Identification of Downregulated Exosome-Associated Gene ENPP1 as a Novel Lipid Metabolism and Immune-Associated Biomarker for Hepatocellular Carcinoma.

Exosome plays an important role in the occurrence and development of tumors, such as hepatocellular carcinoma (LIHC). However, the functions and mechanisms of exosome-associated molecules in LIHC are still underexplored. Here, we investigated the role of the exosome-related gene ENPP1 in LIHC. Comprehensive bioinformatics from multiple databases revealed that ENPP1 was significantly downregulated in LIHC tissues. The patients with downregulated ENPP1 displayed a poor prognosis. Immunohistochemistry (IHC) was used to further confirm the downregulated ENPP1 in LIHC tissues. In addition, the coexpression network of ENPP1 was also explored to understand its roles in the underlying signaling pathways, including fatty acid degradation and the PPAR signaling pathway. Simultaneously, GSEA analysis indicated the potential roles of ENPP1 in the lipid metabolism-associated signaling pathways in the pathogenesis of LIHC, including fatty acid metabolism, fatty acid synthesis, and so on. Finally, immunological analysis indicated that ENPP1 might also be involved in multiple immune-related features, including immunoinhibitors, immunostimulators, and chemokines. Taken together, these findings could enhance our understanding of ENPP1 in LIHC pathogenesis and immune response and provide a new target for ENPP1-related immunotherapy in clinical treatment.

Identification of ACSF gene family as therapeutic targets and immune-associated biomarkers in hepatocellular carcinoma.

Acyl-CoA synthetases (ACSs) are responsible for acyl-CoA synthesis from nonpolar hydrophilic fatty acids and play a vital role in many metabolic processes. As a category of ACS isozymes, members of ACS family (AACS, ACSF2-3, AASDH) participate in lipid metabolism; however, their expression patterns, regulatory mechanisms and effects in hepatocellular carcinoma (HCC) are poorly understood. Here, through evaluating the expression profiles of ACSF gene family, we found that upregulated AACS might be more significant and valuable in development and progression of HCC. Consequently, the mRNA expression levels of AACS and ACSF2 was accordantly increased in HCC. Kaplan-Meier plotter revealed that HCC patients with high level of AACS were highly related to a shorter overall survival time and relapse-free survival. Genetic alterations using cBioPortal revealed that the alteration rate of AACS were 5%. We also found that the functions of ACSF gene family were linked to several cancer-associated pathways, including long-term potentiation, phospholipase D signaling pathway and purine metabolism. TIMER database indicated that the AACS and ACSF2 had a strong relationship with the infiltration of six types of immune cells (macrophages, neutrophils, CD8+ T-cells, B-cells, CD4+ T-cells and dendritic cells). Next, Diseasemeth database revealed that the global methylation levels of ACSF2 was higher in HCC patients. In conclusion, this study firstly demonstrated that Acyl-CoA synthesis gene family, in particular, AACS, could be associated with immune microenvironment, thereby influencing the development and prognosis of patients with HCC.

Upregulation of nuclear division cycle 80 contributes to therapeutic resistance via the promotion of autophagy-related protein-7-dependent autophagy in lung cancer.

Lung cancer remains the leading cause of malignant mortality worldwide. Hence, the discovery of novel targets that can improve therapeutic effects in lung cancer patients is an urgent need. In this study, we screened differentially expressed genes using isobaric tags for relative and absolute quantitation (iTRAQ) analysis and datasets from the cancer genome atlas database, and found that nuclear division cycle 80 (NDC80) might act as a novel prognostic indicator of lung cancer. The expression of NDC80 was significantly increased in lung cancer tissues, as compared to normal tissues, and high expression levels of NDC80 were correlated with unfavorable survival rates. Furthermore, an in vitro analysis showed that the stable knockdown of NDC80 decreased the cell viability and increased therapeutic sensitivity in two lung cancer cell lines, A549-IRR and H1246-IRR. Moreover, gene set enrichment analysis results showed that NDC80 was enriched in autophagy-related pathways. The downregulation of NDC80 inhibited the formation of autophagosomes, and reduced the expression of autophagy-related proteins such as LC3II, Beclin-1, and p62 in lung cancer cells. To further clarify the role of NDC80 as a downstream regulator of autophagy, we validated autophagic mediators through iTRAQ analysis and real-time polymerase chain reaction arrays. Autophagy-related protein7 (ATG7) was observed to be downregulated after the knockdown of NDC80 in lung cancer cells. Immunohistochemistry assay results revealed that both NDC80 and ATG7 were upregulated in an array of lung adenocarcinoma samples, compared to normal tissues, and the expression of NDC80 was identified to be positively associated with the levels of ATG7. Our findings suggest that NDC80 promotes the development of lung cancer by regulating autophagy, and might serve as a potential target for increasing the therapeutic sensitivity of lung cancer.

