LncRNA HOXA11-AS promotes hepatocellular carcinoma progression by repressing miR-214-3p.

Accumulating studies supported that lncRNAs played important roles in tumorigenesis. LncRNA HOXA11-AS was a novel lncRNA that has been proved to involved in several tumours. However, the role of HOXA11-AS in the development of hepatocellular carcinoma (HCC) remains to be explained. In our study, we showed that HOXA11-AS expression was up-regulated in the HCC tissues, and the higher expression of HOXA11-AS was associated with the advanced stage in the HCC samples. In addition, we indicated that the expression of HOXA11-AS was up-regulated in HCC cell lines (Hep3B, SMMC-7721, MHCC97-H and BEL-7402) compared with normal liver cell lines (HL-7702). Overexpression of HOXA11-AS promoted HCC proliferation and invasion and induced the epithelial-mesenchymal transition (EMT) and knockdown of HOXA11-AS suppressed the HCC cell proliferation and invasion. However, we showed that miR-214-3p expression was down-regulated in the HCC tissues and cell lines. Ectopic expression of miR-214-3p suppressed HCC cell proliferation and invasion. Furthermore, we indicated that overexpression of HOXA11-AS decreased the miR-214-3p expression and the expression of miR-214-3p was negatively related with the HOXA11-AS expression in HCC samples. Ectopic expression of HOXA11-AS increased HCC proliferation and invasion and induced EMT through inhibiting miR-214-3p expression. These data suggested that HOXA11-AS/miR-214-3p axis was responsible for development of HCC.

SSRP1 Contributes to the Malignancy of Hepatocellular Carcinoma and Is Negatively Regulated by miR-497.

The aim of this study is to clarify the clinical implication and functional role of structure specific recognition protein 1 (SSRP1) in hepatocellular carcinoma (HCC) and explore the underlying mechanism of aberrant high expression of SSRP1 in cancers. In the present investigation, we validated that SSRP1 was upregulated in HCC samples. We also demonstrated that its upregulation was associated with several clinicopathologic features such as higher serum AFP level, larger tumor size, and higher T stage of HCC patients; and its high expression indicated shorter overall survival and faster recurrence. To investigate the role of SSRP1 in HCC progression, both loss- and gain-function models were established. We demonstrated that SSPR1 modulated both proliferation and metastasis of HCC cells in vitro and vivo. Furthermore, we demonstrated that SSRP1-modulated apoptosis process and its knockdown increased the sensitivity of HCC cells to doxorubicin, 5-Fluorouracil, and cisplatin. We also identified microRNA-497 (miR-497) as a posttranscriptional regulator of SSRP1. Ectopic expression of miR-497 inhibited 3'-untranslated-region-coupled luciferase activity and suppressed endogenous SSRP1 expression at both messenger RNA and protein levels. For the first time, we proved that SSRP1 upregulation contributed to HCC development and the tumor-suppressive miR-497 served as its negative regulator.

The Regulation Network of Glycerolipid Metabolism as Coregulators of Immunotherapy-Related Myocarditis.

Background: To date, immunotherapy for patients with malignant tumors has shown a significant association with myocarditis. However, the mechanism of metabolic reprogramming changes for immunotherapy-related cardiotoxicity is still not well understood. Methods: The CD45+ single-cell RNA sequencing (scRNA-seq) of the Pdcd1-/-Ctla4+/- and wild-type mouse heart in GSE213486 was downloaded to demonstrate the heterogeneity of immunocyte atlas in immunotherapy-related myocarditis. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) spectrum metabolomics analysis detects the metabolic network differences. The drug prediction, organelle level interaction, mitochondrial level regulatory network, and phosphorylation site prediction for key regulators have also been screened via multibioinformatics analysis methods. Results: The scRNA analysis shows that the T cell is the main regulatory cell subpopulation in the pathological progress of immunotherapy-related myocarditis. Mitochondrial regulation pathway significantly participated in pseudotime trajectory- (PTT-) related differential expressed genes (DEGs) in the T cell subpopulation. Additionally, both the gene set enrichment analysis (GSEA) of PTT-related DEGs and LC-MS/MS metabolomics analysis showed that mitochondrial-regulated glycerolipid metabolism plays a central role in metabolic reprogramming changes for immunotherapy-related cardiotoxicity. Finally, the hub-regulated protease of diacylglycerol kinase zeta (Dgkz) was significantly identified and widely played various roles in glycerolipid metabolism, oxidative phosphorylation, and lipid kinase activation. Conclusion: Mitochondrial-regulated glycerolipid metabolism, especially the DGKZ protein, plays a key role in the metabolic reprogramming of immunotherapy-related myocarditis.

