Wanchai Assavalapsakul

Repertoire of bovine miRNA and miRNA-like small regulatory RNAs expressed upon viral infection.

MicroRNA (miRNA) and other types of small regulatory RNAs play a crucial role in the regulation of gene expression in eukaryotes. Several distinct classes of small regulatory RNAs have been discovered in recent years. To extend the repertoire of small RNAs characterized in mammals and to examine relationship between host miRNA expression and viral infection we used Illuminas ultrahigh throughput sequencing approach. We sequenced three small RNA libraries prepared from cell line derived from the adult bovine kidney under normal conditions and upon infection of the cell line with Bovine herpesvirus 1. We used a bioinformatics approach to distinguish authentic mature miRNA sequences from other classes of small RNAs and short RNA fragments represented in the sequencing data. Using this approach we detected 219 out of 356 known bovine miRNAs and 115 respective miRNA* sequences. In addition we identified five new bovine orthologs of known mammalian miRNAs and discovered 268 new cow miRNAs many of which are not identifiable in other mammalian genomes and thus might be specific to the ruminant lineage. In addition we found seven new bovine mirtron candidates. We also discovered 10 small nucleolar RNA (snoRNA) loci that give rise to small RNA with possible miRNA-like function. Results presented in this study extend our knowledge of the biology and evolution of small regulatory RNAs in mammals and illuminate mechanisms of small RNA biogenesis and function. New miRNA sequences and the original sequencing data have been submitted to miRNA repository (miRBase) and NCBI GEO archive respectively. We envisage that these resources will facilitate functional annotation of the bovine genome and promote further functional and comparative genomics studies of small regulatory RNA in mammals.

Studies of the in vitro cytotoxic, antioxidant, lipase inhibitory and antimicrobial activities of selected Thai medicinal plants

BackgroundTraditional folk medicinal plants have recently become popular and are widely used for primary health care. Since Thailand has a great diversity of indigenous (medicinal) plant species, this research investigated 52 traditionally used species of Thai medicinal plants for their in vitro cytotoxic, antioxidant, lipase inhibitory and antimicrobial activities.MethodsThe 55 dried samples, derived from the medicinally used parts of the 52 plant species were sequentially extracted by hexane, dichloromethane, ethanol and water. These 220 extracts were then screened for in vitro (i) cytotoxicity against four cell lines, derived from human lung (A549), breast (MDA-MB-231), cervical (KB3-1) and colon (SW480) cancers, using the MTT cytotoxicity assay; (ii) antioxidant activity, analyzed by measuring the scavenging activity of DPPH radicals; (iii) lipase inhibitory activity, determined from the hydrolytic reaction of p-nitrophenyllaurate with pancreatic lipase; and (iv) antimicrobial activity against three Gram-positive and two Gram-negative bacteria species plus one strain of yeast using the disc-diffusion method and determination of the minimum inhibitory concentration by the broth micro-dilution assay.ResultsThe crude dichloromethane and/or ethanol extracts from four plant species showed an effective in vitro cytotoxic activity against the human cancer cell lines that was broadly similar to that of the specific chemotherapy drugs (etoposide, doxorubicin, vinblastine and oxaliplatin). In particular, this is the first report of the strong in vitro cytotoxic activity of Bauhinia strychnifolia vines. The tested tissue parts of only six plant species (Allium sativum, Cocoloba uvifera, Dolichandrone spathacea, Lumnitzera littorea, Sonneratia alba and Sonneratia caseolaris) showed promising potential antioxidant activity, whereas lipase inhibitory activity was only found in the ethanol extract from Coscinum fenestratum and this was weak at 17-fold lower than Orlistat, a known lipase inhibitor. The highest antimicrobial activity was observed in the extracts from S. alba and S. caseolaris against Pseudomonas aeruginosa and Candida albicans, respectively.ConclusionThe Thai medicinal plant B. strychnifolia is first reported to exert strong in vitro cytotoxic activities against human cancer cell lines and warrants further enrichment and characterization. The broad spectrum of the biological activities from the studied plant extracts can be applied as the guideline for the selection of Thai medicinal plant species for further pharmacological and phytochemical investigations.