The downregulated drug-metabolism related ALDH6A1 serves as predictor for prognosis and therapeutic immune response in gastric cancer.

Drug metabolism-associated genes have been clarified to play a vital role in the process of cancer cell growth and migration. Nevertheless, the correlation between drug metabolism-associated genes and gastric cancer (GC) has not been fully explored and clarified. This paper has focused on the role of aldehyde dehydrogenase 6 family member A1 (ALDH6A1), a drug metabolism-associated gene, in the immune regulation and prognosis of GC patients. Using several bioinformatics platforms and immunohistochemistry (IHC) assay, we found that ALDH6A1 expression was significantly down-regulated in GC tissues. Moreover, higher expression of ALDH6A1 was related to the better prognosis of GC patients. ALDH6A1 was also found to be involved in the regulation of several immune-associated signatures, including immunoinhibitors. In conclusion, the above results have concluded that aberrant expression of ALDH6A1 might be served as the promising predictor for prognosis and clinical immunotherapy response in GC patients.

Spectrum of BRAF Aberrations and Its Potential Clinical Implications: Insights From Integrative Pan-Cancer Analysis.

B-Raf proto-oncogene serine/threonine-protein kinase (BRAF) is frequently altered in multiple cancer types, and BRAF V600 mutations act as a prime target for precision therapy. Although emerging evidence has investigated the role of BRAF, the comprehensive profiling of BRAF expression, alteration and clinical implications across various cancer types has not been reported. In this study, we used the TCGA dataset, covering 10,967 tumor samples across 32 cancer types, to analyze BRAF abnormal expression, DNA methylation, alterations (mutations and amplification/deletion), and their associations with patient survival. The results showed that BRAF expression, alteration frequency, mutation site distribution, and DNA methylation patterns varied tremendously among different cancer types. The expression of BRAF was found higher in PCPG and CHOL, and lower in TGCT and UCS compared to normal tissues. In terms of pathological stages, BRAF expression was significantly differentially expressed in COAD, KIRC, LUSC, and OV. The methylation levels of BRAF were significantly lower in LUSC, HNSC, and UCEC compared to normal tissue. The expression of BRAF and downstream gene (ETS2) was negatively correlated with methylation levels in various cancers. The overall somatic mutation frequency of BRAF was 7.7% for all cancer samples. Most fusion transcripts were found in THCA and SKCM with distinct fusion patterns. The majority of BRAF mutations were oncogenic and mainly distributed in the Pkinase_Tyr domain of THCA, SKCM, COADREAD, and LUAD. The BRAF mutations were divided into five levels according to the clinical targeted therapy implication. The results showed level 1 was mainly distributed in SKCM, COADREAD, and LUAD, while level 3B in THCA. The overall BRAF CNV frequency was about 42.7%, most of which was gain (75.9%), common in GBM, TGCT, and KIRP. In addition, the forest plot showed that increased BRAF expression was associated with poor patient overall survival in LIHC, OV, and UCEC. Taken together, this study provided a novel insight into the full alteration spectrum of BRAF and its implications for treatment and prognosis.

Comprehensive analysis of the potential cuproptosis-related biomarker LIAS that regulates prognosis and immunotherapy of pan-cancers.

Lipoic acid synthetase (LIAS) has been demonstrated to play a crucial role in the progression of cancer. Exploring the underlying mechanisms and biological functions of LIAS could have potential therapeutic guidance for cancer treatment. Our study has explored the expression levels and prognostic values of LIAS in pan-cancer through several bioinformatics platforms, including TIMER2.0, Gene Expression Profiling Interactive Analysis, version 2 (GEPIA2.0), and Human Protein Atlas (HPA). We found that a high LIAS expression was related to the good prognosis in patients with kidney renal clear cell carcinoma (KIRC), rectum adenocarcinoma (READ), breast cancer, and ovarian cancer. Inversely, a high LIAS expression showed unfavorable prognosis in lung cancer patients. In addition, the genetic alteration, methylation levels, and immune analysis of LIAS in pan-cancer have been evaluated. To elucidate the underlying molecular mechanism of LIAS, we conduct the single-cell sequencing to implicate that LIAS expression was related to hypoxia, angiogenesis, and DNA repair. Thus, these comprehensive pan-cancer analyses have conveyed that LIAS could be potentially significant in the progression of various cancers. Moreover, the LIAS expression could predict the efficacy of immunotherapy in cancer patients.

