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Background: Inherited long QT syndrome type 2 results from variants in the KCNH2 gene encoding the human Ether-à-go-go related gene 1 (hERG1) potassium channel. Two main isoforms, hERG1a and hERG1b, assemble to form tetrameric channel. The N-terminal PAS (Per/Arnt/Sim) domain, present only on hERG1a subunits, is a hotspot for pathogenic variants, but it is unknown whether PAS domain variants impact hERG1b expression to contribute to the long QT syndrome type 2 phenotype. We aimed to use patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to investigate the pathogenesis of the hERG1a PAS domain variant hERG1-H70R. Methods: Human iPSCs were derived from a patient with long QT syndrome type 2 carrying the PAS domain variant hERG1-H70R. CRISPR/Cas9 gene editing produced isogenic control iPSC lines. Differentiated iPSC-CMs were evaluated for their electrophysiology, hERG1a/1b mRNA expression, and hERG1a/1b protein expression. Results: Action potentials from single hERG1-H70R iPSC-CMs were prolonged relative to controls, and voltage clamp studies showed an underlying decrease in I Kr with accelerated deactivation. In hERG1-H70R iPSC-CMs, transcription of hERG1a and hERG1b mRNA was unchanged compared with controls based on nascent nuclear transcript analysis, but hERG1b mRNA was significantly increased as was the ratio of hERG1b / hERG1a in mRNA complexes, suggesting posttranscriptional changes. Expression of complex glycosylated hERG1a in hERG1-H70R iPSC-CMs was reduced due to impaired protein trafficking, whereas the expression of the complex glycosylated form of hERG1b was unchanged. Conclusions: Patient-specific hERG1-H70R iPSC-CMs reveal a newly appreciated mechanism of pathogenesis of the long QT syndrome type 2 phenotype due to both impaired trafficking of hERG1a and maintained expression of hERG1b that produces subunit imbalance and reduced I Kr with accelerated deactivation. Graphic Abstract: A graphic abstract is available for this article.

Aim: We aim to identify the key long noncoding RNAs (lncRNAs) in early-stage colon adenocarcinoma (COAD). Patients & methods: Compared with colonic intraepithelial neoplasia, differentially expressed lncRNAs (DElncRNAs) in early-stage COAD were obtained by RNA-sequencing. Our previous work has obtained the differentially expressed mRNAs and miRNAs (DEmRNAs and DEmiRNAs) in early-stage COAD. DEmiRNA–DElncRNA–DEmRNA interaction analysis and functional annotation were performed. Validation of expression and receiver-operating characteristic analyses were performed based on The Cancer Genome Atlas. Results: Seventy-nine significantly DElncRNAs in early-stage COAD were obtained. MiR-153-3p-TUG1-DAPK1/ARNT2/KLK3/PLD1/SMAD2 and miR-153-3p-SNHG17-COL11A1/IGFBP3/KLF6 interactions were associated with early-stage COAD. Five DElncRNAs (ELFN1-AS1, LINC01234, SNHG17, UCA1 and LOC101929549) involved in early-stage COAD with potential diagnostic value. Conclusion: LncRNAs involve in early-stage COAD by interaction with COAD-regulated genes and miRNAs.

Catastrophic arrhythmias and sudden cardiac death can occur with even a small imbalance between inward sodium currents and outward potassium currents, but mechanisms establishing this critical balance are not understood. Here, we show that mRNA transcripts encoding INa and IKr channels (SCN5A and hERG, respectively) are associated in defined complexes during protein translation. Using biochemical, electrophysiological and single-molecule fluorescence localization approaches, we find that roughly half the hERG translational complexes contain SCN5A transcripts. Moreover, the transcripts are regulated in a way that alters functional expression of both channels at the membrane. Association and coordinate regulation of transcripts in discrete ‘microtranslatomes’ represents a new paradigm controlling electrical activity in heart and other excitable tissues.

