Study On The Suppressive Mechanism Of Testin Gene In Non-small Cell Lung Cancer

Objectives:1. To Testint the expression of Testin gene in non-small cell lung cancer(NSCLC)tissues and adjacent normal tissues.2. To Testint the expression of Testin gene in NSCLC cell lines and human bronchial epithelial cell lines.3. To study the correlation of Testin expression in NSCLC tissues with clinical pathological parameters.4. To use lentivirus transfection technique to transfect different lung cancer cells with Testin overexpression vectors and build lung cancer cell lines with stable Testin gene overexpression by constructing the overexpression vector targeted at Testin gene, and to study the influence of Testin gene on malignant biological behaviors like lung cancer cell proliferation, invasion, cloning, apoptosis and transfer through cell experiment.Methods:1. The expressions of Testin gene in NSCLC tissues and adjacent normal tissues were Testinted: patients’(N=30) NSCLC tissues and corresponding adjacent normal tissues were collected and conserved. RT-PCR, western blot and immunohistochemistry were employed to examine Testin expression in NSCLC tissues. The correlation of Testin gene expression with the clinical pathological features of NSCLC was analyzed to provide a theoretical basis for clinical diagnosis and treatment.2. The difference in Testin gene expression between NSCLC cell lines(A549,LTEP-a-2, NCI-H1650) and human bronchial epithelial cell line 16 HBE was Testinted and NSCLC cell lines were cultured. Semi-quantitative PCR and Western blot were employed to Testint the expression of Testin gene in NSCLC cell lines.3. The genetic functions of Testin were preliminarily studied: Testin eukaryotic expression vector was constructed and transfected into the cells of human NSCLC cell lines cultured in vitro(A549, NCI-H1650). A stable expression clone was screened out through Puromycin, with non-transfected group and empty virus transfected group as the control groups. RT-PCR and Western b1 ot were used to Testint Testin expression in cells. Flow cytometry, MTT kit and Transwell method were applied to Testint cell apoptosis and cell clone formation assay, cell proliferation and cell invasion,respectively. The influence of Testin on the malignant biological functions of lung cancer cells was analyzed.Results: According to the results of RT-PCR, the expression levels of Testin in lung cancer cell lines LTEP-a-2, NCI-H1650 and A549 were all lower than that in 16 HBE,showing a significant difference(P<0.01). When compared to adjacent normal tissues,the expression level of Testin m RNA in 70%(21/30) lung cancer tissues exhibited a significant decline, showing a significant difference(P=0.000). According to the results of Western blotting Testint, Testin protein expression in 66%(20/30) lung cancer tissues decreased(P=0.000). Comparing immunohistochemical results with adjacent normal tissues, we discovered a significant reduction of Testin expression in lung cancer tissues(P<0.05). Testin gene expression showed no correlation with patients’ age, gender,tumor size, smoking and pathological type in different groups(P>0.05). Testin gene expression was related to the differentiation degree of lung cancer, lymphatic metastasis and clinical stage(r=0.546, r=0.602, r=0.520, P<0.05). Testin negative expression rate showed a positive correlation with lymphatic metastasis(presence), clinical stage(Ⅲ)and differentiation degree(medium and low). The lower differentiation degree, the less Testin expression was. High differentiation, medium differentiation and low differentiation showed a significant difference(P<0.05). Testin overexpression lentiviral expression vector was constructed and then transfected in lung cancer cell lines A549 and NCI-H1650. The transfection was confirmed to be successful through RT-PCR and Western blotting Testint, which screened out the lung cancer cell line of stable overexpression Testin gene. And overexpression cell line showed a good growth state. It could be seen from MTT results that overexpression Testin in lung cancer cell lines A549 and NCI-H1650 with less Testin expression could weaken the proliferation ability of lung cancer cells(P<0.05). Cell clone formation assay indicated a weaker ability of cell clone formation caused by overexpression Testin gene(P<0.05). According to Transwell Testint results, overexpression Testin genes could reduce the invasiveness of lung cancer cells(P<0.01) and the apoptosis-Testing flow cytometry showed that overexpression Testin gene could significantly promote cell apoptosis(P<0.01).Conclusions: Testin expression shows a significant decrease in NSCLC and has correlations with differentiation degree, lymphatic metastasis and clinical stage. Lung cancer cells successfully transfected with Testin overexpression vector in vitro prove that Testin gene as a tumor suppressive gene can inhibit malignant tumor biologicalbehaviors such as lung cancer cell proliferation, invasion and clone formation while promoting lung cancer cell apoptosis.