Detection Of Livin Isoforms In NSCLC And Research On Induced Apoptosis Of Lung Cancer Cell By Isoform-Specific Gene Silencing

Non-Small Cell Lung Cancer (NSCLC) is so far a hard-to-cure disease, to which increasing anti-apoptosis gene expression, decreasing tumor cell apoptosis, and increasing tumor cell drug resistance may contribute much. One strategy for tumor gene therapy is to inhibit expression of anti-apoptosis gene, selectively induce tumor cell apoptosis with the purpose of eliminating tumor cell rapidly and effectively. Yet, poor targeting rate and specificity weaken the apoptosis-inducing therapy. To induce tumor cell apoptosis effectively, we should select tumor cell- specific gene and inhibit its expression specifically. As a new technology of gene knock-down, RNA interference(RNAi)may be a optimized means of antisense nucleotide technology with following important features: ① High stability. ② High efficiency. ③High specificity. Hence, it may become a new means of tumor gene therapy. Livin, a novel inhibitor of apoptosis protein family member, has two isoforms (i.e. Livinα& Livinβ), which have 54bp difference in sequence and differ in anti-apoptosis function. Livin gene expresses restrictedly in tumor tissues and embryonic tissue. Therefore, analyzing the relationship between Livin and genesis, development, treatment and prognosis of lung cancer and setting up Livin isoform-specific RNAi expression system are important in research on biological function of Livin isoforms and specifically inducing apoptosis of tumor cell.Objective1. To research the expression of two Livin isoforms in NSCLC, and analyze the relationship between livin expression and tumor classification and therapy.2. To set up Livin isoforms expression system, and explore their different anti-apoptosis functions in radiotherapy and chemotherapy for tumor.3. To set up Livin isoform-specific small interfering RNA (siRNA) expression system, and explore its value in inducing apoptosis of tumor cell and clinical significance. Materials and Methods 1. The expression of Livin isoforms in NSCLC and its significanceSpecimens from operation of patients with NSCLC and their medical records were collected. The expression of livin in lung cancer tissue and two cell lines (A549& SPC-A1) were detected by RT-PCR & western blot, then analyzed with regard to pathological classification of tumor, radiotherapy and chemotherapy before operation. 2. Expression of livin isoforms in A549 and research on its functionTotal RNA of SPC-A1 was extracted, full-length cDNA of livin isoforms was gained by RT-PCR, then inserted into T vector by T-A cloning. Positive recombinants were gained. Sequencing was performed to guarantee correct sequence insertion. Subclone the inserted sequence into pcDNA3.1,to get expression vectors of the isoforms(pcDNA3.1-Livinα& pcDNA3.1-Livinβ).Transfect A549 with pcDNA3.1-Livinα& pcDNA3.1-Livinβ,respectively. Get positive clones by G418-resistance screening. Pick up monoclone for culture. MTT was performed to detect effects of chemotherapy drugs and rays on tumor cell growth. The transfected cells were exposed to 10Gy60CO. Then FCAS was used to analyze the effects of livin isoforms on A549 cell cycle with existence of X-rays. The untransfected cells were exposed to 2Gy60CO.Then RT-PCR was performed to detect Livin expression induced by X-rays.3. Set-up of Livin isoform-specific siRNA expression vector and research on its pro-apoptosis effectAccording to different sequence of Livinα& Livinβ cDNA and mutual BIR domain in mRNA, siRNA gene sequences were screened, designed and synthesized and inserted into expression vector pAVU6+27-Livin and pSilencer-Livin, respectively after being ligated with adaptors. After sequencing, the recombinant was transfected into SPC-A1.Positive clones were picked up and cultured. The specificity and silencing efficiency of siRNA in inhibiting the expression of livin isoforms were detected by RT-PCR and western blot. Inverted microscope and electron microscope were used to observe changes in cell morphology. MTT was performed to test effects of different chemotherapy drugs and rays on survival rate o...