Selenium-sensitive histone deacetylase 2 is required for forkhead box O3A and regulates extracellular matrix metabolism in cartilage.

INTRODUCTION: Selenium (Se) as well as selenoproteins are vital for osteochondral system development. Se deficiency (SeD) has a definite impact on the expression and activity of histone deacetylases (HDACs). Abnormal expression of some HDACs affects cartilage development. This current study aims to explore the relationship between differentially expressed HDACs and cartilage development, especially extracellular matrix (ECM) homeostasis maintenance, under SeD conditions. MATERIALS AND METHODS: Dark Agouti rats and C28/I2 cell line under SeD states were used to detect the differently expressed HDAC by RT-qPCR, western blotting and IHC staining. Meanwhile, the biological roles of the above HDAC in cartilage development and homeostasis maintenance were confirmed by siRNA transfection, western blotting, RNA sequence and inhibitor treatment experiments. RESULTS: HDAC2 exhibited lower expression at protein level in both animals and chondrocytes during SeD condition. The results of cell-level experiments indicated that forkhead box O3A (FOXO3A), which was required to maintain metabolic homeostasis of cartilage matrix, was reduced by HDAC2 knockdown. Meanwhile, induced HDAC2 was positively associated with FOXO3A in rat SeD model. Meanwhile, knockdown of HDAC2 and FOXO3A led to an increase of intracellular ROS level, which activated NF-κB pathway. Se supplementary significantly inhibited the activation of NF-κB pathway with IL-1ß treatment. CONCLUSION: Our results suggested that low expression of HDAC2 under SeD condition increased ROS content by decreasing FOXO3A in chondrocytes, which led to the activation of NF-κB pathway and ECM homeostasis imbalance.

Evaluation and perception of online teaching of molecular biology using DingTalk for international medical students during the COVID-19 pandemic.

The COVID-19 pandemic has greatly impacted the education of international students. The authors taught a molecular biology course using the DingTalk platform for international medical students (IMS) in the autumn semester of 2020. We assessed the effect of this online teaching based on an online questionnaire and by analysis of the final examination scores. Our findings demonstrate that the DingTalk platform is a free, effective and convenient online teaching tool for international students. The students' feedback showed that most of them were satisfied with this live teaching with DingTalk. They considered who viewed that the questions used in the live classroom setting were helpful for their learning. There is nonetheless still scope to improve this online teaching mode for international students, such as providing more pre-recorded teaching videos for offline application and use of a virtual simulation experimental molecular biology course. We hope that our findings regarding the experience of IMS with this teaching mode will be of value to other academic faculty.

Identification of a cartilage specific novel miRNA which directly targets PRMT3 in rats.

Through experiments to testify a candidate novel miRNA previously discovered by us is a real miRNA and involved in cartilage development. DESIGN: The miR-novel and the newly hairpin miRNA transcribed sequence (pre-miR-novel) was verified as a genuinely existing miRNA by northern blotting. The predicted secondary structure, sequence alignment and targets of pre-miR-novel were performed by "RNAstructure 5.3" program, LASTN2.8.0+/miRbase22 program and RNA hybird program, respective. GO/KEGG pathway analysis also were performed. The miR-novel expression in cartilage tissue during development was detected by RT-qPCR and dot blotting. The chondrocyte differentiation model was established to examine whether miR-novel is involved in cartilage development. The regulation of PRMT3 expression by novel miRNA was determined with the luciferase reporter gene assay and Western blotting after novel miRNA mimic or inhibitor transfection. RESULTS: It's potential role in specifically regulating rodent cartilage development and associated cellular processes. Furthermore, the expression of protein arginine N-methyltransferase 3 (PRMT3), as a predicted target of the novel miRNA, was found consistently downregulated at rat cartilage during developmental stages and RCJ3.1C5.18 (C5.18) cells during the proliferating and hypertrophic phases of the cartilage development, where the miR-novel expression was significantly up-regulated. Both the dual-luciferase reporter gene assay and the up- or down-regulation of miR-novel suggest that the later can specifically bind with the Prmt3 3'-UTR. CONCLUSION: Overall, this study provides the first comprehensive evidence that a genuine cartilage-specific novel miRNA directly targets PRMT3 and may regulate multitudinous cellular processes and signal transduction during cartilage development.

