HCCLM3 Cells Research Articles

Daidzin inhibits hepatocellular carcinoma survival by interfering with the glycolytic/gluconeogenic pathway through downregulation of TPI1.

Daidzin (DDZ) is a natural brassin-like compound extracted from the soybean, and has been found to have therapeutic potential against tumors in recent years. This study investigates the therapeutic effect of DDZ on hepatocellular carcinoma cells and elucidates the possible mechanisms of action. The viability of HCCLM3 and Hep3B cells was detected by MTT assay. Western blots and qPCR were used to detect the protein and mRNA levels of proliferation and apoptosis related genes. Gas chromatography-mass spectrometry (GC-MS) was used for metabolome analysis. In vivo antitumor effects were assessed in nude mice engrafted with HCC cell lines. Our results show that DDZ treatment dose-dependently inhibited cell viability, migration, and survival. The expressions of CDK1, BCL2, MYC, and survivin were reduced, while the expressions of BAX and PARP were increased in DDZ treated cells. The differentially expressed metabolites detected in DDZ treated cultures are associated with glycolysis/gluconeogenesis pathways. Bioinformatic analysis identified TPI1, a gene in the glycolysis pathway with prognostic value for hepatocellular carcinoma (HCC), and DDZ treatment downregulated this gene. In vivo experiments show that DDZ significantly reduced the tumor volume and weight, and inhibited Ki67 expression within tumors. This study shows that DDZ interfered with the survival and migration of hepatocellular carcinoma cells, likely via TPI1 and the gluconeogenesis pathway.

The lncRNA CASC2 Modulates Hepatocellular Carcinoma Cell Sensitivity and Resistance to TRAIL Through Apoptotic and Non-Apoptotic Signaling.

The immune cytokine tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been widely concerned as a tumor therapy because of its ability of selective triggering cancer cell apoptosis; nevertheless, hepatocellular carcinoma (HCC) exhibits acquired resistance to TRAIL-induced apoptosis. In the present study, tumor-suppressive lncRNA cancer susceptibility candidate 2 (CASC2) was downregulated in HCC tissues and cell lines; HCC patients with lower CASC2 expression predicted a shorter overall survival rate. In vitro, CASC2 overexpression dramatically repressed HCC cell proliferation and inhibited cell apoptosis; in vivo, CASC2 overexpression inhibited subcutaneous xenotransplant tumor growth. CASC2 affected the caspase cascades and NF-κB signaling in TRAIL-sensitive [Huh-7 (S) and HCCLM3 (S)] or TRAIL-resistant cell lines [Huh-7 (R) and HCCLM3 (R)] in different ways. In Huh-7 (S) and HCCLM3 (S) cells, CASC2 affected cell apoptosis through the miR-24/caspase-8 and miR-221/caspase-3 axes and the caspase cascades. miR-18a directly targeted CASC2 and RIPK1. In Huh-7 (R) and HCCLM3 (R) cells, CASC2 affected cell proliferation through the miR-18a/RIPK1 axis and the NF-κB signaling. RELA bound to CASC2 promoter region and inhibited CASC2 transcription. In conclusion, CASC2 affects cell growth mainly via the miR-24/caspase-8 and miR-221/caspase-3 axes in TRAIL-sensitive HCC cells; while in TRAIL-resistant HCC cells, CASC2 affects cell growth mainly via miR-18a/RIPK1 axis and the NF-κB signaling. These outcomes foreboded that CASC2 could be a novel therapeutic target for further study of HCC-related diseases.

CircCBFB is a mediator of hepatocellular carcinoma cell autophagy and proliferation through miR-424-5p/ATG14 axis.

