MicroRNA-34c-5p provokes isoprenaline-induced cardiac hypertrophy by modulating autophagy via targeting ATG4B

Pathological cardiac hypertrophy serves as a significant foundation for cardiac dysfunction and heart failure. Recently, growing evidence has revealed that microRNAs (miRNAs) play multiple roles in biological processes and participate in cardiovascular diseases. In the present research, we investigate the impact of miRNA-34c-5p on cardiac hypertrophy and the mechanism involved. The expression of miR-34c-5p was proved to be elevated in heart tissues from isoprenaline (ISO)-infused mice. ISO also promoted miR-34c-5p level in primary cultures of neonatal rat cardiomyocytes (NRCMs). Transfection with miR-34c-5p mimic enhanced cell surface area and expression levels of foetal-type genes atrial natriuretic factor (Anf) and β-myosin heavy chain (β-Mhc) in NRCMs. In contrast, treatment with miR-34c-5p inhibitor attenuated ISO-induced hypertrophic responses. Enforced expression of miR-34c-5p by tail intravenous injection of its agomir led to cardiac dysfunction and hypertrophy in mice, whereas inhibiting miR-34c-5p by specific antagomir could protect the animals against ISO-triggered hypertrophic abnormalities. Mechanistically, miR-34c-5p suppressed autophagic flux in cardiomyocytes, which contributed to the development of hypertrophy. Furthermore, the autophagy-related gene 4B (ATG4B) was identified as a direct target of miR-34c-5p, and miR-34c-5p was certified to interact with 3' untranslated region of Atg4b mRNA by dual-luciferase reporter assay. miR-34c-5p reduced the expression of ATG4B, thereby resulting in decreased autophagy activity and induction of hypertrophy. Inhibition of miR-34c-5p abolished the detrimental effects of ISO by restoring ATG4B and increasing autophagy. In conclusion, our findings illuminate that miR-34c-5p participates in ISO-induced cardiac hypertrophy, at least partly through suppressing ATG4B and autophagy. It suggests that regulation of miR-34c-5p may offer a new way for handling hypertrophy-related cardiac dysfunction.

The generation of PD-L1 and PD-L2 in cancer cells: From nuclear chromatin reorganization to extracellular presentation

The immune checkpoint blockade (ICB) targeting on PD-1/PD-L1 has shown remarkable promise in treating cancers. However, the low response rate and frequently observed severe side effects limit its broad benefits. It is partially due to less understanding of the biological regulation of PD-L1. Here, we systematically and comprehensively summarized the regulation of PD-L1 from nuclear chromatin reorganization to extracellular presentation. In PD-L1 and PD-L2 highly expressed cancer cells, a new TAD (topologically associating domain) (chr9: 5,400,000-5,600,000) around CD274 and CD273 was discovered, which includes a reported super-enhancer to drive synchronous transcription of PD-L1 and PD-L2. The re-shaped TAD allows transcription factors such as STAT3 and IRF1 recruit to PD-L1 locus in order to guide the expression of PD-L1. After transcription, the PD-L1 is tightly regulated by miRNAs and RNA-binding proteins via the long 3'UTR. At translational level, PD-L1 protein and its membrane presentation are tightly regulated by post-translational modification such as glycosylation and ubiquitination. In addition, PD-L1 can be secreted via exosome to systematically inhibit immune response. Therefore, fully dissecting the regulation of PD-L1/PD-L2 and thoroughly detecting PD-L1/PD-L2 as well as their regulatory networks will bring more insights in ICB and ICB-based combinational therapy.

The 3'-untranslated region contributes to the pregnane X receptor (PXR) expression down-regulation by PXR ligands and up-regulation by glucocorticoids

