RNA Interference-Mediated Knockdown Of DNA Methyltransferase (DNMT1) In Human Lung Cancer Cell

To evaluate the relationship between DNMTl overexpression and tumor malignant phenotype and discuss the significance with DNMTl as the target in tumor therapy, RNAi was applied to knockdown DNMT1 expression.Basing on DNA methylation analysis by MSP or COBRA and gene expression detection by semi-quantitative RT-PCR, Real-time PCR and Western blot, Human lung cancer cell NCI-H157 was chose as in vitro model, in which DNMTl was overexpressed and RASSF1A was downregulated due to hypermethylation. After knockdown of DNMTl expression by two of five candidate targets, RASSF1A expression was restored revealed by RT-PCR, and proliferation of NCI-H157 was inhibited revealed by MTT. FACS analysis indicated that inhibition of NCI-H157 proliferation was due to G1 arrest which possibly induced by upregulation of RASSF1A. To further examine the effect of RNAi-mediated knockdown of DNMT1 on NCI-H157, tetracycline inducible shRNA expressing system was established. Through cotrasfection with luciferase expressing vector, the cell line stably expressing high level of tetracycline repressor was obtained. And the construct expressing SDl and SD5 was established.In a word, all these result suggested that overexpression of DNMTl lead to hypermethylation of tumor suppressor gene. Downregulation of DNMTl by RNAi may reverse the malignant phenotype of tumor in some extent. Evaluation of the role of...