The Research Of The Function And Signal Pathway Of Id1 In H460 By RNAi-mediated Gene Silence And Proteomics Method

Id(inhibitor of DNA binding and /or differentiation) is a member of the family ofhelix-loop-helix transcriptional regulatory proteins. The Id family of helix-loop-helix proteins sharea conserve sequence of a helix-loop-helix dimerization domain,and it does not possess a basicDNA binding domain and functions as a dominant-negative regulator of basic HLH proteins throughthe formation of inactive heterodimers with intact bHLH transcription factors. Hence, the name Idrefers to both inhibition of differentiation and inhibition of DNA binding. The present studyprovides evidence that the HLH protein Id is an important factor in cell differentiation andtranscriptional modulation of various cell cycle .And high levels of Id expression are frequentlydetected in various cancer cells. The Id1 had been detected at high level in lung cancer by quantity RT-PCR. We have studied thefunction of Id1 on human lung cancer big cell line (H460) by sense RNA technique and anti-senseRNA technique primarily .But the effect of the latter was indistinctive. So we silence the expressionof Id1 by RNAi to further study the function of Id1 gene in H460. We established the clonal cellstrain of stable silencing of Id1 by RNAi, and detected the changes between the control cell cloneand the RNAi cell clone by many cell biology techniques as well as proteomics method. Our research suggests that the growth and proliferation of the cancer cell were significantlyinhibited .the index of clone forming on low melting-point agar shows that the RNAi cell hardlygrow on the low melting-point agar .Id1 maybe directly induce cancer. we found that the RNAi cellcycle had be affected as the S period was increased 10% by the FCM .This results were consistentwith the growth index of RNAi cell . The present study provides evidence that the HLH protein Id1is an important factor in regulating cell cycle progression in H460. The expression of p21and p16 were increased in RNAi cell by Western Blotting. The cellperhaps be prevented into S period by the p21-CDK pathway after the silence of Id1 in H460. Onthe other hand, the pRb/p16 pathway may be actived by the up-regulation of p16 to inhibit the overproliferation of the lung cancer cell.However, there are more work to do to illustrate the interactionbetween Id1 and Rb. The results of sub-quantity RT-PCR show that the mRNA level of Id1 was reduced significantly,whereas Id3 and Id4 had no changes. Interestingly, the mRNA level of Id2 were increased .So wepresume that there may be some association between the Id1 and Id2.The low expression level ofId1 could be a signal acting on Id2 in the RNAi cell. So our research suggested the gene therapy oflung cancer may not work if we inhibit the expression of Id1 only. To further study the effect of Id1 and its possible pathway, we use proteomics method to identifiedfifteen proteins of the differentiation expressed proteins in RNAi cell. some proteins were related toprotein degradation pathway such as UBL1 and others may involved in the regulation ofreplication and transcription such as ZBED1 and PSF etc. Thus, Id1 protein may play an important role in H460 cell proliferation and regulation of cellcycle. Nevertheless, our results is critical to understanding the molecular mechanisms of cell cycleregulation in lung cancer cell and intervening in lung cancer growth. At the same time, it is animportant academic foundation and reference to the gene therapy of cancer by RNAi.