LncRNA LINC00460 facilitates the proliferation and metastasis of renal cell carcinoma via PI3K/AKT signaling pathway

Abstract

Renal cell carcinoma (RCC) is one of the most prevalent cancers diseases in the worldwide. Long noncoding RNAs (LncRNAs) have been indicated as a mediator acted in tumorigenesis of RCC. LINC00460 has been reported to participate in many kinds of malignancies and promotes cancer progressions. However, the mechanism of LINC00460 on RCC is yet to be investigated. This study aimed to explore the potential function and regulation mechanism of LINC00460 in RCC. We analysed the LINC00460 expression and the prognosis in RCC patients using Gene Expression Profiling Interactive Analysis (GEPIA) and The Cancer Genome Atlas (TCGA) databases. LINC00460 level in normal renal cell line and RCC cell lines were examined by qRT-PCR. We study the effects of LINC00460 on proliferation, migration, invasion, apoptosis in RCC cells lines using a series of in vivo and in vitro experiments. RNA sequencing (RNA-seq) analysis was applied to searching potential LINC00460 related signal pathway in RCC. We identified the significant up-regulated expression of LINC00460 both in RCC tissues and cell. RCC patients with elevated LINC00460 expression have shorter survival. Up-expression of LINC00460 promoted cell proliferation, invasion and migration, meanwhile down-regulation of LINC00460 exerted inhibitory effect on these activities. We crucially identified that LNC00460 promotes development of RCC by influencing the PI3K/AKT pathway. Knockdown of LNC00460 decreased the phosphorylation of AKT and mTOR. The key finding of our study showed that LINC00460 functions as an oncogene in RCC pathogenesis by mediating the PI3K/AKT.

Keywords: LINC00460; PI3K/AKT pathway; RCC; metastasis; proliferation.

Figure 1

Expression of LINC00460 in RCC tissues based on the data from the GEPIA and TCGA. (A, B) Expression level of LINC00460 in KIRP tissues (n=286) and normal tissues (n=60), KIRC tissues (n=523) and normal tissues (n=100), analyzed in TGCA database (fold change>2.0, * p<0.05). (C) LINC00460 was gradually elevated with advanced staging in RCC. (D, E) OS and DFS rate of RCC patients with lowly or highly LINC00460 analyzed using the Kaplan-Meier analyses and log-rank test.

Figure 2

LINC00460 expression level in RCC cell lines and effects on cell proliferation. (A) Expression patterns of LINC00460 in RCC cell lines and normal renal cell lines detected by qRT-PCR (*** p<0.001). (B, C) Expression patterns of LINC00460 in RCC cells treated with over-expressed or silenced LINC00460 detected by qRT-PCR (*** p<0.001). (D, E) CCK-8 assay of ACHN and 786-O cells transfected with over-expressed or silenced LINC00460 (*** p<0.001).

Figure 3

LINC00460 facilitated RCC cells migration, invasion in vitro. (A, B, C, D) The cell migration and invasion abilities of ACHN and 786-O cells transfected with overexpressed or silenced LINC00460 were determined by Transwell assays (*** p<0.001). (E, F, G, H) Wound healing assay was performed to examine the effect of LINC00460 overexpression or knockdown on ACHN and 786-O cells migration (* p<0.05, *** p<0.001). (I, J) EMT markers was detected by Western blot analysis when silenced LINC00460 was transfected in RCC cells. Data statistics was also shown (GAPDH as negative control, *p < 0.05, *** p<0.001).

Figure 4

Influence of LINC00460 on RCC cells apoptosis and EMT phenotype. (A, B) Cell apoptosis was determined using flow cytometry analysis after LINC00460 overexpression in RCC cell lines (** p<0.01). (C, D) Cell apoptosis markers were detected by Western blot analysis when silenced LINC00460 was transfected in RCC cells. Data statistics was also shown (GAPDH as negative control, *p < 0.05, *** p<0.001).

Figure 5

LINC00460 promoted RCC cells growth and metastasis in vivo. (A) 786-O cells were injected into nude mice after the transfection of shLINC00460 and shCtrl vector. (B) The growth curves of tumors from subcutaneously injected nude mice treated with shLINC00460 or shCtrl (*** p<0.001). (C) The weights of tumors from subcutaneously injected nude mice treated with shLINC00460 or shCtrl (* p<0.05). (D) The levels of LINC00460 expression in paired tumor tissues formed from subcutaneously injected nude mice treated with shLINC00460 or shCtrl, determined by qRT-PCR (*** p<0.001). (E) Representative bioluminescence images and statistical analysis of lung metastases in mice via tail vein injection of indicated cells (**p < 0.01, ***p < 0.001). (F) Representative H&E images and statistical analysis of resected lung xenografts with metastatic loci (*** p < 0.001).

Figure 6

Effect of LINC00460 knockdown on activation of PI3K/AKT pathway in RCC cell lines. (A) Hierarchically clustered heatmap of the upregulated and downregulated genes in 786-O cells after shLINC00460 and shCtrl transfections. (B) Pathway classification of differentially expressed genes (DEGs). Bubble plots represented the number of DEGs, x axis represented rich factor, y axis represented the functional classification of KEGG. (C, D, E, F) The Western blot assay was used to detect PI3K, AKT, p-AKT, mTOR and p-mTOR expression in cells transfection with shLINC00460 or overexpressed LINC00460 as relative to the control groups. (GAPDH as negative control, *p < 0.05, *** p<0.001).