Mechanism Research Of PLZF In Regulating Apoptosis Of Lung Squamous Cell Carcinoma And Adenocarcinoma Cells

PLZF(promyelocytic leukaemia zinc finger), or ZBTB16 (zinc finger and BTB domain containing 16) is a transcription factor that plays a role in the signaling networks that regulate cell cycle, proliferation differentiation and apoptosis. Recent studies indicate that down regulation of PLZF gene expression not only contributes to acute promyelocytic leukemia but links to occurrence of malignant solid tumors such as prostatic csarcinoma and malignant melanoma. Evading apoptosis through down-regulated PLZF expression constitutes a mechanism of tumor growth. The function of PLZF in lung cancer is unknown. Microarray was used to examine the expression of PLZF mRNA in five non small cell lung carcinoma tissues and adjacent normal tissues. The microarray results showed that the level of PLZF mRNA expression in the lung cancer tissues was significantly lower than that in adjacent normal tissues. In this study,it was found PLZF was down-regulated in non small cell lung cancer tissue compared with that in adjacent normal tissue. In addition, there was a significant correlation between PLZF expression and apoptosis, tumor size and pathological types. The mechanism whereby PLZF induced apoptosis and growth inhibition in lung squamous cell carcinoma and adenocarcinoma was also investigated.Part 1 PLZF expression in non small cell lung cancer and its clinical significanceObjectives:To examine the expression level of PLZF in NSCLC tissuses, and determine associations between PLZF expression and age, gender, nodal involvement, clinical phasing, pathological types and apoptosis.Methods:1) Total RNA of 119 pairs of NSCLC samples (tumor tissues and their corresponding normal tissues) was extracted and reversely translated into cDNA. Using real-time quantitative polymerase chain reaction, the relative level of PLZF mRNA was detected.2) 119 pairs of NSCLC samples (tumor tissues and their corresponding normal lung tissues) were made into three tissue microarrays. PLZF protein expression was detected by immunohistochemistry assay using the monoclonal antibody against the extracellular domain of PLZF via the two-step immunohistochemical staining. All samples were examined by light microscopy and scored semi-quantitatively on the basis of the intensity of the staining reaction and the percentage of cells that displayed immunoreactivity. PLZF expression was predominantly in cell nuclei. The samples were divided into four grades according to the staining intensity:0 (negative staining),1 (weak staining),2 (medium staining), and 3 (strong staining). The percentage of positive staining cells was defined as 0 for 0-25%,1 for 26-50%,2 for 51-75%, and 3 for 76～100%. The final score was determined as the product of intensity score and the proportion. A cancer sample was defined as a reduced or a preserved type of PLZF expression if its final score was less than or the same as that of its corresponding normal tissue.3) TUNEL (TdT-mediated biotinylated-dUTP nick end labling method) was adopted to analyze apoptosis of lung squamous cell carcinoma and adenocarcinoma cells. In each case,100 cells which need to detected each in 5 different fields, totaling 500 cells, were evaluated at high magnification (×400), and the apoptotic index (AI) was determined as the number of apoptotic cells per 500 cells.4) The results were analyzed statistically.Results:1) 103 (86.6%) of these lung cancer tissues showed lower levels of PLZF mRNA compared with the corresponding normal tissues. Immunohistochemistry indicated that protein changes were coincident with the PLZF mRNA expression.2) There was no significant difference between PLZF expression and age, gender, nodal involvement and clinical phasing, but PLZF expression was significantly associated with advanced T stages and pathological types.3) AI of lung squamous cell carcinoma and adenocarcinoma tissues with downregulated PLZF expression was significantly lower than that of cancer tissues with normal PLZF expression.Conclusions:1) Abnormal expression of PLZF may participate in the carcinogenesis and progression of lung squamous cell carcinoma and adenocarcinoma.2) Down-regulation of PLZF gene expression may be the possible mechanism whereby cells evade apoptosis in lung squamous cell carcinoma and adenocarcinoma.Part 2 PLZF promotes the course of apoptosis in lung squamous cell carcinoma and adenocarcinoma cellsObjective:To verify the function