Cell cycle genes expression in human corneal endothelium: study by microarray and qRT‐PCR

Abstract Purpose To use microarray and qRT‐PCR to identify changes in cell cycle genes expression in human corneal endothelial cells (CEC) from “ in vivo”, post mortem, and organ cultured (OC) corneas, and also in confluent primary culture and immortalized CEC Methods Total RNAs were extracted. “in vivo” corneas were obtained during penetrating grafts for keratoconus, immediately after trephination, post mortem corneas (body donation to Science) within 24H after death. OC corneas were stored for 15D. Descemet was peeled off to avoid contamination with other cell types. Biotin‐labeled cRNA probe was synthesized from RNAs extract and hybridized to the pretreated Oligo GEArray Human Cell Cycle Microarrays, which covered 112 key genes of cell cycle. Microarrays were performed in duplicate. Gene expression of 'in vivo' CEC served as a reference. A difference >1,5 fold of the transcriptional level was considered significant. qRT‐PCR were done on cyclin E1; cyclin D1; p16INK4a; p21CIP1 and p27Kip1 to confirm the microarrays Results Compared to ‘in vivo’, CEC from OC corneas had 1 upregulated gene (CDK7) and 26 downregulated (CCNE1; CDKN1B; MCM2; RB1…), post mortem corneas had 2 up(CDC28; CDK7) genes and 16 downregulated (ATM; BAX; CDC16; CDKN1B…), primary culture had 1 up(CKS2) and 9 downregulated (BAX; CCNE1; CDK2; MCM2…), and the immortalized cell line had 16 up (ATM; CCNH; CKS2; MCM2…) and 2 downregulated genes (BAX; CDK5R2). qRT‐PCR of the 5 aforementioned genes validated the Microarrays data Conclusion Microarray seems to be a powerful tool to better understand the proliferative status of human CEC. It will help to choose the targets we need to alter in order to trigger CEC proliferation. Grant: Fondationdel’Avenir2007 ET7‐468