[Retracted] MicroRNA­449 suppresses proliferation of hepatoma cell lines through blockade lipid metabolic pathway related to SIRT1.

Following the publication of this paper, it was drawn to the Editor's attention by a concerned reader that several of the protein bands featured in the western blot assay data shown in Fig. 3A­D on p. 2147 were strikingly similar to other protein bands, both comparing the data within the same gel slices and comparing the data across the four different parts of the figure. In addition, the control blots featured in Fig. 3A, B and D had already appeared in a different form written by (largely) different authors at different research institutes. After having conducted an independent review of the data in this Figure in the Editorial Office, the concerns of the reader were found to be validated. Therefore, since contentious data in the above article had already been published prior to its submission to International Journal of Oncology, and owing to an overall lack of confidence in the presented data, the Editor has decided that this paper should be retracted from the Journal. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive a reply. The Editor apologizes to the readership for any inconvenience caused. [International Journal of Oncology 45: 2143­2152, 2014; DOI: 10.3892/ijo.2014.2596].

Distinct binding pattern of EZH2 and JARID2 on RNAs and DNAs in hepatocellular carcinoma development.

Hepatocellular carcinoma (HCC) is one of the most malignant cancers worldwide, with high mortality. However, the molecular regulatory mechanisms of liver cancer, especially transcriptional and post-transcriptional mechanisms, should be further studied. Here we used chromatin and cross-linking immunoprecipitation with high throughput sequencing methods (ChIP-seq and CLIP-seq) to capture the global binding profiles on RNAs and DNAs of Enhancer of zeste homolog 2 (EZH2) and its partner Jumonji And AT-Rich Interaction Domain Containing 2 (JARID2) in liver carcinoma cell lines (HepG2) and normal liver cell line (THLE-2), respectively. We also integrated HCC transcriptome data from the TCGA to analyze the expression pattern of bound genes. We found that EZH2 and JARID2 both showed distinct binding profiles between HepG2 and THLE-2 cells. By binding to the primary RNAs, bound transcripts of EZH2 and JARID2 in HepG2 showed significantly increased transcriptional levels in HCC patients. By performing gene set enrichment analysis (GSEA), the bound transcripts were also highly related to HCC development. We also found EZH2 and JARID2 could specifically bind to several long noncoding RNAs (lncRNAs), including H19. By exploring the DNA binding profile, we detected a dramatically repressed DNA binding ability of EZH2 in HepG2 cells. We also found that the EZH2-bound genes showed slightly increased transcriptional levels in HepG2 cells. Integrating analysis of the RNA and DNA binding profiles suggests EZH2 and JARID2 shift their binding ability from DNA to RNA in HepG2 cells to promote cancer development in HCC. Our study provided a comprehensive and distinct binding profile on RNAs and DNAs of EZH2 and JARID2 in liver cancer cell lines, suggesting their potential novel functional manners to promote HCC development.

MiR-3188 regulates the proliferation and apoptosis of hepatocellular carcinoma cells by targeting CXCL14.

