TMSB10 acts as a biomarker and promotes progression of clear cell renal cell carcinoma

Abstract

Clear cell renal cell carcinoma (ccRCC) is one of the most common urological malignancies. Identifying novel biomarkers and investigating the underlying mechanism of ccRCC development will be crucial to the management and treatment of ccRCC in patients. Thymosin b10 (TMSB10), a member of the thymosin family, is involved in various physiological processes, including tissue regeneration and inflammatory regulation. Moreover, it has been found to be upregulated in many types of carcinoma. However, its roles in ccRCC remain to be elucidated. The present study aimed to explore the expression of TMSB10 in ccRCC through mining The Cancer Genome Atlas (TCGA) and Oncomine databases, and to investigate the association between TMSB10 expression and clinicopathological factors. Furthermore, immunohistochemistry assays and western blotting were conducted to verify TMSB10 expression levels in human ccRCC tissues and cell lines. Functional analyses were also performed to identify the roles of TMSB10 in vitro. The results revealed that TMSB10 was significantly upregulated in RCC tissues and cell lines. The expression of TMSB10 was closely associated with various clinicopathological parameters. In addition, high expression of TMSB10 predicted poor clinical outcome. The receiver operating characteristic curve revealed that TMSB10 could sufficiently distinguish the tumor from normal kidney (area under the curve = 0.9543, P<0.0001). Furthermore, knockdown of TMSB10 impaired the proliferation of ccRCC cells, and attenuated cell and invasion in vitro. In addition, TMSB10 knockdown downregulated reduced the phosphorylation of PI3K and the expression of vascular endothelial growth factor. In conclusion, the present study demonstrated that high expression of TMSB10 could serve as a useful diagnostic and prognostic biomarker and a potential therapeutic target for ccRCC.

Keywords: TMSB10; renal cancer; biomarker; prognosis; diagnosis.

Figure 1

Venn diagram of upregulated genes in different datasets. Upregulated genes common to more than two datasets were extracted.

Figure 2

Overall survival analysis of 19 commonly upregulated genes conducted using the OncoLnc database. (A) TMSB10, (B) ENO2, (C) NNMT, (D) ST8SIA4, (E) CAV1, (F) ANGPTL4, (G) BHLHE41, (H) CA9, (I) CAV2, (J) EGLN3, (K) HILPDA, (L) IGFBP3, (M) NDUFA4L2, (N) PRKCDBP, (O) SAP30, (P) SCARB1, (Q) SLC15A4, (R) SLC16A3 and (S) SPAG4. P<0.05 was regarded as statistically significant by log-rank test. Blue lines indicate low gene expression, while red lines represent high gene expression.

Figure 3

TMSB10 expression level is upregulated in ccRCC. mRNA expression datasets for TMSB10 were acquired from TCGA database, which contained 72 adjacent normal tissues and 539 ccRCC tissues. (A) The expression level of TMSB10 was significantly higher in ccRCC tissues than in normal tissues in TCGA datasets. (B) In 72 paired tissues from patients with ccRCC, TMSB10 expression was higher in the tumor tissues compared with corresponding normal tissue. (C-G) The expression level of TMSB10 was higher in ccRCC tissues than normal tissues in the Oncomine database. (C) Yusenko Renal, (D) Lenburg Renal, (E) Jones Renal, (F) Gumz Renal and (G) Beroukhim Renal datasets. TMSB10, thymosin β10; TCGA, The Cancer Genome Atlas; KIRC, kidney renal clear cell carcinoma; ccRCC, clear cell renal cell carcinoma.

Figure 4

TMSB10 expression level is closely associated with various clinicopathological factors in ccRCC tissues. (A) Sex, (B) vital status, (C) distant metastasis, (D) lymph node metastasis, (E) G grade, (F) TNM stage and (G) T stage. ccRCC, clear cell renal cell carcinoma; TMSB10, thymosin β10.

Figure 5

High TMSB10 mRNA expression predicts poor OS in subgroup patients with ccRCC. Patient samples from The Cancer Genome Atlas were divided into two groups: Those with low TMSB10 expression and those with high TMSB10 expression. (A) Female, (B) male, (C) age ≥60 years, (D) M0 stage and (E) N0 stage. ccRCC, clear cell renal cell carcinoma; OS, overall survival; TMSB10, thymosin β10.

Figure 6

High TMSB10 mRNA expression predicts poor DFS in patients with ccRCC. Patient samples from The Cancer Genome Atlas were separated into two groups: Those with low TMSB10 expression and those with high TMSB10 expression. (A) DFS of patients with ccRCC was associated with TMSB10 expression. DFS subanalysis with regard to TMSB10 expression was preformed in subgroups of patients with ccRCC. (B) age <60 years, (C) age ≥60 years, (D) female, (E) male, (F) G1+G2 grade, (G) G3+G4 grade, (H) N0 stage, (I) M0 stage, (J) T1+T2 stage and (K) stage I+II. ccRCC, clear cell renal cell carcinoma; DFS, disease-free survival; TMSB10, thymosin β10.

Figure 7

TMSB10 expression may act as a diagnostic biomarker in patients with ccRCC. (A) TMSB10 effectively distinguished between ccRCC and paired normal tissues. ROC curve subanalysis was conducted regarding the following subgroups of patients with ccRCC: (B) Vital status, (C) DFS status, (D) distant metastases, (E) lymph node metastasis, (F) TNM stage, (G) T stage and (H) G grade. ccRCC, clear cell renal cell carcinoma; AUC, area under the curve; ROC, receiver operating characteristic; DFS, disease-free survival; TMSB10, thymosin β10.

Figure 8

TMSB10 is upregulated in RCC cells and tissues. (A) Representative western blots and (B) the associated quantified TMSB10 expression levels in 30 paired tissue samples from patients with ccRCC. (C) Western blots and (D) the associated quantified TMSB10 expression levels in normal renal tubular epithelial cells (HK-2) and in renal cancer cell lines (ACHN, 786-O, Caki-1 and A498). (E) Immunohistochemical analysis of TMSB10 expression in ccRCC tissues and adjacent normal tissues; representative images are shown (magnification, x400). TMSB10 expression in the western blots was normalized to β-actin expression. The values of each group are presented as the mean ± SD. Tukey's test was used to analysis the differences among groups. ccRCC, clear cell renal cell carcinoma; N, normal; T, tumor; TMSB10, thymosin β10. ^*P<0.05, ^**P<0.01, ^***P<0.001 and ^****P<0.0001.

Figure 9

TMSB10 regulates various tumor-associated pathways. (A) Biological processes, (B) KEGG pathways, (C) Reactome pathways and (D) the protein-protein interaction network of TMSB10. Enrichment curves are shown for activated gene sets related to (E) DNA replication and (F) P53 signaling pathway. KEGG, Kyoto Encyclopedia of Genes and Genomes; TMSB10, thymosin β10.

Figure 10

Knockdown of TMSB10 impaired the proliferation, invasion and migration of RCC cell lines in vitro. (A) TMSB10 protein expression was successfully knocked down in ACHN cells. (B) Cell Counting kit-8 assays detected the effects of TMSB10 knockdown on the proliferation of ACHN cells. Representative images of (C) migration and (D) invasion assays performed using ACHN cells (magnification, ×100). Data are presented as the mean ± standard deviation from three independent experiments. ^**P<0.01, ^***P<0.001 and ^****P<0.0001. cRCC, clear cell renal cell carcinoma; ns, no statistical significance; NC, negative control; si, small interfering RNA; TMSB10, thymosin β10.