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Supplementary Fig. 1 Confirmation of cell synchronisation at the G1/S boundary. H1299 cells with or without prior transfection with plasmids expressing GFP-tagged CDC6 or GFP alone were transfected twice with the indicated miRNA mimics or control RNA. Untreated cells and those treated with the transfection reagent alone were used for comparison. Some 48 hrs after the final transfection, cells were synchronised at the G1/S boundary with mimosine, and the cell synchrony was determined by flow cytometry. Supplementary Fig. 2 MicroRNA-26a/b inhibit pre-replicative complex formation and cell cycle progression by suppressing CDC6 in human lung cancer cells. (A) SPAC cells with or without prior transfection with plasmids expressing GFP-tagged CDC6 or GFP alone were transfected twice with the indicated miRNA mimics or control RNA. Untreated cells and those treated with the transfection reagent alone were used for comparison. Some 48 hrs post transfection, cells were synchronized at the G1/S boundary with mimosine, and chromatin binding assay was performed with an aliquot of each cell sample. (B) The remaining cells were then released into fresh medium and harvested at different time points for FACS analysis.

Cancer is characterized by mutations, genome rearrangements, epigenetic changes, and altered gene expression that enhance cell proliferation, invasion, and metastasis. To accommodate deregulated cellular proliferation, many DNA replication-initiation proteins are overexpressed in human cancers. However, the mechanism that represses the expression of these proteins in normal cells and the cellular changes that result in their overexpression are largely unknown. One possible mechanism is through miRNA expression differences. Here, it is demonstrated that miR26a and miR26b inhibit replication licensing and the proliferation, migration, and invasion of lung cancer cells by targeting CDC6. Importantly, miR26a/b expression is significantly decreased in human lung cancer tissue specimens compared with the paired adjacent normal tissues, and miR26a/b downregulation and the consequential upregulation of CDC6 are associated with poorer prognosis of patients with lung cancer. These results indicate that miR26a/b repress replication licensing and tumorigenesis by targeting CDC6.Implications: The current study suggests that miR26a, miR26b, and CDC6 and factors regulating their expression represent potential cancer diagnostic and prognostic markers as well as anticancer targets.Visual Overview: http://mcr.aacrjournals.org/content/12/11/1535/F1.large.jpg.Mol Cancer Res; 12(11); 1535–46. ©2014 AACR.