HOX transcript antisense intergenic RNA represses E-cadherin expression by binding to EZH2 in gastric cancer

AIM To clarify the mechanisms of HOX transcript antisense intergenic RNA(HOTAIR) in gastric cancer(GC) migration and invasion.METHODS Quantitative real-time polymerase chain reaction(qp CR) was used to detect the expression level of HOTAIR in GC tissues. The correlation of its expression with clinicopathological features was analyzed. Area under receiver operating characteristic curve(AUCROC) was constructed to evaluate the diagnostic value of HOTAIR. Wound-healing assay and Transwell assay were performed to detect the biological effects of HOTAIR in GC cells. qp CR,western blot and immunohistochemistry were used to evaluate the m RNA and protein expression of E-cadherin. RNAbinding protein immunoprecipitation was used for the analysis of EZH2 interactions with HOTAIR. Chromatin immunoprecipitation assay was performed to investigate direct interactions between EZH2 and E-cadherin.RESULTS The expression of HOTAIR was up-regulated in GC tumorous tissues compared with the para-tumorous tissues(p < 0.001). Its over-expression was correlated with tumor-node-metastasis(TNM) stage(p = 0.024),tumor invasion(p = 0.018),lymph node metastasis(p = 0.023),and poor prognosis(p < 0.001). Multivariate Cox regression analysis confirmed expression of HOTAIR as an independent predictor of overall survival(p = 0.033),together with TNM stage(p = 0.002) and lymph node metastasis(p = 0.002). The AUCROC was up to 0.709(95%CI: 0.623-0.785,p < 0.001). Knockdown of HOTAIR by si RNA in GC cells suppressed the migration and invasion of GC cells. Significantly negative correlation between HOTAIR and E-cadherin was found in GC tissues and cell lines,and HOTAIR contributed to the regulation of E-cadherin through binding to EZH2 with the E-cadherin promoter. CONCLUSION HOTAIR may play a pivotal role in tumor cell migration and invasion. It can be used as a potential diagnostic and prognostic biomarker for GC.

Long noncoding RNA X-inactive specific transcript regulates NLR family pyrin domain containing 3/caspase-1-mediated pyroptosis in diabetic nephropathy

BACKGROUND NLRP3-mediated pyroptosis is recognized as an essential modulator of renal disease pathology.Long noncoding RNAs(lncRNAs)are active participators of diabetic nephropathy(DN).X inactive specific transcript(XIST)expression has been reported to be elevated in the serum of DN patients.AIM To evaluate the mechanism of lncRNA XIST in renal tubular epithelial cell(RTEC)pyroptosis in DN.METHODS A DN rat model was established through streptozotocin injection,and XIST was knocked down by tail vein injection of the lentivirus LV sh-XIST.Renal metabolic and biochemical indices were detected,and pathological changes in the renal tissue were assessed.The expression of indicators related to inflammation and pyroptosis was also detected.High glucose(HG)was used to treat HK2 cells,and cell viability and lactate dehydrogenase(LDH)activity were detected after silencing XIST.The subcellular localization and downstream mechanism of XIST were investigated.Finally,a rescue experiment was carried out to verify that XIST regulates NLR family pyrin domain containing 3(NLRP3)/caspase-1-mediated RTEC pyroptosis through the microRNA-15-5p(miR-15b-5p)/Toll-like receptor 4(TLR4)axis.RESULTS XIST was highly expressed in the DN models.XIST silencing improved renal metabolism and biochemical indices and mitigated renal injury.The expression of inflammation and pyroptosis indicators was significantly increased in DN rats and HG-treated HK2 cells;cell viability was decreased and LDH activity was increased after HGtreatment. Silencing XIST inhibited RTEC pyroptosis by inhibiting NLRP3/caspase-1. Mechanistically,XIST sponged miR-15b-5p to regulate TLR4. Silencing XIST inhibited TLR4 by promotingmiR-15b-5p. miR-15b-5p inhibition or TLR4 overexpression averted the inhibitory effect ofsilencing XIST on HG-induced RTEC pyroptosis.CONCLUSIONSilencing XIST inhibits TLR4 by upregulating miR-15b-5p and ultimately inhibits renal injury inDN by inhibiting NLRP3/caspase-1-mediated RTEC pyroptosis.

