Functional evidence that Drosha overexpression in cervical squamous cell carcinoma affects cell phenotype and microRNA profiles

Abstract

Although gain of chromosome 5p is one of the most frequent DNA copy-number imbalances in cervical squamous cell carcinoma (SCC), the genes that drive its selection remain poorly understood. In a previous cross-sectional clinical study, we showed that the microRNA processor Drosha (located on chromosome 5p) demonstrates frequent copy-number gain and overexpression in cervical SCC, associated with altered microRNA profiles. Here, we have conducted gene depletion/overexpression experiments to demonstrate the functional significance of up-regulated Drosha in cervical SCC cells. Drosha depletion by RNA interference (RNAi) produced significant, specific reductions in cell motility/invasiveness in vitro, with a silent RNAi-resistant Drosha mutation providing phenotype rescue. Unsupervised hierarchical clustering following global profiling of 319 microRNAs in 18 cervical SCC cell line specimens generated two groups according to Drosha expression levels. Altering Drosha levels in individual SCC lines changed the group into which the cells clustered, with gene depletion effects being rescued by the RNAi-resistant mutation. Forty-five microRNAs showed significant differential expression between the groups, including four of 14 that were differentially expressed in association with Drosha levels in clinical samples. miR-31 up-regulation in Drosha-overexpressing samples/cell lines was the highest-ranked change (by adjusted p value) in both analyses, an observation validated by northern blotting. These functional data support the role of Drosha as an oncogene in cervical SCC, by affecting expression of cancer-associated microRNAs that have the potential to regulate numerous protein-coding genes.

Proteomic identification of target proteins following Drosha knockdown in cervical cancer

“…It is well known that Drosha is one of the key enzymes involved in the maturation of miRNA, and the expression level of this gene is robustly associated with the miRNA profile (11). In the present study, for the first time, we demonstrated the significant influence of Drosha-KD on the proteomic profile.…”

“…In Drosha overexpressing cervical cancer cells, 45 microRNAs exhibited a significant association with Drosha levels. The majority of these miRNAs (n=40, 88.9%) were upregulated, with only five (11.1%) being downregulated (11). In the present study, we determined that Drosha-KD altered the entire cell proteomic profile, an effect that can be explained by the potent biological functions of Drosha and miRNAs.…”

“…Drosha overexpression occurs frequently in cervical cancer (8)(9)(10) and is associated with substantial differences in miRNA profiles (11). However, the effects of Drosha dysregulation on the proteomic profile of cervical cancer cells following changes in their miRNA expression profile are unknown.…”

Altered microRNA expression associated with chromosomal changes contributes to cervical carcinogenesis

“…HPV-encoded genes have been shown to influence the miRNA expression of its host cell. 9 --14 Muralidhar et al 15,16 showed that a frequently occurring gain of chromosome 5p in cervical cancer is associated with functionally relevant elevated expression of the miRNA-processing enzyme Drosha, and altered miRNA expression profiles. Furthermore, a panel of miRNAs able to predict cervical cancer survival was recently described.…”

MicroRNA Dysregulation in Squamous Cell Carcinoma of Head and Neck

“…Zhang noticed that the microRNA processing enzymes Dicer and Drosha are overexpressed in salivary gland tumor . Expression of Dorsha (micoRNA processor) will affect the phenotype of squamous epithelial cells (Muralidhar et al, 2011).…”