RNAs Research Articles

Plant epigenetics: From genomes to epigenomes

Epigenetics is the study of heritable changes in gene expression that occur without a change in the DNA sequence. In recent years, this field has attracted increasing attention as more epigenetic mechanisms affecting gene activity are being discovered. Such processes involve a complex interplay between DNA methylation, histone modifications, and non-coding RNAs, notably small interfering RNAs (siRNAs) and micro RNAs (miRNAs). Epigenetic regulation is not only important for generating differentiated cell types during plant development, but also in maintaining the stability and integrity of their respective gene expression profiles. Although epigenetic processes are essential for normal development, they can become misdirected which leads to abnormal phenotypes and diseases, especially cancer. Sensing environmental changes and initiating a quick, reversible and appropriate response in terms of modified gene expression is of paramount importance for plants which are sessile autotrophs. Although epigenetic mechanisms help to protect plant cells from the activity of parasitic sequences such as transposable elements, this defense can complicate the genetic engineering process through transcriptional gene silencing. Epigenetic phenomena have economic relevance in the case of somaclonal variation: a genetic and phenotypic variation among clonally propagated plants from a single donor genotype. The success of sequencing projects on model plants has created widespread interest in exploring the epigenome in order to elucidate how plant cell decipher and execute the information stored and encoded in the genome. New high-throughput techniques are making it easier to map DNA methylation patterns on a large scale and results have already provided surprises.

Effect of extracellular vesicles and microRNAs in follicular fluid on follicular development

Extracellular vesicles (EVs) refer to bilayer membrane transport vesicles secreted by cells. EVs can take macromolecules from cells and transfer them to receptor cells. Among these macromolecular substances, the most studied are microRNAs (miRNAs). miRNA is non-coding RNA involved in the regulation of gene expression. It has been confirmed that there are different non-coding RNAs in mammalian follicular fluid EVs. EVs carrying miRNA can act as an alternative mechanism for autocrine and paracrine, affecting follicular development. This paper systematically introduced the kinds, characteristics and methods of isolation and identification of EVs, focusing on the effects of EVs and miRNAs on follicular development, including early follicular development, oocyte maturation, follicular dominance and effects on granulosa cell function. At the same time, the authors prospected the future research of EVs and microRNAs in follicular fluid, and provided ideas and directions for the research and application of EVs and miRNA functions in follicular fluid.

Curcumin improves the behavior and memory in mice by modulating the core circadian genes and their associated micro-RNAs

Objective: To investigate the neuroprotective effects of curcumin in albino mice focusing on memory and behavior. Materials and Methods: Four groups of 7 mice each, different concentrations of curcumin were administered for 3 weeks in 3 groups and the remaining group was kept as control. The effect of curcumin on behavior and memory was studied using various tests. The expression of core circadian rhythm genes and micro RNAs were also studied. Results: Our results revealed that curcumin improves the mice's behavior by regulating oxidative stress, hippocampal neurogenesis, and modulation of core circadian genes and their associated micro-RNA. Conclusion: This study shows that curcumin has a potential neuroprotective effect.

The functions and mechanisms of BLACAT1 in malignant tumors

Long non-coding RNAs (lncRNAs) are a kind of transcripts which are longer than 200nt and have not protein-coding ability due to the lack of an open reading frame. However, lncRNAs can be involved in tumorigenesis and progression in various ways at the transcriptional and post-transcriptional levels. Bladder cancer associated transcript 1 (BLACAT1) as a lncRNA located on human chromosome 1q32.1, is ectopic expression in various tumors (bladder cancer, gastric malignant tumor, lung carcinoma, et al) and can regulate tumor cell proliferation, anti-apoptosis, invasion and metastasis by different mechanisms leading to occurrence and development of tumors. In this review, we summarized current studies of the functions and mechanisms of BLACAT1 in malignant tumors. Key words: RNA, long noncoding; Bladder cancer associated transcript 1; Neoplasms; Therapeutic target; Review

Role of microRNA in predictingclinical efficacy of neoadjuvant chemoradiotherapy for rectal cancer

