and Cyclin D1 via NF-êB and PTEN pathways. Finally, inhibition of XIAP using embelin, a XIAP-specific small molecule, leads to an increased apoptosis and decreased cell proliferation via arrest at G1 phase. Taken together, XIAP is a central modulator regulating cell apoptosis and cell cycle progression. Therefore, XIAP together with cell cycle regulatory proteins can be used as prognostic markers and therapeutic targets.     

Active Cyclin A-CDK2 Complex, a Possible Critical Factor for Cell Proliferation in Human Primary Lung Carcinomas

Expression of cyclins A and E and cyclin-dependent kinase 2 (CDK2) was examined immunohistochemically in 190 cases of human lung carcinoma. Cyclin A and CDK2 were expressed in the majority of squamous cell carcinomas, small cell carcinomas, and large cell carcinomas, but in significantly fewer cases of adenocarcinomas. Cyclin E was expressed in a minority of all subtypes. In particular, well differentiated cells in squamous cell carcinoma stained positively for cyclin E; in contrast, cyclin A was expressed in the nonkeratinized proliferating areas of the tumor nests. Immunoblotting revealed that all these proteins were expressed at higher levels in tumor tissues than in adjacent normal tissues. Immunoprecipitation also revealed higher levels of cyclin A and cyclin E associated with CDK2 in tumor tissues. Furthermore, tumor tissues which exhibited higher cyclin A and CDK2 expression also had higher CDK2 kinase activity. However, cyclin E-associated kinase activity was barely detectable even in tumor samples exhibiting higher cyclin E expression. Consistent with these data, elevated expression of cyclin A correlated to shorter survival periods in contrast to expression of cyclin E, which correlated to longer survival periods. These results suggest that in human lung carcinomas, elevated expression of active cyclin A-CDK2 complexes with associated higher CDK2 kinase activity is critical for promoting cell cycle progression and unrestrained proliferation of tumor cells and can be a predictive marker for patients’ prognosis. On the other hand, immunohistochemical detection of cyclin E-CDK2 reflects accumulation of inactive forms of protein complexes, implying differentiation or senescence of the tumor and the better prognosis.     

Bioinformatics analysis of gene expression profiles to diagnose crucial and novel genes in glioblastoma multiform

Therefore, the current study aimed to diagnose the genes associated in the pathogenesis of GBM. The differentially expressed genes (DEGs) were diagnosed using the limma software package. The ToppFun was used to perform pathway and Gene Ontology (GO) enrichment analysis of the DEGs. Protein-protein interaction (PPI) networks, extracted modules, miRNA-target genes regulatory network and miRNA-target genes regulatory network were used to obtain insight into the actions of DEGs. Survival analysis for DEGs carried out. A total of 701 DEGs, including 413 upregulated and 288 downregulated genes, were diagnosed between U1118MG cell line (PK 11195 treated with 1?h exposure) and U1118MG cell line (PK 11195 treated with 24?h exposure). The up-regulated genes were enriched in superpathway of pyrimidine deoxyribonucleotides de novo biosynthesis, cell cycle, cell cycle process and chromosome. The down-regulated genes were enriched in folate transformations I, biosynthesis of amino acids, cellular amino acid metabolic process and vacuolar membrane. The current study screened the genes in PPI network, extracted modules, miRNA-target genes regulatory network and miRNA-target genes regulatory network with higher degrees as hub genes, which included MYC, TERF2IP, CDK1, EEF1G, TXNIP, SLC1A5, RGS4 and IER5L Survival suggested that low expressed NR4A2, SLC7?A5, CYR61 and ID1 in patients with GBM was linked with a positive prognosis for overall survival. In conclusion, the current study could improve our understanding of the molecular mechanisms in the progression of GBM, and these crucial as well as new molecular markers might be used as therapeutic targets for GBM.     

