Heparanase-1 is downregulated in chemoradiotherapy orbital rhabdomyosarcoma and relates with tumor growth as well as angiogenesis

AIM: To determine the role of heparanase-1 (HPSE-1) in orbital rhabdomyosarcoma (RMS), and to investigate the feasibility of HPSE-1 targeted therapy for RMS. METHODS: Immunohistochemistry was performed to analyze HPSE-1 expression in 51 cases of orbital RMS patients (including 28 cases of embryonal RMS and 23 cases of alveolar RMS), among whom there were 27 treated and 24 untreated with preoperative chemoradiotherapy. In vitro, studies were conducted to examine the effect of HPSE-1 silencing on RMS cell proliferation and tube formation of human umbilical vein endothelial cells (HUVECs). RD cells (an RMS cell line) and HUVECs were infected with HPSE-1 shRNA lentivirus at a multiplicity of infection (MOI) of 10 and 30 separately. Real-time PCR and Western blot were applied to detect the mRNA and protein expression levels of HPSE-1. Cell viability of treated or control RD cells was evaluated by cell counting kit-8 (CCK-8) assay. Matrigel tube formation assay was used to evaluate the effect of HPSE-1 RNAi on the tube formation of HUVECs. RESULTS: Immunohistochemistry showed that the expression rate of HPSE-1 protein was 92.9% in orbital embryonal RMS and 91.3% in orbital alveolar RMS. Tissue from alveolar orbital RMS did not show relatively stronger staining than that from the embryonal orbital RMS. However, despite the types of RMS, comparing the cases treated chemoradiotherapy with those untreated, we have observed that chemoradiotherapy resulted in weaker staining in patients' tissues. The expression levels of HPSE-1 declined significantly in both the mRNA and protein levels in HPSE-1 shRNA transfected RD cells. The CCK-8 assay showed that lentivirus-mediated HPSE-1 silencing resulted in significantly reduced RD cells viability in vitro. Silencing HPSE-1 expression also inhibited VEGF-induced tube formation of HUVECs in Matrigel. CONCLUSION: HPSE-1 silencing may be a promising therapy for the inhibition of orbital RMS progression.

The Effect of Direct and Indirect EZH2 Inhibition in Rhabdomyosarcoma Cell Lines

Enhancer of Zeste homolog 2 (EZH2) is involved in epigenetic regulation of gene transcription by catalyzing trimethylation of histone 3 at lysine 27. In rhabdomyosarcoma (RMS), increased EZH2 protein levels are associated with poor prognosis and increased metastatic potential, suggesting EZH2 as a therapeutic target. The inhibition of EZH2 can be achieved by direct inhibition which targets only the enzyme activity or by indirect inhibition which also affects activities of other methyltransferases and reduces EZH2 protein abundance. We assessed the direct inhibition of EZH2 by EPZ005687 and the indirect inhibition by 3-deazaneplanocin (DZNep) and adenosine dialdehyde (AdOx) in the embryonal RD and the alveolar RH30 RMS cell line. EPZ005687 was more effective in reducing the cell viability and colony formation, in promoting apoptosis induction, and in arresting cells in the G1 phase of the cell cycle than the indirect inhibitors. DZNep was more effective in decreasing spheroid viability and size in both cell lines than EPZ005687 and AdOx. Both types of inhibitors reduced cell migration of RH30 cells but not of RD cells. The results show that direct and indirect inhibition of EZH2 affect cellular functions differently. The alveolar cell line RH30 is more sensitive to epigenetic intervention than the embryonal cell line RD.

Isolation and Identification of Andrographis paniculata (Chuanxinlian) and Its Biologically Active Constituents Inhibited Enterovirus 71-Induced Cell Apoptosis