Stomach adenocarcinoma (STAD) is the second leading cause of cancer death and a fuller understanding of its molecular basis is needed to develop new therapeutic targets. miRNA and mRNA data were downloaded from The Cancer Genome Atlas database, and the differentially expressed miRNAs and genes were identified. The target genes of differentially expressed miRNAs were screened by prediction tools. Furthermore, the biological function of these target genes was investigated. Several key miRNAs and their target genes were selected for validation using quantitative real‐time polymerase chain reaction (qRT‐PCR). The Gene Expression Omnibus (GEO) dataset was used to verify the expression of selected miRNAs and target genes. The diagnostic value of identified miRNAs and genes was accessed by receiver operating characteristic analysis. A total of 1248 differentially expressed genes were identified in STAD. Additionally, nine differentially expressed miRNAs were identified and 160 target genes of these nine miRNAs were identified via target gene detection. Interestingly, they were remarkably enriched in the calcium signaling pathway and bile secretion. qRT‐PCR confirmed the expression of several key miRNAs and their target genes. The expression levels of hsa‐miR‐145‐3p, hsa‐miR‐145‐5p, ADAM12,ACAN,HOXC11 and MMP11 in the GEO database were compatible with the bioinformatics results. hsa‐miR‐139‐5p, hsa‐miR‐145‐3p and MMP11 have a potential diagnostic value for STAD. Differential expression of the mature form of miRNAs (hsa‐miR‐139‐5p, hsa‐miR‐145‐3p, hsa‐miR‐145‐5p and hsa‐miR‐490‐3p) and genes including ADAM12,ACAN,HOXC11 and MMP11 and calcium and bile secretion signaling pathways may play important roles in the development of STAD.

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal cancer in humans, with a 5‐year survival rate of

Background The behavioural transition from nurses to foragers in honey bees is known to be affected by intrinsic and extrinsic factors, including colony demography, hormone levels, brain chemistry and structure, and gene expression in the brain. However, the molecular mechanism underlying this behavioural transition of honey bees is still obscure. Results Through RNA sequencing, we performed a comprehensive analysis of lncRNAs and mRNAs in honey bee nurses and foragers. Nurses and foragers from both typical colonies and single-cohort colonies were used to prepare six libraries to generate 49 to 100 million clear reads per sample. We obtained 6863 novel lncRNAs, 1480 differentially expressed lncRNAs between nurses and foragers, and 9308 mRNAs. Consistent with previous studies, lncRNAs showed features distinct from mRNAs, such as shorter lengths, lower exon numbers, and lower expression levels compared to mRNAs. Bioinformatic analysis showed that differentially expressed genes were mostly involved in the regulation of sensory-related events, such as olfactory receptor activity and odorant binding, and enriched Wnt and FoxO signaling pathways. Moreover, we found that lncRNAs TCONS_00356023, TCONS_00357367, TCONS_00159909 and mRNAs dop1, Kr-h1 and HR38 may play important roles in behavioural transition in honey bees. Conclusion This study characterized the expression profile of lncRNAs in nurses and foragers and provided a framework for further study of the role of lncRNAs in honey bee behavioural transition. Electronic supplementary material The online version of this article (10.1186/s12864-019-5664-7) contains supplementary material, which is available to authorized users.

Recently, the crustacean female sex hormone (CFSH), which is considered a female-specific hormone, has been shown to play a crucial role in female phenotypes in crustaceans. In this study, two transcripts (Sp-CFSH1 and Sp-CFSH2) encoding the same CFSH precursor were cloned from the mud crab Scylla paramamosain. Homology and phylogenetic analysis showed that CFSHs were homologous to interleukin-17 and highly conserved among brachyuran crabs. PCR analysis revealed that Sp-CFSH was expressed exclusively in the eyestalk ganglion of both prepubertal males and females, and surprisingly, the abundance of Sp-CFSH transcripts detected in the males were not significantly different from that of the females (P > 0.05). In addition, mRNA in situ hybridization showed that Sp-CFSH was localized in the X-organ of the male eyestalk ganglion. During the development of the androgenic gland (AG), the level of Sp-IAG mRNA in AG remained at low levels from stages I to II (early stage) but had a significant increase at stage III (mature stage). In contrast, the level of Sp-CFSH transcripts in the eyestalk ganglion was high in the early stage but extremely low in the mature stage. To investigate the potential function of CFSH in male S. paramamosain, the recombinant protein (∼20 kDa) was expressed in Escherichia coli and was subsequently added to AG explants in vitro. It was demonstrated that recombinant Sp-CFSH protein significantly reduced the expression of Sp-IAG in the AG explants at a concentration of 10−6 M (P < 0.05). In conclusion, our study provides the first piece of evidence that shows CFSH from the eyestalk ganglion acts as a negative regulator inhibiting the development of AG in crustaceans.

