Deficiency in SLC25A15, a hypoxia-responsive gene, promotes hepatocellular carcinoma by reprogramming glutamine metabolism.

BACKGROUND & AIMS: The role of solute carrier family 25 member 15 (SLC25A15), a critical component of the urea cycle, in hepatocellular carcinoma (HCC) progression remains poorly understood. This study investigated the impact of SLC25A15 on HCC progression and its mechanisms. METHODS: We systematically investigated the function of SLC25A15 in HCC progression using large-scale data mining and cell, animal, and organoid models. Furthermore, we analyzed its involvement in reprogramming glutamine metabolism. RESULTS: SLC25A15 expression was significantly decreased in HCC tissues, and patients with low SLC25A15 levels had a poorer prognosis. Hypoxia-exposed HCC cells or tissues had lower SLC25A15 expression. A positive correlation between HNF4A, a transcription factor suppressed by hypoxia, and SLC25A15 was observed in both HCC tissues and cells. Modulating HNF4A levels altered SLC25A15 mRNA levels. SLC25A15 upregulated SLC1A5, increasing glutamine uptake. The reactive metabolic pathway of glutamine was increased in SLC25A15-deficient HCC cells, providing energy for HCC progression through additional lipid synthesis. Ammonia accumulation due to low SLC25A15 levels suppressed the expression of OGDHL (oxoglutarate dehydrogenase L), a switch gene that mediates SLC25A15 deficiency-induced reprogramming of glutamine metabolism. SLC25A15-deficient HCC cells were more susceptible to glutamine deprivation and glutaminase inhibitors. Intervening in glutamine metabolism increased SLC25A15-deficient HCC cells' response to anti-PD-L1 treatment. CONCLUSION: SLC25A15 is hypoxia-responsive in HCC, and low SLC25A15 levels result in glutamine reprogramming through SLC1A5 and OGDHL regulation, promoting HCC progression and regulating cell sensitivity to anti-PD-L1. Interrupting the glutamine-derived energy supply is a potential therapeutic strategy for treating SLC25A15-deficient HCC. IMPACT AND IMPLICATIONS: We first demonstrated the tumor suppressor role of solute carrier family 25 member 15 (SLC25A15) in hepatocellular carcinoma (HCC) and showed that its deficiency leads to reprogramming of glutamine metabolism to promote HCC development. SLC25A15 can serve as a potential biomarker to guide the development of precision therapeutic strategies aimed at targeting glutamine deprivation. Furthermore, we highlight that the use of an inhibitor of glutamine utilization can enhance the sensitivity of low SLC25A15 HCC to anti-PD-L1 therapy.

Novel insights into small open reading frame-encoded micropeptides in hepatocellular carcinoma: A potential breakthrough.

Traditionally, non-coding RNAs (ncRNAs) are regarded as a class of RNA transcripts that lack encoding capability; however, advancements in technology have revealed that some ncRNAs contain small open reading frames (sORFs) that are capable of encoding micropeptides of approximately 150 amino acids in length. sORF-encoded micropeptides (SEPs) have emerged as intriguing entities in hepatocellular carcinoma (HCC) research, shedding light on this previously unexplored realm. Recent studies have highlighted the regulatory functions of SEPs in the occurrence and progression of HCC. Some SEPs exhibit inhibitory effects on HCC, but others facilitate its development. This discovery has revolutionized the landscape of HCC research and clinical management. Here, we introduce the concept and characteristics of SEPs, summarize their associations with HCC, and elucidate their carcinogenic mechanisms in HCC metabolism, signaling pathways, cell proliferation, and metastasis. In addition, we propose a step-by-step workflow for the investigation of HCC-associated SEPs. Lastly, we discuss the challenges and prospects of applying SEPs in the diagnosis and treatment of HCC. This review aims to facilitate the discovery, optimization, and clinical application of HCC-related SEPs, inspiring the development of early diagnostic, individualized, and precision therapeutic strategies for HCC.

IHGA: An interactive web server for large-scale and comprehensive discovery of genes of interest in hepatocellular carcinoma.

