Prognostic significance of elevated expression levels of protein phosphatase 1 regulatory subunit 3G in thyroid carcinoma

Abstract

The prognosis of the poorly differentiated subtype of thyroid carcinoma (THCA) is less favorable due to its aggressive nature, which also increases the likelihood of metastasis to distant organs. The present study aimed to assess the potential application of protein phosphatase 1 regulatory subunit 3G (PPP1R3G) as a molecular biomarker for early identification and prognosis prediction in THCA. The Cancer Genome Atlas (TCGA) database was used to identify genes associated with prognosis and differentially expressed genes in THCA. Additionally, TCGA data were used to analyze the differential expression of the PPP1R3G gene in THCA tissues compared with that in normal tissues. Immunohistochemistry was used for further confirmation of these findings. A prognostic significance analysis of the PPP1R3G gene was performed in patients diagnosed with THCA using Cox regression models. Both univariate and multivariate approaches were used, incorporating clinical information obtained from the patients. Moreover, to gain insights into the molecular mechanisms underlying the regulatory role of the PPP1R3G gene in THCA, a functional enrichment analysis performed. An assessment of the levels of immune cell infiltration in patients with THCA was also performed and their correlation with the expression level of PPP1R3G gene was assessed. The mRNA and protein levels of PPP1R3G were revealed to be higher in THCA tissues compared with that of noncancerous thyroid tissues. Moreover, there were significant associations between the expression of PPP1R3G in THCA and factors such as sex, histological type, pathological node stage, pathological stage, extrathyroidal extension and overall survival (OS). Univariate and multivariate Cox regression analyses revealed that the independent prognostic value of PPP1R3G could be used to predict the OS of patients with THCA. The outcomes obtained from functional enrichment analysis indicated a potential involvement of PPP1R3G in regulation of biological processes such as keratinization, and differentiation of keratinocytes and epidermal cells. Additionally, an association was demonstrated between the expression levels of PPP1R3G gene and several immune cells present within the tumor microenvironment of the thyroid. In conclusion, PPP1R3G has the potential to serve as a prognostic indicator for patients diagnosed with THCA, and may also be considered a promising target for therapeutic interventions in THCA.

Keywords: bioinformatics analysis; protein phosphatase 1 regulatory subunit 3G; thyroid cancer.

Figure 1.

Detection of mRNA with unique expression profiles in patients with THCA (n=571). (A) Unique mRNA expression pattern. (B) Overlap between DEmRNA and prognosis-related genes. THCA, thyroid carcinoma; DEmRNA, differentially expressed mRNA; sig, significant; P.adj, adjusted P-value.

Figure 2.

Diagnostic predictive value of PPP1R3G expression in patients with THCA. (A) Comparative analysis was performed to evaluate the mRNA expression levels of PPP1R3G in different types of malignancy, comparing tumor tissues with adjacent normal tissues. (B) mRNA levels of PPP1R3G were quantified using RNA-seq from TCGA database data obtained from tumor samples and their corresponding normal tissues. (C) Dissimilar mRNA expression of PPP1R3G was observed when comparing normal thyroid tissues (n=59) with those impacted by THCA (n=512), based on the analysis of the RNA-seq data. (D) Further assessment of PPP1R3G mRNA expression was performed using RNA-seq data, using matched samples of THCA (n=59) and non-cancerous thyroid tissues (n=59). (E) Kaplan-Meier curve showing the comparison of overall survival among different subgroups of patients with THCA based on their mRNA expression levels of PPP1R3G. *P<0.05; **P<0.01; ***P<0.001. THCA, thyroid carcinoma; PPP1R3G, protein phosphatase 1 regulatory subunit 3G; RNA-seq, RNA sequencing; TCGA, The Cancer Genome Atlas; TPM, transcripts per million; HR, hazard ratio; ns, not significant.

Figure 3.

