Differential microRNA-34a expression and tumor suppressor function in retinoblastoma cells.
OBJECTIVE
The role of miR-34a, a p53-regulated microRNA, in retinoblastoma (RB) was investigated.


METHODS
The expression of miR-34 family members in RB cells was determined by semiquantitative RT-PCR and real-time qPCR. Regulation of miR-34a expression by p53-activating compounds was determined by qPCR analysis. The tumor suppressor functions of miR-34a in RB cell lines were determined by tetrazolium-based cell growth assay and by caspase-3/7 and activated caspase-3 apoptotic activity assays. Additive growth inhibitory properties of miR-34a in combination with topotecan were determined by cell growth assay. miR-34a targets in RB cells were identified by real-time qPCR expression analysis of previously reported and GenMiR++-predicted mRNAs.


RESULTS
Differential miR-34a and miR-34b expression was observed in RB cell lines and tumor samples. miR-34a expression could be increased in Y79 cells, but not Weri-Rb1 cells, after p53 activation. This differential regulation was not caused by genomic alterations at the miR-34a p53 binding site or mature gene. Exogenous miR-34a inhibited Y79 and Weri-Rb1 cell growth and increased apoptotic activity in Y79 cells. Increased inhibition of Y79 and Weri-Rb1 cell growth was observed with combination miR-34a and topotecan treatment. mRNA expression changes were observed in 7 of 7 previously reported and 13 of 18 GenMiR++-predicted miR-34a targets after transfection of Y79 cells with miR-34a compared with negative control microRNA.


CONCLUSIONS
miR-34a functions as a tumor suppressor in RB cells and is a potential therapeutic target. Differential expression, regulation, and activity of miR-34a in RB cells may suggest further p53 pathway inactivation in RB.
Dalgard
Clifton L
CL
Ocular Oncology Unit, Department of Ophthalmology, University of California at San Francisco, San Francisco, California 94143, USA.
Gonzalez
Marco
M
deNiro
Jennifer E
JE
O'Brien
Joan M
JM
D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival; D004317:Doxorubicin / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D035683:MicroRNAs / Q000235:genetics*; D010879:Piperazines / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D019572:Retinal Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D012175:Retinoblastoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019772:Topotecan / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000494:pharmacology / Q000502:physiology*
D000970:Antineoplastic Agents; D007093:Imidazoles; C522425:MIRN34 microRNA, human; D035683:MicroRNAs; D010879:Piperazines; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; C482205:nutlin 3; D019772:Topotecan; D004317:Doxorubicin; D020169:Caspases