Aim: The study aimed to probe into the functions and mechanisms of miR-3188 in hepatocellular carcinoma (HCC). Materials & methods: Quantitative real-time PCR and western blot were implemented to detect the expressions of miR-3188 and CXCL14 in HCC tissues and cell lines. CCK-8, 5-ethynyl-2'-deoxyuridine and flow cytometry assays were performed to assess cell viability, proliferation and apoptosis. A dual-luciferase reporter assay was conducted to investigate the relationship between miR-3188 and CXCL14. Results: miR-3188 is up-regulated in HCC tissues. MiR-3188 overexpression promoted cell viability and proliferation but inhibited the apoptosis of HCC cells. CXCL14 was proven to be a target of miR-3188, and CXCL14 reversed the effects of miR-3188 on HCC cells. Conclusion: MiR-3188 regulates the growth and apoptosis of HCC cells by targeting CXCL14.

SSRP1 Is a Prognostic Biomarker Correlated with CD8+ T Cell Infiltration in Hepatocellular Carcinoma (HCC).

BACKGROUND: Hepatocellular carcinoma (HCC), one of the most common primary malignancies, is theoretically an epitope candidate for immune checkpoint inhibitors, and therefore, the identification of HCC biomarkers is important. Structure-specific recognition protein 1 (SSRP1) is involved in almost all chromatin-related processes, including DNA replication, repair, and transcription. However, its role in HCC remains to be elucidated. METHODS: This study investigated the expression of SSRP1 in HCCDB, Oncomine, HPA, and other databases. The prognostic value of SSRP1 in HCC and its relationship with clinical characteristics were then explored using Kaplan-Meier plotter. At the same time, SSRP1 coexpression genes were explored and functionally annotated in the LinkedOmics database. Finally, the correlation between the SSRP1 expression and HCC immune cell infiltration was explored in TIMER and online single-cell sequencing database. RESULTS: Significantly elevated transcriptional and proteomic SSRP1 expressions were found in HCC. Increased SSRP1 mRNA expression was significantly correlated with relevant clinicopathological parameters such as immune cells. Notably, the SSRP1 expression was positively correlated with the infiltration levels of Treg and CD8+ T cells, especially exhausted CD8+ T cells. Interestingly, the SSRP1 expression was higher in both tumor Treg and exhausted CD8+ T cells than in adjacent tissues. CONCLUSION: SSRP1, as a new prognostic marker for HCC, promotes HCC development by influencing the infiltration of depleted CD8+ T cells and may influence the effect of immunotherapy.

Regulation of microRNA-497 expression in human cancer.

MicroRNAs (miRNAs/miRs) are a type of non-coding single-stranded RNA, with a length of ~22 nt, which are encoded by endogenous genes and are involved in the post-transcriptional regulation of gene expression in animals and plants. Studies have demonstrated that miRNAs play an important role in the occurrence, development, metastasis, diagnosis and treatment of cancer. In recent years, miR-497 has been identified as one of the key miRNAs in a variety of cancer types and has been shown to be downregulated in a variety of solid tumors. However, the regulation of miR-497 expression involves a complex network, which is affected by several factors. The aim of the present review was to summarize the mechanism of regulation of miR-497 expression at the pre-transcriptional and transcriptional levels in cancer, as well as the role of miR-497 expression imbalance in cancer diagnosis, treatment and prognosis. The regulatory mechanisms of miR-497 expression may aid in our understanding of the causes of miR-497 expression imbalance and provide a reference value for further research on the diagnosis and treatment of cancer.

Low microRNA150 expression is associated with activated carcinogenic pathways and a poor prognosis in patients with breast cancer.

Breast cancer is the most common type of cancer amongst women worldwide, and numerous microRNAs (miRNAs/miRs) are involved in the initiation and progression of breast cancer. The aim of the present study was to identify hub miRNAs and determine the underlying mechanisms regulated by these miRNAs in breast cancer. Breast invasive carcinoma transcriptome data (including mRNAs and miRNAs), and clinical data were acquired from The Cancer Genome Atlas database. Differential gene expression analysis, co­expression network analysis, gene set enrichment analysis (GSEA) and prognosis analysis were used to screen the hub miRNAs and explore their functions. Functional experiments were used to determine the underlying mechanisms of the hub miRNAs in breast cancer cells. The results revealed that low miR150 expression predicted a more advanced disease stage, and was associated with a less favorable prognosis. Through the combined use of five miRNA­target gene prediction tools, 31 potential miR150 target genes were identified. GSEA revealed that low miR150 expression was associated with the upregulation of several cancer­associated signaling pathways, and the downregulation of several tumor suppressor genes. Furthermore, miR150 independently affected overall survival in patients, and interacted with its target genes to indirectly affect overall and disease­free survival. Functional experiments demonstrated that miR150 positively regulated B and T lymphocyte attenuator (BTLA), and the downregulation of miR150 and BTLA combined promoted cell migration. In conclusion, the present study revealed that low miR150 expression was associated with less favorable clinical features, upregulation of several carcinogenic signaling pathways, and poor patient survival. Additionally, a miR150­BTLA axis was suggested to regulate cell viability and migration.

