MiRNA-335 Modulates Hepatoma Cell Lines Apoptosis and Proliferation by Targeting Forkhead Box O3a (FOXO3a)

This study intends to elucidate MiRNA-335’s role in hepatoma cell lines (HCC). Real-time PCR was used to detect MiRNA-335 expression in HCC, flow cytometry and MTT were used to detect apoptosis and proliferation. Luciferase reporting system analyzed the targeting relationship between Foxo3a and MiRNA-335. HCC (SMMC7721 cell) exhibited significantly reduced MiRNA-335 compared to normal hepatocyte cell (HL7702). MiRNA-335 mimic inhibited HCC proliferation and enhanced apoptosis, which were reversed by MiRNA-335 inhibitor. Luciferase reporter gene system showed that MiRNA-335 significantly inhibited the fluorescent activity of Foxo3a 3′-UTR, indicating that MiRNA-335 could target Foxo3a RNA. In conclusion, the decrease of MiRNA-335 can promote the proliferation of hepatoma cells and inhibit apoptosis possibly through regulating Foxo3a, which provides a new direction for the treatment of liver cancer.

Complementary Omics Strategies to Dissect p53 Signaling Networks Under Nutrient Stress

Signaling trough p53 is a major cellular stress response mechanism and increases upon nutrient stresses such as starvation. Here, we show in a human hepatoma cell line that starvation leads to robust nuclear p53 stabilization. Using BioID, we determine the cytoplasmic p53 interaction network within the immediate-early starvation response and show that p53 is dissociated from several metabolic enzymes and the kinase PAK2 for which direct binding with the p53 DNA-binding domain was confirmed with NMR studies. Furthermore, proteomics after p53 immunoprecipitation (RIME) uncovered the nuclear interactome under prolonged starvation, where we confirmed the novel p53 interactors SORBS1 (insulin receptor signaling) and UGP2 (glycogen synthesis). Finally, transcriptomics after p53 re-expression revealed a distinct starvation-specific transcriptome response and suggested previously unknown nutrient-dependent p53 target genes. Together, our complementary approaches delineate several nodes of the p53 signaling cascade upon starvation, shedding new light on the mechanisms of p53 as nutrient stress sensor. Given the central role of p53 in cancer biology and the beneficial effects of fasting in cancer treatment, the identified interaction partners and networks could pinpoint novel pharmacologic targets to fine-tune p53 activity.

Downregulated Expression of miRNA-130a-5p Aggravates Hepatoma Progression via Targeting PTP4A2

Background. Hepatoma is a leading cause of death worldwide, with high metastasis and recurrence rates. The aberrant expression of miRNA-130a-5p is involved in the development and progression of various cancers. However, there are no studies investigating the role of miRNA-130a-5p in hepatoma. The present study is aimed at clarifying the functional role of miRNA-130a-5p in hepatoma progression. Methods. The expression levels of miRNA-130a-5p in hepatoma tissues and cell lines were detected by qRT-PCR assays. Bioinformatic analysis, gain-/loss-of-function experiments, and luciferase activity assays were conducted to verify whether miRNA-130a-5p is targeted by protein tyrosine phosphatase 4A2 (PTP4A2). The functions of miRNA-130a-5p and PTP4A2 in hepatoma were determined by cell proliferation assays. Results. The expression of miRNA-130a-5p was downregulated in hepatoma tissues and was related to poor prognosis. However, the expression level of PTP4A2 was contradictory to that of miRNA-130a-5p, and PTP4A2 upregulation could aggravate hepatoma progression. The ectopic overexpression of PTP4A2 promoted hepatoma cell proliferation in vitro, which could be reversed by miRNA-130a-5p. Conclusions. Our study implies that miRNA-130a-5p, which is downregulated in hepatoma tissues, can suppress hepatoma cell proliferation via targeting PTP4A2.