Pregnane X receptor (PXR) is the major regulator of xenobiotic metabolism. PXR itself is controlled by various signaling molecules including glucocorticoids. Moreover, negative feed-back regulation has been proposed at the transcriptional level. We examined the involvement of the 3'-untranslated region (3'-UTR) of mRNA and microRNAs in PXR- and glucocorticoid receptor (GR)-mediated regulation of gene expression. PXR ligands were found to significantly downregulate mRNA expression in a set of 14 human hepatocyte cultures. Similarly, PXR was downregulated by PCN in the C57/BL6 mice liver. In mechanistic studies with the full-length 3'-UTR cloned into luciferase reporter or expression vectors, we showed that the 3'-UTR reduces PXR expression. From the miRNAs tested, miR-18a-5p inhibited both expression and gene induction. Importantly, we observed significant upregulation of miR-18a-5p expression 6 h after treatment with the PXR ligand rifampicin, which indicates a putative mechanism underlying negative feed-back regulation in hepatic cells. Additionally, glucocorticoids upregulated expression not only through the promoter region but also 3'-UTR regulation, which likely involves downregulation of miR-18a-5p. We conclude that miR-18a-5p is involved in the down-regulation of expression by its ligands and in the upregulation of mRNA expression by glucocorticoids in hepatic cells.

Identification of novel polymorphism in buffalo stanniocalcin-1 gene and its expression analysis in mammary gland under different stages of lactation

Stanniocalcin-1 (STC1) is secreted by the variety of tissues having a major role in the regulation of calcium ions in the involuting mammary gland. The present work aims to sequence and structural characterization as well as expression profiling of STC1 gene in buffalo. Polymorphism identified in the 3-untranslated region (UTR) was analysed by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) genotyping in riverine and swamp buffaloes. Expression profiling of STC1 was performed in different lactation stages of mammary gland and peripheral blood mononuclear cells to study the impact of 3'-UTR polymorphism on its expression. Different polymorphic sites were detected in the entire coding and noncoding regions of riverine and swamp buffaloes, including two INDELs. An identified polymorphic nucleotide locus A324G, having target sites for two miRNAs, namely bta-miR-2382 and bta-miR-1343, reported in cattle, was genotyped by PCR-RFLP to reveal variable allelic distribution among swamp and riverine buffaloes. Gene expression profiling across buffalo mammary tissues representing different lactation stages showed maximum expression of the STC1 gene in the involuting mammary gland. Ruminants’ specific genetic variation has been observed in STC1 and its implication in buffalo mammary gland involution as well as coregulation of gene expression throughmiRNA binding in the 3'-UTR is suggested.

Bioengineered miR-27b-3p and miR-328-3p modulate drug metabolism and disposition the regulation of target ADME gene expression

Drug-metabolizing enzymes, transporters, and nuclear receptors are essential for the absorption, distribution, metabolism, and excretion (ADME) of drugs and xenobiotics. MicroRNAs participate in the regulation of ADME gene expression imperfect complementary Watson-Crick base pairings with target transcripts. We have previously reported that Cytochrome P450 3A4 (CYP3A4) and ATP-binding cassette sub-family G member 2 (ABCG2) are regulated by miR-27b-3p and miR-328-3p, respectively. Here we employed our newly established RNA bioengineering technology to produce bioengineered RNA agents (BERA), namely BERA/miR-27b-3p and BERA/miR-328-3p, fermentation. When introduced into human cells, BERA/miR-27b-3p and BERA/miR-328-3p were selectively processed to target miRNAs and thus knock down and mRNA and their protein levels, respectively, as compared to cells treated with vehicle or control RNA. Consequently, BERA/miR-27b-3p led to a lower midazolam 1'-hydroxylase activity, indicating the reduction of CYP3A4 activity. Likewise, BERA/miR-328-3p treatment elevated the intracellular accumulation of anticancer drug mitoxantrone, a classic substrate of ABCG2, hence sensitized the cells to chemotherapy. The results indicate that biologic miRNA agents made by RNA biotechnology may be applied to research on miRNA functions in the regulation of drug metabolism and disposition that could provide insights into the development of more effective therapies.

Development of precision medicine approaches based on inter-individual variability of BCRP/

Precision medicine is a rapidly-developing modality of medicine in human healthcare. Based on each patient׳s unique characteristics, more accurate dosages and drug selection can be made to achieve better therapeutic efficacy and less adverse reactions in precision medicine. A patient׳s individual parameters that affect drug transporter action can be used to develop a precision medicine guidance, due to the fact that therapeutic efficacy and adverse reactions of drugs can both be affected by expression and function of drug transporters on the cell membrane surface. The purpose of this review is to summarize unique characteristics of human breast cancer resistant protein (BCRP) and the genetic variability in the BCRP encoded gene in the development of precision medicine. Inter-individual variability of BCRP/ can impact choices and outcomes of drug treatment for several diseases, including cancer chemotherapy. Several factors have been implicated in expression and function of BCRP, including genetic, epigenetic, physiologic, pathologic, and environmental factors. Understanding the roles of these factors in controlling expression and function of BCRP is critical for the development of precision medicine based on BCRP-mediated drug transport.