and clarify the mechanism of PLZF in promoting the course of apoptosis in lung squamous cell carcinoma and adenocarcinoma cells in vitro.Methods:1) PLZF expression plasmid was transfected into lung cancer A549, Calu-3,H226 and H520 cells.2) Cells in untreated and treated groups were evaluated for apoptosis by flow cytometry (FCM). Annexin V binding was evaluated using bivariate FCM, and cell staining was determined by fluorescein isothiocyanate-labelled Annexin V, simultaneously with dye exclusion of propidium iodide (PI). The Annexin FITC-/PI- population was regarded as normal healthy cells, while Annexin FITC+/PI+were taken as late apoptosis, Annexin FITC+/PI-cells as early apoptosis, and Annexin FITC-/PI+ as necrosis.2) The level of the key genes mRNA and protein of apoptosis pathways (including Fas,FasL,Bax,Bcl-2, caspase-3,8,9, XIAP and Survivin) were detected by real-time quantitative polymerase chain reaction and Western blotting.3) Apoptosis inhibitor genes were selected as target genes. New recombinant plasmid was constructed by the Cloning of the target gene promoter, and the new recombinant plasmid and PLZF expression plasmids were co-transfected into lung squamous cell carcinoma and adenocarcinoma cells.Results:1) The highest rate of apoptosis in cells transfected by PLZF expression plasmid was observed by FCM. The apoptosis rate in PLZF gene transfected cells significantly increased compared with the void-vector transfected cells and untreated cells 24 h after transfection. With the transfection time prolonging, the apoptosis rate in PLZF gene transfected cells rose significantly.2) The expression level and activity of PLZF in transfected cells reached the peak 72 h after transfection. Fas, Bax, caspase-3,8,9mRNA expressions were significantly higher in PLZF gene transfected cells than those in void-vector transfected cells and untreated cells, and the expression of Bcl-2 mRNA was significantly lower in PLZF gene transfected cells than that in the other cells. There was no significant difference in FasL, Survivin and XIAP mRNA expression level between treated and untreated cells. The effects of protein expression of the above gene were similar to those of mRNA expression.3.Bcl-2 was a target gene of PLZF.Conclusions:PLZF could promote apoptosis of lung squamous cell carcinoma and adenocarcinoma cells in vitro, and could induce apoptosis of lung squamous cell carcinoma and adenocarcinoma cells through the mitochondrion pathway by down-regulating Bcl-2. In addition, PLZF could induce the death receptor Fas/FasL apoptotic pathway, but the specific mechanism is unknown.Part 3 Research of PLZF in regulating apoptosis and proliferation of lung squamous cell carcinoma and adenocarcinoma cells in vivoObjective:To investigate influences of PLZF on proliferation and apoptosis of lung cells via recombinant adeno-associated virus-mediated PLZF gene transfection and discuss the feasibility of lung cancer therapy with Ad.PLZF.Method:1) Recombinant adenovirus Ad.PLZF was first constructed, and then transfected into lung A549, Calu-3,H226 and H520 cells. Each lung cancer cell line was divided into 3 groups:Ad.PLZF group, Ad.GFP group, and untreated group.2) The effect of PLZF overexpression on the proliferation of lung cancer cells was observed by FCM.3) An animal model of lung adenocarcinoma and squamous cell carcinoma bearing nude mice was established by subcutaneous injection of 2×107 lung cancer cells. All mice were assigned to 3 groups:Ad.PLZF group, Ad.GFP group and PBS group. The length (A) and width (B) of tumor per 10 days were measured, and gross tumor volume was calculated based on the formula V=0.5xAxB2. After 30-day treatment, the mice were sacrificed and their tumors were excised for immunohistochemical staining of PCNA and TUNEL.Result:1) Recombinant adenovirus Ad.PLZF was constructed successfully.2) The reproductive activity of transfected pAd.PLZF group dropped significantly. The size and weight of the transplantable tumor in Ad.PLZF groups were significantly smaller and lower than those of the other groups.3) The proliferation index of the transplanted tumor in Ad.PLZF groups was significantly lower than that of Ad.GFP group and untreated group,but the apoptotic index of the transplanted tumor in Ad.PLZF groups was significantly higher than that of the other groups.Conclusion:PLZF could inhibit cell proliferation in lung squamous cell carcinoma and adenocarcinoma, and may prove to be a potential effective target for gene therapy.