长链非编码RNA TTTY15在骨肉瘤组织中的表达及其对骨肉瘤细胞活力和侵袭的影响

目的:观察长链非编码RNA TTTY15在骨肉瘤组织及细胞株中的表达,并探讨其对骨肉瘤细胞活力和侵袭的影响。方法:应用qPCR法检测11例骨肉瘤组织及其瘤旁组织中TTTY15的水平,同时检测TTTY15在骨肉瘤细胞株(143B、Saos2、MG-63、U2OS和HOS)和人成骨细胞株hFOB1.19中的表达。在表达量最高的细胞株(MG-63)中通过转染小干扰RNA敲减TTTY15的表达,CCK-8法检测TTTY15低表达对细胞活力的影响,流式细胞术检测其对细胞周期的影响,Transwell检测其对细胞侵袭能力的影响,qPCR检测miR-216b-5p和FOXM1 mRNA的表达,Western blot法检测相关蛋白的变化。结果 :与瘤旁正常组织相比,TTTY15在骨肉瘤组织中表达升高(P0.01);与人成骨细胞相比,TTTY15在骨肉瘤细胞株中表达升高(P0.05),其中在MG-63细胞中表达水平最高(P0.01)。敲减TTTY15在MG-63细胞的表达后,细胞活力降低(P0.05),细胞周期被抑制在G_0/G_1期(P0.01),细胞的侵袭能力降低(P0.01),miR-216b-5p mRNA表达水平升高(P0.01),FOXM1的mRNA表达降低(P0.01),同时FOXM1、CDK4、cyclin D1、MMP-2和N-cadherin的蛋白表达降低,而E-cadherin的蛋白表达升高(P0.05)。结论:TTTY15在骨肉瘤组织和细胞株中表达增高,敲减TTTY15的表达可抑制骨肉瘤细胞的活力和侵袭能力,其机制可能与TTTY15低表达导致miR-216b-5p的表达升高,引起FOXM1基因表达下调有关。     

Persistent initiation of DNA replication and chromatin-bound MCM proteins during the cell cycle in cdc6 mutants

Faithful inheritance of genetic information requires that DNA be copied only once each cell cycle. Initiation of DNA replication involves the establishment of a prereplication complex (pre-RC) and subsequent activation by CDK/cyclins, converting the pre-RC to a post-RC. The origin recognition complex (ORC), Cdc6p, and the MCM proteins are required for establishing the pre-RC. We show that all six ORC subunits remain bound to chromatin throughout the cell cycle, whereas the MCM proteins cycle on and off, corresponding precisely to transitions of the RC. A newly isolated cdc6 mutant displays promiscuous initiation of DNA replication, increased nuclear DNA content, and constant MCM protein association with chromatin throughout the cell cycle. This gain-of-function cdc6 mutant ignores the negative controls imposed normally on initiation by the CDK/cyclins, suggesting that Cdc6p is a key mediator of once-per-cell-cycle control of DNA replication.     

Overexpression of UQCRC2 is correlated with tumor progression and poor prognosis in colorectal cancer

Ubiquinol-cytochrome c reductase complex core protein 2 (UQCRC2) is an important subunit of mitochondrial respiratory complex III. However, its role in tumorigenesis and tumor progression remains unknown, especially with regards to colorectal cancer (CRC). In this research, we measured the expression of UQCRC2 protein by immunohistochemistry assay in 89 paired paraffin-embedded tumor tissues and corresponding adjacent normal tissues from patients with colorectal adenocarcinoma and investigated possible correlations of UQCRC2 expression with clinicopathological parameters and prognosis. We found that UQCRC2 was significantly upregulated in CRC tissues compared with adjacent normal tissues, and immunohistochemical UQCRC2 status was correlated to the depth of invasion (T), lymph node metastasis (N), advanced TNM stage. Multivariate analysis indicated that UQCRC2 remained an independent prognostic factor for poorer overall survival. Furthermore, we determined the role of UQCRC2-knockdown in CRC cells (RKO and HCT116) using lentivirus-mediated small hairpin RNAs (shRNAs). The effects of UQCRC2 knockdown on CRC cells (RKO and HCT116) proliferation were analyzed by cell proliferation and colony formation assay, and cell cycle and apoptosis were assessed by flow cytometry. We found that silencing UQCRC2 suppressed cell proliferation and colony formation in RKO and HCT116 cells, led to a cell cycle arrest and induced cell apoptosis in vitro. These results provided novel insights into the potential role of UQCRC2 in the tumorigenesis and progression of CRC, and revealed that UQCRC2 may serve as a new prognostic and therapeutic target in CRC.     