Rapidly accumulating evidence has now suggested that the long non-coding RNAs (LncRNAs), a large and diverse class of non-coding transcribed RNA molecules with diverse functional roles and mechanisms, play a major role in the pathogenesis of many human inflammatory diseases. Although some LncRNAs are overexpressed in plasma, T cell, and synovial tissues of patients with rheumatoid arthritis (RA), there is a dearth of knowledge in what role these transcripts play in fibroblast-like synoviocytes (FLSs) of these patients. Here, our studies showed that GAPLINC, a newly identified functional LncRNA in oncology, displayed a greater degree of expression in FLSs from RA than in patients with traumatic injury. GAPLINC suppression in RA-FLS cells revealed significant alterations in cell proliferation, invasion, migration, and proinflammatory cytokines production. Additionally, we performed a preliminary bioinformatics analysis of GAPLINC gene sequence in order to find its target molecules, using miRanda, PITA, RNAhybrid algorithms, Kyoto encyclopedia of genes and genomes, and gene ontology analysis. Since the results predicted that some of microRNAs and mRNA may interact with GAPLINC, we simulated a gene co-action network model based on a competitive endogenous RNA theory. Further verification of this model demonstrated that silencing of GAPLINC increased miR-382-5p and miR-575 expression. The results of this study suggest that GAPLINC may function as a novel microRNAs sponging agent affecting the biological characteristics of RA-FLSs. Additionally, GAPLINC may also promote RA-FLS tumor-like behaviors in a miR-382-5p-dependent and miR-575-dependent manner. Based upon these findings, LncRNA GAPLINC may provide a novel valuable therapeutic target for RA patients.

Food-borne pathogens have been recognized as a major cause of human infections worldwide. Their identification needs to be simpler, cheaper and more reliable than the traditional methods. Here, we constructed a low-cost paper platform for viable pathogenic bacteria detection with the naked eye. In this study, an effective isothermal amplification method was used to amplify the hlyA mRNA gene, a specific RNA marker in Listeria monocytogenes. The amplification products were applied to the paper-based platform to perform a visual test using sandwich hybridization assays. When the RNA products migrated along the platform by capillary action, the gold nanoparticles accumulated at the designated area. Under optimized experimental conditions, as little as 0.5 pg/μL genomic RNA from L. monocytogenes could be detected. It could also be used to specifically detect 20 CFU/mL L. monocytogenes from actual samples. The whole assay process, including RNA extraction, amplification, and visualization, can be completed within several hours. This method is suitable for point-of-care applications to detect food-borne pathogens, as it can overcome the false-positive results caused by amplifying nonviable L. monocytogenes. Furthermore, the results can be imaged and transformed into a two-dimensional bar code through an Android-based smart phone for further analysis or in-field food safety tracking.

Background Hepatitis B virus (HBV) infection in the body can damage liver cells and cause disorders in blood lipid metabolism. Apolipoprotein C3 (ApoC3) plays an important role in the regulation of lipid metabolism, but no study on the HBV regulation of ApoC3 has been reported. This purpose of this study was to investigate the effect of HBV on ApoC3 expression and its regulatory mechanism. Methods The expression levels of ApoC3 mRNA and protein in the human hepatoma cell lines HepG2 and HepG2.2.15 were determined using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot. The HepG2 cells were co-transfected with the ApoC3 gene promoter and either HBV-infected clone pHBV1.3 or its individual genes. The changes in luciferase activity were assayed. The expression levels of ApoC3 mRNA and protein were determined using RT-qPCR and Western blot. The content of ApoC3 in the supernatant of the cultured cells was determined using an enzyme-linked immunosorbent assay (ELISA). The sera were collected from 149 patients with HBV infection and 102 healthy subjects at physical examination as the normal controls. The serological levels of ApoC3 in the HBV group and the normal control group were determined using ELISA. The contents of serum triglyceride (TG) and very-low-density lipoprotein (VLDL) in the HBV patients and the normal control were determined using an automatic biochemical analyser. Results The expression levels of ApoC3 mRNA and protein were lower in the HepG2.2.15 cells than in the HepG2 cells. pHBV1.3 and its X gene could inhibit the activity of the ApoC3 promoter and its mRNA and protein expression. The serum levels of ApoC3, VLDL and TG were 65.39 ± 7.48 μg/ml, 1.24 ± 0.49 mmol/L, and 0.46 ± 0.10 mmol/L in the HBV patients and 41.02 ± 6.88 μg/ml, 0.76 ± 0.21 mmol/L, 0.29 ± 0.05 mmol/L in the normal controls, respectively, statistical analysis revealed significantly lower serum levels of ApoC3, VLDL and TG in HBV patients than in the normal controls (P