Mining gene expression data is valuable for discovering novel biomarkers and therapeutic targets in hepatocellular carcinoma (HCC). Although emerging data mining tools are available for pan-cancer-related gene data analysis, few tools are dedicated to HCC. Moreover, tools specifically designed for HCC have restrictions such as small data scale and limited functionality. Therefore, we developed IHGA, a new interactive web server for discovering genes of interest in HCC on a large-scale and comprehensive basis. Integrative HCC Gene Analysis (IHGA) contains over 100 independent HCC patient-derived datasets (with over 10,000 tissue samples) and more than 90 cell models. IHGA allows users to conduct a series of large-scale and comprehensive analyses and data visualizations based on gene mRNA levels, including expression comparison, correlation analysis, clinical characteristics analysis, survival analysis, immune system interaction analysis, and drug sensitivity analysis. This method notably enhanced the richness of clinical data in IHGA. Additionally, IHGA integrates artificial intelligence (AI)-assisted gene screening based on natural language models. IHGA is free, user-friendly, and can effectively reduce time spent during data collection, organization, and analysis. In conclusion, IHGA is competitive in terms of data scale, data diversity, and functionality. It effectively alleviates the obstacles caused by HCC heterogeneity to data mining work and helps advance research on the molecular mechanisms of HCC.

Hypoxia-Responsive lncRNA AC115619 Encodes a Micropeptide That Suppresses m6A Modifications and Hepatocellular Carcinoma Progression.

Long noncoding RNAs (lncRNA) regulate a number of aspects of cancer biology. Recent research has shown that lncRNAs can encode micropeptides that mediate their functions in tumors. Here, we revealed that the liver-specific putative lncRNA, AC115619, is expressed at low levels in hepatocellular carcinoma (HCC) and encodes a micropeptide, designated as AC115619-22aa. AC115619 played a crucial role in the regulation of tumor progression and was a prognostic indicator in HCC. The encoded micropeptide AC115619-22aa inhibited the progression of HCC by binding to WTAP and impeding the assembly of the N6-methyladenosine (m6A) methyltransferase complex, which regulates the expression of tumor-associated genes, such as SOCS2 and ATG14. AC115619 was cotranscribed with the adjacent upstream coding gene APOB, and hypoxia induced transcriptional repression of both APOB and AC115619 by controlling HIF1A/HDAC3 and HNF4A signaling. In animal and patient-derived models, AC115619-22aa reduced global m6A levels and suppressed tumor growth. In conclusion, this study establishes AC115619 and its encoded micropeptide as potential prognostic markers and therapeutic targets for patients with HCC. SIGNIFICANCE: A micropeptide encoded by lncRNA AC115619 impedes formation of the m6A methylation complex to lower m6A levels and reduce the growth of hepatocellular carcinoma.

Hypoxia-responsive PPARGC1A/BAMBI/ACSL5 axis promotes progression and resistance to lenvatinib in hepatocellular carcinoma.

Emerging evidence has indicated that peroxisome proliferator-activated receptor-gamma coactivator-1α (PPARGC1A) is involved in hepatocellular carcinoma (HCC). However, its detailed function and up- and downstream mechanisms are incompletely understood. In this study, we confirmed that PPAGC1A is lowly expressed in HCC and is associated with poor prognosis using large-scale public datasets and in-house cohorts. PPAGC1A was found to impair the progression and sensitivity of HCC to lenvatinib. Mechanistically, PPAGC1A repressed bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) by inhibiting WNT/ß-catenin signaling. BAMBI mediated the function of PPARGC1A and regulated ACSL5 through TGF-ß/SMAD signaling. PPARGC1A/BAMBI regulated ROS production and ferroptosis-related cell death by controlling ACSL5. PPARGC1A/BAMBI/ACSL5 axis was hypoxia-responsive. METTL3 and WTAP silenced PPARGC1A in an m6A-YTHDF2-dependent way under normoxia and hypoxia, respectively. Metformin restored PPARGC1A expression by reducing its m6A modification via inhibiting METTL3. In animal models and patient-derived organoids, consistent functional data of PPARGC1A/BAMBI/ACSL5 were observed. Conclusions: These findings provide new insights into the role of the aberrant PPARGC1A/BAMBI/ACSL5 axis in HCC. And the mechanism of PPARGC1A dysregulation was explained by m6A modification. Metformin may benefit HCC patients with PPARGC1A dysregulation.

Downregulation of SLC14A1 Expression Indicates Poor Prognosis and Promotes the Progression of Non-Small Cell Lung Cancer.