This study aims to investigate the role of circCBFB in hepatocellular carcinoma (HCC) cell proliferation and autophagy. qRT-PCR and Western blotting analyses quantified the expression levels of circCBFB, miR-424-5p, and ATG14 in HCC tissues and/or HCC cell lines. After transfection with pcDNA3.1-CircCBFB, sh-CircCBFB, miR-424-5p mimic, miR-424-5p inhibitor, pcDNA3.1-ATG14, sh-ATG14, sh-CircCBFB + miR-424-5p inhibitor, pcDNA3.1-CircCBFB + miR-424-5p mimic, sh-CircCBFB + pcDNA3.1-ATG14, or pcDNA3.1-CircCBFB + sh-ATG14, the proliferation, cell cycle, and apoptosis of Huh-7 and HCCLM3 cells were detected, respectively, through MTT assay and flow cytometry. Western blotting measured the expression levels of ATG14 and autophagy-related proteins (LC3-ΙΙ/LC3-Ι, Beclin1, and p62). The interactions among circCBFB, miR-424-5p, and ATG14 were identified through RNA fluorescence in situ hybridization and RNA immunoprecipitation. In HCC tissues, circCBFB and ATG14 were highly expressed, and miR-424-5p expression was downregulated. Transfection of pcDNA3.1-CircCBFB, miR-424-5p inhibitor, or pcDNA3.1-ATG14 into HCC cells facilitated HCC cell proliferation and autophagy, while suppressing cell apoptosis, evidenced by elevated cell viability, increased protein levels of autophagosome markers (LC3-ΙΙ/LC3-Ι and Beclin1), repressed apoptosis rate, and suppressed protein level of autophagy receptor p62. miR-424-5p was a target gene of circCBFB, and miR-424-5p negatively mediated ATG14. CircCBFB inhibits miR-424-5p and upregulates ATG14, thus promoting HCC cell proliferation and autophagy.

Dexmedetomidine promotes apoptosis and suppresses proliferation of hepatocellular carcinoma cells via microRNA-130a/EGR1 axis

Accumulating evidence has revealed the role of microRNAs (miRs) in hepatocellular carcinoma (HCC). Dexmedetomidine, a highly selective α2-adrenergic agonist, is widely used in perioperative settings for analgesia and sedation. Herein, we aimed to determine whether dexmedetomidine might directly regulate miR-130a/early growth response 1 (EGR1) axis in HCC and explore the related mechanisms. miR-130a and EGR1 expression were determined in HCC tissues and their correlation was evaluated. Human HCC cell line HCCLM3 was selected. Upon the determination of the optimal concentration of dexmedetomidine, HCCLM3 cells were treated with dexmedetomidine, miR-130a- or EGR1-related oligonucleotides or plasmids were transfected into cells to explore their functions in cell biological behaviors. miR-130a and EGR1 levels in cells were tested. The targeting relationship between miR-130a and EGR1 was verified. miR-130a was inhibited while EGR1 was elevated in HCC tissues and they were negatively correlated. EGR1 was targeted by miR-130a. With the increase of dexmedetomidine concentration, HCCLM3 cell viability was correspondingly inhibited, miR-130a expression was elevated and EGR1 expression was decreased. Dexmedetomidine, upregulating miR-130a or downregulating EGR1 inhibited proliferation, invasion and migration, and promoted apoptosis of HCCLM3 cells. MiR-130a upregulation/downregulation enhanced/impaired the effect of dexmedetomidine on cell biological behaviors. Our study provides evidence that raising miR-130a enhances the inhibitory effects of dexmedetomidine on HCC cellular growth via inhibiting EGR1. Thus, miR-130a may be a potential candidate for the treatment of HCC.

MiR-126-5p Promotes Tumor Cell Proliferation, Metastasis and Invasion by Targeting TDO2 in Hepatocellular Carcinoma.

TDO2 is a key enzyme in the kynurenine metabolic pathway, which is the most important pathway of tryptophan metabolism. It has been shown that miRNAs are involved in cell metastasis through interaction with target mRNAs. In this study, we found 645 miRNAs that could be immunoprecipitated with TDO2 through the RNA-immunoprecipitation experiment. miR-126-5p was selected as the research target, which was also confirmed by dual-luciferase reporter assay. Through qRT-PCR analysis, it was verified that the overexpression of miR-126-5p promoted the expression of TDO2, PI3K/AKT and WNT1. Meanwhile, it was verified that overexpression of miR-126-5p can promote intracellular tryptophan metabolism by HPLC. We also verified the effects of miR-126-5p on cell proliferation, migration, and invasion by cck-8, cell colony formation and trans-well assay in both HCCLM3 cells and HepG2 cells. In vivo experiments were also conducted to verify that miR-126-5p promoted tumor formation and growth via immunohistochemical detection of cell infiltration and proliferation to generate markers Ki-67, BAX, and VEGF. In conclusion, our results suggest that miR-126-5p is a biomarker and a potential new treatment target in the progression of HCC via promoting the expression of TDO2.