Characteristics of mRNA dynamic expression related to spinal cord ischemia/reperfusion injury:a transcriptomics study

Following spinal cord ischemia/reperfusion injury,an endogenous damage system is immediately activated and participates in a cascade reaction.It is difficult to interpret dynamic changes in these pathways,but the examination of the transcriptome may provide some information.The transcriptome reflects highly dynamic genomic and genetic information and can be seen as a precursor for the proteome.We used DNA microarrays to measure the expression levels of dynamic evolution-related m RNA after spinal cord ischemia/reperfusion injury in rats.The abdominal aorta was blocked with a vascular clamp for 90 minutes and underwent reperfusion for 24 and 48 hours.The simple ischemia group and sham group served as controls.After rats had regained consciousness,hindlimbs showed varying degrees of functional impairment,and gradually improved with prolonged reperfusion in spinal cord ischemia/reperfusion injury groups.Hematoxylin-eosin staining demonstrated that neuronal injury and tissue edema were most severe in the 24-hour reperfusion group,and mitigated in the 48-hour reperfusion group.There were 8,242 differentially expressed m RNAs obtained by Multi-Class Dif in the simple ischemia group,24-hour and 48-hour reperfusion groups.Sixteen m RNA dynamic expression patterns were obtained by Serial Test Cluster.Of them,five patterns were significant.In the No.28 pattern,all differential genes were detected in the 24-hour reperfusion group,and their expressions showed a trend in up-regulation.No.11 pattern showed a decreasing trend in m RNA whereas No.40 pattern showed an increasing trend in m RNA from ischemia to 48 hours of reperfusion,and peaked at 48 hours.In the No.25 and No.27 patterns,differential expression appeared only in the 24-hour and 48-hour reperfusion groups.Among the five m RNA dynamic expression patterns,No.11 and No.40 patterns could distinguish normal spinal cord from pathological tissue.No.25 and No.27 patterns could distinguish simple ischemia from ischemia/reperfusion.No.28 pattern could analyze the need for inducing reperfusion injury.The study of specific pathways and functions for different dynamic patterns can provide a theoretical basis for clinical differential diagnosis and treatment of spinal cord ischemia/reperfusion injury.

RNA-Seq transcriptome analysis of porcine cloned and in vitro fertilized blastocysts

Somatic nuclear transfer technology has become increasingly promising in biomedicine and agriculture.Whereas the approach remains inefficient and underlying mechanisms remain ambiguous.Although cloned embryos have similar in vitro developmental capacity as in vitro fertilized（IVF） embryos before implantation,they appeared to have much lower full-term developmental efficiency in pig and cattle,and thus it would be reasonable to postulate that profound distinction at the molecular level should exist between them.Herein,RNA sequencing technique was used to screen differentially expressed genes in cloned and IVF blastocysts,and in total 628 differentially expressed transcripts were obtained,among which,280 transcripts are up-regulated and 348 transcripts are down-regulated in cloned blastocysts.Moreover,one statistically significant pathway associated with endoplasmic reticulum（ER） protein processing was enriched,and some ER-stress markers such as ATF4,ATF6,PDIA3,HSPA1 B,HSP40 and HSP90 between cloned and IVF blastocysts were suggested.Additionally,some developmentally important genes such as lipid metabolism related genes（MGLL,DDHD2 and FADS2） and epigenetic modification genes（DNMT1,KDM5 C and MBD3L5） were found differentially expressed between cloned and IVF embryos.

An efficient and rapid method to detect and verify natural antisense transcripts of animal genes

