TXNDC5 is a cervical tumor susceptibility gene that stimulates cell migration, vasculogenic mimicry and angiogenesis by down-regulating SERPINF1 and TRAF1 expression

Abstract

TXNDC5 (thioredoxin domain-containing protein 5) catalyzes disulfide bond formation, isomerization and reduction. Studies have reported that TXNDC5 expression is increased in some tumor tissues and that its increased expression can predict a poor prognosis. However, the tumorigenic mechanism has not been well characterized. In this study, we detected a significant association between the rs408014 and rs7771314 SNPs at the TXNDC5 locus and cervical carcinoma using the Taqman genotyping method. We also detected a significantly increased expression of TXNDC5 in cervical tumor tissues using immunohistochemistry and Western blot analysis. Additionally, inhibition of TXNDC5 expression using siRNA prevented tube-like structure formation, an experimental indicator of vasculogenic mimicry and metastasis, in HeLa cervical tumor cells. Inhibiting TXNDC5 expression simultaneously led to the increased expression of SERPINF1 (serpin peptidase inhibitor, clade F) and TRAF1 (TNF receptor-associated factor 1), which have been reported to inhibit angiogenesis and metastasis as well as induce apoptosis. This finding was confirmed in Caski and C-33A cervical tumor cell lines. The ability to form tube-like structures was rescued in HeLa cells simultaneously treated with anti-TXNDC5, SERPINF1 and TRAF1 siRNAs. Furthermore, the inhibition of TXNDC5 expression significantly attenuated endothelial tube formation, a marker of angiogenesis, in human umbilical vein endothelial cells. The present study suggests that TXNDC5 is a susceptibility gene in cervical cancer, and high expression of this gene contributes to abnormal angiogenesis, vasculogenic mimicry and metastasis by down-regulating SERPINF1 and TRAF1 expression.

Keywords: SERPINF1; TRAF1; TXNDC5; cervical tumor; pathway.

Figure 1. Detection of TXNDC5 expression in cervical carcinoma tissues

(A) Immunohistochemistry detected significant expression of TXNDC5 in cervical carcinoma tissue. (B) TXNDC5 expression was not significantly expressed in normal cervical tissue adjacent to the tumor. Original magnification × 20. (C) The immuno-reactive score analysis indicated significantly increased TXNDC5 expression in cervical squamous cell carcinomas compared with the corresponding adjacent normal tissues. (D) Western blotting detected a 48-kDa band in tissue extracts from all 7 cervical tumor samples and 5 of 7 uterine myoma samples using an anti-TXNDC5 antibody. (E) TXNDC5 expression was normalized to GAPDH expression in each of the tissues. The analysis indicated significantly increased TXNDC5 expression in cervical tumor tissues. *indicates p < 0.05, **indicates p < 0.01 and ***indicates p < 0.001.

Figure 2. The effect of TXNDC5 on the capability of HeLa cells to form tube-like structures

(A) HeLa cells treated with AllStars siRNA. (B) HeLa cells treated with anti-TXNDC5 siRNA. (C) HeLa cells treated with anti-TXNDC5 siRNA and anti- TRAF1 siRNA. (D) HeLa cells treated with anti-TXNDC5 siRNA and anti-SERPINF1 siRNA. (E) HeLa cells treated with anti-TXNDC5 siRNA, anti- TRAF1 siRNA and anti-SERPINF1 siRNA. (F) Quantification of tube-like structures. The number of tubular-like structures was counted using phase contrast microscopy (×10). All test samples were performed in triplicate. *indicates p < 0.05, **indicates p < 0.01, and ***indicates p < 0.001. Full line indicates the difference between AllStars siRNA treatment and other siRNA treatment; dotted line indicates the difference between anti-TXNDC5 siRNA treatment and siRNA mixture treatment.

Figure 3. PCR array analysis of relative gene expressions in anti-TXNDC5 siRNA-treated HeLa cells

(A) Angiogenesis PCR Array, (B) Human Cancer PathwayFinderPCR Array, (C) Signal Transduction PathwayFinder PCR Array and (D) TNF Signaling Pathway PCR Array detected gene expression related to specific signaling pathways. (E) The above PCR array results are depicted in one map. The expression levels of MMP14, EGF and IFRD1 were down-regulated, whereas the expression levels of TRAF1, NGFR, SERPINF1, ITGB3 and KRT14 were up-regulated in the siRNA-treated cells.

Figure 4. Real-time PCR analysis of EGF, IFRD1, ITGB3, KRT14, MMP14, NGFR, SERPINF1 and TRAF1 expression in HeLa cells treated with anti-TXNDC5 siRNA

The transcription levels of the target genes were normalized to GAPDH expression. *indicates p < 0.05, **indicates p < 0.01, and ***indicates p < 0.001.

Figure 5. Western blot analysis of SERPINF1 and TRAF1 expression in HeLa cells treated with anti-TXNDC5 siRNA

The expression levels of the target genes were normalized to GAPDH expression. **indicates p < 0.01.

Figure 6. Real-time PCR analysis of TXNDC5, SERPINF1 and TRAF1 expression in Caski and C-33A cells treated with anti-TXNDC5 siRNA

The expression levels of the target genes were normalized to GAPDH expression. (A–C) mRNA expression levels of target genes in sRNA-treated Caski cells. (D–F) mRNA expression levels of target genes in siRNA-treated C-33A cells. *indicates p < 0.05, and **indicates p < 0.01.

Figure 7. Western blot analysis of SERPINF1 and TRAF1 expression in Caski and C-33A cells treated with anti-TXNDC5 siRNA

Western blot analysis of SERPINF1 and TRAF1 expression in Caski and C-33A cells treated with anti-TXNDC5 siRNA. (A) Western blot detected SERPINF1, TRAF1, TXNDC5 and GAPDH protein expression in the treated Caski cells. (B–D) SERPINF1, TRAF1 and TXNDC5 expression in the treated Caski cells was normalized to GAPDH expression. (E) Western blot detected SERPINF1, TRAF1, TXNDC5 and GAPDH protein expression in the treated C-33A cells. (F–H) SERPINF1, TRAF1 and TXNDC5 expression in the treated C-33A cells was normalized to GAPDH expression. *indicates p < 0.05.

Figure 8. The effect of TXNDC5 on the formation of tube-like structures by HUVECs

(A) HUVECs treated with AllStars siRNA. (B) HUVECs treated with TXNDC5 siRNA. (C) Quantification of tube-like structures. The number of tubes was counted using phase contrast microscopy (× 10). All test samples were performed in triplicate. ***indicates p < 0.001.