Neoadjuvant chemoradiotherapy (NCRT) has become the standard treatment for patients with locally advanced rectal cancer (LARC). However, the response to NCRT varies among LARC patients and a subset of patients show resistance to NCRT. NCRT may delay the timing of surgery and even reduce the overall survival. Therefore, it is of significance to identify biomarkers for predicting the clinical efficacy of NCRT, screen patients who are resistant to NCRT and perform surgery as early as possible, eventually establishing an individualized therapeutic strategy. MicroRNAs are a class of small non-coding RNAs that post-transcriptionally regulate gene expression, which areinvolved in multiple signaling pathways and DNA damage repair process and affect the radiosensitivity of rectal cancer cells. Many recent studies have evaluated the role of microRNA in predicting the response to NCRT. The purpose of this article is to review the research progress and validate the role of microRNA in predicting the clinical efficacy of NCRT for rectal cancer. Key words: Rectal neoplasm/neoadjuvant chemoradiotherapy; MicroRNA; Predict

Tumor-derived urinary exosomal long non-coding RNAs as diagnostic biomarkers for bladder cancer.

Bladder cancer (BC) is the sixth most common malignancy in men and 17th in women. Exosomal long non-coding RNAs (lncRNAs) have been defined as a novel biomarker for BC. The aim of this study is to evaluate the clinical significance of urine exosomal PVT-1, ANRIL and PCAT-1 as a biomarker in BC patients with tumors classified as T1 or T2. Exosomes were isolated from urine of BC patients and healthy donors, then characterized according to their shape, size, and exosome markers by Electron Microscopy, Dynamic light scattering, and Western blotting. Exosomal lncRNAs extraction was done to determine the expression levels of PVT-1, ANRIL and PCAT-1 by qRT-PCR. ANRIL and PCAT-1 expression was significantly higher in BC patients compared to normal subjects. To evaluate the performance of the identified lncRNAs for BC detection, we performed ROC curves analysis. The diagnostic accuracy of ANRIL and PCAT-1, measured by AUC, was 0.7229 (sensitivity = 46.67 % and specificity = 87.5 %) and 0.7292 (sensitivity = 43.33 % and specificity = 87.5 %). Transcript levels of lncRNAs in urinary exosomes are potential diagnostic biomarkers in bladder cancer.

MiR-19a suppress apoptosis of myocardial cells in rats with myocardial ischemia/reperfusion through PTEN/Akt/P-Akt signaling pathway.

To detect differentially expressed micro ribonucleic acids (miRNAs) in rats with myocardial ischemia/reperfusion (MIR), and to explore the influence of miR-19a on MIR rats and its mechanism. Firstly, the Sprague-Dawley (SD) rats were used to prepare MIR models, RNAs were extracted, and miRNA sequencing analysis was carried out to determine differentially expressed miRNAs related to MIR. Secondly, the predicted target genes of miR-19a were collected, and WebGestalt was applied to analyze gene ontology (GO) and pathway enrichment. Thirdly, the expression of the related proteins and the apoptosis of myocardial cells in MIR rats were detected via Western blotting. Fourthly, the interaction between miR-19a and the target gene phosphatase and tensin homolog (PTEN) was examined through Luciferase reporter assay. Compared with that in the Sham operation (Sham) group, the miR-19a expression in rat myocardial tissues in the MIR group was significantly increased (p<0.05). Compared with those in the miR-negative control (miR-NC) group, the messenger RNA (mRNA) and protein expressions of PTEN in the miR-19a group were notably decreased (p<0.05). In comparison with the miR-NC group, miR-19a group had elevated expression of phosphorylated protein kinase B (p-Akt) (p<0.05). The Luciferase reporter gene assay manifested the direct binding of miR-19a to PTEN mRNA. MiR-19a inhibits the PTEN expression by directly binding to the 3'-UTR of PTEN mRNA, thus activating the Akt/p-Akt signaling pathway to suppress the apoptosis of myocardial cells in MIR injury.

LncRNA PROX1-AS1 promotes proliferation, invasion, and migration in prostate cancer via targeting miR-647.