A Prognostic Signature Consisting of Pyroptosis-Related Genes and SCAF11 for Predicting Immune Response in Breast Cancer.

Pyroptosis, characterized as an inflammasome-mediated cell death pathway, may be participated in tumorigenesis and progression. However, the underlying molecular function and mechanism of pyroptosis in BRCA remain unclear. In our study, we aimed to develop a prognostic signature in BRCA based on pyroptosis-associated genes. Data was downloaded from TCGA database, and then we screened 760 female BRCA samples and 104 normal breast tissues as the training set. Seven pyroptosis-related genes (CASP9, GPX4, IL18, NLRC4, SCAF11, TIRAP, and TNF) were identified as the pyroptosis-related prognostic model for BRCA using LASSO Cox regression. We subsequently tested the prognostic value of pyroptosis-associated gene signature in a validation set, GSE 20685. Time-dependent receiver operating characteristic analysis demonstrated the credible predictive capacity of this pyroptosis-associated gene signature. The area under the curves were 0.806 at 3 years, 0.787 at 5 years, 0.775 at 8 years, and 0.793 at 10 years in the training set, and 0.824 at 5 years, 0.808 at 8 years, and 0.790 at 10 years in the validation set. Furthermore, there are currently few data on SCAF11 regulating pyroptosis. To clarify this issue, we performed integrative bioinformatics and experimental analysis. Knocking down SCAF11 possessed an anti-cancer effect in terms of inhibiting cell viability and suppressing colony-formation in in-vitro functional assays. Meanwhile, the biological functions of SCAF11 in BRCA were further validated with several algorithms, such as Xiantao tool, LinkedOmics, GEPIA2, and TISIDB. These findings indicated that the expression of SCAF11 was significantly correlated with diverse tumor-infiltrating lymphocytes (TILs), including T central memory cell (Tcm), and type 2 T helper cell (Th2), etc. Functional enrichment analysis suggested that co-expression genes of SCAF11 primarily participated in inflammation and immune-related signaling pathways, such as oxidative phosphorylation, antimicrobial humoral response, and immunoglobulin complex. Moreover, SCAF11 expression was positively correlated with several immune checkpoints, including PD-L1, B7H3, and PDCD1LG2. Taken together, this study uncovered that pyroptosis-associated gene signature might be applied as an effective independent predictor in patients with BRCA. The pyroptosis-related gene SCAF11 might play potential roles in the regulation of immune microenvironment in BRCA.

Potential Biological Roles of Exosomal Long Non-Coding RNAs in Gastrointestinal Cancer.

Exosomes, a type of extracellular vesicles (EVs), are secreted by almost all cells and contain many cellular constituents, such as nucleic acids, lipids, and metabolites. In addition, they play a crucial role in intercellular communication and have been proved to be involved in the development and treatment of gastrointestinal cancer. It has been confirmed that long non-coding RNAs (lncRNAs) exert a range of biological functions, such as cell metastasis, tumorigenesis, and therapeutic responses. This review mainly focused on the emerging roles and underlying molecular mechanisms of exosome-derived lncRNAs in gastrointestinal cancer in recent years. The biological roles of exosomal lncRNAs in the pathogenesis and therapeutic responses of gastrointestinal cancers were also investigated.

Ferroptosis-Related Gene MT1G as a Novel Biomarker Correlated With Prognosis and Immune Infiltration in Colorectal Cancer.

Ferroptosis, a newly discovered way of cell death, has been proved to be involved in the oncogenesis and development of cancers, including colorectal cancer (CRC). Here, by identifying the differentially expressed genes (DEGs) from three CRC transcriptome microarray datasets (GSE20842, GSE23878, and GSE25070), we found that the expression of MT1G was significantly decreased in CRC tissues, and the patients with a high level of MT1G displayed a poor prognosis. Quantitative PCR (qPCR) further confirmed the downregulated MT1G in two CRC cells, HCT8 and HCT116. The colony-forming assay indicated that the MT1G overexpression exhibited a remarkable inhibition of cell proliferation in HCT8 and HCT116 cells. In addition, we explored the co-expressed genes of MT1G to gain a better understanding of its potential signaling pathways. Aberrantly expressed MT1G also affected the immune response of CRC patients. Collectively, these findings might deepen our comprehension on the potential biological implications of MT1G in CRC.

Downregulated exosome-associated gene FGF9 as a novel diagnostic and prognostic target for ovarian cancer and its underlying roles in immune regulation.