OBJECTIVE: Solute carrier (SLC) genes are known to be involved in the development of human tumors. Here, we identified a group of non-small cell lung cancer (NSCLC) - associated SLC genes and explored the role and potential mechanism of action for SLC14A1 in NSCLC. METHODS: We used public NSCLC gene expression databases to select NSCLC-associated SLC genes. A Cox proportional risk model was used to estimate the hazard rate of selected NSCLC-associated SLC genes. SLC14A1 was selected as a candidate for further study. The expression and effect on survival of SLC14A1 in multiple NSCLC datasets were investigated. The expression of SLC14A1 was modulated in Calu-6 and A549 cells by transfection and a series of proliferation and migration assays were performed. Parameters related to glycolysis and HK2 expression were detected after transfection. RESULTS: Fourteen SLC genes were identified as NSCLC-related SLC genes. Among them, SLC14A1 was downregulated in tumor tissues in multiple datasets. Low expression of SLC14A1 was indicative of poor prognosis in NSCLC. Knockdown of SLC14A1 promoted proliferation and migration in Calu-6 cells. Overexpression of SLC14A1 showed the opposite effect in A549 cells. Additionally, SLC14A1 changes were associated with glycolysis. This was demonstrated by HK2 mRNA and protein levels being regulated by SLC14A1. CONCLUSIONS: In our study SLC14A1 suppressed the proliferation and migration of NSCLC cell lines Calu-6 and A549 and was associated with glycolysis. Therefore, SLC14A1 may be a diagnostic marker or therapeutic target for NSCLC patients.

LncRNA KCNQ1OT1 Participates in Ox-LDL-Induced Proliferation/Apoptosis Imbalance in Vascular Smooth Muscle Cells by Regulating the MiR-196a-5p/FOXO1 Axis.

Backgroud The present study aimed to investigate the function and regulatory mechanisms of lncRNA KCNQ1OT1 in vascular smooth muscle cells under oxidation low lipoprotein stimulation. Methods RNA sequencing was used to detect transcriptome changes of vascular smooth muscle cells treated with oxidation low lipoprotein. KCNQ1OT1, miR-196a-5p, and FOXO1 expression levels in VSMCs after oxidation low lipoprotein treatment were assessed using qRT-PCR and western blotting. RNA immunoprecipitation, RNA pull-down, and dual-luciferase reporter assay were used to confirm the interaction among lncRNA KCNQ1OT1, miR-196a-5p, and FOXO1. The functions of KCNQ1OT1, miR-196a-5p, and FOXO1 were analyzed by CCK-8 and flow cytometry. The serum samples of high fat-feeding mice and atherosclerosis patients were collected, and the levels of KCNQ1OT1 and miR-196a-5p were analyzed. Results In vitro expression of KCNQ1OT1 and FOXO1 decreased in VSMCs treated with oxidation low lipoprotein, accompanied by overexpression of miR-196a-5p. As a ceRNA, KCNQ1OT1 positively regulated FOXO1 and imparted a negative regulatory effect on miR-196a-5p. Interference KCNQ1OT1/miR-196a-5p/FOXO1 could change roliferation/apoptosis imbalance in VSMCs under oxidation low lipoprotein stimulation. Higher levels of KCNQ1OT1 and lower levels of miR-196a-5p can be found in the thoracic aorta tissues of high fat-feeding mice and serum samples from individuals with carotid atherosclerosis. Conclusion Aberrant expression of KCNQ1OT1/miR-196a-5p/FOXO1 pathway mediated oxidation low lipoprotein-induced proliferation/apoptosis imbalance in VSMCs.

ox-LDL regulates proliferation and apoptosis in VSMCs by controlling the miR-183-5p/FOXO1.

BACKGROUND: microRNA-mRNA axes that are involved in oxidized low-density lipoprotein (ox-LDL)-induced vascular smooth muscle cells (VSMCs) proliferation/apoptosis imbalance need to be further investigated. OBJECTIVE: To investigate the functional role of miR-183-5p/FOXO1 in VSMCs and its interaction with ox-LDL. METHODS: RNA sequencing was used to detect transcriptome changes of VSMCs treated with ox-LDL. miR-183-5p and FOXO1 expression levels in VSMCs after ox-LDL treatment were assessed using qRT-PCR and western blotting. The regulatory effect of miR-183-5p on FOXO1 has been tried to prove using a dual-luciferase reporter assay. The functions of miR-183-5p, and FOXO1 were analyzed by CCK-8 assay and flow cytometry assay. The tissue samples or serum samples of high fat-feeding mice and carotid atherosclerosis patients were collected, and the levels of miR-183-5p/FOXO1 were analyzed. RESULTS: RNA sequencing data showed 81 miRNAs including miR-183-5p was significantly changed after ox-LDL treatment in VSMCs. FOXO1, a miR-183-5p's potential target, was also down-regulated in ox-LDL treated cells. qRT-PCR and western blot found that expression of FOXO1 mRNA and protein significantly reduced in VSMCs treated with ox-LDL, accompanied by overexpression of miR-183-5p. miR-183-5p inhibited FOXO1 mRNA by binding to its 3' UTR. Interference miR-183-5p/FOXO1 could change proliferation/apoptosis imbalance in VSMCs under ox-LDL stimulation. Higher levels of miR-183-5p but reduced FOXO1 can be found in the thoracic aorta tissues of high fat-feeding mice. In serum samples from individuals with carotid atherosclerosis, Higher levels of miR-183-5p were observed. the miR-183-5p level was positively related to the level of serum ox-LDL in patients. CONCLUSIONS: Aberrant expression of miR-183-5p/FOXO1 pathway mediated ox-LDL-induced proliferation/apoptosis imbalance in VSMCs. The miR-183-5p/FOXO1 axis can potentially be utilized as the target in the treatment of patients with atherosclerosis.