The present study aims to investigate the effects of miR-146a on the proliferation and apoptosis of human osteoarthritis (OA) chondrocytes by targeting tumour necrosis factor receptor-associated factor 6 (TRAF6) through nuclear factor-κB (NF-κB) signalling pathway. Human normal and OA chondrocytes were selected and divided into the normal group, blank group, negative control (NC) group, miR-146a mimics group, miR-146a inhibitors, miR-146a inhibitor + si-TRAF6 group and si-TRAF6 group. Quantitative real-time PCR (qRT-PCR) was applied to detect the expressions of miR-146a, TRAF6 mRNA and NF-κB mRNA. Western blotting was used to detect the protein expressions of TRAF6 and NF-κB. CCK-8 assay and flow cytometry were used to detect cell proliferation and apoptosis. Compared with normal chondrocytes, the expression of miR-146a decreased, while the mRNA and protein expressions of TRAF6 and NF-κB increased in OA chondrocytes. OA chondrocytes had a lower proliferation rate and a higher apoptosis rate than the normal chondrocytes. Compared with the blank, NC and si-TRAF6 groups, the expression of miR-146a increased in the miR-146a mimics group, but decreased in the miR-146a inhibitors and miR-146a inhibitor + si-TRAF6 groups. Compared with the blank, NC and miR-146a inhibitor + si-TRAF6 groups, the mRNA and protein expressions of TRAF6 and NF-κB decreased, cell proliferation rate increased and cell apoptosis rate decreased in the miR-146a mimics and si-TRAF6 groups, while opposite trends were observed in the miR-146a inhibitors group. Our study suggests that miR-146a could promote proliferation and inhibit apoptosis of OA chondrocytes by inhibiting TRAF6 expression and suppressing the activation of NF-κB signalling pathway.

Nlrp2 is a maternal effect gene specifically expressed by mouse ovaries; deletion of this gene from zygotes is known to result in early embryonic arrest. In the present study, we identified FAF1 protein as a specific binding partner of the NLRP2 protein in both mouse oocytes and preimplantation embryos. In addition to early embryos, both Faf1 mRNA and protein were detected in multiple tissues. NLRP2 and FAF1 proteins were co-localized to both the cytoplasm and nucleus during the development of oocytes and preimplantation embryos. Co-immunoprecipitation assays were used to confirm the specific interaction between NLRP2 and FAF1 proteins. Knockdown of the Nlrp2 or Faf1 gene in zygotes interfered with the formation of a NLRP2–FAF1 complex and led to developmental arrest during early embryogenesis. We therefore conclude that NLRP2 interacts with FAF1 under normal physiological conditions and that this interaction is probably essential for the successful development of cleavage-stage mouse embryos. Our data therefore indicated a potential role for NLRP2 in regulating early embryo development in the mouse.

The incidence of melanoma, the most aggressive and life-threatening form of skin cancer, has significantly risen over recent decades. Therefore, it is essential to identify the mechanisms that underlie melanoma tumorigenesis and metastasis and to explore novel and effective melanoma treatment strategies. Accumulating evidence s uggests that aberrantly expressed long noncoding RNAs (lncRNAs) have vital functions in multiple cancers. However, lncRNA functions in melanoma tumorigenesis and metastasis remain unclear. In this study, we investigated lncRNA and messenger RNA (mRNA) expression profiles in primary melanomas, metastatic melanomas and normal skin samples from the Gene Expression Omnibus database. We used GSE15605 as the training set (n = 74) and GSE7553 as the validation set (n = 58). In three comparisons (primary melanoma versus normal skin, metastatic melanoma versus normal skin, and metastatic melanoma versus primary melanoma), 178, 295 and 48 lncRNAs and 847, 1758, and 295 mRNAs were aberrantly expressed, respectively. We performed Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses to examine the differentially expressed mRNAs, and potential core lncRNAs were predicted by lncRNA-mRNA co-expression networks. Based on our results, 15 lncRNAs and 144 mRNAs were significantly associated with melanoma tumorigenesis and metastasis. A subsequent analysis suggested a critical role for a five-lncRNA signature during melanoma tumorigenesis and metastasis. Low expression of U47924.27 was significantly associated with decreased survival of patients with melanoma. To the best of our knowledge, this study is the first to explore the expression patterns of lncRNAs and mRNAs during melanoma tumorigenesis and metastasis by re-annotating microarray data from the Gene Expression Omnibus (GEO) microarray dataset. These findings reveal potential roles for lncRNAs during melanoma tumorigenesis and metastasis and provide a rich candidate reservoir for future studies.