Aim:Andrographis paniculata (Burm. f.) Nees (also known as Chuanxinlian in Chinese) of Acanthaceae family is one of the Chinese herbs reputed to be effective in the treatment of inflammation, infection, cold, and fever. Enterovirus 71 (EV71) is one of the most important enteroviruses that cause hand, foot, and mouth disease (HFMD) accompanied with neurological complication.Methods: To explore an anti-infective Chinese herb medicine, pure compounds isolated or synthesized analogues from A. paniculata (AP) ethyl acetate (EtOAc) extract are used to explore their anti-EV71-induced cytotoxicity. The antiviral activity was determined by cytopathic effect (CPE) reduction, and sub-G1 assays were used for measuring lysis and apoptosis of EV71-infected rhabdomyosarcoma (RD) cells. IFNγ-driven luciferase reporter assay was used to evaluate their potential roles in activation of immune responses.Results: Our data showed that EV71-induced sub-G1 phase of RD cells was dose dependently increased. Highly apoptotic EV71-infected RD cells were reduced by AP extract treatment. Ergosterol peroxide (4) has the most anti-apoptotic effect among these seven compounds. In addition, 3,19-O-acetyl-14-deoxy-11,12-didehydroandrographolide (8) synthesized from acetylation of compound 7 showed significantly better antiviral activity and the lowest sub-G1 phase of 6%–18%. Further investigation of IFNγ-inducer activity of these compounds showed that compounds 3, 6, 10, 11, and 12 had significantly higher IFNγ luciferase activities, suggesting their potential to promote IFNγ expression and thus activate immune responses for antivirus function.Conclusion: Our study demonstrated that bioactive compounds of AP and its derivatives either protecting EV71-infected RD cells from sub-G1 arrest or possessing IFNγ-inducer activity might be feasible for the development of anti-EV71 agents.

High throughput sequencing of whole transcriptome and construct of ceRNA regulatory network in RD cells infected with enterovirus D68

Background With the advancement of sequencing technologies, a plethora of noncoding RNA (ncRNA) species have been widely discovered, including microRNAs (miRNAs), circular RNAs (circRNAs), and long ncRNAs (lncRNAs). However, the mechanism of these non-coding RNAs in diseases caused by enterovirus d68 (EV-D68) remains unclear. The goal of this research was to identify significantly altered circRNAs, lncRNAs, miRNAs, and mRNAs pathways in RD cells infected with EV-D68, analyze their target relationships, demonstrate the competing endogenous RNA (ceRNA) regulatory network, and evaluate their biological functions. Methods The total RNAs were sequenced by high-throughput sequencing technology, and differentially expressed genes between control and infection groups were screened using bioinformatics method. We discovered the targeting relationship between three ncRNAs and mRNA using bioinformatics methods, and then built a ceRNA regulatory network centered on miRNA. The biological functions of differentially expressed mRNAs (DEmRNAs) were discovered through GO and KEGG enrichment analysis. Create a protein interaction network (PPI) to seek for hub mRNAs and learn more about protein–protein interactions. The relative expression was verified using RT-qPCR. The effects of Fos and ARRDC3 on virus replication were confirmed using RT-qPCR, virus titer (TCID50/ml), Western blotting. Results 375 lncRNAs (154 upregulated and 221 downregulated), 33 circRNAs (32 upregulated and 1 downregulated), 96 miRNAs (49 upregulated and 47 downregulated), and 239 mRNAs (135 upregulated and 104 downregulated) were identified as differently in infected group compare to no-infected group. A single lncRNA or circRNA can be connected with numerous miRNAs, which subsequently coregulate additional mRNAs, according to the ceRNA regulatory network. The majority of DEmRNAs were shown to be connected to DNA binding, transcription regulation by RNA polymerase II, transcription factor, MAPK signaling pathways, Hippo signal pathway, and apoptosis pathway, according to GO and KEGG pathway enrichment analysis. The hub mRNAs with EGR1, Fos and Jun as the core were screened through PPI interaction network. We preliminarily demonstrated that the Fos and ARRDC3 genes can suppress EV-D68 viral replication in order to further verify the results of full transcriptome sequencing. Conclusion The results of whole transcriptome analysis after EV-D68 infection of RD cells were first reported in this study, and for the first time, a ceRNA regulation network containing miRNA at its center was established for the first time. The Fos and ARRDC3 genes were found to hinder viral in RD cells. This study establishes a novel insight host response during EV-D68 infection and further investigated potential drug targets.

Antiproliferative Activity, c-Myc and FGFR1 Genes Expression Profiles and Safety of Annona muricata Fruit Extract on Rhabdomyosarcoma and BALB/c Mice