Genetic variants in the Hedgehog signaling pathway genes are associated with gastric cancer risk in a Chinese Han population

The Hedgehog signaling pathway participates in the occurrence and progression of cancers including gastric cancer.We conducted this study to evaluate whether genetic variants in the Hedgehog signaling pathway genes would affect gastric cancer risk.Multi-marker Analysis of GenoMic Annotation(MAGMA)was used to investigate the aggregated genetic effects of single nucleotide polymorphisms(SNPs)assigned to candidate genes.The relationship between SNPs and gastric cancer risk was estimated by multivariate logistic regression analyses.Gene expression was calculated using databases obtained from The Cancer Genome Atlas(TCGA)and The Gene Expression Omnibus(GEO).Kaplan‐Meier plotter was used to evaluate the association between gene expression with gastric cancer survival.Tumor Immune Estimation Resource 2.0(TIMER 2.0)was applied to determine the correlation between selected gene expression and the immune cell infiltration degree.We identified that the G allele of rs2990912 in KIF27 was associated with higher gastric cancer risk,especially in the young and male subgroups.The expression of KIF27 in gastric cancer tissues was higher than that in normal tissues,leading to poor survival in gastric cancer patients.Besides,KIF27 expression was related to immune cell infiltration and positively correlated with PD-L1 expression.Our findings highlight the key role of genetic variation in the Hedgehog signaling pathway genes in gastric cancer susceptibility,which may provide important insights into the diagnosis,prognosis,and treatment of gastric cancer.     

IFI30 expression predicts patient prognosis in breast cancer and dictates breast cancer cells proliferation via regulating autophagy

Introduction: Incidence and mortality rates of breast cancer are increasing in women worldwide. Immunotherapy is a relatively popular treatment modality for all malignant tumors including breast cancer in recent years. Interferon γ-inducible protein 30 (IFI30) could catalyze the reduction of disulfide bonds and enhance major histocompatibility complex (MHC) class II-restricted antigen processing. Recent studies showed that IFI30 played an important role in the immune response of malignant tumors.Methods: The Cancer Genome Atlas (TCGA) database and clinical proteomic tumor Analysis consortium (CPTAC) database were applied to predict the role of IFI30 in breast cancer and the relationship between IFI30 and prognosis of breast cancer patients. Then we detected the expression of IFI30 in clinical samples of breast cancer patients, and analyzed the relationship between IFI30 and the prognosis of breast cancer patients. We used lentivirus infection method to construct a stable IFI30 knockdown cell line, and detected the effect of IFI30 in breast cancer cells. Nude mice tumor bearing experiment was performed to investigate the effect of IFI30 on breast cancer cells in vivo. Western blot was used to verify the regulation of autophagy related protein LC3 and p62 by IFI30.Results: We found that IFI30 was highly expressed in breast cancer tissues and was associated with poor outcome of patients. The knockdown of IFI30 could inhibit the proliferation, migration and invasion of breast cancer cells and significantly inhibit tumor growth in vivo. Increased accumulation of LC3-II and p62 suggested impaired autophagy in IFI30 knockdown cells.Discussion: As a result, we suggested that IFI30 might play a key role in the initiation and progression of human breast cancer and might be a new therapeutic target in breast cancer.     