The Transmembrane Receptor TIRC7 Identifies a Distinct Subset of Immune Cells with Prognostic Implications in Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a heterogeneous malignancy with a dismal prognosis. Therapeutic options are largely limited to surgery and conventional chemotherapy offers limited benefit. As immunotherapy has proven highly effective in various cancer types, we have undertaken a quantitative immunohistopathological assessment of immune cells expressing the immunoinhibitory T cell immune response cDNA 7 receptor (TIRC7), an emerging immunoinhibitory receptor, in a cohort of 135 CCA patients. TIRC7+ immune cells were present in both the tumor epithelia and stroma in the majority of CCA cases with the highest levels found in intrahepatic CCA. While intraepithelial density of TIRC7+ immune cells was decreased compared to matched non-neoplastic bile ducts, stromal quantity was higher in the tumor samples. Tumors exhibiting signet ring cell or adenosquamous morphology were exclusively associated with an intraepithelial TIRC7+ phenotype. Survival analysis showed intraepithelial TIRC7+ immune cell density to be a highly significant favorable prognosticator in intrahepatic but not proximal or distal CCA. Furthermore, intraepithelial TIRC7+ immune cell density correlated with the number of intraepithelial CD8+ immune cells and with the total number of CD4+ immune cells. Our results suggest the presence and prognostic relevance of TIRC7+ immune cells in CCA and warrant further functional studies on its pharmacological modulation.

lncRNA-SNHG14 Plays a Role in Acute Lung Injury Induced by Lipopolysaccharide through Regulating Autophagy via miR-223-3p/Foxo3a.

lncRNAs play important roles in lipopolysaccharide- (LPS-) induced acute lung injury. But the mechanism still needs further research. In the present study, we investigate the functional role of the lncRNA-SNHG14/miR-223-3p/Foxo3a pathway in LPS-induced ALI and tried to confirm its regulatory effect on autophagy. Transcriptomic profile changes were identified by RNA-seq in LPS-treated alveolar type II epithelial cells. The expression changes of lncRNA-SNHG14/miR-223-3p/Foxo3a were confirmed using qRT-PCR and west blot. The binding relationship of lncRNA-SNHG14/miR-223-3p/and miR-223-3p/Foxo3a was verified using dual-luciferase reporter, RNA immunoprecipitation, and RNA pull-down assays. Using gain-of-function or loss-of-function approaches, the effect of lncRNA-SNHG14/miR-223-3p/Foxo3a was investigated in LPS-induced acute lung injury mice model and in vitro. Increasing of lncRNA-SNHG14 and Foxo3a with reducing miR-223-3p was found in LPS-treated A549 cells and lung tissue collected from the LPS-induced ALI model. lncRNA-SNHG14 inhibited miR-223-3p but promoted Foxo3a expression as a ceRNA. Artificially changes of lncRNA-SNHG14/miR-223-3p/Foxo3a pathway promoted or protected cell injury from LPS in vivo and in vitro. Autophagy activity could be influenced by lncRNA-SNHG14/miR-223-3p/Foxo3a pathway in cells with or without LPS treatment. In conclusion, aberrant expression changes of lncRNA-SNHG14 participated alveolar type II epithelial cell injury and acute lung injury induced by LPS through regulating autophagy. One underlying mechanism is that lncRNA-SNHG14 regulated autophagy by controlling miR-223-3p/Foxo3a as a ceRNA. It suggested that lncRNA-SNHG14 may serve as a potential therapeutic target for patients with sepsis-induced ALI.

lncRNA BSG-AS1 is hypoxia-responsive and promotes hepatocellular carcinoma by enhancing BSG mRNA stability.

