Hypoxia-regulated lncRNA CRPAT4 promotes cell migration via regulating AVL9 in clear cell renal cell carcinomas

Figures & data

Figure 1 The expression of CRPAT4 in ccRCC tissues and cell lines.

Notes: Relative CRPAT4 expression levels in ccRCC tissues and the matched normal renal tissues (n=30 pairs) (A) and in ccRCC cell lines (B) determined by qRT-PCR. (C) Cell fractionation followed by qRT-PCR showed that CRPAT4 mainly localized in the cytoplasm. (D) The expression of CRPAT4 in high Fuhrman nuclear grade tumors (n=11) is significantly higher than in low Fuhrman nuclear grade tumors (n=19, P=0.0357). *.<0.05.
Abbreviations: ccRCC, clear cell renal cell carcinoma; CRPAT4, ccRCC prognosis-associated transcript 4; qRT-PCR, quantitative real-time polymerase chain reaction.

Figure 2 Kaplan–Meier curve analysis of overall survival and progression-free survival according to the CRPAT4 expression.

Notes: TCGA KIRC RNA sequencing data were extracted for survival analysis. ccRCC patients with high CRPAT4 expression (n=135) exhibited significantly worse overall survival (A) and progression-free survival (B) than patients with low CRPAT4 expression (n=136) (all P<0.0001, log rank test).
Abbreviations: ccRCC, clear cell renal cell carcinoma; CRPAT4, ccRCC prognosis-associated transcript 4; TCGA, The Cancer Genome Atlas; Exp, expression.

Figure 3 CRPAT4 is regulated by HIF-1α in ccRCC cell lines.

Notes: (A) The expression of HIF-1α and HIF-2α in 786-O (vhl mut) and Caki-1 (vhl wild-type) cells exposed to hypoxia or normoxia for 24 h, determined by Western blot. (B) The expression of CRPAT4 was significantly downregulated in Caki-1 cells in hypoxia compared to in normoxia, but not in 786-O cells, as determined by qRT-PCR. (C) The expression of PLOD2 showed significant upregulation in Caki-1 cells in hypoxia compared to in normoxia, but not in 786-O cells, as determined by qRT-PCR. (D) siRNAs targeting HIF-1α or HIF-2α were adopted to knockdown the expression of HIF-1α or HIF-2α in Caki-1 cells, determined by Western blot. (E) qRT-PCR showed that si-HIF1α, but not si-HIF-2α, could counteract the inhibitory effect of hypoxia upon CRPAT4 expression. *.<0.05, **.<0.001.
Abbreviations: ccRCC, clear cell renal cell carcinoma; CRPAT4, ccRCC prognosis-associated transcript 4; Mut, mutant; NC, negative control; qRT-PCR, quantitative real-time polymerase chain reaction.

Figure 4 Effects of CRPAT4 knockdown on cell migration and proliferation in vitro.

Notes: (A) The knockdown efficacy of CRPAT4 by two independent siRNAs targeting CRPAT4 compared with the NC siRNA in both 786-O and Caki-1 cells was verified by qRT-PCR. (B) CRPAT4 knockdown significantly inhibited cell proliferation in 786-O; however, only modest inhibitory effect of the CRPAT4 knockdown on cell proliferation was observed in Caki-1 cells. CRPAT4 knockdown significantly inhibited cell migration in 786-O, determined by wound healing assay (C) and Transwell migration assay (D). (E) qRT-PCR showed that no remarked changes of the AVL9 mRNA were observed following CRPAT4 knockdown in both cell lines. (F) Western blot analysis revealed that CRPAT4 knockdown significantly downregulated AVL9 protein in both cell lines, and more downregulation of AVL9 protein was observed in 786-O than in Caki-1 cells. Data are shown as mean ± SEM. *.<0.05, **.<0.0001.
Abbreviations: ccRCC, clear cell renal cell carcinoma; CRPAT4, ccRCC prognosis-associated transcript 4; NC, negative control; qRT-PCR, quantitative real-time polymerase chain reaction; SEM, standard error of the mean.

Figure 5 Schematic diagram of CRPAT4 as an oncogene in ccRCC progression.

Abbreviations: ccRCC, clear cell renal cell carcinoma; CRPAT4, ccRCC prognosis-associated transcript 4.