The Clinical Relevance and Tumor Promoting Function of C19orf10 in Kidney Renal Clear Cell Carcinoma

Abstract

Kidney renal clear cell carcinoma (KIRC) is the most common primary renal neoplasms. Currently, there are few molecular indicators and therapeutic targets that can be used in diagnostic and prognostic assessment. In this study, we identified the C19orf10 expression in KIRC specimens and explored the diagnostic and prognostic value of C19orf10 in KIRC using TCGA and CPTAC database. Loss-of- and gain-of- function of C19orf10 was performed to investigate the roles of C19orf10 on KIRC cell viability, proliferation, migration and invasion via CCK-8, Edu incorporation and Transwell assays respectively. C19orf10 was overexpressed in KIRC tissues and the elevated C19orf10 expression was closely associated with clinicopathological characteristics of KIRC including histological grade, TNM stage, metastatic status. Silencing C19orf10 significantly suppressed the viability, proliferation, migration and invasion ability, while overexpression of C19orf10 promoted the progression and malignant phenotype in KIRC cells. Furthermore, C19orf10 exerted its carcinogenic function by regulating ZO-1 and PTEN/Akt signaling pathway. Moreover, the Kaplan-Meier survival analysis, Cox regression analysis and receiver operating curve analysis showed that patients with C19orf10 overexpression have poor survival time. C19orf10 could discriminate KIRC patients with high-risk from low-risk. Taken together, C19orf10 contributes to KIRC development via ZO-1 and PTEN/Akt signaling pathway and C19orf10 could serve as a potential diagnostic and prognostic candidate and therapeutic target of KIRC.

Keywords: C19orf10; PTEN/Akt; ZO-1; diagnostic biomarker; kidney renal clear cell carcinoma; prognostic biomarker.

Figure 1

C19orf10 expression elevated in KIRC and correlated with clinical characteristics. (A) C19orf10 expression in 10 paired KIRC samples. (B) C19orf10 expression in KIRC in TCGA dataset. (C) C19orf10 expression in 72 paired KIRC in TCGA dataset. (D–J) Relative mRNA expression level of C19orf10 in TCGA dataset with differential histologic grade (D), TNM stage (E), pathologic metastasis (F), lymph node invasion (G), tumor size (H), age (I) and gender (J). The numbers of samples are indicated in figures, Mean ± SD is shown. P values are calculated by Student’s t-test or one-way ANOVA and indicated in figures.

Figure 2

C19orf10 protein up-regulated in CPTAC database and associates with KIRC clinical pathology. (A) C19orf10 protein expression in KIRC in CPTAC dataset. (B) C19orf10 protein expression in 84 paired KIRC in CPTAC dataset. (C–E) Relative protein expression level of C19orf10 in CPTAC dataset with differential histologic grade (C), TNM stage (D) and tumor size (E). (F) C19orf10 expression in 30 paired KIRC samples by qPCR detection, n=30. (G) Representative immunohistochemical images of C19orf10 in differential histologic grade in KIRC tissues. Scale bar, 100 μm. (H) Elevated C19orf10 expression in high grade KIRC tissues (grade 2-3, grade 3) compared with low grade ones (grade 1, grade 1-2 and grade 2). The numbers of samples are indicated in figures, Mean ± SD is shown. P values are calculated by Student’s t-test or one-way ANOVA and indicated in figures.

Figure 3

Silencing C19orf10 reduced KIRC cell viability and proliferation in vitro. (A) C19orf10 mRNA expression in KIRC cell lines and human renal proximal tubular cells (HK-2), n=3. (B) C19orf10 protein expression level in KIRC cell lines and human renal proximal tubular cells. (C, D) The silencing efficiency of siRNAs targeting C19orf10 on protein (C) and mRNA level (D), n=3. (E) Suppression of C19orf10 inhibited the KIRC cell viability in CCK-8 assay, n=3. (F, G) Knockdown of C19orf10 suppressed KIRC cell proliferation in EdU incorporation assay, n=8. (H) Knockdown of C19orf10 induced more cell apoptosis in ACHN cells, n=3. Scale bar, 50 μm. Mean ± SD is shown. **P < 0.01 by Student’s t-test and two-way ANOVA.

Figure 4

Overexpression of C19orf10 enhances the viability and proliferation of KIRC cells. (A, B) The verify of C19orf10 overexpression on mRNA (A) and protein level (B), n=3. (C) C19orf10 promoted the viability of 769-P cells, n=3. (D, E) C19orf10 increased the proliferation of 769-P cell, n=8. Scale bar, 50 μm, Mean ± SD is shown, **P < 0.01 by two-way ANOVA (C), Student’s t-test (A, E).

Figure 5

C19orf10 promotes the migration and invasion of KIRC cells. (A, B) Silencing C19orf10 inhibited the migration and invasion ability of ACHN cell in transwell migration and invasion assay. (C, D) C19orf10 promoted the migration and invasion of 769-P cell. Scale bar, 1000 μm for overall images, 100 μm for partial migration pictures, 500 μm for partial invasion pictures. Mean ± SD is shown, n = 5. **P < 0.01 by Student’s t-test.

Figure 6

C19orf10 promotes KIRC progression via ZO-1 and PTEN/Akt pathway. (A) ZO-1 was the C19orf10 negatively regulated genes. (Pearson correlation analysis, n=18). (B) PTEN was negatively correlated with C19orf10. (Pearson correlation analysis, n=18). (C) Knockdown of C19orf10 increased PTEN and ZO-1 expression in ACHN cells. (D) Overexpression of C19orf10 decreased PTEN and ZO-1 expression in 769-P cells. (E) Knockdown of C19orf10 inhibited the phosphorylation of Akt. (F) Overexpression of C19orf10 activated Akt.

Figure 7

C19orf10 is a potential prognostic and diagnostic biomarker in KIRC. (A, B) Univariate Cox regression analysis (A) and multivariate Cox regression analysis (B) of the hazard ratios revealed that increased C19orf10 could be an independent prognostic biomarker for the disease free survival in KIRC patients. (C) Kaplan-Meier overall survival curve of KIRC patients with low (n = 262) and high (n =262) C19orf10 expression in TCGA dataset. (D) Kaplan-Meier analysis of disease free survival in KIRC patients based on the expression level of C19orf10, low (n = 241) and high (n =221). (E–K) ROC curve analysis of overall survival (E), disease free survival (F), TNM stage (G), histologic grade (H), distant metastasis (I), lymph node metastasis (J), tumor size (K) for C19orf10 as a biomarker to predict the KIRC prognosis and discriminate KIRC patients with high risk from low risk. The calculated AUC and P values are reported in figures.