Purpose: To investigate the effect of epigallocatechin-3-gallate (EGCG) on bone metabolism and osteoblastic activity. Methods: MG-63 human osteoblast-like cells were treated with varied concentrations of EGCG. Alkaline phosphatase (ALP) activity and matrix mineralization assays were carried out on the treated and untreated MG-63 human osteoblast-like cells. Beta-catenin mRNA level was determined by quantitative real-time polymerase chain reaction (q-PCR). Results: The results showed that EGCG treatment significantly increased ALP and mineralization activities at concentrations of 15 and 30 μM, in a dose-dependent manner. Furthermore, EGCG treatment significantly increased beta-catenin mRNA expression by 70.7 ± 11.0 and 126.7 ± 35.1 %, respectively, at EGCG concentration of 15 and 30 μM. In addition, the stimulating effect of EGCG treatment on ALP activity was abolished by co-treatment with ICI 182,780, an antagonist of estrogen receptor (ER). Again, the increase in beta-catenin MRNA, when treated with EGCG, was inhibited by co-treatment with ICI 182,780. Conclusion: EGCG promotes osteoblastic activity in human osteoblast-like cells, by Wnt signaling through estrogen receptor (ER) pathway. Keywords: Epigallocatechin-3-gallate, Osteoporosis, Osteoclast, Proliferation, Estrogen receptor

Due to the small volume and high density of breast tissue in Asian women, particularly younger women, mammographic diagnosis is sometimes non-conclusive, with a Breast Imaging Reporting and Data System (BI-RADS) result of 0. No alternative based on blood biomarkers has yet succeeded in discriminating between patients with breast cancer (BC) and those with benign breast disease (BBD) among BI-RADS 0 patients. In our study, 84 BC and 94 BBD patients with mammographic results and confirmed pathologic information were enrolled and categorized into two groups, namely, 79 BC and 73 BBD patients with BI-RADS 1-5 and 5 BC and 21 BBD patients with BI-RADS 0. RNA extracted from peripheral blood samples collected in PAXgene (TM) tubes was analyzed after NuGEN WT-Ovation (TM) RNA amplification using Affymetrix GeneChip.

Messenger RNA (mRNA) turnover in eukaryotic cells begins with shortening of the poly (A) tail at the 3' end, a process called deadenylation. In yeast, the deadenylation reaction is predominantly mediated by CCR4 and CCR4-associated factor 1 (CAF1), two components of the well-characterised protein complex named CCR4-NOT. We report here that AtCAF1a and AtCAF1b, putative Arabidopsis homologs of the yeast CAF1 gene, partially complement the growth defect of the yeast caf1 mutant in the presence of caffeine or at high temperatures. The expression of AtCAF1a and AtCAF1b is induced by multiple stress-related hormones and stimuli. Both AtCAF1a and AtCAF1b show deadenylation activity in vitro and point mutations in the predicted active sites disrupt this activity. T-DNA insertion mutants disrupting the expression of AtCAF1a and/or AtCAF1b are defective in deadenylation of stress-related mRNAs, indicating that the two AtCAF1 proteins are involved in regulated mRNA deadenylation in vivo. Interestingly, the single and double mutants of AtCAF1a and AtCAF1b show reduced expression of pathogenesis-related (PR) genes PR1 and PR2 and are more susceptible to Pseudomonas syringae pv tomato DC3000 (Pst DC3000) infection, whereas transgenic plants over-expressing AtCAF1a show elevated expression of PR1 and PR2 and increased resistance to the same pathogen. Our results suggest roles of the AtCAF1 proteins in regulated mRNA deadenylation and defence responses to pathogen infections.