Single-walled carbon nanohorn aggregates promotes mitochondrial dysfunction-induced apoptosis in hepatoblastoma cells by targeting SIRT3.

Single-walled carbon nanohorns (SWNHs) can accumulate in a variety of cell types or tissues and exert biological effects, which have been demonstrated to induce apoptosis in hepatoblastoma cells. However, the role and molecular mechanisms of SWNHs remain unclear. The mitochondrion is an important subcellular structure and may contribute to apoptosis that is induced by SWNHs in hepatoblastoma cells. To address this question, the mitochondrial function of HepG2 or L02 cells that were treated with SWNHs was examined. The results indicated that SWNHs were able to decrease the mitochondrial membrane potential and suppress the activity of the Na+/K+-ATPase. Secondly, HepG2 cells and L02 cells were treated with SWNHs in vivo and in vitro. The expression of mitochondrial-associated proteins [acyl-CoA synthetase short chain family member 1, Bax, cytochrome C (CYT-C), sodium channel epithelial 1α subunit, sirtuin 3 (SIRT3) and voltage-dependent anion channel 1] was analyzed by western blotting and immunohistochemical staining. The results revealed that SWNH treatment was able to alter the expression of multiple mitochondrial apoptotic pathway-associated proteins in HepG2 cells. SWNH treatment was able upregulate the expression of SIRT3, CYT-C and VDAC1 and downregulate the expression of AceCS2, but it had a more stable effect on SIRT3. However, similar findings were not observed in L02 cells. Therefore, the data from the present study indicated that SWNHs might be used as a safe anticancer agent, where it is able to trigger mitochondrial dysfunction-induced apoptosis by upregulating SIRT3 expression in HepG2 cells.

Titanium dioxide nanoparticles induce mitochondria-associated apoptosis in HepG2 cells.

Widespread applications of nanosized materials over the past decade have prompted investigations of desirable properties and potential hazards to humans and the environment. Titanium dioxide (TiO2) nanoparticles are one of the most widely used nanoparticles. To investigate the effect of biological functions induced by TiO2 nanoparticles (10 nm: TiO2 NPs) on human liver cell lines, normal liver cell line L02 and hepatoma cell line HepG2 were co-cultured with exogenous TiO2 NPs. Cell growth and proliferation, cell cycle, and the apoptosis rate were analyzed. The effects of TiO2 NPs on the expression levels of apoptosis-associated protein caspase-3 and the membrane channel protein αENaC and caspase-3/7 activity were determined. Moreover, the influence of TiO2 NPs on the expression levels of the mitochondria-related proteins SIRT3, VDAC1, and ACSS1, the mitochondrial membrane potential and the ADP/ATP ratio were also examined. Our results revealed that TiO2 NPs inhibited the growth and proliferation of HepG2 cells, suppressed the S phase of cell cycling, and induced apoptosis of HepG2 cells. Following an increase in concentration, the inhibitory effect induced by TiO2 NPs on proliferation and cell cycle was more evident, and the apoptosis rate increased in a significant concentration-dependent manner, whereas there was no significant effect on the growth, proliferation, apoptosis, and cell cycle of L02 cells. In addition, the results of western blot showed that in HepG2 cells, TiO2 NPs upregulated the expressions of the apoptosis-related protein caspase-3 and the membrane channel protein αENaC in a concentration-dependent manner. However, in L02 cells, there was no significant difference in the expression levels of caspase-3 or αENaC. Furthermore, TiO2 NPs induced depolarization of the mitochondrial membrane, upregulated the expression levels of the mitochondria-related proteins SIRT3 and VDAC1, and downregulated the expression level of the key respiratory chain protein ACSS1 in HepG2 cells. However, in L02 cells, the expressions of SIRT3, VDAC1, and ACSS1 exhibited no clear change. The apoptosis of HepG2 cells induced by TiO2 NPs may be achieved by regulating intracellular osmotic pressure; moreover, upregulating the expression of the channel protein αENaC or the mitochondrial porin VDAC1 and depolarizing the mitochondrial membrane of HepG2 cells resulted in the loss of Cyt-c and ATP and further activated caspase-3. To further confirm the above results, a nude mouse xenograft model was employed. After a certain period of treatment with TiO2 NPs, the nude mice were sacrificed, tumors were removed, and the expression of related proteins was detected. Immunohistochemistry and western blot results showed that the expressions of the proteins VDAC1 and SIRT3 were clearly upregulated in tissues treated to TiO2 NPs, whereas the expression of ACSS1 was downregulated. The results were consistent with the above in vitro results. All the above results confirmed that TiO2 NPs can act as a safe antitumor agent.