Characterization of microRNAs encoded by the bovine herpesvirus 1 genome

Bovine herpesvirus 1 (BoHV-1) is a ubiquitous and important pathogen of cattle worldwide. This study reports the identification of 10 microRNA (miRNA) genes, Bhv1-mir-B1-Bhv1-mir-B10, encoded by the BoHV-1 genome that were processed into 12 detectable mature miRNAs as determined by ultra-high throughput sequencing bioinformatics analyses of small RNA libraries and expression studies. We found that four of the miRNA genes were present as two copies in the BoHV-1 genome, resulting in a total of 14 miRNA encoding loci. Unique features of the BoHV-1 miRNAs include evidence of bidirectional transcription and a close association of two miRNA genes with the origin of replication, including one miRNA that is encoded within the origin of replication. The miRNA gene Bhv1-mir-B5 was encoded on the opposite DNA strand to the latency associated transcript, potentially giving rise to antisense transcripts originating from this locus. The association of herpesvirus miRNAs with latency appears to be a common feature in the alphaherpesviruses. Analyses of the BoHV-5 genome for putative miRNA gene orthologues identified a high degree of evolutionary conservation for nine of the BoHV-1 miRNA genes. The possible roles for BoHV-1 miRNAs in the regulation of known BoHV-1 transcription units and the genetics of the BoHV-1 genotypes are also discussed.

Application of YHV-protease dsRNA for protection and therapeutic treatment against yellow head virus infection in Litopenaeus vannamei.

While farming of the Pacific white shrimp Litopenaeus vannamei is well established in North and South America, the industry has more recently been introduced to Asia, and the Pacific white shrimp is now the most commonly farmed species in Thailand. However, outbreaks of yellow head virus (YHV) disease in the Pacific white shrimp have caused severe economic losses and currently there is no effective prevention or treatment of YHV infections. The YHV-protease double-stranded RNA (YHV-Pro dsRNA) can act as both a prophylactic agent and as a treatment to inhibit YHV replication in infected black tiger shrimp Penaeus monodon. The utility of this methodology to other shrimp species has not, however, been established. The purpose of this study was to determine whether YHV-Pro dsRNA can be applied to the Pacific white shrimp. To assess prophylactic efficiency, YHV-Pro dsRNA was injected into juvenile shrimp 24 h prior to challenge with YHV. Subsequent YHV replication was inhibited by YHV-Pro dsRNA as compared with injection of an unrelated dsRNA. For therapeutic treatment of YHV-infected shrimp, shrimp were challenged with YHV before dsRNA injection. Injection of YHV-Pro dsRNA up to 6 h post-infection resulted in the almost complete elimination of YHV replication. These results suggest that YHV-Pro dsRNA can also be broadly applied as a prophylactic agent to inhibit YHV replication and therapeutic treatment of YHV-infected Pacific white shrimp.

Upregulation of Stat1-HDAC4 confers resistance to etoposide through enhanced multidrug resistance 1 expression in human A549 lung cancer cells

Despite efforts to develop efficient chemotherapeutic drug strategies to treat cancer, acquired drug resistance is a commonly encountered problem. In the present study, to investigate this phenomenon, human A549 lung cancer cells resistant to the topoisomerase inhibitor etoposide (A549RT‑eto) were used and compared with A549 parental cells. A549RT‑eto cells demonstrated increased resistance to etoposide‑induced apoptosis when compared with A549 parental cells. Notably, A549RT‑eto cells were observed to exhibit greater levels of histone deacetylase 4 (HDAC4), phospho‑Stat1 and P‑glycoprotein [P‑gp; encoded by the multidrug resistance 1 (MDR1) gene], compared with A549 cells. To address whether HDAC4 protein is involved in etoposide resistance in A549 cells, A549RT‑eto cells were treated with trichostatin A (TSA; an HDAC inhibitor) during etoposide treatment. The combined treatment was demonstrated to enhance etoposide‑induced apoptosis and reduce expression levels of HDAC4, P‑gp and phospho‑Stat1. In addition, the suppression of Stat1 with siRNA enhanced etoposide‑induced apoptosis and reduced the expression levels of HDAC4 and P‑gp, suggesting that Stat1 is essential in the regulation of resistance to etoposide, and in the upregulation of P‑gp. Notably, TSA treatment reduced P‑gp transcript levels but Stat1 siRNA treatment did not, suggesting that P‑gp is regulated by HDAC at the transcriptional level and by Stat1 at the post‑transcriptional level. These results suggest that the upregulation of Stat1 and HDAC4 determines etoposide resistance through P‑gp expression in human A549 lung cancer cells.