Construction of Dual-luciferase reporter gene vector of 3'UTR of the COL4A3 gene and validation of its targeting relationship with miR-299 / 中华显微外科杂志

Objective To construct a dual-luciferase reporter gene vector and validate the targeting relation ship between miR-299 and the COL4A3 gene,laying a foundation for the study on the effect of miR-299 in the chondrogenic differentiation of stem cells by regulating the COL4A3 gene.Methods This study was made from March,2018 to December,2018.Firstly,the potential binding sites between miR-299 and COL4A3-3'UTR were pre dicted using bioinformatics.Then,the wild and mutant COL4A3-3'UTR sequences were amplified by PCR and cloned into psiCHECK-2 plasmid to construct corresponding recombinant vectors.The vectors were validated by enzyme digestion and gene sequencing.Finally,the cells were resuscitated,amplified,transfected and divided into 4 groups:COL4A3-WT+miR-299/NC group,COL4A3-WT+miR-299-inhibitor/NC-inhibitor group,COL4A3-MUT+miR-299/NC group and COL4A3-MUT+miR-299-inhibitor/NC-inhibitor group.Each group contains 3 holes,respectively.Luciferase activity in each group was determined using a dual-luciferase assay kit.The statistical analysis was conducted and differences between groups were compared by t test.Probabilities lower than 5％(P＜0.05) were considered statistically significant.Results Enzyme digestion and DNA sequencing showed that the dual-luciferase reporter gene vector of psiCHECK-2-COL4A3 was constructed successfully.Luciferase assay demonstrated that in wild COL4A3 gene,luciferase activity reduced in the miR-299 transfection group (The average R/F value was 59.38％) compared with the NC group (The average R/F value was 100.00％),with a statistical significant difference (P＜0.05).In wild COL4A3 gene treated with inhibitor,luciferase activity increased in the miR-299-inhibitor group (The average R/F value was 153.98％) compared with the NC-inhibitor group (The average R/F value was 100.00％),with a statistical significant difference (P＜0.05).In mutant COL4A3 gene treated with inhibitor,no obvious statistical differences in luciferase activity were found between miR-299 transfection group (The average R/F value was 102.09％),miR-299-inhibitor group (The average R/F value was 108.51％) and NC group (The average R/F value was 104.70％),NC-inhibitor group (The average R/F value was 105.13％) and/9＞0.05.Conclusion The dual-luciferase reporter gene vector of the 3'UTR of the COL4A3 gene is constructed successfully.In addition,dual-luciferase assay further verifies the authenticity of miR-299 directly targeting the 3'UTR of the COL4A3 gene.

Asthenozoospermia is not correlated with 3' UTR polymorphisms of the GRP78 gene / 中华男科学杂志

<p><b>Objective</b>To investigate the correlation of the single nucleotide polymorphisms (SNPs) rs12009, rs1140763 and rs16927997 in the 3'-untranslated region (3'UTR) of the glucose-regulated protein 78 (GRP78) gene with the risk of male asthenozoospermia (AZS).</p><p><b>METHODS</b>We included 400 AZS patients in the AZS group and another 400 fertile men as normal controls. Using the SNaPshot technique, we genotyped the rs12009, rs1140763 and rs16927997 polymorphisms in the 3'UTR of the GRP78 gene in all the male subjects and analyzed the association of the three SNPs with AZS.</p><p><b>RESULTS</b>The percentage of progressively motile sperm was significantly lower in the AZS group than in the normal controls (［20.09 ± 8.18］ % vs ［57.16 ± 13.45］ %, P <0.01). Three genotypes of CC, CT and TT and 2 alleles of C and T were found in rs12009 and rs1140763 of the GRP78 gene, and another three genotypes of GG, GA and AA and two alleles of G and A were observed in rs16927997. There were no statistically significant differences between the control and AZS groups in the frequencies of the C and T alleles in rs12009 (44.3% vs 47.3% and 55.7% vs 52.7%, P >0.05) or rs1140763 (50.0% vs 52.0% and 50.0% vs 48.0%, P >0.05) or those of the G and A alleles in rs16927997 (6.0% vs 4.4% and 94.0% vs 95.6%, P >0.05), nor in the genotypes and allele frequencies of the 3 polymorphisms (P >0.05). Furthermore, three haplotypes of C-C-A, T-C-G and T-T-A were observed in the male subjects but showed no evident correlation between the AZS and normal control groups.</p><p><b>CONCLUSIONS</b>The polymorphisms in the 3'UTR of the GRP78 gene are not correlated with the risk of male asthenozoospermia.</p>

