Study Of Differentially Expression Profile Of Long Noncoding RNAs In Human Osteosarcoma

Objective:Long noncoding RNAs （lncRNAs） have a critical role in genome regulation. The contributions of lncRNAs to osteosarcoma remain unknown. Here, we describe the expression profile of lncRNAs in osteosarcomas compared with paired adjacent noncancerous tissue using microarray analysis and study their biology funtions prelininarily.Methods:Firstly, we extracted total RNA from nine clinical samples of primary osteosarcoma and their paired adjacent noncancerous tissues.Labeling and hybridization of RNA were precessed and we got the differentially expression profiles after a series of procedures. Secondly, Six over-regulated lncRNA and four under-regulated from the differentially expression profiles were validated by RT-PCR in nine clinical samples of primary osteosarcoma and their paired adjacent noncancerous tissues, using GAPDH as inter reference. Thirdly, Bioinformatic analysis (gene ontology analysis, pathway analysis was used for further research on the differentially expression profiles based on NCBI RefSeq, UCSC, RNAdb, LncRNAs databases and we constructed a coding-noncoding gene co-expression network that included the differentially expressed lncRNAs and targeted coding genes by calculating PCC （PCC≥0.95）.Results:Compared with adjacent noncancerous tissues, the lncRNA and mRNA expression profiles are significantly differentially expressed in the primary osteosarcoma tissues. An average of3528lncRNA （range from2723-4537） was significantly over-regulated and an average of3958lncRNA （range from3469-4368） was significantly under-regulated （foldchange≥2.0, P≥0.05）.403lncRNAs were consistently over-regulated and798lncRNAs were consistently under-regulated in all samples analyzed. ASLNC02724was the most over-regulated lncRNA （foldchange:7.23） and ASLNC05129was the most under-regulated lncRNA （foldchange:27.39）. Over-regulated lncRNA was more common than under-regulated lncRNA. An average of2604mRNA （range from1394-3710） was significantly over-regulated and an average of2344mRNA （range from1513-2867） was significantly under-regulated （foldchange≥2.0, P≦0.05）.986mRNAs were consistently over-regulated and933mRNAs were consistently under-regulated in all samples analyzed. NM₀13227was the most over-regulated lncRNA （foldchange:33.17） and NM₀05963was the most under-regulated lncRNA （foldchange:170.32）. Over-regulated mRNA was equal to under-regulated mRNA. Six over-regulated lncRNAs:ASLNC21868, ASLNC22124, ASLNC23844, ASLNC24587, BE503655, BC050642and four under-regulated lncRNAs:ASLNC00339, ASLNC11435, ASLNC13387, ASLNC18814were validated by RT-PCR in in nine clinical samples of primary osteosarcoma and their paired adjacent noncancerous tissues. The results demonstrated that ASLNC21868, ASLNC22124, ASLNC23844, ASLNC24587, BE503655and BC050642were over-regulated and ASLNC00339, ASLNC11435, ASLNC13387and ASLNC18814were under-regulated in the osteosarcoma samples compared with normal samples. The RT-PCR results and microarray data are consistent. We found that the highest enriched GOs targeted by the over-regulated lncRNAs were cell adhesion （ontology:biological process）, extracellular matrix （ontology:cellular component） and protein binding （ontology:molecular function） and that the highest enriched GOs targeted by under-regulated lncRNAs were muscle system process （ontology:biological process）, contractile fiber （ontology:cellular component） and structural constituent of muscle （ontology:molecular function）. Pathway analysis indicated that34pathways corresponded to over-regulated transcripts and that the most enriched network was "ECM-receptor interaction-Homo sapiens （human）" composed of22targeted genes. Furthermore, this analysis showed that32pathways corresponded to under-regulated transcripts and that the most enriched network was "Hypertrophic cardiomyopathy-Homo sapiens （human）" composed of24targeted genes. We constructed a coding-noncoding gene co-expression network that included six over-regulated lncRNAs: ASLNC02419, ASLNC21868, ASLNC23844, ASLNC24079, BE050642, BE503655and six under-regulated lncRNAs:ASLNC00339, ASLNC04683, ASLNC08248, ASLNC11435, ASLNC13387, ASLNC23339and227targeted coding genes. The lncRNAs and coding genes that had Pearson correlation coefficients equal to or greater than0.95were chosen to construct the network.Conclusions:（1） LncRNA microarray analysis was ideal and effective way to screen the differentially expression profiles in osteosarcoma;（2） Our results were the first to reveal differentially expressed lncRNAs in osteosarcoma and some of the differentially expressed lncRNAs were validated by RT-PCR to promote the credibility of results. Prelininary bioinformatic analysis revealed that lncRNA was involved in the occurrence and development of osteosarcoma;（3） lncRNAs may be novel candidate biomarkers for the diagnosis of osteosarcoma and potential targets for gene therapy...