Spermatogenesis in mammals is characterized by two waves of piRNA expression: one corresponds to classic piRNAs responsible for silencing retrotransponsons and the second wave is predominantly derived from nontransposon intergenic regions in pachytene spermatocytes, but the function of these pachytene piRNAs is largely unknown. Here, we report the involvement of pachytene piRNAs in instructing massive mRNA elimination in mouse elongating spermatids (ES). We demonstrate that a piRNA-induced silencing complex (pi-RISC) containing murine PIWI (MIWI) and deadenylase CAF1 is selectively assembled in ES, which is responsible for inducing mRNA deadenylation and decay via a mechanism that resembles the action of miRNAs in somatic cells. Such a highly orchestrated program appears to take full advantage of the enormous repertoire of diversified targeting capacity of pachytene piRNAs derived from nontransposon intergenic regions. These findings suggest that pachytene piRNAs are responsible for inactivating vast cellular programs in preparation for sperm production from ES.

Tannins, a group of major active components of Chinese rhubarb and widely distributed in nature, have a significant antidiarrhoeal activity. Aquaporins (AQPs) 2 and 3 play important roles in regulating water transfer during diarrhoea. The present study aims to determine the effect of the total tannins extract of rhubarb on aquaporins (AQPs) 2 and 3 in diarrhoea mice and HT-29 cells both induced by magnesium sulphate (MgSO4). Our results showed that rhubarb tannins extract (RTE) significantly decreased the faecal water content in colon and evaluation index of defecation of diarrhoea mice. Interestingly, RTE could markedly reduce the mRNA and protein expression levels of AQPs 2 and 3 in apical and lateral mucosal epithelial cells in the colons of diarrhoea mice and HT-29 cells both induced by MgSO4in a dose-dependent manner. Furthermore, RTE suppressed the production of cyclic monophosphate- (cAMP-) dependent protein kinase A catalytic subunitsα(PKA C-α) and phosphorylated cAMP response element-binding protein (p-CREB, Ser133) in MgSO4-induced HT-29 cells. Our data showed for the first time that RTE inhibit AQPs 2 and 3 expressionin vivoandin vitrovia downregulating PKA/p-CREB signal pathway, which accounts for the antidiarrhoeal effect of RTE.

We analyzed the expression of plasma glutathione peroxidase (GSHPx-P) messenger RNA (mRNA) in mouse, rat, and human tissues, using a human GSHPx-P cDNA clone as the probe. Unlike the classical cellular glutathione peroxidase (GSHPx-1), GSHPx-P expression appears to be tissue-specific. In the mouse and rat, kidney expresses an mRNA at a high level detected with the human probe. A signal is also detected in mRNA isolated from mouse and rat heart, rat cardiac myocytes, mouse lung, epididymis, and the mammary gland of midpregnant mice. No signal is detected in mRNA isolated from mouse and rat liver, mouse brain, uterus, and testis. In human tissues, an mRNA hybridizing to GSHPx-P cDNA is present in liver, as well as kidney, heart, lung, breast, and placenta. We have shown that human kidney expresses a GSHPx-P mRNA, and not a GSHPx-P-like message, by isolating a cDNA clone from a human kidney library in lambda gt11. From the 412-nucleotide partial sequence of the kidney cDNA, which codes for the 40–170 amino acids of GSHPx-P including the TGA codon for selenocysteine, we found complete sequence identity of the kidney cDNA with GSHPx-P isolated from placenta. The expression of GSHPx-P mRNA in cell lines was also studied. There is some correlation of the expression of GSHPx-P in these cell lines with that in normal tissues. Cell lines that expressed GSHPx-P mRNA or protein included the human hepatocarcinoma HepG2, Hep3B cells, human kidney carcinoma A498 cells, and the human breast cancer SK-BR-3, T47D, MDA-MB-231, and AdrrMCF-7 cells. Cell lines that did not express GSHPx- P included human choriocarcinoma BeWo cells, human breast cancer MCF-7, ZR-75–1, and Hs578T cells, and mouse hepatoma Hepa-1 cells.

No study has been performed on identifying microRNAs (miRNAs) and their targets in cotton although cotton is one of the most important fiber and economic crops around the world. In this study, we found 30 potential cotton miRNAs using a comparative genomic approach based on genomic survey sequence analysis and miRNA secondary structure. These cotton miRNAs belong to 22 miRNA families. Expressed sequence tag (EST) analysis indicated that the predicted miRNAs were expressed in cotton plants. Based on the characteristic that miRNAs exhibit perfect or nearly perfect complementarity with their targeted mRNA sequences, a total of 139 potential miRNA targets were identified in cotton genome. A majority of these targets belong to transcriptional factors which regulate cotton growth and development, including leaf, root, stem, flower, and even fiber development. Those miRNAs may also be involved in other cellular and metabolic processes, such as stress response, signal transduction, and secondary wall synthesis and deposition. Some of the newly identified miRNA targets may be unique to cotton species. In this study, we found that at least 3 miRNA families (miR 396, 414, and 782) target callous synthase, fiber protein Fb23, and fiber quinone-oxidoreductase, suggesting that miRNAs play an important role in cotton fiber differentiation and development.

