Peranan RNA Bulat dalam Kanser Kolorektal / Jurnal Sains Kesihatan Malaysia

@#Circular RNAs (circRNAs) are a class of non-coding RNAs formed by covalently closed loops through backsplicing. From previous studies, RNA was found to be involved in the development and formation of colorectal cancer. By emphasizing on the true function of circRNAs through focusing on its biogenesis, roles in disease treatment and roles as biomarkers, we are able to utilize circRNAs as therapy for cancer. Cancer is a chronic disease that contributes to high mortality worldwide. Colorectal cancer is one of the most common cancer in the world and in Malaysia in general. The incidence and mortality rates of colorectal cancer worldwide show a drastic and alarming increase. Colorectal cancer occurs due to mutations from certain genes involved in proliferation regulation, cell survival and cell death. This article aims to discuss the role and importance of Circular RNA in colorectal cancer. By identifying the true function of circRNAs, it can help us to develop our understanding on the real biological roles of circRNAs in colorectal cancer.

Construction and expression of circular RNA in vitro / 中国生物制品学杂志

@#Objective To construct encoding RNA that can be cyclized in vitro by using the permuted intron exon(PIE)strategy in the maturation process of eukaryotic mRNA,and transfect it into HEK-293T cells for expression.Methods The sequences of 5'and 3'cyclic arms with groupⅠcatalytic intron,the internal ribosome entry sites(IRES)of Coxsackievirus B3(CVB3)and the target gene were selected to construct the template plasmid. Linearization plasmid template obtained by PCR was used to synthesize linear RNA through in vitro transcription(IVT),which then started in vitro cyclization(IVC)by the addition of cyclization reagents to obtain circular RNA(circRNA). RNA cyclization was confirmed by agarose gel electrophoresis and ribonuclease R(RNase R)digestion. HEK-293T cells were transfected with circRNAs respectively carrying enhanced green fluorescent protein(EGFP),firefly luciferase(Fluc),and influenza virus hemagglutinin(HA)IVR-180 genes,to verify their expression with in vitro.Results With RNA cyclization,the main band of agarose gel electrophoresis became smaller and small fragments appeared. After RNase R digestion,only some circRNA bands remained.HEK-293T cells transfected with EGFP-circRNA showed significant green fluorescence under the fluorescence microscope.The Fluc expression values of HEK-293T cells transfected with Fluc-circRNA were on average 20 times higher than non cyclized RNA,and the relative light unit(RLU)scaled up with the increase of Fluc-circRNA transfection dose. Western blot analysis showed that HA protein was successfully expressed in HEK-293T cells transfected with HA-circRNA.Conclusion In this study,linear RNA was successfully cyclized in vitro and different proteins were expressed,which lays a foundation of the research of new influenza vaccines and mRNA vaccines.

Promising roles of non-exosomal and exosomal non-coding RNAs in the regulatory mechanism and as diagnostic biomarkers in myocardial infarction / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Non-exosomal non-coding RNAs (non-exo-ncRNAs) and exosomal ncRNAs (exo-ncRNAs) have been associated with the pathological development of myocardial infarction (MI). Accordingly, this analytical review provides an overview of current MI studies on the role of plasma non-exo/exo-ncRNAs. We summarize the features and crucial roles of ncRNAs and reveal their novel biological correlations via bioinformatics analysis. The following contributions are made: (1) we comprehensively describe the expression profile, competing endogenous RNA (ceRNA) network, and "pre-necrotic" biomarkers of non-exo/exo-ncRNAs for MI; (2) functional enrichment analysis indicates that the target genes of ncRNAs are enriched in the regulation of apoptotic signaling pathway and cellular response to chemical stress, etc.; (3) we propose an updated and comprehensive view on the mechanisms, pathophysiology, and biomarker roles of non-exo/exo-ncRNAs in MI, thereby providing a theoretical basis for the clinical management of MI.