Emerging evidence indicates that aberrant changes of lncRNAs expression induced by hypoxia participate in the development of HCC. The present study aimed to identify novel hypoxia-responsive lncRNAs and reveal its role and mechanism in HCC. Hypoxia exposure in HCC tissues was comprehensively estimated based on public data using multiple hypoxia gene signatures. Huh7 cells were treated with hypoxia and RNA-seq was performed. Then we analyzed the changes of lncRNAs in HCC tissues and cells exposed to hypoxia. We found that lncRNA BSG-AS1 was highly expressed in tissues with high hypoxia score. Then we verified the response of lncRNA BSG-AS1 to hypoxia in the cell hypoxia model in vitro. Through functional phenotypic analysis, we found that lncRNA BSG-AS1 can mediate the promoting effect of hypoxia on the proliferation and migration in HCC cells. RNA-seq was used to find the downstream target genes of lncRNA BSG-AS1. Sequencing data and wet experiments showed that mRNA of BSG decreased after knockout of lncRNA BSG-AS1, and mediated the promotive effect of lncRNA BSG-AS1 on proliferation and migration in HCC cells. The mechanism is that lncRNA BSG-AS1 can enhance the stability of BSG mRNA as antisense lncRNA. Finally, the data based on the public cohort and the cohort we collected suggested that the overexpression of lncRNA BSG-AS1 and BSG are related to the poor prognosis. In conclusion, lncRNA BSG-AS1 is a novel hypoxia-responsive lncRNA. LncRNA BSG-AS1 can positively regulate BSG, by maintaining the mRNA stability of BSG, thus promoting the proliferation and migration of HCC. High expression of lncRNA BSG-AS1 and BSG are risk factors for prognosis.

The E2F1/USP11 positive feedback loop promotes hepatocellular carcinoma metastasis and inhibits autophagy by activating ERK/mTOR pathway.

Deubiquitinase ubiquitin-specific protease 11 (USP11), a member of the deubiquitinating family, plays an important but still controversial role in cancer development. Namely, USP11 has been shown to promote the proliferation and metastasis of hepatocellular carcinoma (HCC), but the underlying molecular basis is poorly understood. This study aimed to unravel novel functions of USP11 in HCC, especially those related to autophagy. Here, EdU, migration and colony formation assays, and mouse models showed that USP11 played a crucial role in HCC cell proliferation and metastasis in vitro and in vivo. Results from co-immunoprecipitation and ubiquitination assays demonstrated that USP11 interacted with E2F1 and maintained E2F1 protein stability by removing its ubiquitin. Notably, E2F1 regulated USP11 expression at the transcriptional level. Thus, the E2F1/USP11 formed a positive feedback loop to promote the proliferation and migration of HCC cells. Moreover, E2F1/USP11 inhibited autophagy by regulating ERK/mTOR pathway. In addition, the combination treatment inhibition of USP11 and autophagy enhanced the apoptosis of HCC cells and inhibited the tumor growth in mice more effective than either treatment alone. Taken together, these results indicate that the E2F1/USP11 signal axis promotes HCC proliferation and metastasis and inhibits autophagy, which provides an experimental basis for the treatment of HCC.

Hypoxia associated multi-omics molecular landscape of tumor tissue in patients with hepatocellular carcinoma.

The present study was designed to update the knowledge about hypoxia-related multi-omic molecular landscape in hepatocellular carcinoma (HCC) tissues. Large-size HCC datasets from multiple centers were collected. The hypoxia exposure of tumor tissue from patients in 10 HCC cohorts was estimated using a novel HCC-specific hypoxia score system constructed in our previous study. A comprehensive bioinformatical analysis was conducted to compare hypoxia-associated multi-omic molecular features in patients with a high hypoxia score to a low hypoxia score. We found that patients with different exposure to hypoxia differed significantly in transcriptomic, genomic, epigenomic, and proteomic alterations, including differences in mRNA, microRNA (miR), and long non-coding RNA (lncRNA) expression, differences in copy number alterations (CNAs), differences in DNA methylation levels, differences in RNA alternative splicing events, and differences in protein levels. HCC survival- associated molecular events were identified. The potential correlation between molecular features related to hypoxia has also been explored, and various networks have been constructed. We revealed a particularly comprehensive hypoxia-related molecular landscape in tumor tissues that provided novel evidence and perspectives to explain the role of hypoxia in HCC. Clinically, the data obtained from the present study may enable the development of individualized treatment or management strategies for HCC patients with different levels of hypoxia exposure.