KDM5B基因在乳腺癌中的表达及其临床意义

目的:研究KDM5B基因在乳腺癌组织中的表达情况,并探讨其表达差异与患者临床病理资料和预后的关系。方法:从肿瘤基因组图谱(TCGA)数据库中收集113例正常乳腺组织和1 090例乳腺癌数据集,下载KDM5B mRNA表达谱资料;收集中山大学附属第三医院甲乳外科2015年～2016年乳腺癌切除标本共90例,采用real-time PCR的方法检测乳腺癌及其癌旁组织中KDM5B mRNA的表达量,取中位数分为高表达组与低表达组,分析其与临床资料及病例特征的关系;利用Kaplan-Meier plotter分析KDM5B mRNA表达与乳腺癌患者预后的相关性。结果:KDM5B在乳腺癌中的表达均显著高于正常乳腺组织(P 0. 01)。在TCGA的乳腺癌数据中,KDM5B的表达与人表皮生长因子受体2(HER2)、雌激素受体(ER)、年龄、病理组织类型及淋巴结转移显著相关(P 0. 01),与孕激素受体(PR)、绝经及远处转移无显著相关。在我们收集的乳腺癌样本中,KDM5B的表达与HER2、年龄及淋巴结转移显著相关(P 0. 05),而与ER、PR、绝经、病理组织类型和远处转移无显著相关。KDM5B表达越高,乳腺癌患者总生存时间(HR=1. 39,P=0. 005)和无病生存时间(HR=1. 32,P 0. 001)越短。结论:在乳腺癌组织中KDM5B高表达,且与患者的预后相关; KDM5B的表达与乳腺癌患者HER2表达、年龄和淋巴结转移显著相关。     

HMGB2对肺腺癌细胞周期和增殖的影响

目的:探讨高迁移率族蛋白B2(HMGB2)对肺腺癌细胞周期和增殖的影响。方法:从Cancer RNA-Seq Nexus (CRN)数据库分析肺腺癌组织HMGB2表达情况;从OncoLnc数据库分析HMGB2与肺腺癌患者预后的相关性;从肿瘤单细胞数据库(CancerSEA)分析HMGB2与肺腺癌细胞14种功能状态的相关性;利用siRNA技术下调人肺腺癌A549细胞中HMGB2表达,通过real-time PCR和Western blot验证沉默效果,CCK8和EdU实验检测细胞的增殖。结果:HMGB2在肺腺癌中高表达;HMGB2高表达组肺腺癌患者的总生存期明显低于HMGB2低表达患者(log-rank检验P=0.017 3);HMGB2表达与肺腺癌细胞周期和增殖呈正相关;敲减HMGB2表达后A549细胞的活力和增殖能力显著降低(P<0.05)。结论:HMGB2的表达与肺腺癌细胞周期和增殖显著正相关,可以作为潜在的评估肺腺癌患者预后的标志物和治疗靶标。     

LETM1 is a potential cancer stem-like cell marker and predicts poor prognosis in colorectal adenocarcinoma

The leucine zipper-EF-hand containing transmembrane protein 1 (LETM1) is highly expressed in many human malignancies and is correlated with poor prognosis. However, LETM1 has rarely been explored as a cancer stem-like cell marker for the prognostic evaluation of colorectal adenocarcinoma (CRA). Herein, we assessed the expression of LETM1 and its relationship with cancer stemness genes, cell cycle markers, PI3K/Akt/NFκB signaling pathway genes, and HIF1α in 102 paraffin-embedded CRA tissue samples using immunohistochemistry (IHC). Additionally, we further confirmed the correlation between LETM1 and cancer stemness genes in CRA cell lines using immunofluorescence (IF) imaging and Western blotting. LETM1 expression was remarkably upregulated in human fetal sagittal sections and CRA tissues. The expression of LETM1 in CRA tissue was correlated with clinical stage, lymph node metastasis, distant metastasis, and microvessel density. LETM1 expression was significantly associated with lower overall survival and disease-free survival. Moreover, the expression of LETM1 positively correlated with SOX9, LSD1, CD44, CD133, LGR5, SOX2, and HIF1α. IF revealed that LETM1 co-localized with CD44, SOX9, and LGR5 in HCT116. Moreover, LETM1 expression was also strongly linked to the expression of cell cycle regulators (cyclinD1, CDK4, p27) and PI3K/Akt/NFκB pathway genes (pPI3K-p85, pAkt-Ser473, pAkt-Thr308, pNFκB-p65). LETM1 may therefore be a cancer stem-like cell marker and an indicator of poor prognosis in patients with CRA.