Scientists have found that knocking out miR-21 significantly reduces tumorigenesis in an established mouse model of skin cancer. They also identified a number of target genes that are suppressed by miR-21 upregulation, which then allows cancer formation to occur.
The team was led by Yong Li, Ph.D., associate professor of biochemistry and molecular biology at University of Louisville. The researchers report their findings in PNAS in a paper titled “Loss of the miR-21 allele elevates the expression of its target genes and reduces tumorigenesis.”
miR-21 is overexpressed in virtually all types of carcinomas and various types of hematological malignancies, supporting its putative role as a ubiquitous oncogene, Dr. Li and colleagues report. However, the pathological involvement of miRNAs in skin cancer has yet to be explored.
To gain further insights into the role of miR-21 in skin cancer, the researchers knocked out the miR-21 gene in a well-established mouse model that develops chemically induced skin carcinomas that mirror skin carcinogenesis in humans and particularly squamous cell carcinoma.
When the miR-21-knockout animals were subjected to the carcinogenic chemicals, they developed fewer papillomas than their wild-type littermates, and one of the knockouts remained tumor-free. The experimental animals exhibited normal overall epidermal morphology and thickness but reduced levels of cell proliferation and higher levels of apoptosis, both in normal areas of skin and in the tumors themselves.
Keratinocytes from miR-21-null mice that hadn’t been chemically treated to induce carcinogenesis also displayed higher levels of apoptosis than those from untreated wild-type animals, validating a role for miR-21 in apoptosis, the researchers note. Further supporting this finding, the authors confirmed that wild-type keratinocytes transfected with miR-21 inhibitors displayed higher rates of apoptosis than untransfected keratinocytes, while miR-21-null keratinocytes transfected with miR-21 precursors displayed lower rates of cellular apoptosis.
miRNAs normally act to suppress target genes. Carrying out a global expression profile of mRNAs in keratinocytes from both wild-type and miR-21-null mice, the researchers initially identified 153 putative or validated miR-21 targets. Using techniques including qPCR to analyze selected genes including those differentially expressed in wild-type or miR-21-null keratinocytes, the team then narrowed down their search to 30 validated or predicted miR-21 targets that were up-regulated in miR-21-null cells. The vast majority of these were then down-regulated when miR-21 was reintroduced, corroborating the notion that their upregulation in miR-21-null cells was directly due to the allele knockout.
More specifically, the miR-21 target genes Spry1, Pen, and Pdcd4 were upregulated in the miR-21-null cells. Upregulation of these genes coincided with reduced phosphorylation of ERK, AKT, and JNK, which are three major downstream effectors of Ras activation, which plays a predominant role in the chemically induced initiation of skin cancers in the mouse model.
“Collectively, these data provide a strong link between elevated expression of miR-21 to attenuate Ras signaling and reduced skin tumorigenesis,” the authors conclude.