Correlation of interaction between genetic polymorphisms with thrombin activity in plasma and pathological stages of esophageal carcinoma / 西安交通大学学报(医学版)

Objective To investigate the correlation of interaction between polymorphisms of prothrombin gene G20210A in 3' untranslated region and tissue factor pathway inhibitor (TFPI) gene C399T in 5' untranslated region with thrombin activity in plasma and the pathological stages of esophageal carcinoma.Methods Based on TNM method,we selected 198 patients with stage Ⅰ esophageal carcinoma,198 with stage Ⅱ,198 with stage Ⅲ,and 198 with stage Ⅳ from the First Affiliated Hospital of Xinxiang Medical College from May 2011 to August 2015 for this study;198 patients with esophageal carcinoma of stage 0 served as the control group.The thrombin activity in plasma were determined by chromogenic substrate assay.The genetic polymorphisms of prothrombin gene G20210A in 3' untranslated region and TFPI gene C399T in 5' untranslated region in peripheral blood leukocytes of the above-mentioned patients were analyzed by PCR-RFLP technique.Unconditional logistic regression model and single factor analysis were performed to calculate the adjusted odds ratios (OR) and 95％ confidence intervals (95％ CI) of polymorphisms prothrombin gene G20210A and TFPI gene C399T polymorphisms and to analyze the interaction of nucleotide polymorphisms with thrombin activity in plasma and the pathological stages of esophageal carcinoma.Results The frequencies of G20210A (GA),G20210A (AA),C399T (CT) and C399T (TT) were 24.24％,26.77％,24.24％ and 25.76％ in stage Ⅰ group;34.34％,37.37％,34.85％ and 36.36％ in stage Ⅱ group;39.90％,42.93％,40.41％ and 41.92％ in stage Ⅲ group;45.45％,46.97％,45.35％ and 46.46 in stage Ⅳ group;and 13.64％,14.14％,13.13％ and 13.64％ in stage 0 group,respectively.Statistical tests showed significant difference in the frequencies among each group (all P＜0.01).The risks of invasion and metastasis of esophageal carcinoma significantly increased in the subjects with G20210A,in those with G20210A(AA) genotype,in those with C399T (CT) genotype and in those with C399T (TT) genotype.Combined analysis of the polymorphisms showed that percentage of G20210A (AA)/C399T (TT) in stage Ⅰ group,stage Ⅱ group,stage Ⅲ group,stage Ⅳ group and stage 0 group was 7.07％,14.14％,18.18％,21.71％ and 1.52％,respectively,and statistical tests showed significant difference in the frequency among each group (all P＜0.01).People who carried G20210A(AA)/C399T(TT) had higher risks of invasion and metastasis of esophageal carcinoma,and statistical analysis suggested a positive interaction between G20210A (AA) and C399T (TT) in increasing the risks of invasion and metastasis of esophageal carcinoma (All γ＞ 1).Likewise,there were also positive interactions in the pathogenesis of invasion and metastasis of esophageal carcinoma between G20210A (GA) and C399T (TT),G20210A (GA) and C399T(CT),G20210A (AA) and C399T (CT) (All γ＞1).The thrombin activities in plasma in stage Ⅰ,Ⅱ,Ⅲ and Ⅳ groups were all significantly higher than those in stage 0 group,and there were significant differences among stage Ⅰ,stage Ⅱ,stage Ⅲ and stage Ⅳ in thrombin activities (all P＜0.01).Patients with mutation genotype had significantly higher thrombin activities than those with wild homozygous in the same TNM stage.Conclusion G20210A and C399T gene mutations are the risk factors in the invasion and metastasis of esophageal carcinoma.Significant interactions between G20210A and C399T mutations increase the risk of invasion and metastasis of esophageal carcinoma,which may be closely related to their increased thrombin activities in plasma.