High-throughput sequencing has identified a large number of sense-antisense transcriptional pairs, which indicates that these genes were transcribed from both directions. Recent reports have demonstrated that many antisense RNAs, especially lnc RNA（long non-coding RNA）, can interact with the sense RNA by forming an RNA duplex. Many methods, such as RNA-sequencing, Northern blotting, RNase protection assays and strand-specific PCR, can be used to detect the antisense transcript and gene transcriptional orientation. However, the applications of these methods have been constrained, to some extent, because of the high cost, difficult operation or inaccuracy, especially regarding the analysis of substantial amounts of data. Thus, we developed an easy method to detect and validate these complicated RNAs. We primarily took advantage of the strand specificity of RT-PCR and the single-strand specificity of S1 endonuclease to analyze sense and antisense transcripts. Four known genes, including mouse β-actin and Tsix（Xist antisense RNA）, chicken LXN（latexin） and GFM1（Gelongation factor, mitochondrial 1）, were used to establish the method. These four genes were well studied and transcribed from positive strand, negative strand or both strands of DNA, respectively, which represented all possible cases. The results indicated that the method can easily distinguish sense, antisense and sense-antisense transcriptional pairs. In addition, it can be used to verify the results of high-throughput sequencing, as well as to analyze the regulatory mechanisms between RNAs. This method can improve the accuracy of detection and can be mainly used in analyzing single gene and was low cost.

How to make the end of a gene,the simple way

Transcription termination of nearly all protein-coding genes in mammals requires 3’end processing by a multiprotein complex that will cleave and polyadenylate the messenger RNA precursor.Because a variety of enzyme complexes intervene,3’end processing was thought to be fundamentally complex and subject to a multitude of regulatory effects.The possibility to select just one out of several polyadenylation sites,in particular,has caused much questioning and speculation.What appear to be separate mechanisms however can be combined into a defined set of rules,allowing for a relatively simple interpretation of 3’end processing.Ultimately,readiness of the terminal exon splice site determines when a transcript reaches the maturity to select a nearby polyadenylation signal.Transcriptional pausing then acts in concert,extending the timeframe during which the transcription complex is close to polyadenylation sites.Since RNA polymerase pausing is governed by the same type of sequences in bacteria and metazoans,mammalian transcription termination resembles its prokaryote counterpart more than generally thought.

The long non-coding RNA expression profile of Coxsackievirus A16 infected RD cells identified by RNA-seq

Coxsackievirus A16(CVA16) is one of major pathogens of hand, foot and mouth disease(HFMD) in children. Long non-coding RNAs(Inc RNAs) have been implicated in various biological processes,but they have not been associated with CVA16 infection. In this study, we comprehensively characterized the landscape of Inc RNAs of normal and CVA16 infected rhabdomyosarcoma(RD)cells using RNA-Seq to investigate the functional relevance of Inc RNAs. We showed that a total of 760 Inc RNAs were upregulated and 1210 Inc RNAs were downregulated. Out of these dysregulated Inc RNAs, 43.64% were intergenic, 22.31% were sense, 15.89% were intronic, 8.67% were bidirectional, 5.59% were antisense, 3.85% were s RNA host Inc RNAs and 0.05% were enhancer. Six dysregulated Inc RNAs were validated by quantitative PCR assays and the secondary structures of these Inc RNAs were projected. Moreover, we conducted a bioinformatics analysis of an Inc RNAs(ENST00000602478) to elucidate the diversity of modification and functions of Inc RNAs. In summary, the current study compared the dysregulated Inc RNAs profile upon CVA16 challenge and illustrated the intricate relationship between coding and Inc RNAs transcripts. These results may not only provide a complete picture of transcription in CVA16 infected cells but also provide novel molecular targets for treatments of HFMD.

Biological Function and Mechanism of Long Noncoding RNAs Nuclear-Enriched Abundant Transcript I in Development of Cervical Cancer

