Role of miR-31 in Metastatic Phenotypes of Cutaneous Squamous Cell Carcinoma
Loading...
Date
2020-05
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
The Ohio State University
Abstract
Metastatic cutaneous squamous cell carcinoma (cSCC) is rare but deadly, leading to an estimated 3,932 to 8,791 deaths in the United States annually. Changes in microRNA (miRNA) expression have been found in every cancer and are associated with tumor development and metastasis. A Toland lab study investigated miRNA expression in cSCCs isolated from sites associated with metastasis (aggressive sites), cSCCs at sites with lower risk for metastasis (non-aggressive sites), and non-tumor skin tissue. miR-21, let-7g, miR-93, miR-22, and miR-31 showed different expression between aggressive and non-aggressive sites. A literature search suggested that these miRNAs could play a role in the aggressiveness of tumors. miR-31 was one of the miRNAs that was upregulated in anatomical sites associated with cSCC metastasis. The upregulation of miR-31 at sites associated with metastasis led to the further investigation of its role in tumor and metastatic phenotypes in this study. Through literature and miRNA target database searches, we identified genes regulated or predicted to be regulated by miR-31. Many of these genes are part of the p38/MAPK pathway which has been reported to control some common metastatic phenotypes. Based on these studies, I hypothesized that an increase in miR-31 expression would lead to an increase in metastatic phenotypes via the p38/MAPK pathway. The goal of this study was to evaluate metastatic phenotypes and determine whether miR-31 regulates p38/MAPK genes in keratinocyte or cSCC cells and if these genes were involved in any observed phenotypes. miR-31 expression was measured in HaCat (an immortalized keratinocyte cell line) and SRB-12 (a cSCC cell line) cells using quantitative real-time PCR (qPCR). Using transfection of miR-31 precursors or antagonists, miR-31 was overexpressed or inhibited in these cells. Western blot analysis was used to measure any post-translation effects on candidate miR-31 targets. Migration and proliferation assays were performed to measure metastatic characteristics. Endogenous miR-31 was upregulated in SRB-12 cells 2.6-fold (p-value=0.06) compared to HaCaT cells. qPCR analysis showed no significant expression differences between cells with or without miR-31 expression and the predicted miR-31 target genes: MAPKAPK2, DUSP7, STAT3, MAP3K1, MAPK14, MAP2K4, MEF2A, and CREB1. qPCR results showed a link between increased miR-31 levels and decreased LIMK2 expression in the HaCaT cell line. MTT assays showed no conclusive differences in proliferation between HaCaT cells with or without miR-31 expression. However, migration assays showed that HaCaT and SRB-12 cells transfected with miR-31 experienced gap closure before cells transfected with anti-miR-31. In summary, increased miR-31 expression leads to an increase in the migration of HaCaT and SRB-12 cells, suggesting that miR-31 may contribute to this metastasis-related phenotype. Of the target genes, we only observed a weak correlation between LIMK2 expression and miR-31. Future studies will determine if MAPK pathway gene, LIMK2, or other untested miR-31 target genes are important for metastatic phenotypes.
Description
Keywords
squamous cell carcinoma, microRNA, metastasis, miR-31, cancer biology