Silencing of PmYPR65 receptor prevents yellow head virus infection in Penaeus monodon.

The suppression of viral replication by double-stranded RNAs (dsRNA) specific to mRNAs of either virus or host genes has been widely investigated as a possible shrimp disease therapy. PmYRP65, a yellow head virus (YHV) receptor, was previously identified and characterized in the black tiger shrimp, Penaeus monodon. In our previous study, entry of YHV into cells of the Oka organ of P. monodon required the host receptor PmYRP65 and silencing of PmYRP65 in vitro led to a complete suppression of YHV replication in the cells. In this study, PmYRP65 was shown to be in vivo suppressed by dsRNA specific for PmYRP65, leading to inhibition of YHV replication and almost complete abolition of shrimp mortality following YHV challenge. Targeting PmYRP65 could be an effective YHV antiviral shrimp strategy.

Extract of Bryophyllum laetivirens reverses etoposide resistance in human lung A549 cancer cells by downregulation of NF-κB

Since multidrug resistance (MDR) is one of the main reasons for failure in cancer treatment, its suppression may increase the efficacy of cancer therapy. In the present study we attempted to identify a new and effective anticancer drug against MDR cancer cells. We first found that lung cancer A549 cells resistant to etoposide (A549RT-eto) exhibit upregulation of NF-κB and SIRT1 in comparison to A549 parental cells. During a search for anticancer drug candidates from medicinal plant sources, we found that an extract fraction (F14) of Bryophyllum laetivirens leaves downregulated expression of NF-κB and SIRT1, sensitizing the levels of A549RT-eto cells to apoptosis through downregulation of P-glycoprotein (P-gp), which is encoded by the MDR1 gene. To address whether NF-κB is involved in resistance to etoposide through P-gp, we treated A549RT-eto cells with Bay11-7802, an inhibitor of NF-κB. We then observed that Bay11-7802 treatment reduced P-gp expression levels, and furthermore combined treatment with the F14 extract and Bay11-7802 accelerated apoptosis through a decrease in P-gp levels, suggesting that NF-κB is involved in MDR. To address whether upregulation of SIRT1 is involved in resistance to etoposide through P-gp, we treated A549RT-eto cells with SIRT1 siRNA or nicotinamide (NAM), an inhibitor of SIRT1. we found that suppression of SIRT1 did not reduce P-gp levels. furthermore, the combined treatment with the F14 extract, and SIRT1 siRNA or NAM did not accelerate apoptosis, indicating that SIRT1 is not involved in the regulation of P-gp levels in A549RT-eto cells. Taken together, we suggest that upregulation of NF-κB determines etoposide resistance through P-gp expression in human A549 lung cancer cells. We herein demonstrated that B. laetivirens extract reverses etoposide resistance in human A549 lung cancer cells through downregulation of NF-κB.

Feroniellin A-induced autophagy causes apoptosis in multidrug-resistant human A549 lung cancer cells

During the screening of natural chemicals that can reverse multidrug resistance in human A549 lung cancer cells resistant to etoposide (A549RT-eto), we discovered that Feroniellin A (FERO), a novel furanocoumarin, shows toxicity toward A549RT-eto cells in a dose- and time-dependent manner. FERO reduced the expression of NF-κB, leading to downregulation of P-glycoprotein (P-gp), encoded by MDR1, which eventually sensitized A549RT-eto cells to apoptosis. FERO specifically diminished transcription and promoter activity of MDR1 but did not inhibit the expression of other multidrug resistance genes MRP2 and BCRP. Moreover, co-administration of FERO with Bay11-7802, an inhibitor of NF-κB, accelerated apoptosis of A549RT-eto cells through decreased expression of P-gp, indicating that NF-κB is involved in multidrug resistance. Conversely, addition of Z-VAD, a pan-caspase inhibitor, blocked FERO-induced apoptosis in A549RT-eto cells but did not block downregulation of P-gp, indicating that a decrease in P-gp expression is necessary but not sufficient for FERO-induced apoptosis. Interestingly, we found that FERO also induces autophagy, which is characterized by the conversion of LC3 I to LC3 II, induction of GFP-LC3 puncta, enhanced expression of Beclin-1 and ATG5, and inactivation of mTOR. Furthermore, suppression of Beclin-1 by siRNA reduced FERO-induced apoptosis in A549RT-eto cells and activation of autophagy by rapamycin accelerated FERO-induced apoptosis, suggesting that autophagy plays an active role in FERO-induced apoptosis. Herein, we report that FERO reverses multidrug resistance in A549RT-eto cells and exerts its cytotoxic effect by induction of both autophagy and apoptosis, which suggests that FERO can be a useful anticancer drug for multidrug-resistant lung cancer.