SWCNH (Single walled carbon nanohorn) supervises ER (Endoplasmic reticulum) stress through triggering autophagy process of hepatocytes, especially in hepatoma cell line HepG2

Backgrounds: The cellular homeostasis is major maintained by the catabolic pathway of autophagy. Our previous work indicated that SWCNH were associated with endoplasmic reticulum (ER) stress mediated by calcium flow and autophagic response. But, its mechanism was unclear. Methods: The regulation of SWCNH on the calcium flow then autophagy of liver cells were investigated through inducing ER stress with tunicamycin and SWCNH. The calcuim flow was determined using Fluo-3, then autophagy was examined with immunofluorescence or western blot for LC3, Beclin-1, ATG-5, and p62. Moreover, the apopototic protein of Bax and Bcl-2 was detected, too. Results: Tunicamycin-induced ER stress in hepatocytes was related to calcium flow, especially for hepatoma cell line HepG2. Moreover, SWCNH participated in the regulation of endoplasmic reticulum stress-related calcium flow. Besides, SWCNH induced hepatocyte autophagy and inhibited cell apoptosis, then mediated the process of hepatocyte autophagy. Conclusions: Tunicamycin-induced ER stress in hepatocytes was related to calcium flow. Moreover, SWCNH induced hepatocyte autophagy, inhibited cell apoptosis, and participated in the autophagy regulation of hepatocyte, especially for hepatoma cell line.

Transcriptome Analysis Reveals the Anti-cancerous Mechanism of Licochalcone A on Human Hepatoma Cell HepG2

Hepatocellular carcinoma is a malignancy with a low survival rate globally, and there is imperative to unearth novel natural phytochemicals as effective therapeutic strategies. Licochalcone A is a chalcone from Glycyrrhiza that displayed various pharmacological efficacy. A globally transcriptome analysis was carried out to reveal the gene expression profiling to explore Licochalcone A's function as an anti-cancer phytochemical on HepG2 cells and investigate its potential mechanisms. Altogether, 6,061 dysregulated genes were detected (3,414 up-regulated and 2,647 down-regulated). SP1 was expected as the transcription factor that regulates the functions of most screened genes. GO and KEGG analysis was conducted, and the MAPK signaling pathway and the FoxO signaling pathway were two critical signal pathways. Protein-protein interaction (PPI) network analysis based on STRING platform to discover the hub genes (MAPK1, ATF4, BDNF, CASP3, etc.) in the MAPK signaling pathway and (AKT3, GADD45A, IL6, CDK2, CDKN1A, etc.) the FoxO signaling pathway. The protein level of essential genes that participated in significant pathways was consistent with the transcriptome data. This study will provide an inclusive understanding of the potential anti-cancer mechanism of Licochalcone A on hepatocellular, signifying Licochalcone A as a promising candidate for cancer therapy.

Isolation and Characterization of SARS-CoV-2 strains circulating in Eastern India.

Emergence of SARS-CoV-2 as a serious pandemic has altered the global socioeconomic dynamics. The wide prevalence, high death counts and rapid emergence of new variants urge for establishment of research infrastructure to facilitate rapid development of efficient therapeutic modalities and preventive measures. In agreement with this, five SARS-CoV2 strains (ILS01, ILS02, ILS03, ILS15 and ILS24) of four different clades (19A, 19B, 20A and 20B) were isolated from patient swab samples collected during the 1st COVID-19 wave in Odisha, India. The viral isolates were adapted to in-vitro cultures and further characterized to identify strain specific variations in viral growth characteristics. All the five isolates showed substantial amount of virus induced CPE however ILS03 belonging to 20A clade displayed highest level of CPE. Time kinetics experiment revealed spike protein expression was evident after 16th hours post infection in all five isolates. ILS03 induced around 90% of cytotoxicity. Further, the susceptibility of various cell lines (human hepatoma cell line (Huh-7), CaCo2 cell line, HEK-293T cells, Vero, Vero-E6, BHK-21, THP-1 cell line and RAW 264.7 cells) were assessed. Surprisingly, it was found that the human monocyte cells THP-1 and murine macrophage cell line RAW 264.7 were permissive to all the SARS-CoV-2 isolates. The neutralization susceptibility of viral isolates to vaccine-induced antibodies was determined using sera from individuals vaccinated in the Government run vaccine drive in India. The micro-neutralization assay suggested that both Covaxin and Covishield vaccines were equally effective (100% neutralization) against all of the isolates. The whole genome sequencing of culture adapted viral isolates and viral genome from patient oropharyngeal swab sample suggested that repetitive passaging of SARS-CoV2 virus in Vero-E6 cells did not lead to emergence of many mutations during the adaptation in cell culture. Phylogenetic analyses revealed that the five isolates clustered to respective clades. The major goal was to isolate and adapt SARS-CoV-2 viruses in in-vitro cell culture with minimal modification to facilitate research activities involved in understanding the molecular virology, host-virus interactions, application of these strains for drug discovery and animal challenge models development which eventually will contribute towards the development of effective and reliable therapeutics.

