Overexpressed pseudogenes, DUXAP8 and DUXAP9, promote growth of renal cell carcinoma and serve as unfavorable prognostic biomarkers

Abstract

Background: Growing studies have reported that pseudogenes play key roles in multiple human cancers. However, expression and roles of pseudogenes in renal cell carcinoma remains absent.

Results: 31 upregulated and 16 downregulated pseudogenes were screened. Higher expression of DUXAP8 and DUXAP9 indicated poorer prognosis of kidney cancer. 33 and 5 miRNAs were predicted to potentially binding to DUXAP8 and DUXAP9, respectively. miR-29c-3p was identified as the most potential binding miRNAs of DUXAP8 and DUXAP9 based on expression, survival and correlation analyses. 254 target genes of miR-29c-3p were forecast. 47 hub genes with node degree >= 10 were identified. Subsequent analysis for the top 10 hub genes demonstrated that COL1A1 and COL1A2 may be two functional targets of DUXAP8 and DUXAP9. Expression of DUXAP8, DUXAP9, COL1A1 and COL1A2 were significantly increased in cancer samples compared to normal controls while miR-29c-3p expression was decreased. Luciferase reporter assay revealed that miR-29c-3p could directly bind to DUXAP8, DUXAP9, COL1A1 and COL1A2. Functional experiments showed that DUXAP8 and DUXAP9 enhanced but miR-29c-3p weakened growth of renal cell carcinoma.

Conclusions: In conclusion, upregulated DUXAP8 and DUXAP9 promote growth of renal cell carcinoma and serve as two promising prognostic biomarkers.

Methods: Dysregulated pseudogenes were obtained by dreamBase and GEPIA. The binding miRNAs of pseudogene and targets of miRNA were predicted using starBase and miRNet. Kaplan-Meier plotter was utilized to perform survival analysis, and Enrichr database was introduced to conduct functional enrichment analysis. Hub genes were identified through STRING and Cytoscape. qRT-PCR, luciferase reporter assay, cell counting assay and colony formation assay were performed to validate in silico analytic results.

Keywords: DUXAP8; DUXAP9; kidney cancer; pseudogene; renal cell carcinoma (RCC).

Figure 1

Expression and prognostic values of 7 potential pseudogenes in clear cell renal cell carcinoma (ccRCC) determined by GEPIA database. (A1) Expression of AC007326.9 in ccRCC compared with normal controls; (A2) expression of DUXAP8 in ccRCC compared with normal controls; (A3) expression of DUXAP9 in ccRCC compared with normal controls; (A4) expression of NUDT4P2 in ccRCC compared with normal controls; (A5) expression of RP11-255H23.2 in ccRCC compared with normal controls; (A6) expression of AF186192.5 in ccRCC compared with normal controls; (A7) expression of SLC2A3P1 in ccRCC compared with normal controls; (B1) expression of AC007326.9 among major stages in ccRCC; (B2) expression of DUXAP8 among major stages in ccRCC; (B3) expression of DUXAP9 among major stages in ccRCC; (B4) expression of NUDT4P2 among major stages in ccRCC; (B5) expression of RP11-255H23.2 among major stages in ccRCC; (B6) expression of AF186192.5 among major stages in ccRCC; (B7) expression of SLC2A3P1 among major stages in ccRCC; (C1) prognostic role (overall survival) of AC007326.9 in ccRCC; (C2) prognostic role (overall survival) of DUXAP8 in ccRCC; (C3) prognostic role (overall survival) of DUXAP9 in ccRCC; (C4) prognostic role (overall survival) of NUDT4P2 in ccRCC; (C5) prognostic role (overall survival) of RP11-255H23.2 in ccRCC; (C6) prognostic role (overall survival) of AF186192.5 in KIRC; (C7) prognostic role (overall survival) of SLC2A3P1 in ccRCC; (D1) prognostic role (disease free survival) of AC007326.9 in ccRCC; (D2) prognostic role (disease free survival) of DUXAP8 in ccRCC; (D3) prognostic role (disease free survival) of DUXAP9 in ccRCC; (D4) prognostic role (disease free survival) of NUDT4P2 in ccRCC; (D5) prognostic role (disease free survival) of RP11-255H23.2 in ccRCC; (D6) prognostic role (disease free survival) of AF186192.5 in ccRCC; (D7) prognostic role (disease free survival) of SLC2A3P1 in ccRCC. “*” represents P-value less than 0.05. Three horizontal lines in the box plot represent minimum, median and maximum, respectively.

