Long noncoding RNA LINC00641 promotes renal cell carcinoma progression via sponging microRNA-340-5p

Abstract

Background: Emerging evidences have revealed that long non-coding RNAs (lncRNAs) have played critical roles in tumor occurrence and progression. LINC00641 has been reported to be involved in the initiation and development of several cancers in the recent years. However, the detailed biological role of LINC00641 in renal cell carcinoma (RCC) remains largely unclear.

Methods: In this study, the expression and biological function of LINC00641 were assessed in renal carcinoma both in vitro and in vivo. Cell proliferation, migration and colony formation assay were performed to explore the effect of LINC00641on growth, progression and invasion of RCC cell. qRT-PCR, flow cytometry and luciferase reporter assay and in vivo tumorigenicity assay were also carried out.

Results: The expression of LINC00641 was overexpressed in RCC tissues and cell lines, and high LINC00641 expression was correlated with tumor-node-metastasis stage. Furthermore, Silencing of LINC00641 remarkably inhibited the ability of cell proliferation, colony formation, and invasive capacities, as well as increasing the apoptotic rates of RCC cells in vitro. Mechanistically, miR-340-5p was validated to be targeted by LINC00641 and knockdown of miR-340-5p counteracted LINC00641 silencing-mediated inhibition of RCC progression. In addition, in vivo experiment confirmed the findings discovered in vitro.

Conclusions: These results suggested that LINC00641 promoted the progression of RCC by sponging miR-340-5p.

Keywords: Cancer progression; LINC00641; Long non-coding RNA; MiR-340-5p; MicroRNA; Renal cell carcinoma.

Fig. 1

Expression of LINC00641 and its prognostic significance in RCC patients. a LINC00641 expression level was determined in tumor and paired adjacent non-tumor tissues by qRT-PCR. b Relative LINC00641 expression in RCC with different TNM stages. c Kaplan–Meier survival analysis according to LINC00641 expression in 48 pair RCC patients. d Relative LINC00641 expression in 5 RCC cell lines and human renal tubular epithelial cell line (HK-2) were detected by qRT-PCR. **P < 0.01

Fig. 2

Knockdown of LINC00641 inhibits RCC cell proliferation and invasion in vitro. a Analysis of the LINC00641 expression level in A498 and ACHN cells expressing shLINC00641 by qRT-PCR. b Growth curve of RCC cells transduced with SCR or shRNA by CCK-8 assays. c Proliferation and d apoptosis of RCC cells transduced with SCR or shRNA as determined by colony formation assay or flow cytometry assay, respectively. e Representative photographs of migrated or invaded cells transduced with SCR or shRNA as determined by transwell migration assay and invasion assay. **P < 0.01

Fig. 3

LINC00641 serves as a sponge of miR-340-5p. a Schematic of the predicted miR-340-5p binding site on LINC00641. b Luciferase activity of LINC00641-WT or LINC00641-MUT was assessed in A498 and ACHN cells transfected with miR-340-5p mimics or miR-NC. c Relative LINC00641 expression in A498 and ACHN cells transfected with miR-340-5p mimics or miR-NC. d miR-340-5p expression in A498 and ACHN cells transduced with SCR or shRNA by qRT-PCR. e qRT-qPCR assays were utilized to measure miR-340-5p expression in RCC tissues and paired normal tissues. f The inverse correlation between LINC00641 and miR-340-5p levels in RCC tissues by Pearson’s correlation. **P < 0.01

Fig. 4

Inhibition of miR-340-5p abolished LINC00641 effects on cell apoptosis and invasion. a The expression of miR-340-5p was detected after transfected with anti-miR-340-5p by qRT-PCR. Cell apoptosis (b) and cell invasion (c) of RCC cells as indicated treatment. d The expression of c-Myc, CyclinD1 and MMP-2 protein in A498 cell lines as indicated treatment. *P < 0.05, **P < 0.01

Fig. 5

In vivo experiments confirmed the effects of LINC00641 silencing on tumor growth. a Photographs of tumors excised 4 weeks after inoculation of stably transduced with SCR or shRNA ACHN cells into nude mice. Tumor growth curve (b) and weight (c) of mouse xenografts subcutaneously injected with ACHN cells with stable LINC00641 knockdown. The expression levels of LINC00641 (d) and miR-340-5p (e) in dissected tumour tissues were detected by qRT-PCR assay. f Xenografts tissues were immunostained for Ki-67. **P < 0.01