| Objective:Lnc-RI is a novel long chain non coding RNA,which was identified in our laboratory as one of the radiation-induced differentially expressed genes screened by gene chip technology. Up to now, there is still no research report about the function of this gene. Our previous study has found that lnc-RI plays an important role in mitosis and spindle formation, indicating that it is a novel mitotic regulatory molecule, but the mechanism is unclear. In this study, the gene chip technology was used to screen lnc-RI downstream target genes, and from which the target genes of mitosis regulation were searched. The detail mechanism has been clarified for the target gene in mediating the regualtion of lnc-RI on mitosis.Methods:1. First of all, a cellular model of knocked-down lnc-RI expression in He La cells was constructed by RNA interference, then gene chip technology was used to screen differentially expressed genes in the lnc-RI knockdown cells. The direct target genes of lnc-RI were identified by bioinformatics analysis, and from which the cell cycle(mitosis)related genes were searched. Using PCR and Western Blot analysis the target molecule involved in the cell cycle regulation of lnc-RI.2. Using lentiviral packaging system(LV-sh RNA-NC and LVsh RNA-lnc-RI) infection of HEK293 and He La cells, three days later,transfection of exogenous PLK1 gene, analyzing cell cycle and mitotic index by flow cytometry.3. Using luciferase reporter assay and RT-PCR analysis the promoter activity of PLK1 gene and the stability of m RNA, after depressed lnc-RI expression in cells.4. Using luciferase reporter assay combined with sequence mutation experiments to verify the binding sequence of lnc-RI and mi RNA-210.Results : 1. From data of the genechip expression profiling, 149 common targeted genes with more than 1.5 times of expression changes were found for two effective RNA interference sequences of lnc-RI,including 116 up-regulated genes, 33 down-regulated genes.2. The major biological pathways for those differentially expressed genes include: MAPK signaling pathway, cytoskeletal formation, cancer pathways, calcium signaling pathway, proteasome pathway, DNA damage repair, cell mitosis, spindle formation, etc.3. The analyses of genes interaction networks predicted that BCL2,PLK1, KDM2 A, THBS1, MAP3K1, FGFR3, etc were identified in the core nodes of the interaction networks, suggesting that they may play an important role in the biological effects produced by lnc-RI.4. Down-regulated lnc-RI significantly inhibited the expression of PLK1 on both m RNA and protein levels, which was in good agreement with the genechip data. Transfection of exogenous PLK1 gene was found to be able to attenuate the mitotic arrest induced by down-regulation of lnc-RI expression.5. The data of the dual luciferase reporter assay and RT-PCR analysis indicated that the down-regulation of lnc-RI expression did not change promoter activity of PLK1 gene, but promoted the degradation of PLK1 m RNA. lnc-RI was confirmed to be the target gene of mi RNA-210-3p. Downregulation of PLK1 resulted in a decreased expression of lnc-RI.Conclsions : 1. Using gene chip technology we have identified a number of lnc-RI downstream target genes, which are involved in a broad biological pathways, such as DNA damage repair, cell cycle regulation, chromosome modification, mitosis, spindle formation, etc.2. Focused on the molecular functional mechanism analysis of these critical genes, and presumed that PLK1, an important mitotic regulation molecular, may be the key molecular in mediating the regulation of lnc-RI on mitosis.3. It is confirmed that PLK1 is a critical downstream target of lnc-RI,through which regulates mitosis. Finally,it has been found that lnc-RI regulates PLK1 expression through competitively binding mi RNA-210-3p, and relieves its inhibition on PLK1. |
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