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. 2018 Aug 28;9(9):861.
doi: 10.1038/s41419-018-0908-z.

Long noncoding RNA NEAT1, regulated by LIN28B, promotes cell proliferation and migration through sponging miR-506 in high-grade serous ovarian cancer

Wu Yong  1   2 , Deng Yu  2   3 , Zhu Jun  1   2 , Duan Yachen  1   2 , Weng Weiwei  2   3 , Xu Midie  2   3 , Ju Xingzhu  1   2 , Wu Xiaohua  4   5
Affiliations

Long noncoding RNA NEAT1, regulated by LIN28B, promotes cell proliferation and migration through sponging miR-506 in high-grade serous ovarian cancer

Wu Yong et al. Cell Death Dis. .

Abstract

The aberrant expression of long noncoding RNAs (lncRNAs) has been reported frequently in specific cancers, including high-grade serous ovarian cancer (HGSOC). The purpose of the present study was to explore the clinical significance and underlying mechanisms of a significantly dysregulated lncRNA (NEAT1) in HGSOC. Our results showed that elevated NEAT1 expression in human HGSOC specimens correlated with a poor prognosis. Functional experiments demonstrated that knockdown of NEAT1 significantly prohibited ovarian cancer cell proliferation and invasion in vitro and restrained tumor growth in vivo. LIN28B was identified by bioinformatics analysis along with experimental evidence as a direct actor that enhanced NEAT1 stability. A rescue functional assay confirmed that the LIN28B/NEAT1 axis contributed to oncogenic functions in ovarian cancer cells. Moreover, gene expression profile data and dual luciferase reporter assay results demonstrated that NEAT1 functioned as a competing endogenous RNA (ceRNA) for miR-506 to promote cell proliferation and migration. Taken together, our results showed that NEAT1, stabilized by LIN28B, promoted HGSOC progression by sponging miR-506. Thus, NEAT1 can be regarded as a vital diagnostic biomarker for HGSOC and a therapeutic target.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. NEAT1 is upregulated in HGSOC and is correlated with patient prognosis.
a NEAT1 expression levels in HGSOC and unpaired normal ovarian tissues determined using the NEAT1 primer (which can detect both transcripts). b NEAT1 expression in HGSOC and normal tissues was determined using the NEAT1-2 primer (which can detect the long transcript). β-actin was used as an endogenous control to normalize the data. c A total of 75 HGSOC patients were divided into high- and low-expression groups based on the median expression value. d Kaplan–Meier analysis of overall survival in all patients with HGSOC according to NEAT1 expression. e Kaplan–Meier curves for PFS of patients based on NEAT1 expression. f NEAT1 expression was quantitated in seven EOC cell lines using qRT-PCR
Fig. 2
Fig. 2. Attenuation of NEAT1 expression inhibits cell proliferation, invasion, and migration in vitro.
a The EOC cell lines OVCAR3 and A2780 were transfected with siRNAs, and the efficiency of knockdown was verified by qRT-PCR using two NEAT1 primers. b CCK-8 assays were performed to determine the proliferation of NEAT1-knockdown cells. c The cell colony formation ability of OVCAR3 and A2780 cells was assessed to determine the effects of NEAT1 knockdown on cell growth. d The cell invasion potential of the OVCAR3 and A2780 cells was assessed using a Transwell assay. Scale bar is 50 μm. e The cell migration ability was evaluated using a wound-healing assay; images of OVCAR3 and A2780 cells were taken at 0 and 36 h postscratch. Scale bar is 200 μm. f Western blotting analysis was used to determine the MMP2, MMP9, N-cadherin, and E-cadherin expression levels. Actin was used as a reference. The data are shown as the mean ± SD of three replicates; *P < 0.05, **P < 0.01 vs. NC
Fig. 3
Fig. 3. NEAT1 interacts with the RBP LIN28B.
a A schematic of the RNA pulldown experiment for the identification of proteins associated with LIN28B. b Two creditable NEAT1 binding sites were chosen to design the RNA probes and the corresponding mutated probes. c RNA pulldown assays using the two groups of biotinylated probes and the mutated probes indicated that NEAT1 interacted with LIN28B. d RIP assays showing the association of LIN28B with NEAT1 in A2780 cells. *P < 0.05, ***P < 0.001
