| Objective and backgroundLung cancer is one of the most common malignancies with rapid growth rates, and nearly 80% of lung cancer is non-small cell lung cancer (NSCLC), although surgical resection is the most effective method in the treatment of early NSCLC, nearly 60% of the patients in the hospital has belonged to late and lost optimal opportunity by surgery and multidisciplinary radical therapy. Therefore chemotherapy has become the main means in the treatment of lung cancer. Owing to chemotherapy resistance or chemotherapy process resistance occurs in NSCLC cell, the prognosis of NSCLC is poor, and one-year survival rate of NSCLC is only 30～35 %. Many studies have showed that apoptosis block in lung cancer cells is a kind of important resistance to chemotherapy. Many chemotherapy drugs induce apoptosis through damaging DNA, tumor suppressor p53 is extremely important genes, missing or mutations in the p53 genes mediated apoptosis function defects is the important mechanism of chemotherapy drug resistance in small cell lung cancer. Apoptosis-stimulating proteins of p53 (ASPP) are regulative proteins which mediate apoptosis, ASPP can not only specifically activate apoptosis function of p53, but also activate apoptotic function p63 and p73 protein which are in the same group with p53, thus to adjust cell apoptosis of tumor cell which express p53 or not. The wild-type p53 is very important tumour suppressor, which regulate the proliferation of cells through either cell-cycle arrest or apoptosis. Understanding why p53 is unable to perform its role as a tumour suppressor in wild-type p53 tumours has been the focus of extensive research. Studies have showed that the apoptotic function of wild-type p53 is stimulated by two members of the ASPP family, ASPP1 and ASPP2. Binding to the DNA domains of p53, ASPP1 and ASPP2 specifically stimulate the transactiva- tion function of p53 on promoters of proapoptotic genes (such as Bax, Fax, PIG3). Therefore, the apoptotic function absence of wild-type p53 may be associated with expression of ASPP1, ASPP2.At present, much of current knowledge of ASPP family and cancer, especially the knowledge of ASPP family and NSCLC, is from in vitro studies, the investigation of ASPP expression in NSCLC tissues and monitoring chemo- therapeutic patients with NSCLC is not reported in domestic. Therefore, this study discussed the expression of ASPP in different p53 genetic background of NSCLC cell lines, and investigated the expression change of ASPP in different cancer cell lines acting with cis- diamine-dichloroplatinum(CDDP).We also detected the ASPP expression of NSCLC tissue, paracar- cinomatous tissue, normal lung tissue, and monitored the ASPP expression in peripheral blood of chemotherapy patients with NSCLC. In order to find new molecular targets for the therapy of NSCLC, the relationship between ASPP expresstion and NSCLC was discussed, and the influence of chemotherapy drugs on the expression of ASPP in NSCLC treatment was also investigated.Material and MethodsLung adenocarcinoma cell line A549 (wild-type p53) and lung squamous carcinoma cell line NCI-H157 (Mutant-type p53) were selected to tudy.Non-small cell lung cancer tissues, paracarcinomatous tissues, normal lung tissues from 37 patients were selected from our hospital, which have 15cases of lung squamous carcinoma, 16 cases of lung adenocarcinoma, 4 cases of differentiated cell carcinoma and 2 cases of giantcellcarcinoma.1.Building high concentration transcription plasmid in vitro as ASPP1 standard, and cultivate the HL-60 cells, high concentration RNA was ex- tracted and purified as ASPP2 standard. According to the known sequences, specific primers were designed, and real-time quantitative fluorescence PCR method was established for the detection of ASPP1 and ASPP2 mRNA expression.2. The expression of ASPP was detected in NSCLC tissue, paracarcino- matous tissue, normal lung tissue by real-time fluorescence quantitative PCR, and the expression of p53 was observed by immunohistochemistry staining.3. NSCLC cell lines A549 (wild-type p53) and NCI-H157 (Mutant-type p53) with p53 different genetic background were cultured, chemosensitivity of the cell lines to CDDP was analyzed by MTT assay, and cell morphology change was detected by electron microscopy. Real-time fluorescence quanti- tative PCR was also used to measure the expression levels of ASPP mRNA.4. ASPP expression was detected in peripheral blood of NSCLC patien- ts and controls, and monitoring ASPP expression in patients with NSCLC chemotherapy.5.Constructed the eukaryotic expression vector of iASPP, and trans- fected them into A549 and NCI-H157 cells, at the same time the mRNA and protein of iASPP were detected by RT-PCR and western blot respectively. SPSS13.0 software was adopted to analyze the experiment data.Results1. Building high concentration transcription plasmid in vitro as ASPP1 standard, and cultivate the HL-60 cells, high concentration RNA was extracted and purified as ASPP2 standard, According to known sequences, specific primers were designed, and real-time quantitative fluorescence PCR method was established for the detection of ASPP1 and ASPP2 mRNA expression. The satisfactory amplification curve and standard curve were obtained in the quantitative standard substance with the single melting peak, which has good stability and repeatability.2. The expression levels of ASPP1 and ASPP2 mRNA in NSCLC tissue, paracarcinomatous tissue, normal lung tissue increased gradually, the levels of ASPP1 and ASPP2 mRNA in cancer tissue were lower than that in paracarcinomatous tissue and normal lung tissue . The levels of ASPP1 and ASPP2 mRNA in p53 negative cancer tissue were significantly lower than that in p53 positive cancer tissue respectively.3. The IC50 values of NCI-H157 and A549 cells were 3.7, 10.5μg/ml respectively. This significant difference suggested that NCI-H157 has high chemosensitivity to CDDP. Mean ASPP1 levels were a statistically signifi- cant 2.66-fold higher in NCI-H157 cell compared to A549 cell, and mean ASPP2 levels were a statistically significant 6.98-fold higher in NCI-H157 cell compared to A549 cell.4. The levels of ASPP1 and ASPP2 mRNA were (2.49±0.32)×103, (7.00±1.17)×103 in peripheral blood of NSCLC patients, and were signifi- cantly lower than those of control group. The levels of ASPP1 and ASPP2 mRNA in patients with NSCLC after 2 periods of postoperative chemotherapy were higher than those of chemotherapy before.5. Compared with the group without transfecting, the expression of iASPP mRNA and protein were lower than that in the group of transfecting.Conclusion1. The standard substances were selected successfully and can be applied to the quantitative test. It was a sensitive, specific and stable method to analyse the ASPP1 and ASPP2 mRNA by real-time quantitative fluorescence PCR.2. The decreased levels of ASPP1 and ASPP2 mRNA expression in NSCLC tissue had close relationship with the development of NSCLC. The levels of ASPP1 and ASPP2 mRNA in p53 negative cancer tissue were significantly lower than that in p53 positive cancer tissue repectively, which gave us a clue that the development of NSCLC with wild-type p53 was associated with decrease of ASPP expression.3. The expression of ASPP1, ASPP2 mRNA in p53 mutant-type cell lines was higher than that in p53 wild-type cell lines, and ASPP protein expression was stronger in NCI-H157 than that in A549 cell line.4. The expression of ASPP had close relationship with chemotherapeu- tic sensitivity of NSCLC. The NSCLC patients with elevated ASPP expres- sion had higher sensitity to chemotherapeutic drug and better prognosis. The relationship between chemosensitivity and ASPP mRNA expression made it possible that ASPP may be a new-brand target for cancer therapy.5. The construction of recombinant expression vector iASPP-shRNA could inhibit the expression of iASPP gene, which results in the cell apoptosis increasing.
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