GmWRKY16 Enhances Drought and Salt Tolerance Through an ABA-Mediated Pathway in Arabidopsis thaliana.

The WRKY transcription factors (TFs) are one of the largest families of TFs in plants and play multiple roles in plant development and stress response. In the present study, GmWRKY16 encoding a WRKY transcription factor in soybean was functionally characterized in Arabidopsis. GmWRKY16 is a nuclear protein that contains a highly conserved WRKY domain and a C2H2 zinc-finger structure, and has the characteristics of transcriptional activation ability, presenting a constitutive expression pattern with relative expression levels of over fourfold in the old leaves, flowers, seeds and roots of soybean. The results of quantitative real time polymerase chain reaction (qRT-PCR) showed that GmWRKY16 could be induced by salt, alkali, ABA, drought and PEG-6000. As compared with the control, overexpression of GmWRKY16 in Arabidopsis increased the seed germination rate and root growth of seedlings in transgenic lines under higher concentrations of mannitol, NaCl and ABA. In the meantime, GmWRKY16 transgenic lines showed over 75% survival rate after rehydration and enhanced Arabidopsis tolerance to salt and drought with higher proline and lower MDA accumulation, less water loss of the detached leaves, and accumulated more endogenous ABA than the control under stress conditions. Further studies showed that AtWRKY8, KIN1, and RD29A were induced in GmWRKY16 transgenic plants under NaCl treatment. The expressions of the ABA biosynthesis gene (NCED3), signaling genes (ABI1, ABI2, ABI4, and ABI5), responsive genes (RD29A, COR15A, COR15B, and RD22) and stress-related marker genes (KIN1, LEA14, LEA76, and CER3) were regulated in transgenic lines under drought stress. In summary, these results suggest that GmWRKY16 as a WRKY TF may promote tolerance to drought and salt stresses through an ABA-mediated pathway.

Proapoptotic Role of Potassium Ions in Liver Cells.

Potassium channels are transmembrane proteins that selectively promote the infiltration of potassium ions. The significance of these channels for tumor biology has become obvious. However, the effects of potassium ions on the tumor or normal cells have seldom been studied. To address this problem, we studied the biological effects of L02 and HepG2 cells with ectogenous potassium ions. Cell proliferation, cell cycle, and apoptosis rate were analyzed. Our results indicated that potassium ions inhibited proliferation of L02 and HepG2 cells and promoted their apoptosis. Potassium ions induced apoptosis through regulating Bcl-2 family members and depolarized the mitochondrial membrane, especially for HepG2 cell. These biological effects were associated with channel protein HERG. By facilitating expression of channel protein HERG, potassium ions may prevent it from being shunted to procancerous pathways by inducing apoptosis. These results demonstrated that potassium ions may be a key regulator of liver cell function. Thus, our findings suggest that potassium ions could inhibit tumorigenesis through inducing apoptosis of hepatoma cells by upregulating potassium ions transport channel proteins HERG and VDAC1.