17ß-Oestradiol Attenuates the Photoreceptor Apoptosis in Mice with Retinitis Pigmentosa by Regulating N-myc Downstream Regulated Gene 2 Expression.

Retinitis pigmentosa (RP) is a heterogeneous group of retinal degenerative diseases in which the final pathological feature is photoreceptor cell apoptosis. Currently, the pathogenesis of RP remains poorly understood and therapeutics are ineffective. 17ß-Oestradiol (ßE2) is universally acknowledged as a neuroprotective factor in neurodegenerative diseases and has manifested neuroprotective effects in a light-induced retinal degeneration model. Recently, we identified N-myc downstream regulated gene 2 (NDRG2) suppression as a molecular marker of mouse retinal photoreceptor-specific cell death. ßE2 has also been reported to regulate NDRG2 in salivary acinar cells. Therefore, in this study, we investigated whether ßE2 plays a protective role in RP and regulates NDRG2 in photoreceptor cells. To this end, we generated RP models and observed that ßE2 not only reduced the apoptosis of photoreceptor cells, but also restored the level of NDRG2 expression in RP models. Then, we showed that siNDRG2 inhibits the anti-apoptotic effect of ßE2 on photoreceptor cells in a cellular RP model. Subsequently, we used a classic oestrogen receptor (ER) antagonist to attenuate the effects of ßE2, suggesting that ßE2 exerted its effects on RP models via the classic ERs. In addition, we performed a bioinformatics analysis, and the results indicated that the reported oestrogen response element (ERE) sequence is present in the promoter region of the mouse NDRG2 gene. Overall, our results suggest that ßE2 attenuated the apoptosis of photoreceptor cells in RP models by maintaining NDRG2 expression via a classic ER-mediated mechanism.

The impact and evaluation of COVID-19 pandemic on the teaching model of medical molecular biology course for undergraduates major in pharmacy.

Molecular biology is a very important basic course for undergraduates major in pharmacy. During the novel coronavirus epidemic, we first adopted an online teaching of molecular biology course with rain class and tencent meeting for undergraduates major in pharmacy, following a blended teaching mode. Finally, we evaluated the effect of this special-time teaching by analyzing the anonymous questionnaire and final examination scores. Student feedback showed that most of students were satisfied with this online teaching, classroom teaching, and experimental teaching, and considered that postlecture quizzes were very helpful for their study. The majority of students supported that classroom teaching should be integrated with online teaching. Analysis of final examination scores showed that the effect of 2020-year teaching was not worse than that of 2019-year teaching, but even better in the excellence rate and rate of poor and failure. Here, we share the experience and thinking of blended teaching of medical molecular biology course during the novel coronavirus epidemic, and hope it helpful for other teachers' teaching.

Intervening upregulated SLC7A5 could mitigate inflammatory mediator by mTOR-P70S6K signal in rheumatoid arthritis synoviocytes.

OBJECTIVE: The disruption of metabolic events and changes to nutrient and oxygen availability due to sustained inflammation in RA increases the demand of bioenergetic and biosynthetic processes within the damaged tissue. The current study aimed to understand the molecular mechanisms of SLC7A5 (amino acid transporter) in synoviocytes of RA patients. METHODS: Synovial tissues were obtained from OA and RA patients. Fibroblast-like synoviocytes (FLS) were isolated, and SLC7A5 expression was examined by using RT-qPCR, immunofluorescence, and Western blotting. RNAi and antibody blocking treatments were used to knockdown SLC7A5 expression or to block its transporter activities. mTOR activity assay and MMP expression levels were monitored in RA FLS under amino acid deprivation or nutrient-rich conditions. RESULTS: RA FLS displayed significantly upregulated expression of SLC7A5 compared to OA FLS. Cytokine IL-1ß was found to play a crucial role in upregulating SLC7A5 expression via the NF-κB pathway. Intervening SLC7A5 expression with RNAi or blocking its function by monoclonal antibody ameliorated MMP3 and MMP13 protein expression. Conversely, upregulation of SLC7A5 or tryptophan supplementation enhanced mTOR-P70S6K signals which promoted the protein translation of MMP3 and MMP13 in RA FLS. CONCLUSION: Activated NF-κB pathway upregulates SLC7A5, which enhances the mTOR-P70S6K activity and MMP3 and MMP13 expression in RA FLS.