Introduction: Rhabdomyosarcoma is an aggressive solid tumour of skeletal muscles origin whose current treatment is associated with high expenses, severe side effects, drug resistance and tumour regrowth. There is a need to develop safer and more effective chemotherapeutic agents. Annona muricata is one of the widely used plants in treating various diseases due to its reported effectiveness. However, there is a dearth of scientific information regarding the efficacy of Annona muricata on rhabdomyosarcoma and its safety. This study aimed to evaluate the effects of Annona muricata ethanolic fruit extract on the antiproliferative activity and gene expression in RD cell line, including its biosafety in BALB/c mice. Materials and Methods: The resazurin metabolic assay was used to assess the antiproliferative and cytotoxic activities of Annona muricata ethanolic fruit extract on RD and Vero cells. Quantitative real-time polymerase chain reaction was used to assess the gene expression profiles on c-Myc and FGFR1 genes. To evaluate the safety of the Annona muricata ethanolic fruit extract, an acute oral toxicity study was conducted on BALB/c mice. Results: Annona muricata ethanolic fruit extract significantly inhibited the growth of RD cells in a concentration and time-dependent manner while being highly selective on the Vero cells (selectivity index of 6.10 at 72h) compared to a reference cancer drug, doxorubicin (Selectivity index of 1.38 at 72hr). The c-Myc and FGFR1 genes were under expressed in RD cells treated with Annona muricata ethanolic fruit extract with (3.4 and 6.1 fold), respectively, compared to untreated cells. Acute oral toxicity studies revealed no significant difference (p ≥ 0.05) between the treated mice and the control group, indicating the safety of the fruit extract. Conclusion: Annona muricata ethanolic fruit extract can serve as effective and safe anticancer agents against rhabdomyosarcoma and further develop into standard drugs. Non-human primate studies need to be undertaken to step towards the clinical utilization of the Annona muricata ethanolic fruit extract in the management of rhabdomyosarcoma.

Enterovirus 71 Induces INF2 Cleavage via Activated Caspase-2 in Infected RD Cells

Enterovirus 71 (EV71) is the major causative pathogen of hand, foot, and mouth disease. The lack of understanding of the virus’s pathogenesis hinders the development of anti-virus drugs and the control of EV71 infection. Our previous studies have demonstrated that both mitochondria and endoplasmic reticulum (ER) were altered significantly in EV71 infected cells, but the mechanism is still unclear. In this study, we investigated the effects of EV71 infection on the expression of INF2, a key regulator factor in ER-Mitochondria communication and mitochondrial fission. We found that INF2 was cleaved in EV71 infected RD cells. The INF2 cleavage occurred at Aspartic 1,051 of INF2 and is mediated by activated caspases, predominantly by activated caspase-2. The subcellular localization of INF2 and caspase-2 was significantly altered in infected cells. We speculate that caspase-2-mediated INF2 cleavage is involved in forming viral replication organelles (ROs) and is a positive feedback regulatory mechanism of mitochondrial disorders caused by EV71 infection.

VGLL4 Promotes EV71 Virus Proliferation and Accelerates the Apoptosis of Infected RD Cells

Enterovirus 71 (EV71) is one of the major pathogens causing hand, foot and mouth disease (HFMD) which affects public health increasingly. Apoptosis plays an important role in EV71 infection, but the molecular mechanism involved in EV71 induced apoptosis is not completely clear. VGLL4 is a multifunctional protein in host cells, which has been studied in tumor and cell apoptosis, but has not been reported in pathogen. In this study, the mammalian eukaryotic expression plasmid of VGLL4 fused with HA tag (HA-VGLL4) and the model of overexpression VGLL4 RD cells were successfully constructed. The effect of VGLL4 on the proliferation of EV71 was detected by western-blot assay, fluorescence quantitative PCR and cytotoxicity assay (CCK assay), and the mechanism of its effect on the proliferation of the virus was researched. The experimental results showed that VGLL4 may promote the replication of EV71 by promoting the apoptosis of infected cells. VGLL4 can be an important target for prevention and treatment of EV71 infection.

Evaluation of expression of messenger RNA (RNAm) of the key proteins of the route of the microRNA (miRNA) in RD cells infected with the Mayaro Virus

Introduction: The Mayaro virus (MAYV) is an arbovirus belonging to the Togaviridae family of the alphavirus genus, which is transmitted by hematophagous arthropods to vertebrate hosts, whereas non-human primates are considered primary reservoirs. Objective: To evaluate the expression of messenger RNAs (mRNA) of crucial proteins in the microRNA pathway and inflammatory cytokines in human cells during infection by the MAYV. Materials and Methods: Viral samples propagated in RD cell cultures. The RNAs were collected and extracted at predetermined times. The total RNA was then used to detect mRNA by PCR in real time (qRT-PCR). Results: the Mayaro virus RNA was verified in the cells during all the days of infection. The AgR2 mRNA protein dropped sharply by 2 dpi and 3 dpi. Conclusion: The present study demonstrated the reduction of proteins in infection with the Mayaro virus.