Analysis of m / 中国生物化学与分子生物学报

In this study, we performed high-throughput sequencing technology methylated RNA immunoprecipitation sequencing (MeRIP-seq), transcriptome sequencing (RNA-seq) and bioinformatics to analyze the differentially m6A-methylated and differentially expressed profile of circular RNA (circRNA) in middle cerebral artery occlusion/reperfusion (MCAO/R) model, which provided some scientific evidences for revealing the relationship between RNA epigenetic modification and cerebral ischemia reperfusion injury. The neurological deficit scores of mice were evaluated by the Longa score standard. TTC staining was used to detect cerebral infarction volumes, and dot blot was used for the quantification of m

Porcine Circular RNA Insulin-like Growth Factor 1 Receptor Promotes Adipocyte Differentiation / 中国生物化学与分子生物学报

Circular RNA (circRNA), as a competitive endogenous RNA (ceRNA), plays an importantrole in the regulation of cell differentiation. The purpose of this study was to identify and analyze porcinecircular RNA insulin-like growth factor 1 receptor (circIGF1R), explore its expression patterns, construct a ceRNA regulatory network related to circIGF1R, and explore the regulation of its ectopicexpression on adipogenic differentiation of mouse mesenchymal stem cells (C3H10T1 / 2) effect. Forwardand reverse PCR, Sanger sequencing, RNase R enzyme digestion tests, and qRT-PCR were used toverify that circIGF1R is a circRNA formed by the second exon of insulin-like growth factor 1 receptor(IGF1R). It was expressed in all tissues of pigs, and its expression level increased with age in adiposetissues. miRDB, TargetScan and miRWalk online software were used to predict circIGF1R target genes. RNAhybrid software was used for binding site prediction. DAVID bioinformatics functional analysissoftware was used to perform GO and KEGG enrichment analysis on candidate target genes. Cytoscapesoftware was used to construct the ceRNA network diagram. Based on the gene expression correlation andpredicted target relationship, the GO and KEGG enrichment analysis was drawn and the ceRNA networkwas constructed; the dual luciferase reporter gene test was used, and we found that circIGF1R andFABP4 can bind to ssc (Sus scrofa chromosome) -miR-133a-5p. The circIGF1R overexpression vectorwas successfully constructed and expressed in C3H10T1/ 2 cells. It was found that after overexpression ofcircIGF1R, the expression of key adipogenic regulatory factors CEBPa, CEBPß, FABP4 and PPAR? increased significantly(P<0. 01), and the number of lipid droplets increased significantly. The results ofthis study show that circIGF1R exists in pig adipose tissues, and may positively regulate the adipogenicdifferentiation of C3H10T1/ 2 cells through the ceRNA mechanism, which lays a theoretical foundation forfurther research on circIGF1R regulating the adipogenic differentiation of pig precursor intramuscularadipocytes.

The Role of m6A Modification of Circular RNA in Tumors / 中国生物化学与分子生物学报

Circular RNA (circRNA) is a novel RNA with circular structures. It is conserved in various species, and characterized by high stability, high expression levels and tissue specificity. Meanwhile, it could serve as microRNA (miRNA) sponges, bind to RNA-binding proteins, or regulate transcription and protein translation. With the development of high-throughput sequencing and bioinformatics, circRNA was reported to participate in the pathogenesis of cancer. N6-methyladenosine (m6A) modification is the most common type of RNA modification in eukaryote RNA, which is a dynamic and reversible process regulated by m6A methyltransferase, m6A demethylase and m6A-binding proteins. M6A modification isinvolved in the regulation of RNA nuclear export, splicing, stability, translation and degradation, thus playing a key role in the occurrence and development of multiple human diseases, such as cancers, cardiovascular diseases. Recently, some researches demonstrated that m6A modification of circRNA was essential in the occurrence and development of malignant tumors, such as cervical cancer, colorectal carcinoma, hepatocellular carcinoma, non-small cell lung cancer, poorly differentiated adenocarcinoma of the stomach. In the current manuscript, we summarized the mechanism of m6A RNA modification, the roles of m6A modification in regulation of circRNA, and the effects of circRNA m6A modification on tumor. The potential clinical application value of m6A-modified circRNA was further discussed, as to provide some new ideas and ways for early diagnosis, clinical treatment and prognosis of tumors.

Exosomal Circular RNAs Mediates Chemotherapy Resistance in Tumors / 中国生物化学与分子生物学报

As an important member of the non-coding RNA family, circRNA is a kind of single-stranded RNA with a covalently closed loop structure without a polyadenylated acid tail and 5'-3' end, showing high stability, abundance and conservation across species characteristics. Recent studies have shown that circRNA plays an important role in many biological processes, including chemotherapy resistance and malignant progression. Exosomes are small extracellular phospholipid bilayer vesicles with a diameter of30-150 nm that are secreted by living cells. They can be used as carriers to encapsulate and transfer functional molecules. Exosomes are important mediators of communication between tumor cells and stromal cells. They can play a role in the transmission of chemoresistance by transferring circRNA. Aschemotherapy resistance is still a huge obstacle to the prognosis of cancer, the research of exosomalcircRNA-mediated tumor chemotherapy resistance is at the forefront of academic research, which is a blueocean with important significance. In this paper, we summarized the latest research progress in the aspects of exosome delivery of circRNA, the mechanism of exosome sorting non-coding RNA cargo, circRNA-mediated tumor chemotherapy resistance, exosome delivery of circRNA-mediated tumor chemotherapy resistance and its potential clinical application, which may provide a reference for the research of tumor chemotherapy resistance.

