The Effect Of MiR-146a On The Proliferation Of A549 Cells And Its Correlation Research Of Nucleotide Polymorphism With Lung Cancer

Background and Objective: Lung cancer is the most common of malignancies in global. It has reported that its mortality rate is the highest. In many countries around the world, including our country, the death rate of lung cancer has been distantly higher than other malignant neoplasms, which has been ranked first in cause of death of all malignant tumors. Many studies have found that single nucleotide polymorphisms determines individual genetic variation, which is currently one of the genetic markers of the most important. However, the present study recognized micro RNA(mi RNA) is a kind of small within endogenous non-coding single-stranded RNA molecules. Under normal conditions, mi RNA mainly in a highly conservative manner involved in the regulation of its downstream gene expression. Studies have confirmed that mi RNA SNPs may occur in any link of the process that mi RNA from beginning translation to maturation. Single nucleotide polymorphisms SNP has become one of the important signs inspecting information to change, which has slowly become a critical step towards application of the Human Genome Project. And a growing number of studies have found that it can participate affect drug sensitivity. Numerous studies confirm the presence of significant abnormal expression of Micro RNA in a number of malignancies, including obvious abnormalities Micro RNA expression in lung cancer, and are closely related with the degree of malignancy, resistant lung cancer. Which found that mi R-146 a expression in tumors was significantly lower than the corresponding adjacent cancer tissue. Interestingly, a recent study found that a large number of mi R-146 a is closely related to the occurrence and development of NSCLC, but the exact mechanism of action is unknown. In addition, a large number of studies have found that MIF is highly expressed in melanoma, NSCLC, liver cancer, prostate cancer, renal cancer, colorectal cancer and other carcinomas. MIF is particularly involved in lung cancer development and progression of NSCLC, Kamimura et overexpression of MIF by immunohistochemistry lung tissue of NSCLC patients, and studies confirm that there is a correlation of MIF expression and prognosis. A large number of studies have reported MIF in normal lung tissue of many benign lesions, NSCLC tissue, lymph node tissue metastases showed a gradual enhancement of expression, and studies have confirmed that high expression of the overall survival rate is lower than the low expression of MIF. Tip NSCLC in mi R-146 a expression and MIF are abnormal. The issue on this basis, by RT-PCR method and immunohistochemistry were used to detect mi R-146 a, the expression of MIF in NSCLC lung tissue and through by luciferase reporter assay verification MIF whether it is a downstream target of mi R-146 a in gene, further transfected with mi R-146 a mimics alter the expression of intracellular mi R-146 a, thus further observe special NSCLC cell biology, and to explore its effect on NSCLC cells MIF expression and cell proliferation in, and the specific role mechanism.Methods: 1. For lung cancer patients and patients with benign lesions surgically resected specimens paraffin DNA extraction and PCR detection, analysis of the differences between single nucleotide polymorphism of benign and malignant lesions of mi R-146 a and clinical features of benign and malignant lesions or no correlation. Analysis of gene polymorphism in patients with lung cancer population expression. 2. RT-PCR method to detect mi R-146 a in non-small cell lung cancer(NSCLC) lung tissue and adjacent normal lung tissue and five NSCLC cell lines and normal human embryonic lung cells expressed immunohistochemical method to detect MIF in NSCLC adjacent normal lung tissue and cancer tissue. 3. Through experimental methods luciferase reporter gene and the like to further explore whether the MIF gene-specific downstream target of mi R-146 a, and will build a good through transfection reagent mi R-146 a mimics and mi R-146 a negative control(NC) transfer to NSCLC cells, changing the expression mi R-146 a, which detect changes in the expression of MIF m RNA and protein by q PCR and Western blotting. In addition, we further by using the MTT assay changes in cell viability after mi R-146 a, plate cloning experiments such as the seizure detection of tumor cell proliferation, but also use Annexin V-FITC/PI apoptosis detection cells, also observed the aspartic protease 3(Caspase 3) change in activity, and analyzed by Western blotting change change in lung cancer cells NF-κB-p65 protein expression after mi R-146 a.Result: 1. This study found that mi R-146 a gene in patients with lung cancer detection rate was higher, and the single nucleotide polymorphisms and lung cancer correlated. 2. NSCLC cells mi R-146 a than normal fetal lung tissue. By liposome mi R-146 a mimics transfected into A549 cells, with the NC group, MIF protein and m RNA expression levels were significantly decreased(p<0.01), decreased cell viability(p<0.01), reduce the number of clones(p<0.01) increase the number of apoptotic cells(p<0.01), Caspase 3 activity increased(p<0.01), reduced NF-κB p65 phosphorylation levels(p<0.01). 3. NSCLC lung tissue mi R-146 a expression is lower than in adjacent normal lung tissue. MIF protein expression in NSCLC lung tissue was higher than in adjacent normal lung tissue, and the luciferase reporter gene confirmed that MIF is a downstream target genes of mi R-146 a.Conclusion: In patients with lung cancer mi R-146 a single nucleotide polymorphisms correlated with lung cancer, mi R-146 a and low expression in NSCLC cell lines, MIF overexpressed in NSCLC, mi R-146 a upregulation by target to inhibit the expression of MIF, thereby inhibiting A549 tumor cell proliferation, induction of apoptosis, possibly through NF-κB signaling pathway.