Long noncoding RNAs (lncRNAs) act as an important role in many diseases. In this research, lncRNA PROX1-AS1 was explored to identify how it functioned in the development of prostate cancer (PC). Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) was utilized to detect PROX1-AS1 expression in PC patients. Then, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay, colony formation assay, and transwell assay were performed to identify its function in PC cells. Furthermore, the potential mechanism was also explored using mechanism assays. PROX1-AS1 expression level was significantly higher in PC tissue samples and cell lines. Results of MTT assay, colony formation assay, and transwell assay showed that cell proliferation and invasion were inhibited through the silence of PROX1-AS1 in PC cells, while cell proliferation and invasion were promoted through the overexpression of PROX1-AS1 in PC cells. Furthermore, the expression of miR-647 was upregulated via the silence of PROX1-AS1 in PC cells, while the expression of miR-647 was downregulated via the overexpression of PROX1-AS1 in PC cells. Further mechanism assays showed that miR-647 was a direct target of PROX1-AS1 in PC. Correlation analysis showed that miR-647 expression was negatively correlated with PROX1-AS1 expression in PC tissues. Results above suggested that PROX1-AS1 could enhance cell proliferation and invasion of PC cells by sponging miR-647 and might be applied as a novel target for the treatment of PC.

Effects of microRNAs polymorphism in cancer progression.

MicroRNAs (miRNAs) are known as a new class of small RNAs (18-25 nucleotides) that regulate gene expression at multiple levels from transcription to translation. Considering the important role of miRNAs in cell proliferation, differentiation, and apoptosis, any variations in their expression can contribute to various anomalies, such as tumorigenesis. Single-nucleotide polymorphisms (SNPs) have received much attention as potential genetic markers for diseases due to their advantage of being present at a high frequency in the human genome. SNPs can occur in different parts of the miRNA genes (primary, precursor, and mature) which result in pathological conditions. In this study, recent findings related to the effects of SNPs in miRNAs on their biogenesis and functions and their role in cancer development and progression are discussed. This review was performed using PubMed to search for related reports. The identified effects may be useful for clinical decision-making and providing important new information about the pathophysiology of miRNAs.

LncRNA CHRF promotes cell invasion and migration via EMT in gastric cancer.

Long non-coding RNAs (lncRNAs) have been verified to involve in the development and progression of gastric cancer (GC). However, the expression of lncRNA CHRF level in GC has not been mentioned before. Here, we focused on the function of lncRNA CHRF played in GC. A total of 103 GC tissues and paired para-tumor tissues from GC patients were collected. The quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was applied to measure the lncRNA CHRF level in these samples and GC cell lines. The Wound-healing experiment, transwell assay, and Matrigel assay were employed to study the migration and invasion abilities of GC cells. The underlying molecular of lncRNA CHRF was measured using Western-blot. LncRNA CHRF expression was significantly higher in 103 GC tissue samples compared with the adjacent para-tumor samples. In GC cells, lncRNA CHRF showed increased expression levels than the human fetal gastric epithelial cells (GES-1). Inhibition of lncRNA CHRF reduced the invasion and migration of MKN-7 cells while the over-expression of lncRNA CHRF promoted HGC-27 cells metastasis. Furthermore, we found that lncRNA CHRF could promote the progression of epithelial-mesenchymal transition (EMT) to promote the GC cell metastasis. Our current study demonstrated that lncRNA CHRF functioned as an oncogene in GC and promoted cell invasion and migration via EMT. This might furnish a potential target for the GC biological diagnosis and therapy.

Long non-coding RNA XIST protects chondrocytes ATDC5 and CHON-001 from IL-1β-induced injury via regulating miR-653-5p/SIRT1 axis.

