Serum KIAA1199 is an advanced-stage prognostic biomarker and metastatic oncogene in cholangiocarcinoma

Abstract

Background: Cell proliferation and migration are the determinants of malignant tumor progression, and a better understanding of related genes will lead to the identification of new targets aimed at preventing the spread of cancer. Some studies have shown that KIAA1199 (CEMIP) is a transmembrane protein expressed in many types of noncancerous cells and cancer cells. However, the potential role of KIAA1199 in the progression of cholangiocarcinoma (CCA) remains unclear.

Results: Analysis of cancer-related databases showed that KIAA1199 is overexpressed in CCA. ELISA, immunohistochemistry, Western blotting and qPCR indicated high expression levels of KIAA1199 in serum, CCA tissues and CCA cell lines. In the serum (n = 41) and large sample validation (n = 177) cohorts, higher KIAA1199 expression was associated with shorter overall survival and disease-free survival times. At the cellular level, KIAA1199 overexpression (OE) promoted CCA growth and metastasis. Subcutaneous tumor xenograft experiments showed that KIAA1199 enhances CCA cell proliferation. Additionally, the expression levels of components in the EMT-related TGF-β pathway changed significantly after KIAA1199 upregulation and silencing.

Conclusion: KIAA1199 is a promising new diagnostic molecule and therapeutic target in CCA. The serum KIAA1199 level can be used as a promising clinical tool for predicting the overall postoperative outcomes of patients with CCA.

Methods: CCA-related KIAA1199 data were downloaded from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. To assess the prognostic impact of KIAA1199, an enzyme-linked immunosorbent assay (ELISA) was used to measure the serum level of KIAA1199 in 41 patients who underwent surgical resection. Immunohistochemical staining, Western blotting and qPCR were used to verify and retrospectively review the expression levels of KIAA1199 in cancer tissue specimens from 177 CCA patients. The effect of KIAA1199 on CCA was evaluated by cell-based functional assays and subcutaneous tumor xenograft experiments. The expression levels of proteins associated with epithelial-mesenchymal transition (EMT) and activation of relevant signaling pathways were measured via Western blotting.

Keywords: KIAA1199; bile; biomarker; cholangiocarcinoma; serum.

Figure 1

Bioinformatics prediction. (A) GEO: (GSE76297), Normal: n=92, CCA: n=91, (p<0.001). (B) TCGA database, Normal: n=9, CCA: n=36, (p<0.001). Protein and mRNA expression of KIAA1199 in CCA tissues cell lines (C) The relative expression levels of KIAA1199 in Pathological sections. (D) The relative protein expression levels of KIAA1199 in 20 pairs of CCA and adjacent normal tissues(p<0.001). (E) The relative mRNA expression levels of KIAA1199 in 20 pairs of CCA and adjacent normal tissues(p<0.001). (F, G) The relative protein and mRNA expression of KIAA1199 in four CCA cell lines.>

Figure 2

(A) Immunofluorescence localization of KIAA1199 and CK-18 in cholangiocarcinoma tissue (red, KIAA1199; green, CK-18; blue, DAPI). (B) Immunofluorescence localization of KIAA1199 and Phalloidin in Hucct-1 cell line (red, KIAA1199; green, Phalloidin; blue, DAPI). (C) Microscopic determination of KIAA1199 cellular localization using QBC939 cells transfected with green fluorescent protein (GFP), KIAA1199-GFP chimeric cDNAs. (D) KIAA1199 had signal peptides. (E) KIAA1199 has 602 amino acids, all of which are extracellular, and there is no transmembrane domain (TMD). (F) Western blot analysis was performed to detect KIAA1199 in cell lysates and conditioned culturing medium from Hucct-1 and Hucct-1 knockdown KIAA1199 (siKIAA1199).

Figure 3

(A) Comparison of bile KIAA1199 levels in CCA patients (n=33) and healthy controls (n=15), p<0.001. (B) Comparison of serum KIAA1199 levels in CCA patients (n=41) and healthy controls (n=41), p<0.001. (C) ROC curve was used to evaluate serum KIAA1199 and ca199 diagnostic performances. (D, E) Kaplan-Meier analyses for overall survival (OS) and disease-free survival (DFS) in 41 CCA patients according to serum KIAA1199 levels. The quartile method is used to divide the data into four parts, and the dotted line in the figure is the dividing line of each part.