A novel hypoxia gene signature indicates prognosis and immune microenvironments characters in patients with hepatocellular carcinoma.

Due to the lack of a suitable gene signature, it is difficult to assess the hypoxic exposure of HCC tissues. The clinical value of assessing hypoxia in HCC is short of tissue-level evidence. We tried to establish a robust and HCC-suitable hypoxia signature using microarray analysis and a robust rank aggregation algorithm. Based on the hypoxia signature, we obtained a hypoxia-associated HCC subtypes system using unsupervised hierarchical clustering and a hypoxia score system was provided using gene set variation analysis. A novel signature containing 21 stable hypoxia-related genes was constructed to effectively indicate the exposure of hypoxia in HCC tissues. The signature was validated by qRT-PCR and compared with other published hypoxia signatures in multiple large-size HCC cohorts. The subtype of HCC derived from this signature had different prognosis and other clinical characteristics. The hypoxia score obtained from the signature could be used to indicate clinical characteristics and predict prognoses of HCC patients. Moreover, we reveal a landscape of immune microenvironments in patients with different hypoxia score. In conclusion, we identified a novel HCC-suitable 21-gene hypoxia signature that could be used to estimate the hypoxia exposure in HCC tissues and indicated prognosis and a series of important clinical features in HCCs. It may enable the development of personalized counselling or treatment strategies for HCC patients with different levels of hypoxia exposure.

A novel machine learning derived RNA-binding protein gene-based score system predicts prognosis of hepatocellular carcinoma patients.

BACKGROUND: Although the expression of RNA-binding protein (RBP) genes in hepatocellular carcinoma (HCC) varies and is associated with tumor progression, there has been no overview study with multiple cohorts and large samples. The HCC-associated RBP genes need to be more accurately identified, and their clinical application value needs to be further explored. METHODS: First, we used the robust rank aggregation (RRA) algorithm to extract HCC-associated RBP genes from nine HCC microarray datasets and verified them in The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) cohort and International Cancer Genome Consortium (ICGC) Japanese liver cancer (ICGC-LIRI-JP) cohort. In addition, the copy number variation (CNV), single-nucleotide variant (SNV), and promoter-region methylation data of HCC-associated RBP genes were analyzed. Using the random forest algorithm, we constructed an RBP gene-based prognostic score system (RBP-score). We then evaluated the ability of RBP-score to predict the prognosis of patients. The relationships between RBP-score and other clinical characteristics of patients were analyzed. RESULTS: The RRA algorithm identified 30 RBP mRNAs with consistent expression patterns across the nine HCC microarray datasets. These 30 RBP genes were defined as HCC-associated RBP genes. Their mRNA expression patterns were further verified in the TCGA-LIHC and ICGC-LIRI-JP cohorts. Among these 30 RBP genes, some showed significant copy number gain or loss, while others showed differences in the methylation levels of their promoter regions. Some RBP genes were risk factors or protective factors for the prognosis of patients. We extracted 10 key HCC-associated RBP genes using the random forest algorithm and constructed an RBP-score system. RBP-score effectively predicted the overall survival (OS) and disease-free survival (DFS) of HCC patients and was associated with the tumor, node, metastasis (TNM) stage, α-fetoprotein (AFP), and metastasis risk. The clinical value of RBP-score was validated in datasets from different platforms. Cox analysis suggested that a high RBP-score was an independent risk factor for poor prognosis in HCC patients. We also successfully established a combined RBP-score+TNM LASSO-Cox model that more accurately predicted the prognosis. CONCLUSION: The RBP-score system constructed based on HCC-associated RBP genes is a simple and highly effective prognostic evaluation tool. It is suitable for different subgroups of HCC patients and has cross-platform characteristics. Combining RBP-score with the TNM staging system or other clinical parameters can lead to an even greater clinical benefit. In addition, the identified HCC-associated RBP genes may serve as novel targets for HCC treatment.

Hypoxia-Induced lncRNA-NEAT1 Sustains the Growth of Hepatocellular Carcinoma via Regulation of miR-199a-3p/UCK2.

