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. 2020 Sep 15;12(9):5670-5682.
eCollection 2020.

lncRNA USP2-AS1 promotes colon cancer progression by modulating Hippo/YAP1 signaling

Dongying Li  1 , Jie Bao  1 , Jiayuan Yao  1 , Jiansheng Li  1
Affiliations

lncRNA USP2-AS1 promotes colon cancer progression by modulating Hippo/YAP1 signaling

Dongying Li et al. Am J Transl Res. .

Abstract

Dysregulation of Hippo signaling by long non-coding RNA (lncRNA) contributes to colon adenocarcinoma (COAD) progression, while the underlying mechanisms remain elusive. Our study shows that lncRNA USP2-AS1 is a Yes-associated protein 1 (YAP1) binding lncRNA, and inactivates Hippo signaling in COAD cells. Moreover, our data indicated that USP2-AS1 lowered the phosph-YAP (S127), elevated the total level of YAP1, and triggered the expression of downstream target genes in COAD cells. The loss- and gain-of function assays demonstrated that USP2-AS1 promotes cellular proliferation and metastasis of COAD cells. Clinically, the USP2-AS1 levels were significantly elevated in COAD tissues and were positively correlated with tumor grade, size, and TNM stage. Collectively, these findings demonstrated that USP2-AS1 modulates and regulates Hippo signaling in COAD and could be a valuable therapeutic target.

Keywords: Hippo signaling; USP2-AS1; cell proliferation; lncRNA; metastasis.

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Conflict of interest statement

None.

Figures

Figure 1
Figure 1
The USP2-AS1 is highly expressed in COAD and predicts poor prognosis. A. The expression of USP2-AS1 in COAD (n = 275) and normal tissues (n = 349), data from Gepia analysis. B. Data from TCGA showed that a higher expression of Linc00337 in COAD is a marker for poor prognostic outcomes. C. The qRT-PCR analysis of USP2-AS1 expression in 43 paired fresh COAD patients. D. Pie chart illustration of the proportions of different USP2-AS1 levels in 43 patients. E. The expression levels of USP2-AS1 in indicated cell lines. C-E. Data is represented as mean ± SD for n = 3.
Figure 2
Figure 2
The USP2-AS1 binds to YAP1 in SW620 cells. A. Two transcripts of USP2-AS1 (T1, T2) are enriched by YAP1, data from Wu et al. [15]. B. USP2-AS1 binding to YAP1. C. The qRT-PCR analysis showed that USP2-AS1 is mainly located in the nucleus in SW620 cells. The GAPDH was used as a cytoplasm marker while U1 as a nuclear marker. D. The RNA-FISH results revealed that USP2-AS1 is mainly located in the nucleus in SW620 cells. The 18S was used as a cytoplasm marker whereas U1 was the nuclear marker. Scale bar, 10 μm. E. Flow chart of RIP assays. F. The RIP-WB results indicated that YAP1 binds to itself, while RIP-qPCR analysis showed that YAP1 binds to USP2-AS1, but not to GAPDH or U1, the negative controls. G. Flow chart of biotin-labeled RNA pulldown assays. H. The RNA-pulldown assays showed that USP2-AS1 could pulldown YAP1 and TAZ. Each bar represents the mean ± SD for n = 3.
Figure 3
Figure 3
The USP2-AS1 promotes cellular proliferation and invasion in COAD cells. (A, E) The qRT-PCR assays showed that the level of USP2-AS1 is significantly inhibited by two shRNAs in LoVo (A) and SW620 (E) cells. (B, F) The CCK8 cell viability assays showed that silencing USP2-AS1 using two shRNAs significantly inhibited cellular proliferation in LoVo (B) and SW620 (F) cells. (C, G) Cellular colony formation assays revealed that USP2-AS1 knockdown inhibited LoVo (C) and SW620 (G) cell growths. (D, H) Matrigel-coated transwell invasion assays showed that USP2-AS1 knockdown significantly inhibited cellular invasion in LoVo (D) and SW620 (H) cells. Scale bar, 50 μm. (I) Overexpression of USP2-AS1 considerably increased the levels of USP2-AS1 in SW480 cells. (J) The CCK8 cell viability assays showed that higher expressions of USP2-AS1 significantly promoted cellular proliferation in SW480 cells. (K) Cellular colony formation assays showed that a higher expression of USP2-AS1 significantly promoted cellular proliferation and growth. (L) Matrigel-coated transwell invasion assays showed that USP2-AS1 overexpression significantly elevated the ability of cellular invasion in SW480 cells. Scale bar, 50 μm. (M, N) Cell cycle assays showed that depletion of USP2-AS1 triggered cell cycle arrest at the G0/G1 stage in LoVo cells (M) and SW620 cells (N). (O) Overexpression of USP2-AS1 accelerated cell cycle progression in SW480 cells. Each bar represents the mean ± SD for n = 3.
Figure 4
Figure 4
USP2-AS1 knockdown significantly inhibited COAD cellular proliferation and metastasis in vivo. (A) Xenograft Tumor formed by SW620 stable cell lines. (B, C) Tumor growth curve (B) and weight (C) showed that USP2-AS1 knockdown significantly inhibited tumor formation in SW620 in vivo. (D) The IHC staining of Ki67 in indicated tumors. Scale bar, 50 μm. (E-G) Caudal vein lung metastasis assays showed that USP2-AS1 silencing was significantly reduced in SW620 cells. (E) The fluorescence intensity, while (F and G) is the number of intrapulmonary metastasis nodes. Scale bar, 50 μm.
Figure 5
Figure 5
The USP2-AS1 regulates Hippo-YAP1 signaling in COAD cells. (A-C) Luciferase reporter assays showed that USP2-AS1 silencing significantly inhibited TEAD transcriptional activity in LoVo (A) and SW620 (B), while USP2-AS1 overexpression enhanced TEAD transcriptional activity (C). (D) Western blot analysis showed that USP2-AS1 regulated the level of p-LAST1 (T1079), LAST1, LAST2, p-YAP1 (S127), and the total YAP1 in indicated cell lines. (E, F) The silencing of USP2-AS1 inhibited the expression of indicated Hippo signaling targets in SW620 cells (E), while its overexpression elevated the levels of these genes (F). (G-I) The expression of USP2-AS1 was positively correlated with CTGF (G), CYR61 (H) and SOX9 (I) in fresh COAD tissues. (J) Constitutively activated YAP1 (S127A) partly improved the proliferation of inhibitory effects through USP2-AS1 knockdown. Each bar represents the mean ± SD for n = 3.
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