RETRACTED ARTICLE: Repressing phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma by microRNA-142-3p restrains the progression of hepatocellular carcinoma

Statement of Retraction Chuanli Zeng, Gang Yuan, Yaoren Hu, Donghui Wang, Xiaojun Shi, Dedong Zhu, Airong Hu, Yina Meng & Jialin Lu (2022) Repressing phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma by microRNA-142-3p restrains the progression of hepatocellular carcinoma, Bioengineered, 13:1, 1491-1506, DOI: 10.1080/21655979.2021.2020549 Since publication, concerns have been raised about the integrity of the data in the article. When approached for an explanation, the authors have been unable to fully address the concerns raised and have not been able to provide their original data in the appropriate format. As verifying the validity of published work is core to the integrity of the scholarly record, we are therefore retracting the article. All authors listed in this publication have been informed. We have been informed in our decision-making by our policy on publishing ethics and integrity and the COPE guidelines on retractions. The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as ‘Retracted’.

The Efficacy of Combined Therapy of Regorafenib with Detoxicating and Stasis Softening Chinese Herbal Spleen Tonics in Mid-/Late-Stage Hepatocellular Carcinoma.

Objective To explore the efficacy of combined therapy of Regorafenib with detoxicating and stasis softening Chinese herbal spleen tonics (DSS-splenic tonics) in mid-/late-stage hepatocellular carcinoma. Methods Retrospective observational data of 120 patients were obtained, 60 of which received combined therapy of DSS-splenic tonics and regorafenib. Adverse event, overall survival (OS), and time-to-progress (TTP) were analyzed. Synergistic effect of DSS-spleen tonics was found and validated in human hepatocellular carcinoma HCCLM3 cell line and xenograft mouse models. Results Combination of regorafenib and DSS-splenic tonics also slightly extended the TTP and OS compared with treatment of regorafenib alone, suggesting DSS-splenic tonics has synergistic effect with regorafenib. Both Regorafenib and DSS-spleen tonics exerted inhibitory effect on cell viability and invasion capability of HCCLM3 cells, and combining both could enhance the antitumor effect. At molecular level, we found that VEGF, HIF-1α, MVD, and VEGF2 were all suppressed by regorafenib and DSS-splenic tonics. These results suggest that DSS-spleen tonics function synergistically with regorafenib in HCC by enhancing the regulation of regorafenib on VEGF, MMP-2, HIF-1α, and MVD, and may diminish angiogenesis during HCC progression. Conclusion DSS-spleen tonics could exert synergistic antitumor effect with regorafenib via targeting VEGF.

Profiling Cystathionine β/γ-Lyase in Complex Biosamples Using Novel Activatable Fluorogens.

Cystathionine lyase, the key enzyme in transsulfuration and reverse transsulfuration pathways, is involved in a wide array of physiological and pathophysiological processes in both mammals and nonmammals. Though the biological significance of the hydrogen sulfide/cystathionine lyase system in disease states is extensively discussed, the absence of molecular methods for direct monitoring of cystathionine lyase in complex biosamples renders the result unreliable and perplexing. Here, we present the first attempt at designing and developing effective activatable fluorescent probes for cystathionine lyase based on the naphthylamide scaffold. CBLP and CSEP were designed based on the catalytic preference of cystathionine β-lyase (CBL) and cystathionine γ-lyase (CSE). Briefly, incorporation of cysteine/homocysteine as the recognition moiety and a carbamate ethyl sulfide group as a self-immolated linker proved to be an effective strategy for cystathionine lyase fluorescence reporting. CBLP exhibits high selectivity and sensitivity in vitro in semiquantifying CBL levels in roots of wild-type Arabidopsis thaliana and cbl mutants (cbl knockout: SALK_014740C, overexpressed: OE-CBL). Meanwhile, CSEP successfully detected CSE levels in HCC-LM3 cells, zebrafish models, and upregulated CSE in frozen section slides from the liver tissue of cecal ligation and puncture (CLP)-induced septic rats, which was also validated by Western blotting and immunohistochemical analysis. In summary, the practical design strategy facilitates profiling of cystathionine lyase activity in biological processes. It may pave the way for the development of accurate and efficient methods for the direct estimation of cystathionine lyase.