Background： Accumulating documents have demonstrated that long noncoding RNAs （lncRNAs） play critical roles in tumorigenesis. As an lncRNA, nuclear-enriched abundant transcript 1 （NEAT1） has been identified to be involved in the progression of many types of cancers. However, the biological function of NE.4T1 in cervical cancer is not fully investigated. The aim of this study was to disclose the specific biological function of lncRNA NEATI in cervical cancer progression. Methods： Quantitative real-time polymerase chain reaction （qRT-PCR） was utilized to identify the expression of lncRNA NE,4 T1 in the cervical cancer tissues and cell lines. All cervical cancer samples used in this study were collected from the Affiliated Suzhou Hospital of Nanjing Medical University between September 2012 and September 2017. The correlation between NE,4T1 expression and the overall survival rate of cervical cancer patients was analyzed by Kaplan-Meier analysis. The effects of NEAT1 knockdown or overexpression on cell proliferation were tested by performing MTT assays and colony formation assays. Transwell assays were conducted to detect the migratory ability of cervical cancer cells, in which NEAT1 was silenced or overexpressed. Western blotting was utilized to validate whether NEAT1 promotes cervical cancer progression through activating PI3K-Akt signaling pathway. Results： High expression of NE,4T1 predicted poor prognosis of cervical cancer patients （χ^2= 0.735, P = 0.005）. Knockdown of NE,4T1 decreased the number of colonies in CaSki cell from 136.667 ± 13.503 to 71.667 ± 7.506 （t = -18.76, P = 0.003） and decreased the number of colonies in HeLa cell from 128.667 ± 13.317 to 65.667 ± 7.024 （t = -5.54, P = 0.031）. However, overexpression of NEA T1 increased the number of colonies in SiHa cell from 84.667 ± 12.014 to 150.667 ± 18.037 （t = 7.27, P = 0.018）. Knockdown of NEAT1 decreased the migratory number of CaSki cell from 100.333 ± 9.866 to 58.333 ± 5.859 （t = -8.08, P = 0.015） and reduced the migratory number in HeLa cell from 123.667± 12.097 to 67.667 ± 7.095 （t = -6.03, P = 0.026）. Overexpression of NEAT1 increased the migratory number of SiHa cell from 127.333 ±16.042 to 231.333 ±31.786 （t = 4.92, P = 0.039）. Conclusion： NEAT1 may exert oncogenic function in cervical cancer and serve as a novel therapeutic target for cervical cancer.

Target Recycling Transcription of Lighting-Up RNA Aptamers for Highly Sensitive and Label-Free Detection of ATP

We describe here a target recycling transcription of lighting-up aptamer strategy for detecting ATP in human serums in a label-free means with high sensitivity.ATP molecules specifically recognize the binding aptamer and result in the structure switching of the DNA assembly probes to imitate the target ATP molecule recycling cycles through the toehold-mediated strand displacement reaction,which causes the formation of many dsDNAs containing the RNA promoter sequences for subsequent transcription generation of large amounts of lighting-up aptamers.The organic dye,malachite green,then associates with these lighting-up aptamers to produce significantly enhanced fluorescence signals,which can sensitively detect ATP within a dynamic range from 10 to 500 nM in a label-free way.The sensing approach shows a detection limit of 7.3 nM and also has an excellent selectivity for ATP analogue molecules.In addition,this method can detect ATP molecules in diluted human serum samples sensitively,which proves the promising potential to diagnose ATP-related diseases.

RNA silencing movement in plants

Multicellular organisms, like higher plants, need to coordinate their growth and development and to cope with environmental cues. To achieve this, various signal molecules are transported between neighboring cells and distant organs to control the fate of the recipient cells and organs. RNA silencing produces cell non-autonomous signal molecules that can move over short or long distances leading to the sequence specific silencing of a target gene in a well defined area of cells or throughout the entire plant,respectively. The nature of these signal molecules, the route of silencing spread, and the genes involved in their production, movement and reception are discussed in this review. Additionally, a short section on features of silencing spread in animal models is presented at the end of this review.

Homogeneous label-free fluorescent assay of small molecule-protein interactions using protein binding-inhibited transcription nanomachine

Quantitative analysis of interactions between small molecules and proteins is a central challenge in chemical genetics, molecular diagnostics and drug developments. Here, we developed a RNA transcription nanomachine by assembling T7 RNA polymerase on a small molecule-labeled DNA heteroduplex. The nanomachine, of which the RNA transcription activity can be quantitatively inhibited by protein binding, showed a great potential for small molecule-protein interaction assay. This finding enabled us to develop a novel homogeneous label-free strategy for assays of interactions between small molecules and their protein receptors. Three small molecule compounds and their protein receptors have been used to demonstrate the developed strategy. The results revealed that the protein-small molecule interaction assay strategy shows dynamic responses in the concentration range from 0.5 to 64 nM with a detection limit of 0.2 nM. Due to its label-free, homogeneous, and fluorescence-based detection format, besides its desirable sensitivity this technique could be greatly robust, cost-efficient and readily automated, implying that the developed small molecule-protein interaction assay strategy might create a new methodology for developing intrinsically robust, sensitive and selective platforms for homogeneous protein detection.