Cytotoxic Synergism between a Proprietary Commiphora mukul Gum Extract GU-MCT810, 2-deoxy-D-glucose, and Metformin in Human Alveolar Rhabdomyosarcoma and Hepatoma Cell Lines In vitro

In this investigation we have analyzed the synergism for the cytotoxic effect of a proprietary guggul gum extract (GU), 2-deoxy-D-glucose (2-DG) and metformin (Met) in SJRH30 human alveolar rhabdomyosarcoma and HepG2 hepatoma cell lines in vitro. 2-DG and Met as single agents have weak cytotoxic effects in both cell lines. However, the combination of GU+2-DG, GU+Met and 2-DG+Met showed synergism for cytotoxic effect by CompuSyn analysis. Therefore, GU can be included in the combination of drugs involving 2-DG and Met to have synergistic effect. GU also showed a dose-dependent increase in cellular glucose uptake in HepG2 cells like the antidiabetic drug 2,4-thiozolidine dione (TZ). The demonstration of synergism of anticancer effects between GU, metformin and 2-DG, suggest that their mechanisms are in general complementary, though further studies are required to delineate the mechanism of GU, 2-DG and metformin combinations.

Exploration of the Effect on Genome-Wide DNA Methylation by miR-143 Knock-Out in Mice Liver

MiR-143 play an important role in hepatocellular carcinoma and liver fibrosis via inhibiting hepatoma cell proliferation. DNA methyltransferase 3 alpha (DNMT3a), as a target of miR-143, regulates the development of primary organic solid tumors through DNA methylation mechanisms. However, the effect of miR-143 on DNA methylation profiles in liver is unclear. In this study, we used Whole-Genome Bisulfite Sequencing (WGBS) to detect the differentially methylated regions (DMRs), and investigated DMR-related genes and their enriched pathways by miR-143. We found that methylated cytosines increased 0.19% in the miR-143 knock-out (KO) liver fed with high-fat diet (HFD), compared with the wild type (WT). Furthermore, compared with the WT group, the CG methylation patterns of the KO group showed lower CG methylation levels in CG islands (CGIs), promoters and hypermethylation in CGI shores, 5′UTRs, exons, introns, 3′UTRs, and repeat regions. A total of 984 DMRs were identified between the WT and KO groups consisting of 559 hypermethylation and 425 hypomethylation DMRs. Furthermore, DMR-related genes were enriched in metabolism pathways such as carbon metabolism (serine hydroxymethyltransferase 2 (Shmt2), acyl-Coenzyme A dehydrogenase medium chain (Acadm)), arginine and proline metabolism (spermine synthase (Sms), proline dehydrogenase (Prodh2)) and purine metabolism (phosphoribosyl pyrophosphate synthetase 2 (Prps2)). In summary, we are the first to report the change in whole-genome methylation levels by miR-143-null through WGBS in mice liver, and provide an experimental basis for clinical diagnosis and treatment in liver diseases, indicating that miR-143 may be a potential therapeutic target and biomarker for liver damage-associated diseases and hepatocellular carcinoma.

Multitargeting Strategy Using Tetrathiomolybdate and Lenvatinib: Maximizing Anti-Angiogenesis Activity in a Preclinical Liver Cancer Model