Exosome has been demonstrated to be secreted from cells and seized by targeted cells. Exosome could transmit signals and exert biological functions in cancer progression. Nevertheless, the underlying mechanisms of exosome in ovarian cancer (OC) have not been fully explored. In this study, we wanted to explore whether Fibroblast growth factor 9 (FGF9), as an exosome-associated gene, was importantly essential in OC progression and prognosis. Firstly, comprehensive bioinformatics platforms were applied to find that FGF9 expression was lower in OC tissues compared to normal ovarian tissues. Meanwhile, downregulated FGF9 displayed favorable prognostic values in OC patients. The gene enrichment of biological functions indicated that abnormally expressed FGF9 could be involved in the OC-related immune signatures, such as immunoinhibitors and chemokine receptors. Taken together, these findings could provide a novel insight into the significance of FGF9 in OC progress and supply a new destination of FGF9-related immunotherapy in clinical treatment.

Integrative bioinformatics and experimental analysis revealed TEAD as novel prognostic target for hepatocellular carcinoma and its roles in ferroptosis regulation.

OBJECTIVE: Transcriptional enhanced associate domain (TEAD) family consists of four members TEAD1/2/3/4 that regulate cell growth, stem cell functions and organ development. As the downstream of Hippo signaling pathway, TEAD family is involved in the progression of several cancers. However, the precise biology functions of TEAD family in hepatocellular carcinoma (HCC) have not been reported yet. METHODS: We apply bioinformatics analysis based on databases including UALCAN, Oncomine, GEPIA, Kaplan-Meier plotter, WebGestalt, cBioPortal, TIMER2.0, and in vitro experimental evidence to identify the exact roles of TEAD family in HCC. RESULTS: The results indicated that TEAD2/4 were significantly upregulated in HCC compared with normal tissues. Downregulated of TEAD2 could promote the death of HCC cells through inducing ferroptosis by iron accumulation and subsequent oxidative damage. According to the Kaplan-Meier plotter database, we found that the high expression of TEAD2 was significantly associated with poor disease-specific survival, overall survival, progression-free survival and relapse-free survival. In aspect of cancer immunity, Tumor Immune Estimation Resource algorithm showed that the expression of TEAD family members was obviously related to multiple of infiltrating immune cells including macrophages, neutrophils, dendritic cells, B cells, CD8+ T cells and CD4+ T cells. Finally, we conducted the functional enrichment analysis including protein-protein interaction network, gene ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathway based on the TEAD family-associated coexpression genes. CONCLUSION: The study provided deep insight information of TEAD family in the diagnostic and prognostic evaluation of HCC patients.

The protective effect of atractylenolide I on systemic inflammation in the mouse model of sepsis created by cecal ligation and puncture.

CONTEXT: Atractylenolide I (AT-I), an active compound isolated from Atractylodes macrocephala Koidz (Compositae), shows several pharmacological activities. OBJECTIVE: Our present study is designed to investigate the protective effect of AT-I on systemic inflammation in the mouse model of sepsis created by cecal ligation and puncture (CLP), and explore the possible mechanism. MATERIALS AND METHODS: Sepsis mouse model was established by CLP, and the tested dosages of AT-I were 10, 20, and 40 mg/kg (ip). Pro-inflammatory cytokines in serum (TNF-α, IL-1ß and IL-6) were determined by the ELISA method; serum lipopolysaccharide (LPS) level was measured by the Limulus Amebocyte Lysate (LAL) test; white blood cells (WBC) were counted by Blood cell analyzer; contents of alanine transaminase (ALT), aspartate transarninase (AST), creatinine (Cre), and blood urea nitrogen (BUN) in serum were determined by automatic biochemistry analyzer. For survival rate tests, CLP mice were observed within 7 days, and body temperature was measured at 0, 4, 8, 12, 24, 48 and 72 h after surgery. RESULTS: Our results indicated that AT-I significantly increased the survival rate of mice with sepsis (p < 0.05), whereas the WBCs and levels of LPS, pro-inflammatory cytokines (TNF-α, IL-1ß and IL-6), ALT, AST, Cre, and BUN decreased significantly after treatment with AT-I (p < 0.05). CONCLUSION: In conclusion, the AT-I ameliorates sepsis syndrome by reduction of pro-inflammatory cytokines and LPS, and provides an improvement in liver and kidney functions.

[Effect of mild to moderate hypothermia on casepase-3, Bcl-2 and Bax expressions in brain tissue of rats after cardiopulmonary resuscitation].

