Study On Genetic Instability Of Nm23H1 Gene In Chinese With The Squamous Cell Lung Carcinoma

1. ObjectivesLung carcinoma is one of the most common human cancers, and the incidence of the cancer and its rate of death is high. Suamous cell lung carcinoma (SLC) is the most common type of lung carcinoma. In SLC, lymphatic metastasis is both the most common diffusion path and the main cause of death. Thus, an understanding of the mechanism of lymphatic metastasis in squamous cell lung carcinoma (SLC) is important to improve clinical therapy and prognosis.Several human cancers are thought to arise through a multistep process involving the inactivation of recessive tumor suppressor genes and the activation of dominant proto-oncogenes. Genetic instability such as microsatellite instability (MSI) and loss of heterozygosity (LOH) may be one of the most important molecular invent resulting in the inactivation of recessive tumor suppressor genes. Nm23-H1 is considered as a tumor metastases suppressor gene, and the lower expression of nm23H1 protein caused by the gene inactivation have been correlated with the presence of distant metastases and tumor progression in several tumor types.So far, little is known about the relationship between genetic instability of nm23H1 gene and the mechanism of lymphatic metastasis in SLC in Chinese. In the present study, we analyzed MSI and LOH of the repetitive untranslated 5' region of the nm23-H1 gene and their influence on the expression of nm23H1 in 50 Chinese patients with SLC by fluorescence-based Polymerase chain reaction single-strand conformation polymorphism (FPCR-SSCP) and immunohistochemistry, and to assess any correlation between genetic instability of nm23H1 gene and the development and lymphatic metastasis of SLC. 2. Materials And Methods2.1 SpecimensBoth paired normal and tumor samples were obtained from each of 50 patients with squamous cell lung carcinoma. The median age of the patients at diagnosis was 61 years (range: 42-76), and the male to female ratio was 47:3. Each frozen tumor biopsy was checked by a pathologist for the presence of tumor cells in the sample. The samples that were at least 80% tumor cells were considered for molecular analysis.2.2 primers and antibodywe used Monoclonal mouse anti-human nm23H1 body and a pair of primers (TAT GAG TTC AAC TAC GCA CG and CTC GAG CAC AGG AGC AGG TT) that flanked the repetitive untranslated 5'region of the nm23-H1 gene.2.3 DNA extractionDNA was extracted from each tumor and corresponding normal tissue using DNA Tissue Extraction Kit.2.4 Fluorescence-based Polymerase chain reaction single-strand conformation polymorphism (FPCR-SSCP) analysisFPCR-SSCP analysis was carried out by fluorescent PCR using a pair of fluorescence-labeled primers. Repeat sequences (110-120bp) were amplified and the PCR products were electrophoresed using an automated DNA sequencer and visualized using Genescan Analysis software.2.5 ImmunohistochemistryImmunohistochemical analysis was performed on formalin-fixed, paraffin-embedded tissue sections, using indirect method technique.Briefly, 5-μm sections were incubated with monoclonal mouse anti-human nm23H1 bodies at a 1:80 dilution. The bound antibodies were detected using goat anti-mouse IGG-HRP, with diaminogenzidine as the chromogen. Staining time was contrloed by microscope. For negative control, PBS was used to replace the first antibody.2.6 Assessment of MSI and LOH and immunohistochemistryThe normal DNA sample was used to determine the allele sizes for those patients. For heterozygous samples, alleles were defined as the 2 peaks of the greatest height. We classified tumors as MSI positive when the PCR product using tumor revealed the presence of at least one peak that was not visible in the PCR product of the corresponding normal tissue DNA, or when the product of tumor DNA showed a mobility shift of at least one peak. A judgment of LOH was excluded when the case showed MSI.The ratio of alleles was calculated for each paired normal and tumor sample, and the tumor ratio was then divided by the normal ratio. A ratio of ≤0.67 or ≥1.5 was taken to be indicative of LOH.2.7 Image analysisWe selected 20 images from each tumor tissue section, and these images did not overlap one another. Using Leica-Qwin Computer Image Analysis System, we analyzed G_A(the grey level of overall background), G_a (the grey level of nm23H1 protein positive particles stained by immunohistochemistry in the field of view), A_Aa (the ratio of the positive area of nm23H1 divided by the total area of the field of view) on a uniform grey-level background.2.8 Data processingPU value (positive unit) was gived using Excel function in mathematics, which represent the expression density of nm23H1 protein in SLC.2.9 Statistical AnalysisThe One-Way ANOVA, Student's t test and X² test were used.3.Results3.1 The relationship between SLC and MSI, LOHUsing FPCR-SSCP analysis, we have found that 44(88%) of 50 cases were informative (heterozytous).Of these informative tumors, the frequency of MSI and LOH were 11.36% and 25.00% respectively. Total 5 cases with MSI positive was belong to moderate (G2) or good (G1) histological grade and all are alive in a year. However, the frequency of MSI and LOH was not relate to tumor differentiation, TNM stage, lymph node metastasis, survival status of patient, and other clinicopathological parameters in SLC (p>0.05).3.2 The relationship between SLC and the expression of nm23Hl protein Neoplastic cells show cytoplasmic positivity. Brown grains locate within cytoplast. Sometimes,the nucleolus and / or cell membrane was also stained.Of 44 informative tumors, the frequency of nm23-H1 protein reacted positive was 50.00% (22/44). The positive frequency of nm23H1 protein in lymph node metastasis cases (30.00%) was significantly lower than those without metastasis (66.67%)(p<0.05). TNM stage III (18.18%) also exhibited lower positive frequency of nm23H1 protein compared with stage I (65.22%)(p<0.05). 3.3 The relationship between the expression of nm23H1 protein and MSI and LOH The positive frequency of nm23H1 protein in MSI positive group and negative group was 40.00% and 51.28%, respectively. The positive frequency of nm23H1 protein in LOH positive group was 54.55% compared with 48.48% in LOH negative group. However, the frequency of MSI and LOH was not relate to the expression of nm23H1 protein (p>0.05). Furthermore, There was also no difference in nm23H1 protein expression intensity analyzed by computer imaging (p>0.05).4.Conclusionswe conclude that increasing the amount of nm23H1 protein expression could play an important role in restraining metastasis of squamous cell lung carcinoma. The heredity instability (MSI and LOH) of nm23H1 gene might not be implicated in expression of the gene and pathogenesis and progression of squamous cell lung carcinoma.