TMEM44 as a Novel Prognostic Marker for Kidney Renal Clear Cell Carcinoma is Associated with Tumor Invasion, Migration and Immune Infiltration

Abstract

Transmembrane (TMEM) proteins are integral membrane proteins that traverse biological membranes. Several members of the TMEM family have been linked to the development and progression of various tumors. However, the specific role and mechanism of TMEM44 in tumor biology remain largely unexplored. In this study, we initially conducted an extensive analysis using the TCGA database to investigate the expression patterns and survival associations of TMEM44 across various human tumors. Subsequently, we focused on KIRC and found a significant correlation between TMEM44 expression and this particular cancer type. To validate our findings, we performed western blot and quantitative polymerase chain reaction (qPCR) assays to confirm the expression levels of TMEM44 in KIRC. Following this, we employed a series of functional assays, including CCK8 viability assay, EDU incorporation assay, wound healing assay, and transwell migration assay, to investigate the biological role of TMEM44 in KIRC. We observed a significant upregulation of TMEM44 expression in KIRC, indicating its potential involvement in the pathogenesis of this cancer. We intervened in the expression of TMEM44 in KIRC cells and found significant inhibitory effects on cell proliferation, migration, and invasion in KIRC cells. Furthermore, our findings indicated that TMEM44 could serve as an independent prognostic factor in KIRC, highlighting its potential clinical significance. Consequently, TMEM44 holds promise as both a prognostic biomarker and a prospective therapeutic target for KIRC.

Keywords: Immune Infiltration; Kidney clear cell carcinoma; Prognostic factor; TMEM44; Transmembrane proteins family.

Fig. 1

Differential expression and overall survival analysis (COX and KM) of TMEM44 in pan-cancer. (A) Pan-cancer difference analysis by TISIDB. (B) Overall survival analysis of pan-cancer (COX). (C-I) KM regression analysis of overall survival differences between high and low expression of TMEM44. (C) KIRC; (D) ACC; (E) ESCA; (F) LGG; (G) LIHC; (H) LUAD; (I) MESO

Fig. 2

Analysis of Progression-free survival (PFS) and Disease-specific survival (DSS) in pan-cancer. (A) Progression-free survival (PFS) of pan-cancer (COX). (B-E) KM regression analysis of PFS differences between high and low expression of TMEM44. (B) KIRC; (C) ACC; (D) LUAD; (E) MESO. (F) Disease-specific survival (DSS) in pan-cancer (COX). (G-K) KM regression analysis of DSS differences between high and low expression of TMEM44. (G) KIRC; (H) ACC; (I) ESCA; (J) MESO; (K) PRAD

Fig. 3

Diagnostic value and clinical relevance of TMEM44 in KIRC. (A) Diagnostic ROC curve of TMEM44 in KIRC. (B) Heat map of clinical data differences between high and low expression of TMEM44. (C) Differences in the expression of TMEM44 between different ages; (D) Differences in the expression of TMEM44 between the sexes; (E) Differences in the expression of TMEM44 between different Grade stages; (F) Differences in the expression of TMEM44 between different Stage stages; (G) Differences in the expression of TMEM44 between different T stage; (H) Differences in the expression of TMEM44 between different N stage; (I) Differences in the expression of TMEM44 between different M stages

Fig. 4

Construction of Nomogram and Clinical Data Validation. (A) Construction of Nomogram. (B) calibration plots of survival probabilities at 1, 3, and 5 years. (C) Survival analysis of patients with Grade1-2 between high and low TMEM44 expression groups. (D) Survival analysis of patients with Grade 3–4 between high and low TMEM44 expression groups. (E) Survival analysis of patients with Stage I-II between high and low TMEM44 expression groups. (F) Survival analysis of patients with Stage III-IV between high and low TMEM44 expression groups

Fig. 5

Differential analysis of TMEM44 in KIRC tissues and cells. (A) Immunohistochemical (IHC) analysis of TMEM44 in KIRC tissue and paraneoplastic tissue. (B) Western blot assay to analyze the difference in protein expression of TMEM44 in 3 pairs of KIRC tissues and paraneoplastic tissues. (C) Western blot assay to analyze the difference in protein expression of TMEM44 in KIRC cells (769-P, ACHN, 786-O) and normal cell(HK2). (D) q-PCR analysis of differential expression of TMEM44 in KIRC cells and normal cells. (E) q-PCR analysis of TMEM44 expression differences in 20 pairs of KIRC tissues and paraneoplastic tissues

Fig. 6

Validation of the proliferative function of TMEM44 after knockdown. (A) q-PCR validates the efficiency of three inhibitors to knock down TMEM44. (B) Western blot assay to analyze the difference in protein expression after TMEM44 knockdown. (C) CCK-8 experiments to analyze the proliferation between si-TMEM44 and si-Con. (D) EdU experiments to analyze the proliferation between si-TMEM44 and si-con

Fig. 7

Cell scratch assay and Transwell assay. (A) Cell migration ability of si-Con and si-TMEM44 analyzed by cell scratching assay. (B) Transwell analysis of cell migration ability of si-Con and si-TMEM44