Analysis of Neuroprotective Effect of Danggui Shaoyaosan on APPswe/PS1ΔE9 Transgenic Mice Based on Circular RNA Sequencing / 中国实验方剂学杂志

Objective:To investigate the neuroprotective effects of Danggui Shaoyaosan （DSS） on APP_swe/PS1_ΔE9transgenic （APP/PS1） mice and its mechanism related to circular RNA （circRNA）. Method:Totally twenty 6-month-old APP/PS1 mice were divided into model group and DSS group， and 10 C57BL/6 wild-type mice were set as the normal control group. The normal group and model group received the same volume of normal saline， and DSS group received drug by gavage administration， all for 8 weeks. The differentially expressed circRNA of APP/PS1 mice before and after DSS intervention was analyzed by circRNA sequencing to construct circRNA-miRNA mRNA interaction network. The results of cricRNA sequencing were then verified by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. The protein expression levels of phosphoinositide 3-kinase （PI3K）， p-PI3K， protein kinase B1 （Akt1）， p-Akt1， B lymphocytoma-2 （Bcl-2）， and Bcl-2-Associated X protein （Bax） in the hippocampus were detected by immunoblotting （Western blot）. The protein expression of Caspase-3 in the hippocampus was detected by immunohistochemistry and the level of apoptosis in the hippocampus was detected by the TUNEL method. Result:Compared with the model group， there were 90 differentially expressed circRNA after intervention with DSS， of which 46 were up-regulated and 44 down-regulated. Compared with the normal group， the expression levels of circRNA1398 and circRNA1399 in the model group decreased， and the expression levels of miR-103-3p， miR-153-3p， miR-143-3p， and miR-143-5p increased. Compared with the model group， the expression levels of circRNA1398 and circRNA1399 in the DSS group were up-regulated， while the expression levels of miR-103-3p， miR-153-3p， miR-143-3p， and miR-143-5p were down-regulated. Compared with the normal group， the expression of p-PI3K， Akt1， p-Akt1， and Bcl-2 in the model group decreased （<italic>P</italic><0.05，<italic> P</italic><0.01）， and the expression of Bax and Caspase in the model group increased （<italic>P</italic><0.01）. Compared with the model group， the expression of p-PI3K， Akt1， p-Akt1， and Bcl-2 in the hippocampus of the DSS group increased （<italic>P</italic><0.01）， and the protein expression of Bax and Caspase decreased （<italic>P</italic><0.01）. Compared with the normal group， the apoptosis level in the hippocampus of the model group increased， with an apoptosis rate of （43.76±2.92）%. Compared with the model group， the apoptosis rate of DSS group was （24.64±3.39）%. Conclusion:DSS can activate PI3K/Akt pathway and inhibit apoptosis in hippocampal neurons of APP / PS1 mice， and play a neuroprotective role. The specific mechanism may be related to the regulation of circRNA1398 and circRNA1399 expression and the corresponding miRNA expression.

CircHMGCS1 Activates the NF-κB Signaling Pathway and Promotes Proliferation and Invasion in Cervical Cancer Cells / 中国生物化学与分子生物学报

Circular RNAs play an important role in tumorigenesis. In this study, we explored the role of aberrant expression of circHMGCS1 in cervical cancer cells with TNF-α treatment. RT-qPCR assays showed that circHMGCS1 was highly expressed after TNF-α treatment. And then we investigated the cyclization and stabilization of circHMGCS1 by RNase R and half-life analysis. We further revealed that overexpression of circHMGCS1 promoted the malignancy of cervical cancer cells and the expression of the epithelial-mesenchymal transition (EMT) markers by Transwell analysis, MTT, clone formation and Western blotting. Furthermore, we found that circHMGCS1 activated the NF-κB signaling pathway. Altogether, this study provides an experimental basis for elucidating the role of circHMGCS1 in cervical cancer cells, and provides a potential biomarker for the diagnosis and treatment of cervical cancer.