First isolation and identification of Getah virus SC1210 in Sichuan / 中华实验和临床病毒学杂志

Objective@#To study the genome molecular characteristics of Getah virus (SC1210) which isolated in Sichuan province in 2012.@*Methods@#Reverse transcriptase polymerase chain reaction (RT-PCR) was used to identify the isolate and the genome was sequenced by the second Ion Torrent PGM. Computer softwares, including Mega Align and Mega 6, were used to analyze the nucleotide and deduced amino acid sequence, and draw phylogenetic trees.@*Results@#SC1210 was identified as Getah virus. The full genome sequence was 11 690nt, the nucleotide and amino acid homology of the full sequence with other strains were 99.2%-99.7% and 96.5%-99.4%.The capsid protein of SC1210 consisting of 804 nucleotides, encoding 268 amino acids and the full-length of E2 protein, had 1 266 nucleotides, encoding 422 amino acids. The nucleotide homology of the capsid protein and the E2 protein with other strains were 94.9%-99.2% and 94.6%-99.6%, and the amino acid were 97%-99.6% and 97.1%-99.5%. The 3′ UTR of the virus included 402 nucleotides and there were three repeat sequence elements and 19 nucleotides conservation sequence.@*Conclusions@#The first GETV isolate SC1210 in Sichuan province has a closer relationship with Yunnan strain YN040 and a far genetic relationship with MM2021.

Advances in the changes of the regulatory regions of congenital heart disease related genes / 国际儿科学杂志

Congenital heart disease is defined as a large group of structural and functional deficits that arise during cardiac embryogenesis,which poses serious threat to children's health.Genetic factors play an important role in the pathogenesis of congenital heart disease.Except for disease-related gene coding regions,recent research suggests that regulatory regions of these genes also participate in its occurrence.Regulatory elements include promoter,enhancer and attenuator,etc.This article reviews the relationship between the changes of these elements and the pathogenesis of congenital heart disease in order to better clarify the underlying mechanism and bring new ideas for clinical managements.

Mutation screening of SCN1A 3′ untranslated region on Dravet syndrome patients and functional analysis of the variant / 中华神经科杂志

Objective To conduct mutation screening of SCN1A 3′ untranslated region (UTR) on Dravet syndrome (DS) patients without mutations in the SCN1A coding region and promoter region, and functional analysis of the variant from DS patients.Methods Twenty-eight DS patients without mutations in the SCN1A coding region and promoter region were screened for SCN1A 3′ UTR mutations using PCR and direct sequencing.Functional analysis of the detected mutation was done via luciferase assay, mRNA stability analysis and RNA electrophoretic mobility shift assay (RNA-EMSA).Results A novo variant (c.*20A>G) in SCN1A 3′ UTR was found in one DS patient.The variant (c.*20A>G) reduced the luciferase gene xpression by 30% through increasing the affinity of pluripotent embryonal carcinoma cell line NT2/cytoplasmic protein binding and reducing luciferase gene mRNA stability (t=8.5,P<0.01).Conclusions A functional variant was detected from one patient with DS.This variant negatively regulated the gene expression by increasing the affinity of pluripotent embryonal carcinoma cell line NT2/cytoplasmic protein binding and reducing mRNA stability.

Construction and function identification of luciferase reporter gene vectors containing SNPs in NFKBIA gene 3'UTR / 药学学报

This study aims to investigate the function of two SNPs (rs8904C > T and rs696G > A) in 3' untranslated region (3'UTR) of NFKBIA gene by constructing luciferase reporter gene. A patient's genomic DNA with rs8904 CC and rs696 GA genotype was used as the PCR template. Full-length 3'UTR of NFKBIA gene was amplified by different primers. After sequencing validation, these fragments were inserted to the luciferase reporter vector, pGL3-promoter to construct recombinant plasmids containing four kinds of haplotypes, pGL3-rs8904C/rs696G, pGL3-rs8904C/rs696A, pGL3-rs8904T/rs696G and pGL3-rs8904T/rs696A. Then these plasmids were transfected into LS174T cells and the luciferase activity was detected. Compared with pGL3- vector transfected cells (negative control), the luciferase activity of the four kinds of recombinant plasmids was significantly decreased (P A, the luciferase activity of the recombinant plasmids containing A allele (pGL3-rs8904C/rs696A and pGL3-rs8904T/rs696A) was about 45.1% (P P T, there were no significant differences in the luciferase activity between the recombinant plasmids containing T allele and those with C allele. Together, the luciferase reporter gene vectors containing SNPs in NFKBIA gene 3'UTR were constructed successfully and rs696G > A could decrease the luciferase activity while rs8904C > T didn't have much effect on the luciferase activity.

