Screening And Identification Of Genes Related To Osteoporosis In Human Periosteum

Primary osteoporosis(OP) is considered as a polygenic skeletal-related disorder, characterized by compromised bone strength predisposing to an increased fracture, especially for postmenopausal and old population, representing the major public health concems. In the last 20 years, extensive research has been devoted to studying the expression of genes related to osteoporosis. These studies have been primarily focused on three principle areas: (1) finding differences in gene expression profiles between normal and diseased tissues, (2) determining effects of various inherent regulators(hormones, receptors, etc.), and (3) ascertaining effects of therapeutic agents in the treatment of osteoporosis. Conventional gene expression studies(Northern blot, Southem blot, Western blot, RT-PCR, etc., for example) have provided important information on possible mechanisms of onset and development of osteoporosis and confirmed that osteoporosis has a complex mode of regulation involving a large number of candidate genes through multiple inherent factors. However, further progress in this field is hampered by the inherent limitations of these methods. The main shortcoming is that, using traditional methods to assay gene expression, researchers are able to survey only a relatively small number of genes at a time and thus can not determined a large number of genes contributing to this disease. In fact, genes expressed by many tissues and cell types contribute simultaneously to the BMD variation and may thus be involved in the development or prevention of osteoporosis. Further, the identified genes related to osteoporosis can not elucidate its complete pathophysiology. Therefore, there are still unidentified genes contributing to onset and development of OP. In terms of limited variety of medications in treatment and prevention of OP, identification and characterization of specific loci or genes involving in determining OP and associated phenotypes leaves broad space for developing gene-targeted therapeutic options for OP.For a long time, researchers took great importance attached to development, differentiation and regulation of bone marrow mesenchymal stem cells in undertaking OP related gene studies, whether in vitro or in vivo, while the marrow has long been considered as a unique source of osteoprogenitors, another source of osteoblastgenitors from periosteum, under which bone forming maintains bone strength, is neglected. In fact, the latter in adults plays a key part in maintaining bone strength. This study aims to characterize the genes expressed in human periosteum, and screen new functional genes related to osteoporosis and give one of them a validation.1. Construction and identification of human periosteum full-length cDNA library. Switch mechanism at 5'end of rnRNA template(SMART) technology was used to construct human periosteum full-length cDNA library. Then the library was tittered, and the average exogenous insertions of the recombinants were determined by use of clone-based PCR. Large-scale colons selected at random from the library were sequenced.2. Functional classification and characterization of genes expressed in human periosteum. Transcripts sequenced from pedosteum cDNA library were clustered and subject to identification by the techniques of bioinformatics. The BLAST search results were used to get further information on function, mortif, chromosomal location, code of gene ontology(GO), as well as novel genes without annotation through database on Genbank, Expasy and Ensemble. In addition, Gene expression profile was analyzed against the skeletal gene database.3. According structure characteristic of full length gene, we chose an uncharacterized gene, named LOC221143 as target gene. Firstly, we cloned this gene's code domain sequence(CDS) into a report plasmid, plasmid of enhanced green fluroscent protein(pEGFP) and then transfected U2OS cell, by which subcelluar location of the translated protein was determined. The over-expression effects of LOC221143 on proliferation of U2OS were also observed. Secondly, we explored this gene's tissue distribution by use of RT-PCR. At same time, Using bioinformatic methods, the structure of LOC221143 were analyzed and its function was predicted, which was in turn, validate the results of biological experiments. Main results1. The titter of library from human periosteum was 5.2×10~6, and the average insertion length was 1.2kb. 5230 contigs and 4714 singlets after initial alignment were obtained from randomly sequencing.2. After removing shorter (equal or less than 100 bp) and redundant sequences, 5659 transcripts, including 2796 contigs and 4714 singlets, were BLAST against public database, in which altogether 5453 sequences (96.4%) were localized on chromosomes. 838 sequences that can match GO code were annotated, in which 30.67% has catalytic activity, 6.8%, signal transducer activity. In comparison with skeletal gene database, we found some genes also expressed in periosteum and some, novel with unidentified function.3. According to bioinformatic results, LOC221143 seemed to be a full-length gene with unidentified function, localized on human chromosome 13 qter, containing 5 exons. The molecular weight of its product is 24.564kD,and isoelectric point (IP) 4.191. It is suggested from a global alignment against homologous sequence that this homologous sequence presents in many kinds of model organisms and keeps high conservation. Results from structural prediction showed that it is possible that LOC221143 is a sort of intracellular enzyme with some similar role as DNA methylation. Rich expressions were found in such tissues as heart, small intestine, skeletal muscle and periosteum, so was in U2OS cell line. pEGFP report systems also revealed that protein translated from LOC221143 expressed intracellularly, which was consistent with results from bioinformatic prediction. In addition, Over-expression of LOC221143 seemed not to affect on U2OS proliferation.Conclusions1. The successful construction of human periosteum full-length cDNA library was the first step in elucidation of mechanisms of bone-related disorders and identification of new genes for targeted therapeutic options.2. Classification of genes expressed in human periosteum provided an expression profile of this tissue at this developmental stage. Also, the uncharacterized genes are the materials for consequent studies on identification of new functional gene related to OP.3. Gene LOC221143 is high conservative, the product of which is a kind of intracellular enzyme with function of DNA methylation expressed in multiple tissues. These data suggest its protein has a critical role with basic function in cell and accordingly primary process of life.