Purpose To investigate the suppressing tumor-promoting effects via multi-anti-angiogenesis activity of the copper chelator (Ammonium Tetrathiomolybdate, TM) combined with lenvatinib for hepatocellular carcinoma. Material and methods Fifty-five C57 mice were injected subcutaneously with Hepa1-6 hepatoma cell suspensions into the right posterior thigh. Seven days later, all subcutaneous tumors were formed and the mice were randomly distributed into 5 groups: TM Group (G1), Lenvatinib Group (G2), TM+Lenvatinib Group (G3), Control Group (G4), and Copper (II) Gluconate Group (G5). And copper concentrations in serum and tumors were measured at the predetermined times. After fourteen days of treatments, tumor weight and volumes were analyzed, histology was observed, and the expressions of VEGF and microvessel density (MVD) in tumor tissues were measured by immunohistochemistry (IHC). Results Average concentration of copper in serum was 405.14 ug/L, 480.44 ug/L, and 679.80 ug/L in normal mice, in mice on 7 days after implantation, and in the control group, respectively. Similarly, intratumoral copper concentrations were greater in G4 mice (1511.90 ug/L) than mice on 7 days after implantation (852.80 ug/L) (p < 0.05). And the serum concentration of copper was 363.65 ug/L, 508.83 ug/L, 370.52 ug/L, 822.12 ug/L in G1, G2, G3, and G5 [G5 vs other Groups, all p < 0.05; (G1, G2, and G3) vs G4, p < 0.05; G1 vs G2, p = 0.013; G2 vs G3, p = 0.018; G1 vs G3, p = 0.903] while intratumoral copper concentrations was 674.31 ug/L, 988.91 ug/L, 550.52 ug/L, and 3004.95 ug/L in G1, G2, G3, and G5 And the average tumor weight was 0.55 g, 0.44 g, 0.08 g, 1.37 g, 3.11 g in the mice of G1, G2, G3, G4, and G5, respectively. [G5 vs other Groups, all p < 0.05; (G1, G2, and G3) vs G4, p < 0.05; G1 vs G3, p < 0.05; G2 vs G3, p < 0.05; G1 vs G2, p > 0.05]. Furthermore, tumors were collected for HE staining and IHC examination. The expression levels of VEGF in G1, G2, and G3 were 43.75, 32.48, and 15, and all of them were significantly lower than those in G4 (64.28) and G5 (89.03) (G4 vs G5, p < 0.05). Similarly, the trend of MVD was just like that of VEGF in the five groups whereas no significant difference occurred in G1 and G2. Conclusion The study shows that there is a significant positive correlation between tumor load and copper. Administration of copper can promote tumor progression, and copper chelating could suppress tumor growth. The combination of TM with lenvatinib can reduce tumor angiogenesis and improved the effect of antitumor treatment. These findings offer basic data support and theoretical foundation for the clinical application of the combination therapy.

Antioxidant supplements promote tumor formation and growth and confer drug resistance in hepatocellular carcinoma by reducing intracellular ROS and induction of TMBIM1

Background Controversy over the benefits of antioxidants supplements in cancers persists for long. Using hepatocellular carcinoma (HCC) as a model, we investigated the effects of exogenous antioxidants N-acetylcysteine (NAC) and glutathione (GSH) on tumor formation and growth. Methods Multiple mouse models, including diethylnitrosamine (DEN)-induced and Trp53KO/C-MycOE-induced HCC models, mouse hepatoma cell and human HCC cell xenograft models with subcutaneous or orthotopic injection were used. In vitro assays including ROS assay, colony formation, sphere formation, proliferation, migration and invasion, apoptosis, cell cycle assays were conducted. Western blot was performed for protein expression and RNA-sequencing to identify potential gene targets. Results In these multiple different mouse and cell line models, we observed that NAC and GSH promoted HCC tumor formation and growth, accompanied with significant reduction of intracellular reactive oxygen species (ROS) levels. Moreover, NAC and GSH promoted cancer stemness, and abrogated the tumor-suppressive effects of Sorafenib both in vitro and in vivo. Exogenous supplementation of NAC or GSH reduced the expression of NRF2 and GCLC, suggesting the NRF2/GCLC-related antioxidant production pathway might be desensitized. Using transcriptomic analysis to identify potential gene targets, we found that TMBIM1 was significantly upregulated upon NAC and GSH treatment. Both TCGA and in-house RNA-sequence databases showed that TMBIM1 was overexpressed in HCC tumors. Stable knockdown of TMBIM1 increased the intracellular ROS; it also abolished the promoting effects of the antioxidants in HCC cells. On the other hand, BSO and SSA, inhibitors targeting NAC and GSH metabolism respectively, partially abrogated the pro-oncogenic effects induced by NAC and GSH in vitro and in vivo. Conclusions Our data implicate that exogenous antioxidants NAC and GSH, by reducing the intracellular ROS levels and inducing TMBIM expression, promoted HCC formation and tumor growth, and counteracted the therapeutic effect of Sorafenib. Our study provides scientific insight regarding the use of exogenous antioxidant supplements in cancers.