Depleting circ_0088364 restrained cell growth and motility of human hepatocellular carcinoma via circ_0088364-miR-1270-COL4A1 ceRNA pathway

ABSTRACT Circular RNA hsa_circ_0088364 (circ_0088364) is a contributory factor in the malignancy of hepatocellular carcinoma (HCC). We aimed to elaborate its role and competing endogenous RNA (ceRNA) mechanism in HCC cell growth and motility. Expression of circ_0088364, microRNA (miR)-1270 and Collagen type IV alpha 1 chain (COL4A1) was measured by real-time quantitative PCR and Western blotting, and their relationships were determined by dual-luciferase reporter assay, RNA immunoprecipitation, biotinylated RNA pull-down, and Spearman’s rank correlation analysis. Cellular programs were measured by cell counting kit-8 assay, flow cytometry and transwell assays, Western blotting, and xenograft experiment. Expression of circ_0088364 and COL4A1 was upregulated, and miR-1270 was downregulated in HCC patients’ tumors; moreover, there were linear correlations among circ_0088364, miR-1270, and COL4A1 expression. Essentially, circ_0088364 and COL4A1 were ceRNAs for miR-1270 via target binding. In function, silencing circ_0088364 or upregulating miR-1270 could suppress cell proliferation, cell cycle entrance, transwell migration and invasion in Huh7 and HCCLM3 cells, as well as promote apoptosis rate. Moreover, above-mentioned effects were accompanied with reduced B-cell lymphoma (Bcl)-2, N-cadherin and Vimentin levels, and elevated Bcl-2-associated X protein (Bax) and E-cadherin levels. Contrarily, exhausting miR-1270 and restoring COL4A1 could severally abrogate the tumor-suppressive roles of circ_0088364 knockdown and miR-1270 overexpression in HCC cells in vitro. In vivo, silencing circ_0088364 retarded xenograft tumor growth in nude mice induced by Huh7 cells by upregulating miR-1270 and downregulating COL4A1. Blocking circ_0088364 suppressed HCC by inhibiting cell growth and motility via targeting miR-1270-COL4A1 axis.

LncRNA LINC00667 aggravates the progression of hepatocellular carcinoma by regulating androgen receptor expression as a miRNA-130a-3p sponge

Emerging studies have found long noncoding RNAs, widely expressed in eukaryotes, crucial regulators in the progression of human cancers, including hepatocellular carcinoma (HCC). Although the long intergenic noncoding RNA 667 (LINC00667) can promote the progression of a variety of cancer types, the expression pattern, the role in cancer progression, and the molecular mechanism involved in HCC remain unclear. This study aims to investigate the function and mechanism of LINC00667 in HCC progression. The effects of LINC00667 silencing in cell proliferation, cell migration, and cell invasion, and androgen receptor (AR) expression were determined with loss-of-function phenotypic analysis in Huh-7 and HCCLM3 cells, and subsequently testified in vivo in tumor growth. We found that the expression of LINC00667 was upregulated in HCC tissues and cell lines. Upregulation of LINC00667 was significantly associated with the unfavorable prognosis of HCC in our study patients. On the other hand, low expression of LINC00667 significantly inhibited the cell proliferation, cell migration and cell invasion of HCC in vitro and tumor growth in vivo. This inhibitory effect could be counteracted by miR-130a-3p inhibitor. LINC00667 reduced the inhibition of AR expression by miR-130a-3p, which correlated with the progression of HCC. Our finding suggests LINC00667 is a molecular sponge in the miR-130s-3p/AR signal pathway in the progression of HCC, in which it relieves the repressive function of miR-130a-3p on the AR expression. This indicates LINC00667 functions as a tumor promotor in promoting HCC progression through targeting miR-130a-3p/AR axis, making a novel biomarker and potential therapeutic target for HCC.

Salvianolic acid B targets mortalin and inhibits the migration and invasion of hepatocellular carcinoma via the RECK/STAT3 pathway

BackgroundTumor migration and invasion is a complex and diverse process that involves the epithelial–mesenchymal transition (EMT) of tumor cells and degradation of the extracellular matrix by matrix metalloproteases (MMPs). Mortalin is an important oncogene. It has been reported to play an important role in tumor migration and invasion through various signaling pathways, but the underlying mechanism is not fully understood.MethodsHere, we investigated the role of mortalin in the migration of the hepatocellular carcinoma (HCC) cell lines HepG2 and HCCLM3.ResultsThe overexpression of mortalin in HepG2 cells decreased the protein level of reversion-inducing cysteine-rich protein with Kazal motifs (RECK) and activated the phosphorylation and acetylation of STAT3, thereby up-regulating matrix metalloproteinase 9 (MMP9) and promoting cell migration and invasion. In contrast, in HCCLM3 cells, mortalin knockdown increased the expression of RECK, inhibited the STAT3 pathway and the activity of MMP9, and inhibited cell migration and invasion. Furthermore, we found that salvianolic acid B, a caffeic acid phenethyl ester analog, specifically bound to mortalin and increased the degradation of mortalin proteasomes through ubiquitination, thereby up-regulating RECK, inhibiting STAT3, and finally inhibiting the migration and invasion of HCC cells.ConclusionOur work suggested that mortalin is a potential therapeutic target for hepatocellular carcinoma.