MicroRNA functional network in pancreatic cancer: From biology to biomarkers of disease.

MicroRNAs (miRs), the 17- to 25-nucleotide-long non-coding RNAs, regulate expression of approximately 30% of the protein-coding genes at the post-transcriptional level and have emerged as critical components of the complex functional pathway networks controlling important cellular processes, such as proliferation, development, differentiation, stress response' and apoptosis. Abnormal expression levels of miRs, regulating critical cancerassociated pathways, have been implicated to play important roles in the oncogenic processes, functioning both as oncogenes and as tumour suppressor genes. Elucidation of the genetic networks regulated by the abnormally expressing miRs in cancer cells is proving to be extremely significant in understanding the role of these miRs in the induction of malignant-transformation-associated phenotypic changes. As a result, the miRs involved in the oncogenic transformation process are being investigated as novel biomarkers of disease detection and prognosis as well as potential therapeutic targets for human cancers. In this \article, we review the existing literature in the field documenting the significance of aberrantly expressed miRs in human pancreatic cancer and discuss how the oncogenic miRs may be involved in the genetic networks regulating functional pathways deregulated in this malignancy

Partial genome molecular characteristics of Getah virus newly isolated in China / 中华微生物学和免疫学杂志

Objective To study the genome molecular characteristics of Getah virus(DY0824)which isolated in Shandong province,2008 by molecular biology methods.Methods Reverse transcriptasepolymerase chain reaction(RT-PCR)was used to amplify the structural gene and 3'UTR fragments then the RT-PCR products were inserted into PGEM-T easy to be sequenced.Computer software was used to analyze the nucleotide and deduced amino acid sequence,and draw phylogenetic trees,including Clustal X1.83 and MegaAlign and Mega4.Results The capsid protein of DY0824 consists of 804 nucleotides,encoding 268 amino acids and the full-length of E2 protein is 1266 nucleotides,encoding 422 amino acids.The nucleotide homology of the capsid protein and the E2 protein with other strains were 95.4%-99.9%and 94.8%-99.5%,and the amino acid were 97.4%-100%and 97.6%-100%.The 3'UTR of the virus include 401 nucleotides and there are three repeat sequence elements.Conclusion Compared with the prototype virus,the Getah virus isolated in Shandong province had 7 amino acid differences in capsid protein genes and 10 amino acid differences in E protein genes.The 3'UTR region had multi-nucleotide changes.

Functions of The 3′ Untranslated Region of Eukaryotic mRNA / 生物化学与生物物理进展

Functions of the 3′untranslated regions (3′UTR) of eukaryotic mRNAs are complicated. They can control the stability and intracellular localization of mRNAs, and direct the translation of special amino acids. Mutations in the 3′UTR of some mRNA can cause serious diseases, and recent studies showed that the 3′UTR of some eukaryotic mRNAs possess tumor-suppression function.

PCR sequence analysis of the 3'UTR of the SON gene of human and mammalian / 中国法医学杂志

Objective To study the specific sequences in the 3'untranslated region (UTR) of the SON gene of human beings and 14 kinds of mammalian. Method The specific sequences in the 3'untranslated region (UTR) of the SON gene of human beings and 14 kinds of mammalians were amplified and directly se-quenced. The sequence data of PCR products of human beings and mammalians were declared. Results The sequences of 3'UTR of SON gene of human beings differed from 14 kinds of mammalians. The sequences of samples came from different human individuals did not show any difference. Conclusion The 3'UTR of SON gene is a good DNA region for specie identification. Using this technology, DNA of human beings can be distinguished from 14 kinds of mammalians. This technology is very useful in of forensic science.