The Mutation Analysis Of MiR-155,miR-32 Gene In Lung Cancer

Objective: To study the miR-155,miR-32 gene mutation by using polymerase chain-reaction single strand conformation polymorphism (PCR-SSCP) in human lung cancer tissue, explore its possible effect and mechanism on the development of lung cancer.Methods: 120 paraffin imbedding specimens from primary site of lung cancer tissues and 42 paraffin imbedding samples of normal adjacent lung tissues was digested by protease K and was extracted genome DNA by phenol-chloroform, The miR-155 and miR-32 was analyzed by using polymerase chain-reaction single strand conformation polymorphism (PCR-SSCP). Data statistics was deal with in SPSS 13.0 software, date was analyzed by x² test and Spearman correlation analysis, P<0.05 has statistical significance.Results:(1)The result of DNA extractionEvery specimen was succeeded extracting genome DNA. The electrophoresis result in 1% agarose gel shows that the genome DNA degrades more seriously from paraffin imbedding lung tissue, than from the whole blood or fresh tissues. All genome DNA samples were determined by the DU640 ultra-spectro luminosity meter, OD₂₆₀/OD₂₈₀>1.75 means pure DNA, the concentration of every sample was adjusted to 20-50ng/μl by adding different amount of TE buffer.(2)The result of PCR Both miR-155 PCR and miR-32 PCR succeeded, PCR product shows single strap in 1% agars gel electrophoresis without obvious insertion or deletion. (3)The result of SSCPNo abnormal of miR-155 PCR product was found in 120 cases of lung cancer tissues and 42 cases of normal adjacent lung tissues. No abnormal of miR-32 PCR product was found in 42 cases of normal adjacent lung tissues. But there were 32 cases of miR-32 gene mutation in 120 cases of lung cancer tissues. The mutation rate is 26.67%, and the analysis revealed that its mutation was positively correlated to the pathological type (rs = 0.200, P = 0.028), and was positively correlated to the status of lymph node metastasis (rs =0.219, P =0.019), but no relationship was found between miR-32 gene mutation and other clinical features.Conclusion:miR-155 gene has not been found any mutation in paraffin imbedding lung cancer tissue. It shows the high expression of miR-155 in lung cancer has other mechanism. The mutation rate of miR-32 in lung cancer is 26.67%, and it is positively correlated to the pathological type and the status of lymph node metastasis. And it is coincident with the effect of its target gene MMP-1 in lung cancer. All of these show miR-32 gene mutation may be associated with the invasion and progression of Lung cancer especially squamous carcinoma.