Objective: The long noncoding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 (NEAT1) has emerged as a novel player in hepatocellular carcinoma (HCC). Hypoxia is a common characteristic of the microenvironment of HCC. This study aimed to investigate whether lncRNA-NEAT1 is induced by hypoxia in HCC, and the mechanism that underlies LncRNA-NEAT1 function. Methods: The expression changes of lncRNA-NEAT1 in HCC cell lines under hypoxic conditions were examined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The regulatory effect of HIF-1α on lncRNA-NEAT1 was confirmed with chromatin immunoprecipitation (ChIP) and luciferase reporter assays. The function of lncRNA-NEAT1 on HCC cell growth under hypoxic conditions was determined by CCK-8 assay and flow cytometry. lncRNA -NEAT1 was predicted to serve as a competing endogenous RNA (ceRNA) within microRNA (miRNA)/mRNA axes based on microarray data, public HCC-related datasets and integrative bioinformatics analysis, and the miR-199a-3p/UCK2 axis was selected and validated by qRT-PCR, western blotting, RNA immunoprecipitation, and luciferase reporter analyses. The role of miR-199a-3p/UCK2 in HCC and its functional association with lncRNA-NEAT1 were assessed both in vitro and in vivo. Results: LncRNA-NEAT1 expression was significantly induced by hypoxia in SNU-182 and HUH7 cells. HIF-1α was shown to regulate lncRNA-NEAT1 transcription. Under hypoxic conditions, lncRNA-NEAT1 maintained the growth of HCC cells and inhibited apoptosis and cell cycle arrest. LncRNA-NEAT1 was predicted to regulate a panel of HCC-associated miRNA-mRNA pairs consisting of 8 miRNAs and 13 mRNAs. LncRNA-NEAT1 was shown to function as a ceRNA of miR-199a-3p/UCK2 both in HCC cells under hypoxic conditions and in an animal model. Conclusion: LncRNA-NEAT1 is a hypoxia-responsive lncRNA in HCC cell lines Insilico evidence suggested that LncRNA-NEAT1 may sustainthe growth of HCC cells by regulating HCC-associated mRNAs that interact with tumor-suppressive miRNAs. The lncRNA-NEAT1/miR-199a-3p/UCK2 pathway may contribute to the progression of HCC cell lines in a hypoxic microenvironment and therefore may represent a novel therapeutic target for HCC.

A panel of collagen genes are associated with prognosis of patients with gastric cancer and regulated by microRNA-29c-3p: an integrated bioinformatics analysis and experimental validation.

Background: The systematic expression characteristics and functions of collagen genes in gastric cancer (GC) have not been reported. Through public data integration, combined with bioinformatics analysis, we identified a panel of collagen genes overexpressed in GC. The functions of these genes were analyzed and validated in a GC-related cohort. microRNAs that may potentially target such genes were investigated in vitro. Methods: Four GC-related datasets retrieved from the Gene Expression Omnibus (GEO) were used to extract differentially expressed genes (DEGs) in GC. Functional annotation was performed to identify the potential roles of the identified DEGs. The association of candidate genes involved in the prognosis of GC patients (n=876) was determined using data provided by the Kaplan-Meier-plotter database, The Cancer Genome Atlas Stomach Adenocarcinoma (TCGA-STAD) repository, and a GC-related dataset (GSE15459). The expression characteristics of candidate genes and their associations with clinical parameters were validated in our in-house cohort (n=58). MicroRNAs able to target the identified candidate genes were predicted and confirmed using qRT-PCR, Western blotting, and dual-luciferase reporter assays in vitro. Results: After the integration of four GEO datasets, 76 DEGs were identified. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that these DEGs were significantly enriched in ECM-related functions and pathways. A group of collagen genes was significantly upregulated in the GC tissues and constituted a protein-protein interaction network as important nodes. Some of these collagen genes were closely associated with poor prognosis in GC patients. Overexpression of COL1A1 and COL4A1 was confirmed in our in-house cohort, and this was related to prognosis and certain clinicopathological parameters. We found that microRNA-29c-3p could directly target COL1A1 and COL4A1 in BGC-823 cells. Conclusions: Collagen genes identified in this study were associated with patient prognosis in GC and may represent diagnostic markers or potential therapeutic targets. Aberrant expression of such candidate genes may be induced by microRNA-29c-3p.

Prognostic Impact of Carboxylesterase 2 in Cholangiocarcinoma.