Upregulated PKM2 in Macrophages Exacerbates Experimental Arthritis via STAT1 Signaling.

Recent studies indicate that glucose metabolism is altered in rheumatoid arthritis. We hypothesize that Pkm2, as a key regulatory enzyme of glycolysis pathway, triggers the activation of macrophages (Mφ), which results in proinflammatory cytokine production during the arthritis progress. In this study, Pkm2 was found to be overexpressed in ED1-positive Mφ in spleens and synovial tissues from arthritic rats via immunofluorescence, Western blotting, and quantitative RT-PCR. To reveal the role of Pkm2, Dark Agouti rats were treated with either Pkm2 enzyme inhibitor shikonin or the RNA interference plasmids of Pkm2 and negative control plasmids, respectively, via i.p. injection. Pkm2 intervention could alleviate the severity of pristane-induced arthritis in aspects of the macroscopic arthritis score, perimeter changes of midpaw, and the synovitis and destruction of the bone and cartilage as well as reduce the ED1 and p-Stat1-positive cell population in rat synovial tissues. Silencing Pkm2 by RNA interference in classical activated rat and mouse Mφ resulted in less Tnf-α, Il-1ß production via Stat1 signaling. Collectively, Pkm2 is highly expressed in ED1-positive Mφ of spleens and synovial tissues from arthritic rats and promotes Mφ activation via Stat1 signaling. Pkm2 might be a promising selective metabolic target molecule for rheumatoid arthritis treatment.

The mechanisms of lncRNA GAS5 in cardiovascular cells and its potential as novel therapeutic target.

Long noncoding RNAs (lncRNAs) are a large class of non (protein)-coding RNAs, which are longer beyond 200 nucleotides. LncRNA GAS5 is widely considered as a tumour suppressor in cell proliferation, apoptosis, cell migration and invasion of tumour cells. Recently, a growing body of evidences indicated that GAS5 was also widely involved in the pathologic process of cardiovascular cells, including regulation of apoptosis and inflammatory injury of cardiomyocytes; proliferation, apoptosis, autophagy and angiogenesis of endothelial cells; and proliferation, migration, apoptosis and differentiation of VSMCs. In this regard, we summarised current studies of GAS5 in cardiovascular cells, which shed light on not only our understanding of the mechanisms of GAS5 in cardiovascular cells but also understanding of the potential of GAS5 as novel therapeutic target.

The mechanism of lncRNA H19 in fibrosis and its potential as novel therapeutic target.

Fibrosis is widely observed in multiple organs, which is generally associated with aging-related diseases, including liver fibrosis, lung fibrosis, cardiac fibrosis and renal fibrosis. The excess deposition of extracellular matrix ultimately leads to the disruption of organ architecture and loss of function. H19 is paternally imprinted maternally expressed lncRNA, which is highly expressed during the development of embryo but quickly downregulated after birth. Recently, a growing body of studies indicates that H19 is widely involved in the pathologic mechanism of fibrosis in various organs. In this review, we summarized current studies of H19 in fibrosis and hopefully aid in a better understanding of the molecular mechanism of fibrosis and the potential of H19 as novel therapeutic target for fibrosis.

The Human Novel Gene LNC-HC Inhibits Hepatocellular Carcinoma Cell Proliferation by Sequestering hsa-miR-183-5p.

