Relationship Between Apoptosis Inhibitor Gene Survivin And Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma(NPC) has a striking geographic and ethnic distribution, It occurs with high frequency in southern China and Southeast Asia. The familial aggregation and genetic susceptibility have been regarded as the most important cause of NPC, virus as EBV infection , diet and environment are the importment cause too.It is a prevalent acquaintance on modern molecular biology that the cancer is closely related to many genes.Survivin,a member of the inhibitor of apoptosis protein(IAP) family, is down-regulated and almost undetectable in normal adult tissues, but it prominently reexpress in all of the most common cancers and human embryonic development tissue.Survivin gene locates to human chromosomes 17q25, contains 4 exons and 3 introns,there is an open reading frame which are 426bps on its coding strand. Survivin precursor mRNA product at least four transcript variants by splicing selectively, but the full-length natures only three of them have been determined on NCBI GenBank.Survivin inhibits cell apoptosis and regulates cell mitosis, it is important factor which contacts the cell cycle and the cell apoptosis interface,and its primary biologic function as follows: accelerating cell division and multiplication;being concerned with building blood vessel; being concerned with virus infection;accelerating tumour development.RNA interference(RNAi) is the process of sequence-specific degradation of homologous mRNA triggered by double-stranded RNA found in many organisms.This is a specifically mechanism involved in kinds of proteins to complete the interference function. Structure of siRNA affects which strand will be assembled into RISC. Another role of siRNA is directing RITS complex to bind with homologue chromosome, and then induces heterochromatinization. Although systemic silence induced by dsRNA is observed in Caenorhabditis elegans and plants,this progress is probably transmembrane protein-dependent, and mostly, the systemic silencing is controlled by multi-factors. As a technically simple and an effective genetic tool which can exert effect on the expression of gene and substitute for gene knock-out technique in some degree,RNAi phenomena have been broadly validated in diverse mode lorganisms such as Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana and Neurospora crassa. Simultaneously, study on molecula rmanchnism of RNAi, which might be involved in the level of post-transcription, translation, genome methylation or conduction of silencing signals, is now making unceasing progress. Clear elucidation of the molecular manchnism could provide important theoretical references and powerful tools for the practical application in this field where RNAi may be put into the use of systematic gene screening,the discovery of new genes and the gene therapy for human tumor or other refractory human diseases.We used immunohistochemical S-P staining, in situ hybridization and fluorescent quantitation RT-PCR to detecte 64 NPC and 30 chronic nasopharyngitis tissue's survivin protein,mRNA and the relative quantification of gene expression. We used fluorescent quantitation RT-PCR to detecte cell line CNE-2 the relative quantification of survivin gene expression.And we used 3 siRNAs to interfere survivin RNA of CNE-2 cell, then detectived cell's inhibition of proliferation and apoptosis.We found that the positive expression rate for survivin protein and mRNA in NPC tissue were 71.9%(46/64)and 65.6%(42/64) respectively, we scored tumor high expression level when >25% cancer cell stained positive, thus the high expression of survivin protein and mRNA were 59.4%(38/64) and 46.9%(30/64) respectively.The positive expression rate for survivin protein and mRNA in chronic nasopharyngitis were 23.3%(7/30) and 26.7%(8/30) respectively.We regarded homekeeping gene GAPDH as intemal control,then the relative quantification of survivin gene expression of CNE-2 was 2.48×10-3,while they were from 0 to 1.56, 4.16×10-2 on average in NPC tissue ,and frome 0 to 2.20×10-3,4.42×10-4 on average in chronic nasopharyngitis tissue.The survivin expression of NPC tissue are higher than chronic nasopharyngitis(P<0.01, respectively),but the difference of expression of survivin protein ,mRNA and the relative quantification of gene expression in NPC of ages and sexes of patients were undistinguished(P>0.05 respectively).The high expression of survivin protein and mRNA inⅢ+Ⅳstage NPC were 61.1%(11/18),72.2%(13/18)while they both were 50.0%(2/4) inⅠ+Ⅱstage NPC.After interfered cell line CNE-2 by siRNA,we found among 3 siRNAs, siRNA3: 5'-GGCAGUGGCCUAAAUCCUUtt-3'(sense);5'-GUGUGACAGAUAAGGAACCtt-3'(antisense) was the most efficient. The result of fluorescent quantitation RT-PCR showed that the relative quantification of survivin gene expression of CNE-2 was 3.42×10-12,which far less negative control and blank control's,they were 4.99×10-5 and 3.39×10-3 respectively.But, the relative quantification of survivin gene expression of CNE-2 interfered by siRNA1 was 4.83×10-5,which is undistinguished to the negative control's.After interfered cell line CNE-2 by siRNA3,we detectived the CNE-2 cell's inhibition rate of proliferation, found in some concentration scope,in the same interval,more siRNA3 interfered, more inhibition of proliferation of cell.After transfered siRNA3 from 12.5nM to 100.0nM into CNE-2 cell 24h,the inhibition rate of proliferation was from 5.38±0.71% to 27.97±2.90% correspondingly. Detectived proliferation of CNE-2 cell,we found in some interval, more the siRNA3 transfered,more inhibition of proliferation of cell.After transfered 50nm/L siRNA3 into CNE-2 cell 24h,48h and 96h,the inhibition rates of proliferation were 16.31±1.12%,52.12±2.90% and 84.21±1.66% respectively. longer after the siRNA3 transfered,more inhibition of proliferation of cell.The analysis of cell apoptosis after transfered siRNA3 by flow cytometry showed that the apoptotic rate increased by degrees correspond to the concentration of siRNA3 when the concentration of siRNA3 was from 12.5nM to 100.0nM. And when 12.5nM siRNA3 transfered into CNE-2 cell,the apoptotic rate was 6.10±0.42%, which was 18.58±0.75% when the concentration of siRNA3 add to 100.0nM。It inhibited proliferation of CNE-2 cell and accelerated it's apoptosis when survivin RNA's physiological process been inhibited and/or the process of survivin RNA→protein have been inhibited.Maybe,survivin could being the target of RNAi—the new bioremediation on NPC clinic cure.Overall, survivin gene may play some roles in pathogenesis of NPC,and it will be helpful for diagnosis, clinical staging ,prognosis and biotherapy of NPC.As the further study on survivin become more and more , It will be important factor of searching NPC's pathogenesis mechanism ,and the biotherapy which target survivin gene will contribute to NPC therapy greatly.