| Circular RNA is a new category of re-understood regulatory non-coding RNA.Although the molecules have been recognized for its circular transcriptional forms for several decades,they were traditionally considered as splicing errors during the RNA processing,or some products specific to a certain pathogens,such as plant viroids or the hepatitis virus[1-5].Until recent years,with the rapid development of technologies in molecular biology and computational biology[6],more and more works revealed that circular RNAs are abundant and stable.They expressed across kinds of species,in a cell-specific,tissue-specific or developmental-specific manner,consisting a big family contributing to the growing list of noncoding RNAs[7-9].They can be divided into two types according to their constituent parts,arising from exons(circRNA)or introns(ciRNA)[1,10].CircRNAs are produced from precursor mRNA through back-splicing manner,and a small part of these also contains intron retention sequences[11].Most of circRNAs expressed at a relative low level,but in some cases,the abundance of these exceeded more than 10 times to their associated linear mRNA[12].ciRNAs still have a low express level,and they were originated from intron lariats with conserved sequences protecting them from degradation by debranching enzymes.Thus,unlike circRNAs,the circular molecule derived from introns is a novel type of circular RNAs,no matter in concepts or generation mechanisms[13,14].But,both of the two type circular RNAs have specific vital roles in gene regulation.Exonic circular RNAs can serve as miRNA sponges to bind miRNAs disturbing the binding of their coding m RNA targets,leading to the increase of the linear RNAs’ expression,such as circRNA CDR1 or circRNA Sry,and so on[16-19].In addition,they also can have potential functions for protein binding(for example,RNA binding proteins),regulation of translation[20,21].By contrast,intronic circular RNAs almost all express at a low level,and they located in the cell nucleus.One of their functions has been observed for regulating Pol II transcription,largely accumulating to the sites of transcription[13].In my paper,the major object of study has been focused on circRNAs,derived from exon sequences[29,30].Accumulating evidences have worked out that circRNAs have some associations to many diseases,such as Alzheimer’s disease,atherosclerotic vascular disease,pathological hypertrophy and heart failure,osteoarthritis,and some kinds of cancers[22-27,35].But circRNAs are not splicing errors,and they have their own specific mechanisms to be generated,which are not that clearly understood[1,28].So,my research topic has focused on the production mechanisms of this kind of circular RNAs.Based on this,more efforts are took out to explore the potential roles of circular RNAs,served as possible diagnostic biomarkers,in terms of translational medicine.Firstly,with the help of modern molecular biology technics,a expression vector of circular RNAs has been successful constructed,and the generic features of the circular molecules are also studied.Enriched by RNase R[31,32],which is a magnesium-dependent 3’→5’ exoribonuclease,we have showed a class of circular RNAs with high confidence from the RNA-seq data of human H9 cell lines,assistant by the circular RNA research tool named CIRCexplorer[29].And further verification tests for canonical circular RNAs were performed through Northern Blot assays.On the other hand,a circRNA constituted of the 9th and 10 th exon derived from gene POLR2 A was picked out,for the construction of the circular RNA expression vector in depth.The associated sequences were inserted into the pZW1 vector,and further confirmed by Sanger sequencing and Northern Blot assays.This has provided a solid foundation for the succeeding work.Secondly,based on the circular RNA expression vector,a series of attempts were carried out aimed at complementary sequences like Alu elements,because our hypothesis is that complementary sequences take the key roles leading the exon circularization.Alu elements are about 300 bp long and frequently arising in genomic sequences,accounted for 11% of the whole genomics[33,34].They have the potential to execute the complementary base-pairing reactions,and may play vital roles forming circular RNAs.So,it was carried out that various combinations of Alu elements were deleted in the circRNA POLR2 A expression vector.Besides,bases substitution mutation were also performed with artificial modified complementary sequences,like other IRAlu pairs and reconstructive intron sequences with the similar length on upstream part of the gene POLR2 A.On conclusion,when the inverted complementary sequences lie in both side of the flanking introns,circular RNA would be generated by the predicted exons.Above all,inverted complementary sequences are the vital factors leading to exon circularization,and we have further proposed the new concept-alternative circularization‖,which extends the complexity of mammalian transcriptional or posttranscriptional regulation.Thirdly,a further effort have performed to explore the potential diagnostic biomarker role of circular RNAs in the Osteoporosis patients combined with degenerative lumbar diseases.The frame of research work was set up for studying the circular RNAs in plasma or serum exosomes.With the help of analysis by the next generation high throughput sequencing technics and molecular biology methods,their expression and distribution characteristics have shown that circular RNAs are stable in both plasma and serum at 4℃or 25℃.There are more circular RNAs in plasma than serum on comparing the RNA-seq data between the set for RNA samples derived from them,about 4 pairs of plasma and serum from the same patient.To reduce the influence of RNAs from the cracked platelet,plasma samples were chosen for further verification by QPCR assay between the cohort composed by patients suffered degenerative lumbar diseases with or without Osteoporosis.Consequently,a set of circular RNA associated to Osteoporosis were screened out.Thus,it is meaningful for develop new tools to diagnose the metabolic bone disease,and find out novel targets for the treatment of the disease.The study is approved by the Committee on Ethics of Biomedicine,Second Military Medical University,and carried out according to the Helsinki Declaration.Conclusively,circular RNAs are identified in many mammal loci,and the detailed mechanisms for their biogenesis have remained elusive.In my paper,by using modern molecular methods and computational approach for the circle molecules recapitulation,we have shown that the formation of the molecules is dependent on inverted complementary sequences in flanking introns,and further proposed the new concept ―alternative circularization‖,increasing the cognitions about the round molecules.Based on previous results,we have taken further effort to figure out if circular RNAs can be served as diagnostic biomarkers for Osteoporosis patients combined with degenerative lumbar diseases.Luckily,several circular RNAs related to Osteoporosis were screened out.This has provided meaningful supports for further investigation on the detailed mechanisms of the diseases.From the basis to the clinics,I hope to set up a bridge,connecting the bench to the bed,and do more as possible as I can to serve people who suffered uncomfortable.It is a perfect experience to have this wonderful scientific journey,and I also wish this would be a new start for further exploration. |
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