Studies On Radiaton Induced Expression Of LRIGx/RIGb And Its Effects On Cell Proliferation

LRIGx (low dose radiation-induced gene) is a novel radiation-induced gene previously discovered by our lab in 0.5Gy v-ray irradiated human embryonic lung (HEL) cell by means of mRNA differential display technique. Then after cDNA cloning identification and bioinformatic analysis, LRIGx gene was mapped on human chromosome 20ql 1.2-12.Previous study revealed that the mRNA level of LRIGx was significantly up-regulated in A549 cells at 2~4 hours after 0.2Gy of y -ray exposure. However, the induced level of LRIGx mRNA by 2.0 Gy was not so significant as that by 0.2 low dose exposure. Using synchronized cells, an expression peak of LRIGx mRNA was observed in G2/M phase cells.Recently, this gene was also isolated by a UK group in searching for new members of SWI2/ SNF2-related genes family. They named the gene CHD5 for chromodomain-helicase-DNA binding-5 (changed to CHD6 lately).For the first time, our lab discovered that LRIGx/CHD6 expressed at least 3 transcripts. Based on the cloned cDNA with the length about 1.8kb (transcript named RIGb), we carried out the following functional studies on this gene. Major results are as follows.Firstly, further investigate its mRNA expression changes induced by differential doses of 7-ray irradiation in Hela cell. Results indicated that 0.5 Gy v-ray irradiation induced significantly mRNA expression and its induced expression level varied greatly with time course after exposure. Similar to our previous study in A549 cell, 4 Gy exposure could not induce any detectable changes in its mRNA expression. However, when irradiation dose was increased to 20 Gy, an increased expression level was alsoobserved.Secondly, compare mRNA expression level of RIGb among eight normal human tissues. We found that it had substantial expression in heart, liver and testis, and moderate expression level in spleen and muscle. In contrast, very low expression was observed in lung, stomach and kidney tissues.Thirdly, we have constructed RIGb sense and antisense expression vectors and obtained their stable transformed HeLa cell lines. Our results demonstrated that up-regulation of the sense gene expression could result in a contacting inhibition effect on Hela cell growth. The cell cycle analysis showed a prolonging G1 progression in the sense RIGb expressing cells. On the other hand, down-regulation of its expression by antisense RNA promoted G1/S transition in the early stage, and resulted in an increased ratio of S phase and G2/M phase cell population but did not display obvious effect on the cell growth.At last, in order to elucidate cellular and molecular mechanisms involved in the regulation of RIGb on the cell cycle and cell growth control, we have successfully constructed the GFP-fused and HA-tagged expression vectors. This may pave the way for the further investigation on the sub-cellular localization of the gene product and its interacting protein(s).