Fig. 4
Fig. 4. LIN28B enhances the stability of NEAT1.
a Immunoblotting to evaluate the LIN28B protein levels after NEAT1 downregulation in OVCAR3 and A2780 cells. b The qRT-PCR analysis of LIN28B interference in EOC cells. c The EOC cell lines A2780 and OVCAR3 were transfected with either LIN28B or a vector control, and LIN28B overexpression was verified by qRT-PCR. Relative RNA levels of NEAT1 in OVCAR3 and A2780 cells with LIN28B knockdown (d) or overexpression (e) based on qPCR. f The half-life of NEAT1 after treatment with 2.5 μM actinomycin D for the indicated times with LIN28B overexpression in A2780 cells. *P < 0.05, **P < 0.01, and ***P < 0.001. PCDH represents empty control. ns not significant
Fig. 5
Fig. 5. Rescue assays confirm that NEAT1 is an important downstream effector of LIN28B.
a CCK-8 rescue assays were performed after NEAT1 knockdown in LIN28B-overexpressing cells. b The cell colony formation ability was used to evaluate cell growth after transfection with NEAT1-siRNA in LIN28B-overexpressing cells. c Transwell invasion rescue assays were performed after NEAT1 knockdown in LIN28B-overexpressing cells. Scale bar is 50 μm. d A wound-healing assay was applied to analyze the migratory capacity of EOC cells after transfection with the NEAT1-siRNA in LIN28B-overexpressing cells. e The differential expression of LIN28B in 75 HGSOC tissues and unpaired normal ovarian tissues was analyzed. f Kaplan–Meier analysis of OS of 75 HGSOC patients based on LIN28B mRNA expression was performed. The cut-off threshold used to divide the patients into high- and low-expression groups was the median value. g Pearson’s correlation curves are shown, revealing the positive relationship between LIN28B and either total NEAT1 or NEAT1-2. Scale bar is 200 μm. *P < 0.05, **P < 0.01, and ***P < 0.001
Fig. 6
Fig. 6. Knockdown of NEAT1 inhibits ovarian tumor growth in vivo.
a NEAT1-knockdown stable cell lines were constructed, and the knockdown efficiency was tested by qRT-PCR. b Downregulation of NEAT1 expression attenuated tumor growth in nude mice. c, d The effect of NEAT1 on EOC tumor growth was evaluated based on the tumor volumes and tumor weights in the two groups. e An immunofluorescence assay was used to detect the Ki-67 expression levels in frozen sections. Scale bar is 50 μm. *P < 0.05
Fig. 7
Fig. 7. NEAT1 promotes cell proliferation and migration via miR-506.
a Gene expression profiling was performed in NEAT1-knockdown HeLa cells. DEGs are presented in the form of a heat map. b DEGs were used to conduct an enrichment analysis. c A subset of representative enriched terms was converted into a network layout. d Relative miR-506 levels in OVCAR3 and A2780 cells with NEAT1 knockdown are shown. e The binding region between miR-506 and NEAT1 was predicted using bioinformatics analysis, and luciferase reporter plasmids containing the wild type (WT-NEAT1) or mutant NEAT1 (MUTNEAT1) sequence are shown. f WT-NEAT1 or MUT-NEAT1 was cotransfected into HEK-293T cells with miR-506 mimics or their corresponding negative controls; then, the luciferase reporter assay was performed. g Western blotting was used to detect the levels of miR-506 target genes, including ZEB1, Vimentin, and Snail2, following miR-506 overexpression in A2780 cells. h The levels of miR-506 target genes were detected by Western blotting when NEAT1 was downregulated in A2780 and OVCAR3 cells. *P < 0.05 and **P < 0.01. ns not significant
Fig. 8
Fig. 8. miR-506 reversed the promoting effect of NEAT1 on the cell migration capacity of ovarian cancer cells.
a, b Transwell and cell wound-healing assays were conducted in OVCAR3 and A2780 cell lines cotransfected with NEAT1 siRNA and miR-506 inhibitor. Scale bars are 50 and 200 μm. c Western blotting was used to detect the levels of miR-506 target genes, including ZEB1, Vimentin, and Snail2, with NEAT1 siRNA and miR-506 inhibitor cotransfected in A2780 and OVCAR3 cells. d Integrated model depicting lncRNA NEAT1 as an oncogene in HGSOC. *P < 0.05
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