Carboxylesterase 2 (CES2) is instrumental for conversion of ester-containing prodrugs in cancer treatment. Novel treatment strategies are exceedingly needed for cholangiocarcinoma (CCA) patients. Here, we assessed CES2 expression by immunohistochemistry in a CCA cohort comprising 171 non-liver fluke associated, intrahepatic (n = 72) and extrahepatic (perihilar: n = 56; distal: n = 43) CCAs. Additionally, 80 samples of high-grade biliary intraepithelial neoplastic tissues and 158 corresponding samples of histological normal, non-neoplastic biliary tract tissues were included. CES2 expression was highest in non-neoplastic biliary tissue and significantly decreased in CCA. Patients showing any CES2 expression in tumor cells had a significantly better overall survival compared to negative cases (p = 0.008). This survival benefit was also maintained after stratification of CES2-positive cases, by comparing low, medium and high CES2 expression levels (p-trend = 0.0006). Evaluation of CCA subtypes showed the survival difference to be restricted to extrahepatic tumors. Correlation of CES2 expression with data of tumor-infiltrating immune cells showed that particularly CD8+ T cells were more frequently detected in CES2-positive CCAs. Furthermore, treatment of CCA cell lines with the prodrug Irinotecan reduced cell viability, increased cytotoxicity and modulated inflammatory gene expression. In conclusion, reduced CES2 expression is associated with poor outcome and low CD8+ T cell infiltration in CCA patients. Further clinical studies could show, whether CES2 expression may serve as a predictive marker in patients treated with prodrugs converted by CES2.

Modulation of the Proliferation/Apoptosis Balance of Vascular Smooth Muscle Cells in Atherosclerosis by lncRNA-MEG3 via Regulation of miR-26a/Smad1 Axis.

The balance between proliferation and apoptosis of vascular smooth muscle cells (VSMCs) plays a critical role in the initiation of atherosclerosis. LncRNA-MEG3 is involved in the pathophysiology of atherosclerosis through regulation of endothelial cell proliferation and migration. Its effect on the dysfunction of VSMCs and the corresponding mechanisms are actively researched. In this study, we observed that downregulated lncRNA-MEG3 expression was inversely correlated with the microRNA-26a level in coronary artery disease tissues. The overexpression of lncRNA-MEG3 could inhibit VSMCs proliferation while facilitating apoptosis. Moreover, alteration in the miR-26a/Smad1 axis could antagonize this effect. Bioinformatic analysis indicated that lncRNA-MEG3 could interact with miR-26a via complementary binding sites. The enforced expression of lncRNA-MEG3 could reduce the level of miR-26a in VSMCs, while the expression of Smad1 increases. Further, the direct binding between lncRNA-MEG3 and miR-26a was confirmed via dual-luciferase reporter assay, which indicated that lnc-MEG3 could sponge miR-26a as a competing endogenous RNA. In summary, we propose that lncRNA-MEG3 modulates the proliferation/apoptosis balance of VSMCs in atherosclerosis by regulating the miR-26a/Smad1 axis.

MicroRNA­21 expression is associated with the clinical features of patients with gastric carcinoma and affects the proliferation, invasion and migration of gastric cancer cells by regulating Noxa.

The expression levels of microRNA­21 (miR­21) are increased in a number of types of solid tumors. However, the association between miR­21 expression and clinical features of patients with gastric carcinoma, and gastric cancer proliferation, invasion and migration remains to be elucidated. The present study investigated the effect of miR­21 on the clinical features, proliferation, invasion and migration of gastric cancer and the underlying mechanisms associated with Noxa. Reverse transcription quantitative polymerase chain reaction (RT­qPCR) was performed to detect the expression levels of miR­21 and Noxa in samples of gastric cancer tissue and matched, adjacent, non­tumor tissue. The association between miR­21 expression and the clinical features of patients with gastric carcinoma, as well as the correlation between the mRNA and protein expression levels of miR­21 and Noxa were analyzed. SGC­7901 gastric cancer cells were cultured in vitro and transfected with an miR­21 mimic. The effect of miR­21 upregulation on proliferation and the cell cycle was determined using the MTT assay and flow cytometry. In addition, migration and invasion of SGC­7901 cells were observed using the Transwell assay. The target gene of miR­21 was identified using bioinformatics software and a dual luciferase reporting system. The effect of miR­21 upregulation on Noxa expression levels in SGC­7901 cells was also analyzed by RT­qPCR and western blotting. Increased levels of miR­21 expression and decreased levels of Noxa expression were observed in gastric cancer tissue samples when compared with the adjacent non­tumor tissue samples. An increased miR­21 expression level was identified as a risk factor for advanced stage gastric cancer, lymph node metastasis and larger primary tumors. Furthermore, the overexpression of miR­21 inhibited Noxa expression levels in SGC­7901 cells. Therefore, high levels of miR­21 expression may induce gastric cancer migration and invasion via the downregulation of Noxa expression.