Chondrocyte apoptosis is linked to cartilage degeneration, and considered as a crucial event during the development of osteoarthritis (OA). X inactive specific transcript (XIST) is an oncogenic long non-coding RNA (lncRNA). However, its role in the pathophysiological process of OA remains largely unknown. In this work, quantitative real-time reverse transcriptase PCR (qRT-PCR) was employed to measure the expression of XIST, miR-653-5p and sirtuin1 (SIRT1) mRNA in OA and normal cartilage tissues. Chondrocyte cell lines, CHON-001 and ATDC5, were treated with different doses of interleukin- 1β (IL-1β) to mimic the inflammatory environment of OA in vitro. Overexpression plasmids, microRNA (miRNA) mimics, miRNA inhibitors and small interfering RNAs (siRNAs) were constructed and transfected into CHON-001 and ATDC5 cells. 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay was adopted to determine the cell viability. Western blot was used to detect the expression of apoptosis-related proteins. Enzyme-linked immunosorbent assay (ELISA) was employed to probe the expression levels of inflammatory factors. Flow cytometry was used to analyze the cell apoptosis. StarBase and TargetScan databases were used to predict the binding sites between XIST and miR-653-5p, miR-653-5p and 3'UTR of SIRT1, respectively, which were then verified by dual luciferase reporter assay. The data in the present study demonstrated that XIST and SIRT1 were down-regulated while miR-653-5p was up-regulated in OA tissues and cell models. The up-regulation of XIST increased the viability of CHON-001 and ATDC5 cells, while it impeded their apoptosis and inflammatory response induced by IL-1β. Conversely, miR-653-5p had opposite effects. It was proved that miR-653-5p could be sponged and suppressed by XIST. Additionally, SIRT1 was identified as a target of miR-653-5p, and SIRT1 could be suppressed by XIST indirectly. In conclusion, down-regulated XIST was involved in the injury of chondrocytes during the pathophysiological process of OA, and XIST up-regulation protected chondrocytes from inflammatory injury via regulating miR-653-5p/SIRT1 axis.

Long noncoding RNA NNT-AS1 functions as an oncogene in breast cancer via repressing ZFP36 expression.

Breast cancer is one of the most common cancers in women. This study focuses on the effects of Long non-coding RNAs (lncRNAs) NNT-AS1 on breast cancer cell growth and metastasis. Fifty-six pairs of breast cancer (BC) tissues and matched paracarcinoma tissues were obtained. The BC cell lines and normal human breast cell line were employed. NNT-AS1 in BC cells was knocked down by shRNA. Cell counting kit-8 assay (CCK-8), colony formation assay, cell cycle analysis, cell apoptosis analysis, cound healing assay, Transwell assay, cioinformatics analysis, Western blot analysis and Xenograft model were used. Quantitative real-time polymerase chain reaction (qRT-PCR) assay indicated that expression of NNT-AS1 was obviously upregulated in breast cancer tissues compared with adjacent tissues (n=56). Knockdown of NNT-AS1 could attenuate breast cancer cell viability, proliferation, invasion and migration, as well as promote cell apoptosis and induce cell cycle arrest at G0/G1 phase. ZFP36 was directly combined with NNT-AS1, and silencing of ZFP36 could rescue tumor suppression role by downregulating NNT-AS1 on cell proliferation and metastasis. Knockdown of NNT-AS1 could suppress cell growth and metastasis via interacting with ZFP36 in vivo. This study demonstrated that knockdown of NNT-AS1 had tumor-suppressive effect on breast cancer progression and metastasis via interacting with ZFP36 in vitro and in vivo, which provides a new insight into the treatment and prognosis evaluation of breast cancer.

Upregulation of circular SMAD7 inhibits tumorigenesis of gastric cancer by reversing epithelial-to-mesenchymal transition.

Gastric cancer (GC) is one of the most ordinary malignant tumors. Recent studies have revealed that circular RNAs (circRNAs) play an important role in the progression of tumorigenesis. In this research, circ-SMAD7 was selected to identify how it functions in the progression of GC. Circ-SMAD7 expression in paired GC patients' tissue samples and cell lines was detected by Real Time-quantitative Polymerase Chain Reaction (RT-qPCR). The role of circ-SMAD7 in the metastasis of GC was detected through wound healing assay and transwell assay. Western blot assay and RT-qPCR were used to discover the function of circ-SMAD7 in epithelial-to-mesenchymal transition (EMT) process. Furthermore, tumor metastasis assay was also performed in vivo. In this study, RT-qPCR results showed that circ-SMAD7 expression was significantly lower in GC tissues compared to that in adjacent ones. Cell migration and invasion of GC were inhibited via upregulation of circ-SMAD7. Moreover, the results of further experiments revealed that the EMT-related proteins were regulated via upregulation of circ-SMAD7 in GC. Furthermore, tumor metastasis of GC was inhibited via upregulation of circ-SMAD7 in nude mice. These results indicate that upregulation of circ-SMAD7 inhibits GC cell migration and invasion via reversing the EMT process.