Hepatocellular carcinoma (HCC) is the most commonly diagnosed cancer and the leading cause of cancer mortality. Several lines of evidence have demonstrated the aberrant expression of long noncoding RNAs (lncRNAs) in carcinogenesis and their universal regulatory properties. A thorough understanding of lncRNA regulatory roles in HCC pathology would contribute to HCC prevention and treatment. In this study, we identified a novel human lncRNA, LNC-HC, with significantly reduced levels in hepatic tumors from patients with HCC. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide) assays as well as colony formation and wound healing experiments showed that LNC-HC significantly inhibited the proliferation of the HCC cell line Huh7. Xenograft transplantation of LNC-HC-overexpressing Huh7 cells in nude mice resulted in the production of smaller tumors. Mechanistically, LNC-HC inhibited the proliferation of HCC cells by directly interacting with hsa-miR-183-5p. LNC-HC rescued the expression of five tumor suppressors, including AKAP12, DYRK2, FOXN3, FOXO1, and LATS2, that were verified as target genes of hsa-miR-183-5p. Overall, human LNC-HC was identified as a novel tumor suppressor that could inhibit HCC cell proliferation in vitro and suppress tumor growth in vivo by competitively binding hsa-miR-183-5p as a competing endogenous RNA (ceRNA). These findings suggest that LNC-HC could be a biomarker of HCC and provide a novel therapeutic target for HCC treatment.

Long noncoding RNAs as novel players in the pathogenesis of hypertension.

Long noncoding RNAs (lncRNAs) are non-(protein)-coding RNAs longer than ~200 nucleotides and have been reported to be involved in multiple human diseases by regulating gene expression. A growing body of evidence has demonstrated that lncRNAs are also widely implicated in mechanisms of hypertension, including regulation of the proliferation, migration, and apoptosis of VSMCs; the production of iNOS and NO; and the angiogenic function of endothelial cells. Several lncRNAs were also differentially expressed in the renal and cardiac tissues of hypertensive rats and even in placental samples from preeclampsia patients. In particular, several circulating lncRNAs have been identified as novel biomarkers of hypertension. In this review, we summarize the current studies of lncRNAs in the pathogenesis of hypertension in order to aid in better understanding the molecular mechanism of hypertension and provide a basis to explore new therapeutic targets.

Long Noncoding RNA lnc-HC Regulates PPARγ-Mediated Hepatic Lipid Metabolism through miR-130b-3p.

Nonalcoholic fatty liver disease (NAFLD) is due to the excessive lipid accumulation within hepatocytes. Metabolic nuclear receptors (MNRs) play great roles in lipid homeostasis. We have identified a novel long noncoding RNA (lncRNA), lnc-HC, which regulates hepatocytic cholesterol metabolism through reducing Cyp7a1 and Abca1 expression. Here, we further elucidate its roles in hepatic fatty acid and triglyceride (TG) metabolism through a novel lncRNA regulatory mechanism. The most prominent target of lnc-HC identified by in vitro study is PPARγ. Further studies revealed that lnc-HC negatively regulates PPARγ at both the mRNA and protein levels and suppresses hepatocytic lipid droplet formation. Importantly, the function of lnc-HC in regulating PPARγ expression depends on modulating miR-130b-3p expression from the transcriptional to the post-transcriptional level, not through lncRNA's critical modulating patterns. In vivo, the reduction of lnc-HC expression significantly decreases miR-130b-3p expression, induces PPARγ expression, and increases TG concentration in rat livers with hyperlipidemia. These findings further help in understanding the regulatory pattern of lnc-HC in hepatic lipid metabolism and might present a possible therapeutic target for improving lipid homeostasis.

Aberrant expression of long noncoding RNAs in the serum and myocardium of spontaneous hypertensive rats.

Circulating long noncoding RNAs as biomarkers of diseases have attracted increasing attention recently. However, circulating lncRNAs in hypertension is still unexplored niche. The levels of lncRNAs GAS5, NR024118, MRAK134679, AX765700 and MRNR026574 were measured in the serum and myocardium of hypertensive rats and normal controls with real time PCR. The levels of GAS5 were significantly higher both in the myocardium (P = 0.0067) and serum (P < 0.0001) of hypertensive rats compared with controls. The levels of NR024118 were remarkably higher in the myocardium of hypertensive rats (P = 0.0202) while the levels of serum NR024118 were not statistically significant in two groups (P = 0.6926). The levels of serum AX765700 (P = 0.0644) and cardiac AX765700 (P = 0.1938) were not statistically significant in hypertensive rats and controls. The levels of MRAK134679 were not different in the myocardium of two groups (P = 0.1692) and were too low in the serum to be detected. The levels of MRNR026574 were significantly higher in the myocardium of hypertensive rats compared with controls (P < 0.0001) and were too low in the serum to be detected. In conclusions, the levels of GAS5, NR024118 and MRNR026574 were increased in the myocardium of hypertensive rats, suggesting that they participate in the pathogenesis of hypertensive cardiac remodeling. Although, the levels of GAS5 in the serum and heart tissue were both significantly increased in SH rats, the potential biomarker capacity of GAS5 for HT needs to be further explored on larger human cohorts.