Figure 2

Survival analysis of DUXAP8 and DUXAP9 in chRCC (chromophobe renal cell carcinoma) and pRCC (papillary renal cell carcinoma). (A) Prognostic value (overall survival) of DUXAP8 in chRCC; (B) prognostic value (disease free survival) of DUXAP8 in chRCC; (C) prognostic value (overall survival) of DUXAP8 in pRCC; (D) prognostic value (disease free survival) of DUXAP8 in pRCC; (E) prognostic value (overall survival) of DUXAP9 in chRCC; (F) prognostic value (disease free survival) of DUXAP9 in chRCC; (G) prognostic value (overall survival) of DUXAP9 in pRCC; (H) prognostic value (disease free survival) of DUXAP9 in pRCCs.

Figure 3

Establishment of the potential DUXAP8/DUXAP9-miRNA regulatory network in renal cell carcinoma.

Figure 4

Expression and survival analysis of miRNAs in clear cell renal cell carcinoma (ccRCC or KIRC). (A) Box-whisker plot showed expression of hsa-miR-29c-3p in ccRCC compared with normal controls (three horizontal lines in the box plot represent minimum, median and maximum); (B) box-whisker plot showed expression of hsa-miR-92b-3p in ccRCC compared with normal controls (three horizontal lines in the box plot represent minimum, median and maximum); (C) box-whisker plot showed expression of hsa-miR-500a-3p in ccRCC compared with normal controls (three horizontal lines in the box plot represent minimum, median and maximum); (D) prognostic value of hsa-miR-29c-3p in ccRCC; (E) prognostic value of hsa-miR-92b-3p in ccRCC; (F) prognostic value of hsa-miR-500a-3p in ccRCC.

Figure 5

KEGG pathway enrichment, GO functional annotation and PPI network analysis for target genes of hsa-miR-29c-3p. (A) The top 10 enriched KEGG pathway items; (B) the top 10 enriched biological process (BP) items; (C) the top 10 enriched cellular component (CC) items; (D) the top 10 enriched molecular function (MF) items; (E) the top 47 hub genes (node degree >= 10) in PPT network of target genes.

Figure 6

COL1A1 and COL1A2 were identified as two potential target genes of DUXAP8 and DUXAP9. (A) Box-whisker plot represented expression of COL1A1 in clear cell renal cell carcinoma (ccRCC or KIRC) compared with normal controls determined by GEPIA database; (B) Box-whisker plot represented expression of COL1A2 in ccRCC compared with normal controls determined by GEPIA database; (C) expression of COL1A1 in ccRCC compared with normal controls determined by UALCAN database; (D) expression of COL1A2 in ccRCC compared with normal controls determined by UALCAN database; (E) expression of COL1A1 among major stages in ccRCC determined by GEPIA database; (F) expression of COL1A2 among major stages in ccRCC determined by GEPIA database; (G) correlation analysis between has-miR-29c-3p and COL1A1 in ccRCC; (H) correlation analysis between has-miR-29c-3p and COL1A2 in ccRCC. “*” represents P-value less than 0.05. Three horizontal lines in the box plot represent minimum, median and maximum, respectively.

Figure 7

Pseudogenes DUXAP8 and DUXAP9 promote tumor growth via suppression of miR-29c-3p-COL1A1/COL1A2 axis in renal cell carcinoma. (A) The expression of pseudogene DUXAP8 in renal cell carcinoma samples and normal controls; (B) the expression of pseudogene DUXAP9 in renal cell carcinoma samples and normal controls; (C) the expression of miR-29c-3p in renal cell carcinoma samples and normal controls; (D) the expression of COL1A1 in renal cell carcinoma samples and normal controls; (E) the expression of COL1A2 in renal cell carcinoma samples and normal controls; (F) miR-29c-3p suppressed Renilla luciferase activity of the reporters containing the wild-type but not mutant DUXAP8 in ACHN cell line; (G) miR-29c-3p suppressed Renilla luciferase activity of the reporters containing the wild-type but not mutant DUXAP9 in A498 cell line; (H) miR-29c-3p suppressed Renilla luciferase activity of the reporters containing the wild-type but not mutant COL1A1 in ACHN cell line; (I) miR-29c-3p suppressed Renilla luciferase activity of the reporters containing the wild-type but not mutant COL1A2 in A498 cell line; (J) the knockdown effect of si-DUXAP8 and si-DUXAP9 in ACHN and A498 cell lines; (K) the overexpression effect of miR-29c-3p mimic in ACHN and A498 cell lines; (L) knockdown of pseudogene DUXAP8/DUXAP9 or/and overexpression of miR-29c-3p inhibited cell growth in vitro; (M) knockdown of pseudogene DUXAP8/DUXAP9 or/and overexpression of miR-29c-3p suppressed the colony formation of cell lines in renal cell carcinoma. “*” represents P-value less than 0.05. Abbreviations: MUT, mutant; WT, wild type.

Figure 8

Model of DUXAP8/DUXAP9-miR-29c-3p-COL1A1/COL1A2 axis in renal cell carcinoma.