Molecular Mechanism of Chrysin in Hepatocellular Carcinoma Treatment Based on Network Pharmacology and in Vitro Experiments

This study elucidated the potential molecular mechanism of chrysin in hepatocellular carcinoma (HCC) treatment using network pharmacology and in vitro experiments. Chrysin and candidate targets of HCC were obtained from the TCMSP and DrugBank databases, followed by mapping and screening of chrysin and HCC targets to identify the core targets of chrysin in HCC treatment. The interaction of chrysin and its targets, including CDK1, CDK5, as well as MMP9, were evaluated by molecular docking. The STRING database and Cytoscape (version 3.8.2) software were used to construct protein interactions and component-target networks of the core targets. Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis of the core target genes were performed using the DAVID database. Network pharmacology results showed that chrysin treatment of HCC was mainly related to cell proliferation and cell cycle. Accordingly, the cell counting kit-8 method and flow cytometry were used to detect the cell viability and cell cycle of hepatocarcinoma cells HCCLM3 and BEL-7402 in vitro. A total of 142 compound targets of chrysin, 12,179 HCC-related targets, and 116 intersecting targets were screened. The first 20 GO biological annotations of 17 core targets and the first 20 KEGG pathways mainly involved cell proliferation and cell cycle. In vitro experiments showed that chrysin inhibits the proliferation of human hepatocarcinoma cells (HCCLM3 and BEL-7402) in a dose-dependent manner. Moreover, chrysin induced cell cycle arrest in HCCLM3 and BEL-7402 cells in the G2 phase, and the expression was downregulated of cyclin-dependent kinases (CDKs), CDK2 and CDK4. Chrysin can offset HCC mainly by regulating the cell cycle and inhibiting cell proliferation. The network pharmacology results were verified, providing the basis for further study on the mechanism of chrysin intervention in HCC.

A New Nanomaterial Based on Extracellular Vesicles Containing Chrysin-Induced Cell Apoptosis Through Let-7a in Tongue Squamous Cell Carcinoma

Although the therapeutic strategy showed significant improvement, the therapeutic effect was poor on metastases in tongue squamous cell carcinoma (TSCC) which is the most malignant tumor found in the head and neck. Chrysin, similar to the flavonoids, plays an antitumor role by regulating the expression of ncRNAs in many kinds of cancers. Compared to flavonoids, gold nanoparticles (AuNPs) provide a novel insight into inhibiting cancer cell growth via photothermal therapy (PPT) which is irradiated by near-infrared radiation (NIR). However, most flavonoids and AuNPs lack specificity of tumor in vivo. The extracellular vesicles (EVs) which were abundant with ncRNAs are isolated from the cellular supernatant fluid and have the ability to carry drugs or nanoparticles to improve specificity. In the present study, we aimed to synthesize a new nanomaterial based on EVs containing chrysin and analyzed cell apoptosis in TSCC cells. Our results demonstrated that EVs-chrysin were isolated from SCC9 cells that were treated with chrysin. To improve the therapeutic effect, AuNPs were carried by EVs-chrysin (Au-EVs). Compared to BGC823 and HCC-LM3 cells, the uptake of Au-EVs was specific in SCC9 cells. Moreover, Au-EVs combined with NIR enhanced cell apoptosis in TSCC cells. To confirm the role of miRNAs in cell apoptosis, the differentially expressed miRNAs between EVs-Con and EVs-chrysin were screened by RNA-seq. The results revealed that the let-7a-3p family, which acts as the tumor suppressor, was upregulated in EVs-chrysin compared to EVs-Con. Thus, let-7a-3p was screened in the apoptosis pathway that was associated with the p53 protein. Furthermore, compared to the Con group, Au-EVs combined with the NIR group effectively inhibited tumor growth in vivo via increasing the expression of let-7a-3p. Together, as a new nanomaterial, Au-EVs induced cell apoptosis and inhibited tumor growth by regulating let-7a-3p expression in TSCC.