Abnormal Expression of DICER1 Leads to Dysregulation of Inflammatory Effectors in Human Synoviocytes.

Dysregulation of multiple microRNAs widely takes place during rheumatoid arthritis (RA) and experimental arthritides. This study is performed to explore the possible mechanism underlying DICER1 deficiency-mediated inflammation in human synoviocytes SW982. Firstly, RNAi of DICER1 led to increased COX2, MMP3, and MMP13 protein production, while DICER1 overexpression could reduce MMP13 expression. Secondly, the increase of IL-8 and decrease of TGF-ß1 and TIMP1 were determined in the supernatant derived from DICER1 siRNA-treated cells, while DICER1 overexpression was found capable to reverse this effect. Ingenuity pathway analysis (IPA) software predicted that the Dicer1 deficiency-induced dysregulated cytokines in synoviocytes could possibly lead to the inflammatory disorders in the synovial tissue. Moreover, DICER1 deficiency could also reduce apoptosis, while DICER1 overexpression was found to decrease the proliferation and enhance apoptosis. In addition, DICER1 deficiency could lower the expression of multiple RA-related miRNAs such as miR-155. Meanwhile, DICER1 overexpression could rescue their low expression levels. And then, gain or loss of miR-155 function could regulate the protein levels of MMP3 and MMP13. These results indicated that DICER1 might play its role through regulating its downstream RA-related miRNAs. Our data demonstrated that DICER1 deficiency could cause multiple proinflammatory events in human synoviocytes SW982. This mechanism study might provide the possible target molecule to modify the inflammatory destruction and overproliferation in synoviocytes.

Circular RNAs as novel regulators, biomarkers and potential therapies in fibrosis.

Fibrosis is the excess deposition of extracellular matrix components which ultimately leads to the disruption of organ architecture and loss of function. Circular RNAs (circRNAs) are a newly discovered type of long noncoding RNAs with single-stranded covalently closed loops. It is known that circRNAs are novel regulators of gene expression via various ways, including miRNA sponge, protein sponge, regulation of transcription and post transcription. Recently, a growing body of evidence suggests that circular RNAs are also involved in tissue fibrosis in several organs. In this review, we summarized current studies of circular RNAs in fibrosis and hopefully aid in better understanding the molecular mechanism of fibrosis and provide a basis to explore new therapeutic targets of fibrosis.

Selenium-sensitive miRNA-181a-5p targeting SBP2 regulates selenoproteins expression in cartilage.

Selenium (Se) deficiency brings about defects in the biosynthesis of several selenoproteins and has been associated with aberrant chondrogenesis. Selenocysteine (Sec) Insertion Sequence (SECIS) and SECIS binding protein 2 (SBP2) interaction is a very critical node for the metabolic balance between Se and selenoproteins. The Gpx1, Gpx4 and SelS have different binding affinities with SBP2 in cells. According to our results, both miR-181a-5p and SBP2 appeared to be selenium-sensitive and regulated the expression of selenoproteins in C28/I2 cells under Se sufficient environment. However, they showed significantly opposite expression trend in Se deficiency rats cartilage and SeD C28/I2 cells. The SBP2 is a direct target gene of miR-181a-5p in C28/I2 cells as determined by reporter gene and off-target experiments. And the miR-181a-5p could regulate SBP2 and the selenoproteins in C28/I2 cells. Depending upon the Se supply levels, C28/I2 cells were divided into three groups, that is normal Se, SeD and SeS, which underwent through a 7-day Se deprivation process, then SBP2 was knocked-down and overexpressed in all the groups. Moreover, the selected selenoproteins were down-regulated in second-generation low Se diet rat cartilage. The selenoproteins expression was decreased by Se deficiency which depended on the Selenium-sensitive miR-181a-5p to participate and regulate SBP2 at post-transcriptional level. It involves a series of antioxidant and ECM (extracellular matrix) genes, to overcome the ROS-related stress for the protection of essential physiological functions and to maintain the balance between anabolism and catabolism of the cartilage.