OBJECTIVE: To explore the effect of mild to moderate hypothermia on the expressions of apoptosis-related genes in the brain tissue of rats after cardiopulmonary resuscitation (CPR). METHODS: CPR models were established by asphyxia in 15 male SD rats, which were randomized equally into normal temperature group, 34 degrees celsius hypothermia group and 32 degrees celsius hypothermia group. The brain tissues of the rats were obtained after treatment for 12 h to observe the pathological changes. The expression of caspase-3 in cerebral cortex neurons was determined with immunohistochemistry, and the expressions of Bcl-2 and Bax were detected by Western blotting. RESULTS: Compared with normal temperature group, the two hypothermia groups (especially 32 degrees celsius group) showed significantly decreased expression of caspase-3 in the cortical neurons (P<0.05). Bcl-2 protein expression was significantly increased in the hypothermia groups, especially in 32 degrees celsius hypothermia group (P<0.05). There was no significant difference in Bax protein expression among the 3 groups. CONCLUSION: Mild hypothermia can relieve brain injury by down-regulating caspase-3 expression and up-regulating Bcl-2 protein expression to inhibit apoptosis of the brain neurons. Hypothermia at 32 degrees celsius offers better protection of the brain tissue than hypothermia at 34 degrees celsius.

Overexpression of sorcin results in multidrug resistance in gastric cancer cells with up-regulation of P-gp.

Sorcin, a calcium-binding protein was found up-regulated in the vincristine-induced multi-drug resistance (MDR) gastric cancer cell line SGC7901/VCR, over its parental SGC7901 cells in our previous proteomic studies. The present study explored the role and mechanism of sorcin in the development of MDR in gastric cancer. We constructed the recombinant plasmids FLAG-sorsin-pcDNA3.1 containing the full open reading frame of sorcin and a FLAG affinity tag. Overexpression of sorcin by gene transfection was able to confer drug resistance to vincristine, adriamycin, taxol and 5-fluorouracil in SGC7901 cells. Down-regulation of sorcin expression by sorcin antisense oligonucleutides, (ASO) increased sensitivity to vincristine. The intracellular concentration of vincristine in SGC7901 cells decreased in sorcin transfected cells and increased in sorcin ASO-transfected cells, indicating that sorcin had a direct or indirect function on pumping the drug out of cells. Overexpression of sorcin up-regulated the expression of P-gp and P-gp inhibitor verapamil partially reversed the sorcin-mediated MDR in SGC7901 cell, suggesting that regulation of P-gp might be one of the mechanisms of sorcin-mediated MDR. The further study of the interaction protein of sorcin may be helpful for understanding the mechanisms of MDR in gastric cancer and developing possible strategies to treat gastric cancer.

[Correlation of sorcin overexpression to multidrug resistance of human gastric cancer cell line SGC7901].

BACKGROUND & OBJECTIVE: Vincristine (VCR)-resistant gastric cancer cell line SGC7901/VCR is a typical multidrug resistant (MDR) cell line with high expression of P-glycoprotein (P-gp). However, verapamil (VRP), the inhibitor of P-gp, can not totally reverse the drug resistance, indicating that additional mechanisms must contribute to the MDR phenotype. Our previous study showed that sorcin, a calcium-binding protein, is significantly up-regulated in SGC7901/VCR cells. This study was to explore the role of sorcin in the development of MDR in human gastric cancer cell line SGC7901. METHODS: The full length sorcin cDNA was isolated by reverse transcription-polymerase chain reaction (RT-PCR). The FLAG-sorsin-pcDNA3.1 plasmid was constructed and transfected into SGC7901 cells. The mRNA and protein levels of sorcin in stable clones were detected by RT-PCR and Western blot. The sensitivity of SGC7901 cells to chemotherapeutic drugs was detected using MTT assay. Then sorcin-transfected SGC7901 cells (SGC-F-Sor) were transfected with sorcin antisense oligonucleotides (ASO). The VCR-sensitivity of SGC7901 cells was determined by MTT assay. RESULTS: The full-length sorcin cDNA (616 bp) was amplified by RT-PCR. The FLAG-sorsin-pcDNA3.1 plasmid was constructed successfully. The mRNA and protein levels of sorcin were up-regulated in SGC-F-Sor cells. Overexpression of sorcin produced 8.87 folds of VCR-resistance, 6.13 folds of adriamycin (ADM)-resistance, 6.67 folds of taxol-resistance, and 2.80 folds of 5-fluorouracil (5-FU)-resistance. However, when SGC-F-Sor cells were transfected with sorcin ASO, down-regulation of sorcin expression and increased sensitivity to VCR were observed. CONCLUSIONS: Overexpression of sorcin could induce low level of MDR in SGC7901 cells, indicating that sorcin is associated with MDR of SGC7901 cells. Sorcin maybe be a target of MDR reversal in gastric cancer cells.