Antiapoptosis Gene, Livin Expression In Non-Small-Cell Lung Cancer And Its Correlation With P53, Bcl-2, Hyperplasia And Apoptosis

Lung cancer was one of the most dangerous diseases to mankind and both the morbidity and mortality of which increasingly rose in the world. In the developed countries and metropolises of China, its mortality was on the top list of tumors. Although interventions such as speration, radiotherapy, chemiotherapy and biological therapy were used, there was no obvious improvement in prognosis , for the development of lung cancer was a complicated course with multi-gene change. So, the further researches of molecular biological feature were important to the diagnosis, therapy and prognosis of lung cancer.Tumor was a kind of diseases mainly resulted from cell's mutation. Uncontrolled proliferation was one of the important characters of the tumors. The activation of oncogene and the inactivation of antioncogene had been proved to be important to the development of tumors in recent researches. Further researches showed that some kinds of these genes caused the normal death of the cells. There was a close relationship between tumor agenesis and the control of abnormal cell death. So, apoptosis and tumor became a new focus in molecular biological researches. Apoptosis was important to the stability of the internal environment of the body. The chaos of the regulation of apoptosis contributed to cell proliferation and the development of tumors, which was one of important mechanisms in the forming of the tumor.The inhibitor of apoptosis protein (LAP) had a main function to inhibit apoptosis. Livin found recently was a novel inhibitor of apoptosis family members. The distribution of Iivin had a high selectivity, tissue-specific expression in human fetal tissues and nearly all solid malignancies. There were little expressionof Livin in normal adult tissues, such as spleen, thymus, prostate, testis, small lintestin, colon, ovary, liver, lung, brain, skeletal muscle, lymph cell and peripheral blood which was different from another antiapoptosis gene Bel-2. Livin expression had correlation with the patient's prognosis and cell proliferation.At present, the reports about livin expression in lung cancer were absence and the correlation between its expression and clinical pathology wasn't clear. There were no reports about the relationship between Livin expression and cell apoptosis and proliferation. RT-PCR method (reverse transcription-polymerase chain reaction) was used to detect livin mRNA in 45 cases of non-small-cell lung cancer(NSCLC);western-blot analysis was used to detected the expression of Livin protein. The expression of livin, p53 and Bcl-2 protein in NSCLC and its neighboring tissues was discussed by immunohistochemical tests and used im-munofluorescence method to display the expression of livin gene in BE1 and LH7 cell line. We used flow cytometry to detect the level of NSCLC cell apoptosis and proliferation and tried to draw a conclusion that the expression of livin correlated with apoptosis and proliferation.Materials and methodsMaterialsAll samples during May 2004 to December 2004 were collected in the department of thoracic surgery, First Affiliated Hospital China Medical University. There were 37 male, 23 female patients with an average age of 48.5 (from 22 to 75) years and 45 cases of NSCLC, 15 cases of benign tumors (inflammatory pseudotumor 12 cases, harmartoma 2 cases and one case of sclerosing hemangio-ma of the lung). 35 cases of normal tissues beside malignant tumors were obtained. According to 1997 UICC staging criteria, 26 cases were classified as stage I ,6 cases II , 10 cases H and 3 cases IV. According to pathological subtype,there were 19 cases of squamas carcinoma,2leases of gland tumor and 5cases of big-cell tumor. According to the differentiation degree, the cases were classified as highly differentiated (13cases) and lowly diferentiated (32cases). No cases were received rediotheropy or chemiotheropy before operation.Methods1. RT-PCR for Livin geneTotal RNA from each sample (contain lOOmg tissue) were isolated by u-sing Trizol solution, then the concentration and purity of RNA PCR Buffer 1 jxl, dNTP ljxl ,MgCl2 2jjd .Random 9mers 0. 5jxl ,RNA ljxl ,RNase inhibitor 0. 25jxl,Reverse Transcriptase 0.5|il,ddH2O 3.75jxlo Followed by the condition;10 min at 30T:, 30 min at 42t ,5 min at 9960% ,many cells were yellow or brown yellow;moderate positive ( ++ ) , positive cell number and color intensity were between weak positive and intense positive. The section having no obvious positive cells was defined negative.4. ImmunofluorescenceBEl and LH7 cell line derived from PG cell line was established from human pulmonary giant cell carcinoma (a gift from Dr. Zheng Jie, Beijing Medical University, China) , and BEl cells have high metastasis ability. The cells (2 x 105) were cultured in 35 cm2 plastic flasks respectively, which put cover slips in advance, with RPMI 1640 medium (GiBCO Inc. ) containing 10% fetal calf serum (GiBCO Inc. ) , 2g/L NaHCO2, lOO.U/ml penicillin G and 100 U/ml streptomycin at 37 C in a humidified atmosphere (5%CO2 and 95% air) for 24 hours. Then the slips were taken out and fixed with dimethylketone for lOmin atAll the sections treated with 0. 