Long Non-Coding RNA USP2-AS1 Accelerates Cell Proliferation and Migration in Ovarian Cancer by Sponging miR-520d-3p and Up-Regulating KIAA1522

Abstract

Background: Ovarian cancer is one of the malignant tumors attacking the female reproductive system. Currently, increasing studies have clearly determined the importance of long non-coding RNAs (lncRNAs) in various human cancers including ovarian cancer. However, the role and in-depth mechanism of ubiquitin specific peptidase 2 antisense RNA 1 (USP2-AS1) in ovarian cancer have been not reported yet.

Purpose: We were absorbed into exploring the character of USP2-AS1 in ovarian cancer.

Methods: RT-qPCR analysis reflected gene expression. The GEPIA database provided further evidences, and bioinformatics tools analyzed the potential molecules downstream USP2-AS1 in ovarian cancer. The changes on ovarian cancer cellular functions were assessed via EdU, TUNEL, JC-1 and transwell assays. RNA pull down, RIP and luciferase reporter assays estimated molecule interactions.

Results: USP2-AS1 was obviously up-regulated in ovarian cancer tissues and cell lines. Inhibiting USP2-AS1 had anti-proliferation, pro-apoptosis, and anti-migration effects on ovarian cancer cells. Furthermore, we confirmed that USP2-AS1 sequestered miR-520d-3p to enhance KIAA1522. In addition, miR-520d-3p silence reversed the effect of depleted USP2-AS1 on ovarian cancer cellular behaviors, while such reversion was then abolished by KIAA1522 knockdown.

Conclusion: USP2-AS1 facilitated ovarian cancer progression via miR-520d-3p/KIAA1522 axis, implying USP2-AS1 as a new perspective for the treatment of ovarian cancer.

Keywords: KIAA1522; USP2-AS1; miR-520d-3p; ovarian cancer.

Figure 1

Knockdown of USP2-AS1 represses the progression of ovarian cancer. (A) RNA-Seq analysis data from GEPIA detected the expression of USP2-AS1 in diverse cancers including ovarian cancer. (B) GEPIA database analyzed the levels of USP2-AS1 in ovarian cancer tissues compared with normal tissues. (C) USP2-AS1 expression in ovarian cancer cell lines (ES-2, CAOV3, A2780, and SKOV3) and normal human ovarian surface epithelial cell line HOSEPiC was assessed via RT-qPCR. (D) The interference efficiency of USP2-AS1 was assessed by RT-qPCR in A2780 and SKOV3 cells treated with sh-USP2-AS1#1and2. (E) Cell proliferation ability was examined by EdU assay in response to USP2-AS1 silence. (F and G). TUNEL assay and JC-1 assay examined the alteration on the apoptosis of A2780 and SKOV3 cells in the case of silencing USP2-AS1. (H) Transwell assay was used to test cell migration under the context of USP2-AS1 silence. *P<0.05, **P<0.01.

Figure 2

USP2-AS1 is capable of targeting miR-520d-3p. (A and B) FISH assay and subcellular fraction assay were adopted to analyze the distribution of USP2-AS1 in A2780 and SKOV3 cells. (C) The potential miRNAs that could bind to USP2-AS1 were predicted with the aid of ENCORI and ReGRNA. (D) RT-qPCR detected miR-520d-3p expression in ovarian cancer cells and HOSEPiC cells. (E) The co-existence of USP2-AS1 and miR-520d-3p in RISCs was confirmed by RIP assay. (F and G) RNA pull down assays was used to assess the interaction between USP2-AS1 and miR-520d-3p. (H) RT-qPCR analysis detected the overexpression efficiency of miR-520d-3p mimics in ovarian cancer cells. (I) The binding sites between USP2-AS1 and miR-520d-3p were displayed by ENCORI. (J) Under the condition of miR-520d-3p mimics, luciferase reporter assay tested the combination between USP2-AS1 and miR-520d-3p. (K) The interference efficiency of miR-520d-3p inhibitor in ovarian cancer cells was analyzed via RT-qPCR. (L) Under the condition of miR-520d-3p inhibitor, luciferase reporter assay tested combination between USP2-AS1 and miR-520d-3p. **P<0.01.

Figure 3

MiR-520d-3p silence abolishes the effect of USP2-AS1 down-regulation on ovarian cancer cell proliferation, apoptosis and migration. (A) The impact of miR-520d-3p inhibition on the proliferation of USP2-AS1-silence cells was determined by EdU assay. (B and C) TUNEL and JC-1 assays evaluated whether miR-520d-3p inhibitor could regulate the apoptosis of USP2-AS1-depleted A2780 and SKOV3 cells. (D) Transwell assay assessed the migration ability of A2780 and SKOV3 cells transfected with sh-NC, sh-USP2-AS1#1 or sh-USP2-AS1#1+ miR-520d-3p inhibitor. **P<0.01.

Figure 4

USP2-AS1 acts as a ceRNA of KIAA1522 by absorbing miR-520d-3p. (A) RNA22, microT and PicTar databases forecasted four latent targets for miR-520d-3p. (B) The binding of miR-520d-3p with the above four candidates was detected via RNA pull down assay. (C) GEPIA data of KIAA1522 expression in ovarian cancer tissues and normal samples. (D) The level of KIAA1522 in ovarian cancer cells was tested by RT-qPCR. (E) RT-qPCR detected the influence of miR-520d-3p on KIAA1522 expression in A2780 and SKOV3 cells. (F) RT-qPCR detected the expression of KIAA1522 in two ovarian cancer cells under indicated conditions. (G) RIP assay confirmed the existence of all USP2-AS1, miR-520d-3p, and KIAA1522 in RISCs. (H) The binding sites between miR-520d-3p and KIAA1522 were exhibited by ENCORI. (I) Luciferase reporter assay was carried out to confirm if miR-520d-3p and KIAA1522 could bind each other. (J) The interference efficiency of KIAA1522 in A2780 and SKOV3 cells was tested by RT-qPCR. (K) RT-qPCR analyzed the alteration of KIAA1522 expression in ovarian cancer cells with appropriate transfection of sh-USP2-AS1#1, miR-520d-3p inhibitor, and sh-KIAA1522. *P<0.05, **P<0.01.

Figure 5

USP2-AS1 accelerates ovarian cancer development by miR-520d-3p/KIAA1522 axis. (A) The proliferation of indicated ovarian cancer cells was measured via EdU assay. (B and C) The apoptosis of transfected cells in different groups was tested by TUNEL and JC-1 assays. (D) The migration of transfected cells from indicated groups was assessed by transwell assay. **P<0.01.