Pristane induces autophagy in macrophages, promoting a STAT1-IRF1-TLR3 pathway and arthritis.

Autophagy is involved in both innate and adaptive immune regulation. We propose that autophagy regulates activation of TLR3 in macrophages and is thereby essential for development of pristane-induced arthritis. We found that pristane treatment induced autophagy in macrophages in vitro and in vivo, in spleen cells from pristane injected rats. The induced autophagy was associated with STAT1 phosphorylation and expression of IRF1 and TLR3. Blocking the pristane activated autophagy by Wortmannin and Bafilomycin A1 or by RNAi of Becn1 led to a downregulation of the associated STAT1-IRF1-TLR3 pathway. Most importantly, the development of arthritis was alleviated by suppressing either autophagy or TLR3. We conclude that pristane enhanced autophagy, leading to a STAT1-IRF1 controlled upregulation of TLR3 expression in macrophages, is a pathogenic mechanism in the development of arthritis.

Circulating long noncoding RNAs as novel biomarkers of human diseases.

Long noncoding RNAs (lncRNAs) are a kind of noncoding RNAs which are longer than ˜200 nucleotides, lacking of protein-encoding capacity and are implicated in the pathogenesis of various diseases. Recently, it was demonstrated that lncRNAs could be released into the circulation and be stable in blood. Circulating lncRNAs have been reported to have potential in distinguishing patients from healthy individuals. Therefore, the detection of circulating lncRNAs may be valuable for improving the diagnosis and prognosis of various diseases. This review summarized the current understanding of circulating lncRNAs as novel biomarkers of various human diseases, such as cancer, cardiovascular diseases, nervous system diseases and other diseases, which highlighted the significance of circulating lncRNAs as novel diagnostic and prognostic biomarkers of human diseases.

Efficacy of tenofovir-based rescue therapy in patients with lamivudine-resistant hepatitis B virus: A systematic review and meta-analysis.

BACKGROUND: Currently, there are no conclusive results on the efficacy of Tenofovir disoproxil fumarate (TDF) monotherapy in chronic hepatitis B (CHB) patients with lamivudine-resistant (LAM-R). OBJECTIVE: The aim of this study was to compare the efficacy between TDF and TDF-based combination therapy against LAM-R HBV in CHB patients. METHODS: Randomized and non-randomized control trials directly comparing TDF and TDF-based therapy for treatment of LAM-R CHB patients, were searched in Pubmed, Medline, EMBASE, database up to June 15, 2015. The data were analyzed with Review Manager (v.5.3). RESULTS: Five articles (683 patients in total) met entry criteria. The overall efficacy of tenofovir-based combination therapy was not significantly better with regard to the rates of virological response (85.5% vs. 81.5%; RR=0.95, 95%CI=0.88-1.03, P=0.25), ALT normalization (61.9% vs.72.0%; RR=1.18, 95%CI=0.96-1.44, P=0.11) and HBeAg loss (17.0% vs. 18.1%; RR=1.40, 95%CI=0.78-2.49, P=0.26) compared with TDF monotherapy through 48-week treatment. Additionally, subgroup analysis showed that no significant difference was determined as TDF group compared to TDF-based group at 48weeks, in terms of rates of HBV DNA undetectability, ALT normalization and HBeAg loss in the treatment of LAM-R patients with prior failure of LAM monotherapy. Moreover, the rates of HBV DNA suppression between groups were similar through 24 or 48weeks of treatment in LAM-R patients with prior failure of LAM/ADV therapy. CONCLUSIONS: TDF monotherapy is as effective as TDF-based combination therapy in maintaining viral suppression in LAM-R patients with prior failure of LAM or LAM/ADV therapy.