2% TritonX-100 for 15 min, and blocked with normal goat serum at 37 °C for 30 min. Then the sections were incubated with livin-specific rabbit monoclonal antibody(1:100,ALEXIS, USA) , Bcl-2-specific mouse monoclonal antibody(1: 100,Boshide, China) and p53-specific mouse monoclonal antibody (1: 100,Maixin, China) respectively overnight at 4X.. Washed in PBS thrice, each for 5 minutes, then sections were incubated with FITC and rhodamine labeled secondary antibody respectively ( 1:200, Zhongshan Golden Bridge, Beijing, China) for30 min at 371. The nuclei were counterstained with DAPI (l:200, Huamei, China) for 30 min at 37X1. The sections were observed with fluorescence microscope.5. Flow cytometry to detect apoptosis and proliferationThe tisues were resected in deep-low temperature. The liquid containingsingle cell was regulated by PBS into 1 X 106 cell/ml. PI liquid (PI 5mg. Rnase 2mg. TritonX-100 lml, normal saline 65ml, sodium citrate lOOmg, add distilled water tolOOml) lml was added to one, 30min at 4X1 in dark, and FTTC-Annexin V and PI each 5jxl were added to the other, 15min at room temperature in dark.Stained cells were analyzed by means of a FACS Calibu ( B. D ,USA) and relative modifat software in Macintosh 9. 5 computer. 10000 cells were counted in every specimen. Before detection, the CV was regulated under 3. 0%. PI could reflect activity of proliferation. The early apoptosis cells were counted on two-dimensional lattice picture.6. Statistical analysisChi-squared test was used in data of quantity. T-rest was used in data of quality. The data was analyzed by the SPSS12.0 software.Results1. The expression of livin mRNA;71.1% in NSCLC (32/45) , 6.67% in benign tumors (1/15) and 5. 71% in normal tissues (2/35) (P <0.01).2. The expression of livin protein: 62.22% in NSCLC(28/45) ,6.67% in benign tumors (1/15) and 2. 86% in normal tissues (1/35) (P <0.01).3. Correlation between expression of Livin and clinicopathological factors;There was no significant correlation between positive livin expression andage, sex, cigarette smoking, histological subtype, tumor differentiation, lymph node metastasis or the TNM staging(P > 0.05).4. The expression of livin protein in NSCLC had close relationship with the expression of p53 and Bcl-2:Among the p53 protein expression, the ration of positive expression of livin was 75.86% (22/29) and the ration of negative expression of livin 24.14% (7/29) (P < 0. 05). Among the Bcl-2 expression, the ratio of positive expression of livin was 80. 65% (25/31), and the ration of negative expression of livin 19.35% (6/31) , with significant deference (P <0.01).5. Expression of livin,p53 and BCL-2 in BE1 and LH7 cell line;The immunofluorescence showed that livin and BCL-2 protein displayed cy-toplasmic expression, and p53 protein displayed nucleus in the cell lines.6. The expression of livin gene in NSCLC had correlation with cell apoptosis:The average ratio of early apoptosis in 45 cases of NSCLC was(3.08 ±0. 94) %. The average apoptosis ratio of 32 cases with positive livin mRNA expression and 13 cases with negvative expression was respectively (2. 88 ±0. 83) % and (3.58 ± 1.02) % , with significant deference (t = 2. 390, P < 0. 05). The average apoptosis ratio of 28 cases with positive livin protein expression and 17 cases with negative expression was respectively (1. 82 ±0.58) % and (3. 65 ± 1.08) % , with significant deference (t = 7.405, p <0.01).7. The expression of livin gene in NSCLC was correlated with tumor cell proliferation:The average proliferation ratio in 45 cases of NSCLC was (33. 92 ± 8. 16)%. The average PI of 32 cases with positive livin mRNA expression and 13 cases with negative expression were respectively (37. 29 ± 11. 35% and (27.18 ±4. 35) % , with significant deference (t =3.095, P <0. 01). The average PI in 28 cases with positive livin protein expression and 17 cases with negative expression were respectively (41. 27 ±11.81)% and (29. 96 ± 9. 55 ) % , with significant deference (t = 3. 337, P < 0.01).Conclusion1. livin gene may play a role in NSCLC development and increased expression of livin mRNA may serve as a new target for lung cancer diagnosis and treatment .2. There was no significant correlation between positive livin expression and age, sex, cigarette smoking, histological subtype, tumor differentiation, lymph node metastasis or the TNM staging.3. The expression of livin protein in NSCLC had close relationship with the expression of p53 and Bcl-2.4. The immunofluorescence showed that livin protein displayed cytoplas-mic expression.5. The expression of Livin gene in NSCLC had correlation with cell apoptosis.6. The expression of Livin gene in NSCLC was correlated with tumor cell proliferation.