High expression of long non-coding RNA SPRY4-IT1 predicts poor prognosis of clear cell renal cell carcinoma

Abstract

Introduction: Long non-coding RNAs (lncRNAs) play a key role in cellular processes, such as cell growth, apoptosis, and carcinogenesis. lncRNAs SPRY4-IT1 has recently been identified to be involved in tumorigenesis of several cancers such as non-small cell lung cancer and esophageal squamous cell carcinoma. However, the role of SPRY4-IT1 in clear cell renal cell carcinoma (ccRCC) remains unclear.

Methods: The expression of SPRY4-IT1 was examined in ccRCC patients and renal cancer cell lines by using quantitative real-time PCR (qRT-PCR). The relationship between SPRY4-IT1 level and clinicopathological parameters of ccRCC was analyzed with the Kaplan-Meier method and Cox proportional hazards model. Small interfering RNA (siRNA) was used to suppress SPRY4-IT1 expression in renal cancer cell line 786-O. In vitro assays were performed to further explore its role in renal cancer progressio.

Results: The relative level of SPRY4-IT1 was significantly higher in ccRCC tissues compared to the adjacent normal renal tissues. And higher expression of SPRY4-IT1 was found in renal cancer cell lines compared with the normal human proximal tubule epithelial cell line HK-2. The ccRCC patients with higher SPRY4-IT1 expression had an advanced clinical stage and poorer prognosis than those with lower SPRY4-IT1 expression. Multivariate analyses by Cox's proportional hazard model revealed that expression of SPRY4-IT1 was an independent prognostic factor in ccRCC. In vitro assays, our results indicated that knockdown of SPRY4-IT1 reduced renal cancer cell proliferation, migration, and invasion.

Conclusions: Our data suggested that lncRNA SPRY4-IT1 might be considered as a potential prognostic indicator and a potential target for therapeutic intervention in RC.

Keywords: Long non-coding RNAs; SPRY4-IT1; clear cell renal cell carcinoma; prognosis.

Figure 1

Expression analysis of SPRY4-IT1 in ccRCC tissues and cell lines. The relative SPRY4-IT1 expression levels were determined using qRT-PCR and demonstrated using the comparative ΔCt method. A. Significant higher expression of SPRY4-IT1 was found in ccRCC tissues than in adjacent normal renal tissues. B. Significant higher expression of SPRY4-IT1 was found in four RCC cell lines than in normal human proximal tubule epithelial cell line HK-2. Results are expressed as mean ± SD for three replicate determination. All data analyzed using Student’s t test. *P < 0.05.

Figure 2

Kaplan-Meier overall survival curves by SPRY4-IT1 level. Patients with higher SPRY4-IT1 expression in tumor tissue were closely correlated with poorer overall survival than patients with lower SPRY4-IT1 expression (P < 0.05).

Figure 3

SPRY4-IT1 is essential for renal cancer cell proliferation. A. The relative expression level of SPRY4-IT1 in 786-O cells is significantly decreased by si-SPRY4-IT1 compared with the si-NC detected by qRT-PCR. B. The proliferation was decreased in si-SPRY4-IT1 group as compared to that of the si-NC group as identified by MTT assays. C. Inhibition of the SPRY4-IT1 promotes apoptosis in 786-O cells. D. Inhibition of the SPRY4-IT1 promotes cell cycle arrest in G0/G1 phase in 786-O cells. Results are expressed as mean ± SD for three replicate experiments. All data analyzed using Student’s t test. *P < 0.05.

Figure 4

The effects of SPRY4-IT1 on 786-O cells migration and invasion. A, B. Wound healing assays showed that 786-O cells transfected with si-SPRY4-IT1 displayed significantly lower migration capacity compared with those transfected with si-NC. C, D. Transwell invasion assays indicated that 786-O cells transfected with si-SPRY4-IT1 displayed significantly lower invasion capacity compared with those transfected with si-NC. Five areas were randomly selected in each chamber. The number of cells in these areas was counted, and results are expressed as means ± SD for three replicate experiments. All data analyzed using Student’s t test. *P < 0.05.