miR-21 promotes dengue virus serotype 2 replication in HepG2 cells

Abstract Infection with the mosquito transmitted dengue virus (DENV) remains a significant worldwide public health problem. While the majority of infections are asymptomatic, infection can result in a range of symptoms. MicroRNAs (miRNAs) are small non‐coding RNAs that regulate gene expression through repression or degradation of mRNAs. To understand the contribution of miRNAs to DENV 2 replication, we screened a number of candidate miRNAs for variations in expression levels during DENV 2 infection of HepG2 (liver) cells. Seven miRNAs were identified as differentially expressed, and one, miR‐21, was differentially expressed at all time points examined. Interestingly, miR‐21 was also differentially regulated in DENV 2 infection under conditions of antibody dependent enhancement of infection, and in direct Zika virus infection, but not in DENV 4 infection. The role of miR‐21 during DENV infection was further examined by treating HepG2 cells with an anti‐miR‐21 (AMO‐21) before DENV infection. The results showed a significant reduction in DENV 2 production, clearly suggesting that miR‐21 plays a key role in DENV 2 replication. To further confirm the role of miR‐21 in DENV infection, a peptide nucleic acid‐21 (PNA‐21) construct with a nucleotide sequence complementary to AMO‐21, was co‐administered with AMO‐21 as an AMO‐21/PNA‐21 complex followed by DENV 2 infection. The results showed that AMO‐21 significantly reduced DENV 2 titer, PNA‐21 significantly increased DENV 2 titer and the combined AMO‐21/PNA‐21 showed no difference from non‐treated infection controls. Taken together, the results show that miR‐21 promotes DENV 2 replication, and this mechanism could serve as a possible therapeutic intervention point. HighlightsmiR‐21 played a key role in DENV 2 replication in HepG2 cells.miR‐21 was differentially expressed at all time points examined.miR‐21 promotes DENV 2 replication in HepG2 cells.Anti‐miRNA‐21 oligonucleotide (AMO‐21) significantly reduced DENV 2 titer.

Cholesterol-based cationic liposome increases dsRNA protection of yellow head virus infection in Penaeus vannamei.

Protection of shrimp from yellow head virus (YHV) infection has been demonstrated by injection and oral delivery of dsRNA-YHV protease gene (dsYHV) or shrimp endogenous gene (dsRab7). However, to achieve complete viral suppression and to prolong dsRNA activity, the development of an effective dsRNA delivery system is required. In this study, four cationic liposomes were synthesized and tested for their ability to increase dsRNA efficiency. The results demonstrated that entrapping dsYHV in a cholesterol-based cationic liposome gave the best protection against YHV infection when compared with other cationic lipids. The cholesterol-based cationic liposome-dsYHV (Chol-dsYHV) complex conferred YHV protection in a dose-dependent manner. Injection with Chol-dsYHV at 0.05μg dsYHV/g shrimp could give comparable level of YHV protection to the injection with 1.25μg naked dsYHV/g shrimp. The shrimp injected with Chol- dsYHV at 1.25μg dsRNA/g shrimp showed only 50% mortality at 60days post injection whereas the naked dsYHV at the same concentration gave 90% mortality. Thus, the liposome-entrapped dsYHV could lower an effective dsRNA concentration in viral protection and prolong dsRNA activity. In addition, encapsulating dsRab7 in the cholesterol-based cationic liposome could protect the dsRab7 from enzymatic digestion, and continuous feeding the shrimp with the diet formulated with the liposome-entrapped dsRab7 for 4days in the total of 960μg dsRab7/g shrimp could enhance YHV protection efficiency compared with the naked dsRab7. Our studies reveal that cholesterol-based cationic liposome is a promising dsRNA carrier to enhance dsRNA efficiency in both injection and oral delivery systems.