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التحري عن التعبير الجيني لل FOXP3 وTGF - ?1 باستخدام الطرائق الجزيئية والمناعية في سرطان الرئة اللاصغير الخلية == Detection of FOXP3 Gene Expression And TGF - ?1 Using Molecular And Immunological Methods In Non - Small Cell Lung Carcinoma
Author name:
سهاد فيصل حاتم المقدادي
Supervisor name:
امنة نصيف جاسم | بان عباس عبد المجيد
General topic:
Biology
Specific topic:
Microbiology
Degree:
Doctorate
University:
University of Baghdad - Ibn Al-Haytham College Of Education For Pure Sciences - Department Of Biology
Language:
English
University location:
Baghdad
First pages:
24T2665 - p.pdf
Abstract:
Studies have linked FOXP3 and TGF - ? expression to the outcome of certain cancers. FOXP3 is a marker known to be expression in T - regulatory cells while TGF - ? is a secreted protein usually detected in the extra cellular matrix.The present study aimed at focusing on the identification of immune markers namely FOXP3 and TGF - ? with their expression patterns in lung cancer patients as a useful tool to predict disease progression.Also it is aimed to design molecular evaluation of m RNA expression of both FOXP3 and TGF - ? in peripheral blood mononuclear cells and bronchial (brush) cells of patients with lung cancer and benign lesions, using qRT PCR; determining the T - reg level in the peripheral blood employing the High Rsolution Melting (HRM) as a novel method to detect Treg - specific demethylated region (TSDR); molecular DNA analysis of somatic mutation of exons 3, 6, 7 of FOXP3 in patients with lung cancer tissue and benign lesions and immunohistochemical (IHC) estimation of FOXP3 and TGF - ?1 in T - reg and cancer cells in formalin fixed paraffin embedded(FFPE)lung cancer tissue and benign lesions.Blood samples were collected from 30 patients with newly diagnosed, non small cell lung carcinoma and 30 patients with benign lesions.Patients were recruited at The Specialized Surgery Hospital and Oncology Teaching Hospital/Baghdad.Samples from 16 apparently healthy donors were used as control during the period from June 2012 to June 2013. The samples preservation with TRIzol reagent were subjected to molecular study including RNA and DNA extraction; reverse transcription; RT - PCR; HRM assay and DNA sequencing were done in the Molecular Oncology Unit/Guys and ST Thomas? s hospital/ King College/London/UK.The expression level of FOXP3 was high in 16(61.5%)in lung cancer cases.A significant difference was noticed between cancer cases from one side and benign lesions or healthy control on the other side p<0.05.Mean of FOXP3 expression(fold change)was significantly high(2.64±0.09)in cancer cases than in benign cases(1.32±0.04)and healthy control(1.38±0.06)with p<0.05.A significant association between high expression level and >60 age and squamous cell carcinoma in cancer cases P<0.05.The expression level of TGF - ? was high in 16(61.5%)in lung cancer cases.A significant difference was noticed between cancer cases from one side and benign lesions or healthy control on the other side p<0.05.Mean of TGF - ? expression (fold change) was significantly high (6.27±0.56) in cancer cases more than healthy control (2.87±0.09) with p<0.05.The association was significant between TGF - ? expression level (high and low) and age>60in cancer and benign groups (p<0.05), while no significant association with gender and cancer types were noticed.For FOXP3 mRNA expression in bronchial brush cells, the result showed no significant difference between the mean fold change of malignant(3.57 ± 0.06) and benign(4.02 ± 0.06) patients. The low expression was predominated both in cancer and benign cases. No significant differences were found between FOXP3 expression (high and low) and age; gender ; cancer types..According to FOXP3 T - reg specific demethylated region detection, results showed that the mean percentage of FOXP3demethylation in lung cancer patients (4.32 ± 0.04) was significantly higher than in benign lesions (3.22 ± 0.02) patients andhealthy controls(3.33±0.04). A positive correlation coefficient with high significant, was found in the group of cancer samples (R² = 0.6653;r = 0.69;P : 0.0017)on correlating percentage of Treg and demethylation of FOXP3 from one side with its m RNA expression on the other side.In benign lesion group was(R² = 0.5334;r= 0.59;p= 0.0027), While in the control group a positive correlation but a weak significance was found (R²= 0.2383;r=0.28;P=0.0437).FOXP3 gene sequencing revealed high frequency of missense mutations c.715 GTA>CTA : V 239 L in 17 (94.44%) in malignant sample and non cancerous cases7 (87.5%)without statistical differences. Missence mutations were also detected in exone 3 in 3(16.67%) cancer cases and in 1(12.5 %) benign lesion.No missense mutations could be detected in exon 6. Intronic mutations and silent mutation were variable in three exons without statistical differences. Many cases of adenocarcinoma have shown multiple mutations either of missense or Intronic types. Missense mutations of exon 7 were correlated significantly with an age of 60?years. Exon 3 mutations were significantly associated with adenocarcinoma. Positive FOXP3 Immunohistochemistry (IHC) staining in tumor cells was associated with high missense mutations frequency 10(55.55%) in exon7, while in exone 3 was 2(11.11%). Negative FOXP3 IHC staining in the tumor cells was associated with seven missense mutations in exon7 and one (5.55%) in exon 3, in addition 4(23.53%) cases of the exon 7 missense mutations were associated with negative FOXP3 expression in lymphocytes.The result showed that FOXP3 by using IHC staining was positive in 21(70%) of nuclei of cancer cells, and 22(73.3%)in Treg infiltrates.The positive cancer cells and Treg infiltrates associated significantly with age>60 (p<0.05).No significant association was found withgender, cancer type, while there is association with moderate differentiation compared to poorly differentiation (p<0.05). High frequence of FOXP3 expression score 3 and high intensity were appeared in nuclear cancer cells compared to benign lesions cells, while Treg infiltrates with score 1 and high intensity was high frequency in malignant and benign.The result showed that TGF - ?1 by using IHC staining was positive in 25(83.3%) in the cancer cells and 21(70%) in stromal cells. No significant difference was noted between positive expression in malignant and benign lesions p>0.05.No significant association was noticed between positive cells expression and age, gender, cancer type and differentiation p>0.05. High frequency of TGF - ?1 expression score 3 and high intensity in malignant cells and benign. Also stromal cell expression score 3 and high intensity were predominated in malignant and benign lesions. The high and moderate intensity expression was more frequent in matrix surrounding cancer cells compared to non cancerous.Total agreement and kappa coefficient between FOXP3 and TGF - ?1were poor in malignant and benign epithelial cells and stroma, while the perfect agreement was between expression of TGF - ?1in stromal cells