Immunohistochemistry and RT-qPCR analysis. (A) Evaluation of PPP1R3G protein expression performed in both the para-cancer (n=67) and tumor samples from patients with THCA (n=67). Magnification, ×200. (B) The expression levels across all 67 cases were quantitatively analyzed by measuring the percentage of positive cells. (C) Additionally, the RT-qPCR results indicated that THCA tissues exhibited significantly elevated RNA expression levels of PPP1R3G compared to their corresponding para-cancer normal tissues.**P<0.01. THCA, thyroid carcinoma; PPP1R3G, protein phosphatase 1 regulatory subunit 3G.

Figure 4.

Fluctuations in PPP1R3G mRNA expression levels across patients with thyroid carcinoma exhibiting diverse clinical attributes. The clinical characteristics assessed were (A) Sex (n=515), (B) pathological N stage (n=465), (C) occurrence of OS event (n=515), (D) tumor pathological stage (n=513), (E) presence of extra-thyroidal extension (n=497) and (F) histological type (n=515). *P<0.05; **P<0.01; ***P<0.001. PPP1R3G, protein phosphatase 1 regulatory subunit 3G; T, tumor; N, nodal; OS, overall survival; TPM, transcripts per million.

Figure 5.

Nomogram curve created to assess the probability of OS in patients with THCA (n=507) for durations of 2, 5 and 10 years. The variables taken into account were the (A) clinical stage, pathological stage and expression of PPP1R3G. (B) The nomogram was used to forecast the OS at 2, 5 and 10 years for the THCA population. OS, overall survival; THCA, thyroid carcinoma; PPP1R3G, protein phosphatase 1 regulatory subunit 3G; T, tumor; M, metastasis.

Figure 6.

Differentially expressed genes were identified and subjected to GO/KEGG and GSEA analysis to cluster in patients with THCA (n=571). (A) Heat map displaying the co-expression of PPP1R3G mRNA with other genes. (B) Ranked list of differentially expressed genes analyzed using GO/KEGG analysis to facilitate clustering. (C) GSEA analysis performed on the gene list for clustering purposes. (D) Key genes were identified by constructing a gene interaction network and calculating their importance based on network topology. PPP1R3G, protein phosphatase 1 regulatory subunit 3G; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; GSEA, Gene Set Enrichment Analysis; TPM, transcripts per million; BP, biological process; CC, cellular component, MF, molecular function; NES, normalized enrichment score.

Figure 7.

Correlation between infiltrated immune cells and the PPP1R3G expression level in patients with THCA (n=571). (A) Correlation analysis of PPP1R3G and immune checkpoint molecule expression. (B) Relationship between the expression levels of PPP1R3G and infiltrated immune cells assessed in clinical samples obtained from patients diagnosed with THCA. THCA, thyroid carcinoma; PPP1R3G, protein phosphatase 1 regulatory subunit 3G; SEMA3F, sema domain, Immunoglobulin Domain (Ig), Short Basic Domain, Secreted, (Semaphorin) 3F; PLXND1, plexin D1; CRLF1, Cytokine Receptor Like Factor 1; TNFRSF12A, TNF Receptor Superfamily Member 12A; CCL26, C-C Motif Chemokine ligand 26; GIPR, Gastric Inhibitory Polypeptide Receptor; PLAUR, Plasminogen Activator, Urokinase Receptor; BID, BH3 Interacting Domain Death Agonist; MARCO, Macrophage Receptor With Collagenous Structure; TNC, Tenascin C; NGFR, Nerve Growth Factor Receptor; FGFR3, Fibroblast Growth Factor Receptor 3; TPM, transcripts per million; DC, dendritic cells; Th, T helper; Treg, regulatory T cells; Tcm, central memory T cells; Tem, effector memory T cell; aDC, activated dendritic cells; iDC, immature DC; pDC, plasmacytoid dendritic cell; Treg, regulatory T cell; TFH, Follicular helper T cell; Tgd, γ/δ T cell; NK, natural killer.