Effect of PLC-\u03b21/CaM signaling pathway mediated by AT1R on the occurrence and development of hepatocellular carcinoma

ObjectiveTo study the roles of AT1R, PLC-β1, CaM and other related signal molecules in the formation and development of hepatocellular carcinoma (HCC) and their correlation.MethodsELISA and immunohistochemistry were used to analyze the expressions of target proteins in serum and liver tissue of HCC patients, and the correlation between AT1R, PLC-β1 and CaM and postoperative survival status of patients was followed up and determined. CCK-8 method was used to screen the doses of Ang II and candesartan sensitive to HepG2 and HCCLM3 cells. Transwell experiment was used to observe the effects of different drugs on the migration and invasion activity of HCC cells. Meanwhile, flow cytometry and Western blot were used to detect the expression levels of AT1R, PLC-β1 and CaM in the cells. Then PLC-β1 siRNA was selected to transfect HCC cells, so as to further clarify the mechanism of the above signal proteins. HepG2 cells were inoculated under the hepatic capsule of mice to induce the formation of HCC in situ. Ang II and candesartan were used to stimulate HCC mice to observe the difference in liver appearance and measure the liver index. Finally, ELISA and immunofluorescence experiments were selected to analyze the levels of target proteins in mouse serum and liver tissue.ResultsThe expression levels of target proteins in serum and liver tissue of HCC patients were significantly increased, and the postoperative survival time of patients with high expression of AT1R, PLC-β1 or CaM was obviously shortened. Ang II and candesartan could significantly promote and inhibit the motility of HCC cells, and had different effects on the levels of AT1R, PLC-β1 and CaM in cells. However, in hepatocellular carcinoma cells transfected with PLC-β1 siRNA, the intervention ability of drugs was obviously weakened. Ang II could significantly promote the formation and progression of mouse HCC, while candesartan had the opposite effect. Meanwhile, medications could affect the expressions of target proteins in mouse serum and liver tissue.ConclusionAT1R, PLC-β1 and CaM may be risk factors affecting the formation and prognosis of HCC, and the PLC-β1/CaM signaling pathway mediated by AT1R is an important way to regulate the migration and invasion activity of HCC cells.

The expression of circ_0090049 in hepatocellular carcinoma and the molecular regulation mechanism of other biological functions.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in liver cancer. Circular RNA_0090049 (circ_0090049) has been shown to be involved in the advance of HCC. However, the interaction between circ_0090049 and microRNA (miRNA) in HCC has not been studied. Quantitative real-time PCR was used to detect the expression of related genes. Through detection of cell proliferation, migration, invasion, and rate of tumor sphere formation, the capping experiment was carried out to verify the regulatory relationship between miRNA and circ_0090049 or circ_0090049 and ubiquitin-conjugating enzyme E2 T (UBE2T). The expression of related proteins was detected by Western blotting. The interaction of miRNA with circ_0090049 or UBE2T was notarized by Dual-luciferase reporter assay. Xenotransplantation experiments confirmed the function of circ_0090049 in vivo. Circ_0090049 and UBE2T were upregulated in liver cancer. Silencing circ_0090049 reduced the proliferation, migration, invasion, and tumor spheroid formation rate of Huh7 and HCCLM3 cells. MiR-605-5p and miR-548c-3p were identified as targets of circ_0090049, and UBE2T was the target of miR-605-5p and miR-548c-3p. Anti-miR-605-5p, anti-miR-548c-3p or UBE2T overexpression restored the inhibitory effect of circ_0090049 knockdown on HCC cells. Animal experiments confirmed the antitumor effect of silence circ_0090049. Circ_0090049 regulates the expression of UBE2T by regulating miR-605-5p or miR-548c-3p, thereby promoting the development of HCC cells.

GJA1 promotes hepatocellular carcinoma progression by mediating TGF-β-induced activation and the epithelial-mesenchymal transition of hepatic stellate cells.