Effects of Competing Endogenous RNA on Proliferation and Differentiation of Skeletal Muscle Cells / 中国生物化学与分子生物学报

The skeletal muscle is an important part of the animal body, which is closely related to body movement, energy consumption, production performance and its development process is regulated by many factors. The molecular mechanism of skeletal muscle growth and development is not only closely related to animal husbandry production, but also provides a theoretical basis for the treatment of muscle dis-eases, such as amyotrophic muscular dystrophy, and so on. Non-coding RNA (ncRNA) is a kind of RNA without coding ability, in which circular RNA (circRNA), long non-coding RNA (lncRNA) and micro RNA (miRNA) have been proved to play an important role in the development of skeletal muscles. miR-NA can specifically bind to the 3′ untranslated region (3′ UTR) of the target gene through its seed se-quence, so as to inhibit the translation process of the target gene or directly degrade the target gene, and become a prominent participant in a variety of biological processes. Studies have shown that circRNA, ln-cRNA, pseudogenes and mRNA with miRNA response elements (MRE) can competitively combine miRNA to regulate gene expression, forming a competing endogenous RNA (ceRNA) regulatory network model. This hypothesis subverts the previous concept of unidirectional regulation of target genes by miRNA, and endows them with new biological functions in transcriptome, which is of great biological significance. In recent years, it has been found that ceRNA plays an important regulatory role in the growth and development of skeletal muscles. This paper reviews the role of miRNA, lncRNA and circRNA in the proliferation and differentiation of animal skeletal muscle cells, which is an important member of ceRNA mechanism, in order to broaden the research direction of skeletal muscle mechanism and provide new ideas for the development of animal husbandry and the treatment of muscle diseases.

Circular RNA CDR1as: A Novel Regulator of Malignant Tumors / 中国生物化学与分子生物学报

Circular RNA (circRNA) is a new type of non-coding RNA with closed circular structures that are widely distributed in various tissues Compared with traditional linear RNA, circRNA does not have 5′ and 3′ ends and will not be easily degraded by exonuclease It can stably exist in a variety of body fluids and is evolutionarily conserved It has become a key research object of clinical non-coding RNA Malignant tumors have the characteristics of late detection, rapid progression, and easy recurrence Currently, effective treatment methods are lacking, and their morbidity and mortality have been high Therefore, how to carry out early diagnosis, treatment intervention and prognosis evaluation is one of the research frontier of contemporary medical research CDR1as is the most widely studied circRNA It can regulate the expression of downstream genes through sponge microRNA (miRNA) or directly bind to RNA-binding proteins (RBPs) to activate related signaling pathways, thereby promoting or inhibiting tumor progression, and even affecting tumor chemotherapy sensitivity CDR1as mainly exists in the cytoplasm and can be released into the blood at the early stage of the disease Therefore, CDR1as may become a bi-omarker for early diagnosis of malignant tumors or an ideal target for therapeutic intervention Focusing on the characteristics and biological functions of circRNA, this article reviews the expression level, mechanism of action and related signaling pathways of CDR1as in the occurrence and development of malignant tumors At the same time, this article analyzes the current research status of CDR1as, preliminarily summarizes the problems it may face in clinical applications in the future, and puts forward ideas and suggestions on the future research direction of CDR1as

The Regulatory Mechanism and Role of CircRNA in Tumors / 中国生物化学与分子生物学报

Circular RNAs (circRNAs) are a class of non-coding RNAs that form closed rings in structure. They contain a high content in eukaryotic transcripts, and are characterized by richness, stability, high conservatism and tissue specificity. In recent years, it has been gradually revealed that circRNA can bind to some miRNAs or proteins and participate in the regulatory mechanisms of biogenesis and molecular functions, including the regulation of miRNAs molecular sponge, protein translation, gene transcription and RNA splicing. With the application of high-throughput sequencing and bioinformatics, circRNA has gradually become a new research hotspot in the field of non-coding RNA due to its special properties. The latest research evidence shows that circRNA plays a key role in the occurrence and development of tumors, and is inextricably linked with cell proliferation, apoptosis, angiogenesis, and metastasis, indicating that targeting circRNA will be attractive treatment strategies and potential biomarkers. In this paper, the characteristics and mechanism of circRNA were briefly described, the mechanism of action and regulation of circRNAs in human tumors were summarized, and the strategies and development prospects of circRNA in tumor research were further discussed. In sum, circRNA plays an important role in early diagnosis, precise treatment and prognosis prediction of tumors.