Long noncoding RNA SNHG8 promotes the proliferation of osteosarcoma cells by downregulating miR-542-3p.

Small nucleolar RNA host genes (SNHGs) as a subset of long noncoding RNAs (lncRNAs) have critical roles in the pathogenesis of multiple malignancies, however, the role and molecular mechanisms of lncRNA SNHG8 in osteosarcoma (OS) remain unclear. In the present study, the correlation of SNHG8 or miR-542-3p with clinicopathological elements and prognosis in OS patents was estimated by TCGA cohort. Cell viability and invasion were assessed by MTT and Transwell assays. The interplay between SNHG8 and miR-542-3p was affirmed by a luciferase report assay. The effects of SNHG8 on miR-542-3p expression were examined in MG-63 and SW-1353 cells by qRT-PCR analysis. The results showed that incremental expression of SNHG8 or reduced expression of miR-542-3p was related to poor survival and tumor recurrence in OS patients. Overexpressing SNHG8 accelerated the growth and invasion of MG-63 cells, but silencing SNHG8 harbored an opposite effect in SW-1353 cells. Additionally, SNHG8 could negatively regulate miR-542-3p expression and bind with miR-542-3p, which attenuated SNHG8 induced cell proliferation. Taken together, these findings indicate that lncRNA SNHG8 promotes the proliferation of OS cells by downregulating miR-542-3p.

Research progress of non-coding RNA in hepatic ischemia-reperfusion injury

Hepatic ischemia-reperfusion injury (HIRI) is the main cause of liver damage and even multiple organ failure after complex liver surgery. When liver ischemia reperfusion occurs, the non-coding RNAs in the liver tissue is dysregulated and part of the non-coding RNAs with abnormal expression is involved in HIRI regulation. Non-coding RNAs to may be the intervention target for reducing HIRI. This article summarized the types and related functions of non-coding RNAs, the role of different non-coding RNAs in HIRI, and the interconnections between various non-coding RNAs in HIRI. Key words: Reperfusion injury; Liver; Non-coding RNA; MicroRNA; Small interfering RNA

Increased Expression Level of Long Noncoding RNA H19 in Plasma of Patients with Myocardial Infarction.

Long noncoding RNAs (lncRNAs) are lengthy noncoding transcripts which are actively involved in crucial cellular pathways. Tissue-specific expression of lncRNAs besides its secretion into the body fluids, has made lncRNAs in attention as biomarkers of the diseases. According to the role of lncRNAs, especially H19 in cardiac regeneration, it is not surprising if their altered expression levels lead to cardiac diseases. In the present study, the relative expression of H19 was compared in the plasma of atherosclerotic myocardial infarction and control individuals by real time-PCR, and data were normalized using GAPDH. The association of plasma level of lipid and homocystine with H19 expression was also considered. The potential of H19 to discriminate the case from control was studied using the ROC analysis. We found that the plasma level of H19 transcript significantly increased in the plasma of patients in comparison with the control group. Additionally, the relative expression level of H19 was directly associated with the plasma homocystine level. The relative expression of H19 at threshold of 0.3 showed 70% sensitivity and 94% specificity to discriminate cases from controls. This study revealed that the expression level of H19 may be considered as a biomarker of myocardial infarction, although further studies are needed to generalize this finding.

Downregulated long noncoding RNA LUCAT1 inhibited proliferation and promoted apoptosis of cardiomyocyte via miR-612/HOXA13 pathway in chronic heart failure.

