Long Non-Coding RNA DUXAP8 Enhances Renal Cell Carcinoma Progression via Downregulating miR-126

Abstract

BACKGROUND Renal cell carcinoma (RCC) is one of the common malignant tumors in the urinary system, which endangers human health for a long time. The past decade, the molecular biology of renal cell carcinoma has made considerable progress, so that we have a more profound understanding of renal cell carcinoma. Molecular biological mechanism of renal cell carcinoma remains to be explored. Evidence indicates that long non-coding RNAs (lncRNAs) may be important players in human cancer progression, including RCC. In this study, we found that a newly discovered pseudogene-derived lncRNA named DUXAP8, a 2107-bp RNA, was remarkably upregulated in RCC. MATERIAL AND METHODS Expression of lncRNA DUXAP8 was determined by a qRT-PCR assay in RCC tissues. The proliferation and invasion of RCC cell were measured by a cell proliferation assay and a Transwell invasion assay. Expression of miR-126 was detected by real-time PCR. Interactions between lncRNA DUXAP8 and miR-126 were measured by a luciferase reporter assay and an RNA-pull down assay. In vivo experiments were used to detect tumor formation. RESULTS Together, our study not only identifies lncRNA DUXAP8 as a negative regulator of renal cancer with potential clinical value, but also reveals a regulatory mechanism by long non-coding RNAs to control tumor development. CONCLUSIONS Results from this study provide evidence that lncRNA DUXAP8 enhances renal cell carcinoma progression via downregulating of miR-126, which offers a new approach for the treatment of RCC.

Figure 1

lncRNA DUXAP8 may have effects on the pathology development of RCC. (A) RT-PCR assay was performed to screen 10 potential lncRNAs. lncRNA DUXAP8 was the most appropriate lncRNA to select. Each column is the ratio of cancer tissue/normal tissue. (B) lncRNA DUXAP8 expression in RCC tissues vs. normal renal tissues. (C) Association of lncRNA DUXAP8 expression of 6 RCC patients. P values are given in the figures.

Figure 2

lncRNA DUXAP8 promoted the invasion of lung adenocarcinoma. (A, B) Verification of lncRNA DUXAP8 knockdown by qRT-PCR assay in A498 and 786-O cells. (C, D) Cell growth was measured by a Transwell assay. Each sample was run in triplicate and in multiple experiments for mean ±SEM. * P<0.05, ** P<0.01 compared to controls.

Figure 3

lncRNA DUXAP8 promotes RCC cell proliferation by downregulating miR-126 expression. (A) qRT-PCR assay was performed to detect the expression level of miR-126. (B) The correlation between miR-126 and DUXAP8 level in RCC tumor tissues was analyzed by q-PCR. (C) qRT-PCR assay was performed to detect the expression of miR-126 after knocking down lncRNA DUXAP8in A498 and 786-O cells. (D) qRT-PCR assay was performed to detect the expression of miR-126 after overexpression of lncRNA DUXAP8 in A498 and 786-O cells. Each sample was run in triplicate and in multiple experiments for mean ±SEM. * P<0.05, ** P<0.01 compared to controls.

Figure 4

miR-126 targets CED-9 to inhibit RCC tumorigenesis. (A) Luciferase reporter assays were performed in 293T cells after co-transfecting CED-9 wild-type or mutant UTR with miR-126. (B) The protein level of CED-9 after knocking down miR-126 in 786-O cells. (C) The protein level of CED-9 after over expression of miR-126 in 786-O cells. (D) The correlation between miR-126 andCED-9 level in RCC tumor tissues were analyzed by q-PCR. Each sample was run in triplicate and in multiple experiments for mean ±SEM. * P<0.05, ** P<0.01 compared to controls.

Figure 5

lncRNA DUXAP8 functions by altering miR-126/CED-9 signals to promote RCC cell proliferation and invasion. (A, B) Protein level of CED-9 was determined after cotransfection with lncRNA DUXAP8-shRNA and miR-126 inhibitor in A498 and 786-O. (C, D) Adding miR-126 inhibitor partially rescued the growth of A498 and 786-O cells transfected with lncRNA DUXAP8-shRNA by a cell proliferation assay. Each sample was run in triplicate and in multiple experiments for mean ±SEM. * P<0.05, ** P<0.01 compared to controls.