Analysis of Enigma Gene-Signaling Pathways in Thyroid Cancer

Abstract

Fine-needle aspiration (FNA) is one of the most accurate modes of obtaining thyroid nodule biopsies, however, up to 25% of biopsies still yield indeterminate results. There is an increasing number of thyroidectomies due to indeterminate nodules by FNA alone. Therefore, more accurate and time efficient diagnostic approaches for analyzing indeterminate thyroid nodules is required. Recent studies showed that Enigma is associated with different cancer types, including thyroid cancer progression and calcification through its interaction with bone morphogenic protein-1 (BMP-1) and tyrosine kinases linked to mitogen-activated protein kinase (MAPK) signaling pathway. Our published data on Enigma protein analysis with immunohistochemistry showed promising findings to discriminate malignant versus benign nodules. We also showed a thyroid cancer stage-dependent enhancement of Enigma protein expression. In this study, we are investigating Enigma at a gene expression level by quantitative reverse transcription polymerase chain reaction (RT-qPCR), which is more time-efficient, quantitative, and requires less tissue than immunohistochemistry. We extracted mRNA/DNA/proteins from fresh malignant and benign thyroid nodules using a Qiagen AllPrep DNA/RNA/Protein Mini Kit. After verification of the quantity and purity by NanoDrop, isolated mRNA was then run through Enigma-RT-qPCR. MAPK assay was done by western blotting using MAPK-antibody. Our initial results found that Enigma-mRNA expression level was 3-fold higher in malignant compared to benign thyroid tissues. This finding supports our previous protein expression data with a relative quantitative difference in Enigma-mRNA expression level between malignant and benign thyroid nodules. MAPK expression was upregulated in thyroid cancer compared to benign nodules. We conclude that Enigma-RT-qPCR can be used effectively in FNA samples derived from thyroid nodules, which could potentially enhance the diagnostic accuracy of indeterminate nodules and decrease unnecessary thyroidectomies. Furthermore, both Enigma and MAPK were highly expressed in advanced tumor in the same tissues. Future study is needed to establish the functional interaction of Enigma-MAPK activity in thyroid cancer cells.