IntroductionGap junction protein, alpha 1 (GJA1), which is correlated with recurrences and unfavorable prognoses in hepatocellular carcinomas (HCCs), is one of the specific proteins expressed by activated hepatic stellate cells (HSCs).MethodsExpression of GJA1 was compared between HCCs and nontumor tissues (NTs), between hepatic cirrhosis and NTs, and between primary and metastatic HCCs using transcriptomic datasets from the Gene Expression Omnibus and the Integrative Molecular Database of Hepatocellular Carcinoma. The in vitro activities of GJA1 were investigated in cultured HSCs and HCC cells. The underlying mechanism was characterized using Gene Set Enrichment Analysis and validated by western blotting.ResultsThe expression of GJA1 was significantly increased in HCCs and hepatic cirrhosis compared to that in NTs. GJA1 was also overexpressed in pulmonary metastases from HCCs when compared with HCCs without metastasis. Overexpression of GJA1 promoted while knockdown of GJA1 inhibited proliferation and transforming growth factor (TGF)-β-mediated activation and migration of cultured HSCs. Overexpression of GJA1 by lentivirus infection promoted proliferation and migration, while conditioned medium from HSCs overexpressing GJA1 promoted migration but inhibited proliferation of Hep3B and PLC-PRF-5 cells. Lentivirus infection with shGJA1 or conditioned medium from shGJA1-infected HSCs inhibited the proliferation and migration of HCCLM3 cells that had a high propensity toward lung metastasis. Mechanistically, GJA1 induced the epithelial–mesenchymal transition (EMT) in HSCs and HCCLM3 cells.ConclusionGJA1 promoted HCC progression by inducing HSC activation and the EMT in HSCs. GJA1 is potentially regulated by TGF-β and thus may be a therapeutic target to inhibit HCC progression.

CircRNA_PTPRA functions as a sponge of miR-582-3p to regulate hepatocellular carcinoma cell proliferation, migration, invasion and apoptosis.

Hepatocellular carcinoma (HCC) is a lethal disease and one of the most common types of cancer. HCC is associated with exponentially increasing morbidity and mortality rates. Accumulating evidence has identified circular RNAs (circRNAs) to be regulators of cancer progression. However, to the best of our knowledge, the potential effect of circRNA protein tyrosine phosphatase receptor type A (circRNA_PTPRA) in HCC and its mechanism remain unknown. The present study aimed to assess the effects and underlying mechanism of circRNA_PTPRA in a HCC Huh-7 cells model. The sites of interaction between circRNA_PTPRA and microRNA (miR)-582-3p were predicted using the StarBase software and verified using dual luciferase reporter and RNA immunoprecipitation (RIP) assays in Huh-7 cells. HCC cell viability, apoptosis, migration and invasion were measured using MTT, flow cytometry and Transwell assays, respectively. The expression levels of circRNA_PTPRA, miR-582-3p, cyclin D1, MMP-9, Bcl-2 and Bax were analyzed using reverse transcription-quantitative PCR and western blotting. The results of the dual luciferase reporter and RIP assays demonstrated that miR-582-3p directly targeted circRNA_PTPRA. Compared with the human normal hepatocyte cell line, THLE-2, the expression levels of circRNA_PTPRA were upregulated, which were found to be inversely correlated with those of miR-582-3p expression in Huh-7 and HCCLM3 cells. miR-582-3p overexpression using mimics suppressed cell proliferation, migration and invasion, whilst downregulating cyclin D1 and MMP-9 expression in Huh-7 cells. In addition, transfection of HCC cells with the miR-582-3p mimic promoted apoptosis by downregulating Bcl-2 expression and upregulating Bax expression in Huh-7 cells. Knocking down circRNA_PTPRA expression using small interfering RNA (siRNA) markedly downregulated circRNA_PTPRA expression levels and upregulated miR-582-3p expression, but was reversed by co-transfection with the miR-582-3p inhibitor. Furthermore, reduced HCC cell proliferation, migration and invasion, increased levels of cell apoptosis, upregulated Bax expression and downregulated cyclin D1, MMP-9 and Bcl-2 expression were all observed after knocking down circRNA_PTPRA. All these effects aforementioned were reversed by co-transfection with the miR-582-3p inhibitor. In conclusion, findings from the present study suggested that circRNA_PTPRA may regulate HCC cell proliferation, invasion, apoptosis and migration by sponging miR-582-3p. This indicates that the circRNA_PTPRA/miR-582-3p axis may represent a potential target for HCC diagnosis and treatment.

CircMRPS35 promotes malignant progression and cisplatin resistance in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the major causes of cancer-related death worldwide. Circular RNAs (circRNAs), a novel class of non-coding RNA, have been reported to be involved in the etiology of various malignancies. However, the underlying cellular mechanisms of circRNAs implicated in the pathogenesis of HCC remain unknown. In this study, we identified a functional RNA, hsa_circ_0000384 (circMRPS35), from public tumor databases using a set of computational analyses, and we further identified that circMRPS35 was highly expressed in 35 pairs of HCC from patients. Moreover, knockdown of the expression of circMRPS35 in Huh-7 and HCC-LM3 cells suppressed their proliferation, migration, invasion, clone formation, and cell cycle invitro, and it suppressed tumor growth invivo as well. Mechanically, circMRPS35 sponged microRNA-148a-3p (miR-148a), regulating the expression of Syntaxin 3 (STX3), which modulated the ubiquitination and degradation of phosphatase and tensin homolog (PTEN). Unexpectedly, we detected a peptide encoded by circMRPS35 (circMRPS35-168aa), which was significantly induced by chemotherapeutic drugs and promoted cisplatin resistance in HCC. These results demonstrated that circMRPS35 might be a novel mediator in HCC progress, and they raise the potential of a new biomarker for HCC diagnosis and prognosis, as well as a novel therapeutic target for HCC patients.