Long non-coding RNAs (lncRNAs) have been reported to play important roles in numerous kinds of cardiovascular disease, including chronic heart failure (CHF). In this study, we mainly focused on investigating the potential roles of lncRNA LUCAT1 patients with CHF. RT-PCR was used to detect the expressions of LUCAT1 and miR-612 in serum samples of CHF patients (n=60) and healthy volunteers. Relationships between the expressions of LUCAT1 and miR-612, LUCAT1 and overall survival (OS) were analyzed using the Kaplan-Meier method. Si-LUCAT1 and miR-612 mimic were constructed and respectively transfected into AC16 cells to explore the functions of LUCAT1 and miR-612. Cell proliferation abilities were detected by CCK-8 assay AC16 cells. Cell apoptotic rates were measured by flow cytometry (FACS) analysis. Western blot (WB) was performed to detect the protein levels of HOXA13, Bcl-2, Bax, Bad and Cleaved Caspase3. In addition, luciferase gene reporter assay was used to prove the relationships between LUCAT1 and miR-612, miR-612 and HOXA13. Firstly, we found that LUCAT1 was decreased for 1.7 folds in CHF patients, which was correlated with poor prognosis patients. LUCAT1 repression inhibited cell proliferation and promoted cell apoptosis in human cardiomyocyte cell line AC16 cells. Furthermore, we found that miR-612 was increased for 2.0 folds in CHF patients, which was negatively interacted with LUCAT1 expression. Luciferase gene reporter assay demonstrated that LUCAT1 could directly bind with miR-612 in AC16 cells. Moreover, miR-612 overexpression also inhibited cell proliferation and promoted cell apoptosis in AC16 cells. Luciferase reporter assay indicated that miR-612 could directly target at HOXA13 in AC16 cells, which was associated with cell proliferation and apoptosis. Finally, miR-612 inhibitor was transfected into AC16 cells with si-LUCAT1. The results showed that the inhibited cell proliferation and promoted cell apoptosis were reversed, which confirmed that LUCAT1 repression inhibited cell proliferation and promoted apoptosis via miR-612/HOXA13 axis in CHF patients. According to the above results, our study revealed that LUCAT1 was decreased in CHF patients, which was correlated with poor prognosis of CHF patients. Furthermore, the downregulation of LUCAT1 inhibited cell proliferation and promoted cell apoptosis via targeting miR-612/HOXA13 axis. Our results elucidated a potential mechanism underlying cardiomyocyte apoptosis, which might be used as a promising prognostic marker and a potential target for CHF patients.

Research progress of microRNAs in cardiovascular diseases

Objective MicroRNAs (miRNAs) are endogenous short stranded RNAs with a length of about 22 NT, which are highly conserved and have no coding function.Mature miRNAs play a role by specifically binding to the 3′untranslated region of the target gene, degrading the mRNA of the gene or hindering its translation at the post transcriptional level, so as to negatively regulate the expression of the target gene and play a biological role.In recent years, the research of miRNAs in cardiovascular diseases is increasingly in-depth.A large number of evidences show that miRNAs play a role in the pathogenesis of many cardiovascular diseases, and will become a potential marker and new treatment target for the diagnosis and prognosis evaluation of cardiovascular diseases. Key words: MicroRNAs; Cardiovascular diseases; Biomarkers

Corrigendum: Single-nucleotide polymorphism screening and RNA sequencing of key messenger RNAs associated with neonatal hypoxic-ischemia brain damage.

[This corrects the article DOI: 10.4103/1673-5374.264469].

Circulating CYTOR as a Potential Biomarker in Breast Cancer.

Long non-coding RNAs (lncRNAs) are newly introduced as tumor-related molecules. They are being considered as potential diagnostic and therapeutic targets in cancer studies. Here, we have assessed the importance of CYTOR (linc00152) as a biomarker for the detection of breast cancer in both tumoral tissue and plasma. The relative expression of breast cancer-associated CYTOR was measured in 20 tumoral and paired margin tissues, and moreover, in 80 plasma samples by real-time-polymerase chain reaction. In addition, plasma levels of CA 15-3 were measured using the ELISA test. The receiver operating characteristic curve (ROC) was applied for assessing the sensitivity and specificity of tested biomarkers. The results of the present study disclosed significantly increases in the CYTOR expression levels in tumor tissue with relative quantitation (RQ) equals to 5.15 (P<0.001) and in plasma (RQ =3.03, P<0.01) of breast cancer patients. The area under the ROC curve (AUC) of circulating CYTOR was 0.907 (95% CI: 0.842-0.972, P<0.001), while it was 0.868 (95% CI: 0.783–0.952, P<0.001) for CA 15-3. Based on these findings, we suggest lncRNA CYTOR as a new potential biomarker for breast cancer detection in both solid tumor tissue and plasma.