Previous studies have shown that up-regulation of transforming growth factor β1 results in neuroprotective effects. However, the role of the transforming growth factor β1 downstream molecule, SMAD2/3, following ischemia/reperfusion remains unclear. Here, we investigated the neuroprotective effects of SMAD2/3 by analyzing the relationships between SMAD2/3 expression and cell apoptosis and inflammation in the brain of a rat model of cerebral ischemia/reperfusion. Levels of SMAD2/3 mRNA were up-regulated in the ischemic penumbra 6 hours after cerebral ischemia/reperfusion, reached a peak after 72 hours and were then decreased at 7 days. Phosphorylated SMAD2/3 protein levels at the aforementioned time points were consistent with the mRNA levels. Over-expression of SMAD3 in the brains of the ischemia/reperfusion model rats via delivery of an adeno-associated virus containing the SMAD3 gene could reduce tumor necrosis factor-α and interleukin-1β mRNA levels, down-regulate expression of the pro-apoptotic gene, capase-3, and up-regulate expression of the anti-apoptotic protein, Bcl-2. The SMAD3 protein level was negatively correlated with cell apoptosis. These findings indicate that SMAD3 exhibits neuroprotective effects on the brain after ischemia/reperfusion through anti-inflammatory and anti-apoptotic pathways.

We examined the in vivo effect of lipopolysaccharide (LPS) on Sp1 (promoter-selective transcription factor 1) DNA binding activity and studied the mechanisms involved in mouse lungs. The Sp1 DNA complex displayed a major band composed of Sp1, Sp2, and Sp3 trimer and a minor band composed of Sp3 homodimer. Compared with control, nuclear proteins from lungs challenged with LPS for 60, 90, 120, 150, 180, and 240 min, respectively, showed a markedly reduced Sp1 binding activity. Down-regulation of Sp1 binding activity was accompanied by a reduced expression of two Sp1-dependent genes (endothelial nitric oxide synthase and cyclooxygenase-1). Immunoprecipitation-Western blot experiments demonstrated that LPS dephosphorylated Sp1 protein at serine and threonine residues but not at the tyrosine residue. Dephosphorylation of Sp1 protein in vitro significantly reduced Sp1 DNA binding activity. Deglycosylation of Sp1 protein also reduced Sp1 binding activity. However, LPS did not cause Sp1 deglycosylation. LPS markedly reduced nuclear Sp1 protein level but had no significant effect on Sp1 mRNA abundance and on Sp1 protein nuclear translocation. Both Sp1 protein dephosphorylation and Sp1 protein degradation are temporally correlated to the reduced Sp1 binding activity. Our results demonstrate that challenge of mice with LPS in vivo down-regulates Sp1 DNA binding activity through promoting Sp1 protein dephosphorylation and degradation.

A long and ever-expanding roster of small (∼20-30 nucleotides) RNAs has emerged during the last decade, and most can be subsumed under the three main headings of microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs), and short interfering RNAs (siRNAs). Among the three categories, miRNAs is the most quickly expanded group. The most recent number of identified miRNAs is 16,772 (Sanger miRbase, April 2011). However, there are insufficient publications on their primary forms, and no tissue-specific small RNAs precursors have been reported in the epididymis. Here, we report the identification in rats of an epididymis-specific, chimeric, noncoding RNA that is spliced from two different chromosomes (chromosomes 5 and 19), which we named HongrES2. HongrES2 is a 1.6 kb mRNA-like precursor that gives rise to a new microRNA-like small RNA (mil-HongrES2) in rat epididymis. The generation of mil-HongrES2 is stimulated during epididymitis. An epididymis-specific carboxylesterase named CES7 had 100% cDNA sequence homology at the 3'end with HongrES2 and its protein product could be downregulated by HongrES2 via mil-HongrES2. This was confirmed in vivo by initiating mil-HongrES2 over-expression in rats and observing an effect on sperm capacitation.