Morphine promotes angiogenesis by activating PI3K/Akt/HIF-1α pathway and upregulating VEGF in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is characterized by the neo-angiogenesis induced by tumor and adjacent cells. It is a leading cancer-related cause of death. Morphine has effects on angiogenesis with pro-angiogenic or anti-angiogenic phonotypes. This study explores the function of morphine on cancer cell growth, angiogenesis and the underlying mechanism in HCC. Morphine was used to treat BEL-7402 or HCC-LM3 cells and human umbilical vein endothelial cells (HUVECs) were subsequently incubated in the conditioned media (CM) of HCC cells. The potential effects of cell proliferation, migration and tube formation of CM-treated HUVECs were investigated. Furthermore, the angiogenesis regulated factors of VEGFA, PIGF, ANG-1, ANG-2, FGF-1 and FGF-2 were assessed. siRNA and LY294002 were further used to explore the mechanism mediating the effects of morphine on the angiogenesis pathway. The neovascularization effect by morphine was confirmed through the use of human HCC cancer heterotopic mouse model in vivo. A significantly increased cell proliferation, migration, and tube formation effect of HUVECs induced by the CM from HCC cell lines treated with morphine was observed. More VEGFA secretion in CM from LM3 or BEL-7402 cell lines was found than the controls (P=0.03 and P=0.027, respectively). VEGFA knock-down could significantly reverse cell proliferation, migration and tube formation induced by the CM from HCC cell lines with morphine treatment. Further molecular experiments indicated that VEGFA secretion was activated by morphine potentially through the PI3K/Akt/HIF-1α pathway. Morphine-induced neovascularization was also observed by the IHC of CD31 and VEGFA. Morphine promotes angiogenesis in hepatocellular carcinoma possibly through the activation of the PI3K/Akt/HIF-1α pathway and VEGFA stimulation.

Green synthesized silver nanoparticles (AgNPs) from Parrotiopsis jacquemontiana (Decne) Rehder leaf extract and its biological activities.

Parrotiopsis jacquemontiana (Decne) Rehder aqueous extract of leaf was used for biosynthesis of AgNPs and characterized through UV-Visible spectroscopy, X-ray diffraction, Fourier-transform-infrared, diffraction light scattering, and scanning electron microscope analysis. Moderate to strong antioxidant activity during in vitro antioxidant assays for scavenging of 2,2-diphenyl-1-picryl hydrazyl (DPPH), hydroxyl (OH), nitric oxide (NO) radicals, iron chelation, and inhibition of β-carotene bleaching was recorded with minimum IC50 value (27.70 ± 2.67 μg/ml) calculated for OH radicals. The AgNPs were evaluated against six multidrug resistant human bacterial strains and minimum inhibitory concentration (MIC) along with minimum bactericidal concentration (MBC) values was determined and all were found remarkably susceptible. The bacterial strain Staphylococcus aureus was the most susceptible with MIC=5μg/ml and MBC=10μg/ml. Among six fungal strains, Fumigatus esculentum was the most susceptible with MIC and MBC of 10μg/ml. 3-(4,5-Dimethylthiazol-2 yl)-2,5-diphenyltetrazolium bromide (MTT) screening assay against cancer cell lines (HCCLM3, HEPG2, MDA-MB 231, and MCF-7) revealed the least IC50 values against HCCLM3 (74.20 ± 5.04) and MCF-7 (91.90 ± 1.17). While no cytotoxicity against normal cell lines; LO2 and MCF-10a was recorded. Parrotiopsis jacquemontiana silver nanoparticles (PJAgNPs) significantly (p > .001) prevented the migration of HCCLM3 cells in a dose-dependent style, relative to control. The wound healing potential of AgNPs in rat was found higher (p < .05) for wound contraction rates, hydroxyproline content, hemostatic and re-epithelization and regeneration efficiency in comparison to the reference group.