Double stranded cDNA was synthesized according to the One Cycle cDNA Syn thesis Kit, starting from 5 ug of the total RNA. The material was purified with a Sam ple Cleanup Module. Purified cDNA was used for an in vitro transcription thoroughly reaction by using an IVT labeling kit. The hybridization cocktail containing fragmen ted biotin labeled target cRNA at a final concentration of 0. 05 ug/uL was transferred into an Affymetrix Human Genome U133A 2. 0 and incubated at 45 C on a rotator in a hybridization oven 640 for 16 h at 60 rpm. The arrays were washed and stained on a Fluid ics Station 450 by using a Hybridization Wash and Stain Kit. In order to increase the signal strength, the antibody amplifica tion protocol was used.

Chip data processing Gene chips were processed with an Affymetrix GeneChip Scanner 3000 7G and DAT image files of the microarrays were generated using GeneChip Operating Software. Within GeneSpring raw data were preprocessed including background adjust ment, normalization, and summarization of probe sets, using the GeneChip Robust Multiarray Analysis. Genes whose signals were lower than background in all gene chips were filtered out, subsequently genes were filtered based on fold change. Statistical analysis on the gene expression profile were performed by using Fish ers analysis of variance. Profiles of genes sig nificantly up or downregulated in CD133 D10 cells as compared to CD133 D10 cells were obtained. In addition, final gene expression data were ana lyzed using the Protein Analysis Through Evolutionary Re lationships classification system.

Background Accumulating evidence showed that Toll like receptors 9, which were mainly expressed on immune cells, were also functional expressed on lung cancer cells. And TLR9 signaling could alter biological character of lung cancer cells including promoting the proliferation and enhancing the metastatic potential of tumor cells, indicating that activation of TLRs signaling in lung cancer cells could contribute to the progression of lung cancer. Recent literatures further showed that miRNAs, a major class of gene expression regulators, played critical roles in regulating the biological effects of TLR9 signaling pathway on various cells. As such, miR 17 92 cluster might regulate the biological effect of CpG ODNs on chronic lymphocytic leukemia cells.

One newly evidence also showed that upregulation of miRNA 574 5p was critical for Batimastat TLR9 signaling enhanced tumor progression of human lung cancer. However, the underlying mechanism regulating the expression of TLR9 signaling associated miRNAs in lung cancer cells remains largely unknown. MicroRNA 7, a unique member of miRNAs, played an important role in the progression of various tumors including lung cancer. Mechanistic evidence showed that miR 7 could regulate the transduction of Akt pathway, which was critical for growth and metastasis of tumor cells.