Figure 4

Immunohistochemical staining for KIAA1199, E-cadherin, N-cadherin and vimentin. (A). A1, A2: Positive KIAA1199 expression in CCA tissue. E1, E2: Negative KIAA1199expression in adjacent tissue. B1, B2: Negative E-cadherin expression in CCA tissue. F1, F2: Positive E-cadherin expression in adjacent tissue. C1, C2: Positive N-cadherin expression in CCA tissue. G1, G2: Positive Vimentin expression in CCA tissue. H1, H2: Negative Vimentin expression in adjacent tissue. (scale bar, 50 μm; magnification: ×200, ×400) (B) Western blot analysis of EMT signaling molecules (N-cadherin, E-cadherin and Vimentin) in KIAA1199 silenced Hucct1 cell line. (C) Western blot analysis of EMT signaling molecules ((N-cadherin, E-cadherin and Vimentin) in KIAA1199 overexpressed QBC939 cell line. Representative of three independent experiments.

Figure 5

(A) Kaplan-Meier analysis of overall survival (OS) and disease-free survival (DFS) in 177 patients with CCA according to KIAA1199 staining. (B, C) Univariate and multivariate analyses of factors associated with survival and recurrence.

Figure 6

KIAA1199 regulates proliferation and invasion in CCA cell lines. (A, B) The relative protein and mRNA expression of four small interfering RNA in siKIAA1199-transfected cells compared with control and parental cells. (F, G) Overexpression of KIAA1199 in QBC939 cells with lentivirus infection was verified by western blotting and qPCR. Proliferation of Hucct-1 cells was detected with CCK-8 after silencing KIAA1199 in Hucct-1 cells (C) and overexpressing KIAA1199 in QBC939 cells (H) in normal medium with 10% FBS. Wound healing assay was applied to evaluate migration of Hucct-1 (D) and QBC939 (I). 24 h after a scratch in the cell monolayer, the wound size was measured again. Migration of Hucct-1 and QBC939 cells was assessed with transwell assay (E, J). After KIAA1199 knockdown and overexpression, cells were seeded in the upper transwell chamber and incubated for 24 h, with FBS in the lower chamber. (original magnification: ×200; scale bar, 20 μm). Data, mean ± S.D., and representative of three independent experiments.

Figure 7

(A) Xenografts were established in nude mice with stable KIAA1199 knockdown or overexpression cell. KIAA1199 knockdown decreased the volume (B) and weight (C) of xenograft tumors. KIAA1199 overexpression increased the volume and weight of xenograft tumors. Tumor diameter was measured every 3 day. (D) Ki67 staining showed that KIAA1199 can improve the proliferation ability of xenograft tumors. (E, F) Immunohistochemistry and WB detected the expression of KIAA1199 in xenograft tumors. *, ** and *** represented P<0.05, P = 0.01 and 0.001 by Student's t-test, between the indicated groups.

Figure 8

The expression of KIAA1199 and TGF-β-PI3K-Akt pathway-associated proteins by western blot analyses. (A) Western blot analysis of KIAA1199 and TGF-β-PI3K-Akt pathway-associated proteins in KIAA1199 silenced Hucct1 cell line. (B) Western blot analysis of KIAA1199 and TGF-β-PI3K-Akt pathway-associated proteins in KIAA1199 overexpressed QBC939 cell line. (C) QBC939 were pretreated with TGF-β inhibitor (SB431542, 5 μM) for 2 h and transwell migration assay was performed in the absence or presence of KIAA1199 conditioned medium (CM). (D) QBC939 were pretreated with PI3K inhibitor (LY294002, 6μM) for 2 h and trans-well migration assay was performed in the absence or presence of KIAA1199 conditioned medium (CM). (E) KIAA1199-mediated EMT may occur through a non-Smad pathway. At least three independent experiments were preformed, data presented as mean ± SD, *, ** and *** represented P<0.05, P= 0.01 and 0.001 by Student's t-test, between the indicated group.