Histone deacetylase inhibitor screening identifies HC toxin as the most effective in intrahepatic cholangiocarcinoma cells.

Histone deacetylases (HDACs) are highly expressed in intrahepatic cholangiocarcinoma (ICC) and are associated with poor prognosis of these patients. The aim of the present study was to explore the inhibitory effects of HDAC inhibitors on ICC cells and identify effective and sensitive drugs for ICC. Effects of 34 HDAC inhibitors were screened through two rounds of cell viability assays, and HC toxin, a cyclic tetrapeptide first isolated from the secondary metabolite of Helminthosporium carbonum, exhibited an antitumor activity superior to that of the other HDAC inhibitors and gemcitabine. The mechanisms involved in the inhibitory effects of HC toxin on CCLP-1 cells were investigated by cell counting, colony formation assay, cell morphological observation, real-time PCR, western blotting and flow cytometry. It was demonstrated that HC toxin inhibited the cell proliferation and clone formation ability of the CCLP-1 cells. HC toxin increased the acetyl-histone H4 level and this was associated with the inhibitory effect of HC toxin on the CCLP-1 cells. We also found that HC toxin reduced the level of HDAC1 protein in a post-transcriptional manner. Morphological observation showed multiple morphological changes and indicated the possibility of cell differentiation owing to HC toxin. With increasing concentration of HC toxin, the cell cycle was gradually arrested at the G0/G1 stage and the percentage of apoptotic cells increased which was not mainly through the caspase-3-dependent ways. These results indicated that HC toxin was the most effective among the various HDAC inhibitors with multiple functions in the suppression of ICC in vitro. Thus, HC may be a potential chemotherapeutic for ICC.

Single-walled carbon nanohorn (SWNH) aggregates inhibited proliferation of human liver cell lines and promoted apoptosis, especially for hepatoma cell lines.

Single-walled carbon nanohorns (SWNHs) may be useful as carriers for anticancer drugs due to their particular structure. However, the interactions between the material itself and cancerous or normal cells have seldom been studied. To address this problem, the effects of raw SWNH material on the biological functions of human liver cell lines were studied. Our results showed that unmodified SWNHs inhibited mitotic entry, growth, and proliferation of human liver cell lines and promoted their apoptosis, especially in hepatoma cell lines. Individual spherical SWNH particles were found inside the nuclei of human hepatoma HepG2 cells and the lysosomes of normal human liver L02 cells, implying that SWNH particles could penetrate into human liver cells_and the different interacted mechanisms on human normal cell lines compared to hepatoma cell lines. Further research on the mechanisms and application in treatment of hepatocellular carcinoma with SWNHs is needed.

MicroRNA-449 suppresses proliferation of hepatoma cell lines through blockade lipid metabolic pathway related to SIRT1.

MicroRNA (miRNA or miR) inhibition of oncogenic related pathways has been shown to be a promising therapeutic approach for cancer. SIRT1 might be a promoter factor on tumorigenesis of hepatocellular carcinoma (HCC). However, the mechanism is unknown. We investigated whether miRNAs regulate the SIRT1 and its downstream SREBP-lipogenesis-cholesterogenesis metabolic pathway in hepatoma cells. Human hepatoma cells were transfected with miR-449 mimics and inhibitors, and the effects of miR-449 on cell proliferation was assessed. We identified the miRNAs, miR-449, that control lipogenesis and cholesterogenesis in hepatoma cells by inhibiting SIRT1 and SREBP-1c expression and downregulating their targeted genes, including fatty acid synthase (FASN) and 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR). MiR-449 repressed DNA synthesis, mitotic entry and proliferation of hepatoma cells. Restoration of miR-449 led to suppression of SIRT1 expression and liver tumorigenesis. The newly identified miRNAs, miR-449 represents a novel targeting mechanism for HCC therapy.