Regulatory and Interacting Partners of PDLIM7 in Thyroid Cancer

Abstract

Enigma protein, encoded by the PDLIM7 gene, is overexpressed in thyroid cancer in a stage-dependent manner, suggesting a potential involvement in the initiation and progression of thyroid cancer. The Enigma interacts with several cellular pathways, including PI3K/AKT, MDM2, and BMP-1. The Enigma is regulated by microRNAs. Specifically, we showed that the Enigma protein upregulation corresponds to the downregulation of Let-7 family genes. There is limited research on the interactions and regulation of the Enigma with other proteins/genes in thyroid cancer tissues, indicating a gap in current knowledge. Our aim is to establish the Enigma as a biomarker. We also aim to study the interacting partners of the Enigma signaling pathways and their probable miRNA regulation in thyroid cancer progression. Using Western blotting, densitometric analysis, immunoprecipitation (IP), and reverse IP, we detected the protein expression and protein-protein interactions in the corresponding papillary thyroid carcinomas (PTCs). Utilizing real-time qPCR assay and Pearson's correlation test, we highlighted the correlation between PDLIM7 and Let-7g gene expression in the same tissues. The results showed the differential upregulations of the Enigma protein in different stages of PTCs compared to benign tissues along with AKT, VDR, BMP-1, and MDM2 proteins. Loss of DBP was observed in a subset of PTCs. Strong interactions of the Enigma with PI3K/AKT and MDM2 were noted, along with a weaker BMP-1 interaction. Pearson's correlation coefficient analysis between PDLIM7 and let-7g gene expression was significant (p < 0.05); however, there was a weak inverse correlation (r = -0.27). The study suggests the potential utility of the PDLIM7-qPCR assay as a biomarker for thyroid cancer. The Enigma's interactions with key signaling pathways may provide valuable insights into the development of thyroid cancer. The study contributes to understanding the molecular mechanisms involving the Enigma protein in thyroid cancer and highlights its potential as a biomarker.

Keywords: BMP-1; Enigma; MDM2; PI3K/AKT; let-7g; thyroid cancer.

Figure 1

(A) Western blotting data of representative protein tissue samples from patients probed with Enigma (55 kDa), BMP-1 (80–100 kDa), PI3K/AKT (56 kDa), MDM2 (75–85 kDa), DBP (52–58 kDa), and GAPDH (37 kDa) antibodies in 41 cancer tissue samples. Pt#, patient number; Mol wt, molecular weight. (B) Densitometric analysis of PDLIM7 protein expression in different staging. B9, benign (n = 3), PT1a (n = 9); PT1b (n = 11); PT2 (n = 6); PT3 (n = 9); PT4 (n = 3). Data was shown as Mean +/− Stdev. pT1aN0-pT1bN0 (Stage I), pT1b pN1a (Stage II), pT1a(m) N1a Mx (Stage III), pT3N1bM1~ (stage IV) [1,23]. Stage I (n = 20); Stage II (n = 6); Stage III (n = 9); Stage IV (n = 3).

Figure 2

Immunoprecipitation scans of patients #1–5 showing interaction levels of MDM2, PI3K/AKT, and BMP-1 with the Enigma antibody as a bait. Two lanes per patient are shown, S (supernatant) and B (beads).

Figure 3

Reverse Immunoprecipitation with VDR antibody, probed with the Enigma, and BMP-1 antibodies.

Figure 4

(A) D-D CT value of PDLIM7 expression (Y-axis) gene was plotted in 87 samples. (B) Scatter plot of D-D CT value of PDLIM7 (n = 84) to staging. There is a positive correlation. PT1 = 37; PT2 = 15; PT3 = 22; PT4 = 9). PTNM classification was shown in result section [1,23]. (C) ∆-∆ CT values of let-7g expression (Y-axis) gene were plotted in 87 samples. (D) Scatter plot of correlation between both gene expressions.