lncRNA CRNDE promotes the proliferation and metastasis by acting as sponge miR-539-5p to regulate POU2F1 expression in HCC

Abstract

Background: This article focuses on the roles and mechanism of lncRNA CRNDE on the progression of HCC.

Methods: We used qRT-PCR to detect the expression of lncRNA CRNDE in HCC cells, normal cells and clinical tissues. MTT assay, FCM analysis, Transwell migration and invasion assay were used to detect the effects of lncRNA CRNDE on cell viability, apoptosis, migration and invasion of HCC cells. The expression of apoptosis-related proteins Bcl-2, Bax, Cleaved Caspase 3, Cleaved Caspase 9, EMT epithelial marker E-cadherin and mesothelial marker Vimentin were analyzed by Western blot. Online prediction software was used to predict the binding sites between lncRNA CRNDE and miR-539-5p, or miR-539-5p and POU2F1 3'UTR. Dual luciferase reporter assay, qRT-PCR and RNA pulldown were used to detect target-relationship between lncRNA CRNDE and miR-539-5p. Dual luciferase reporter assay, qRT-PCR, Western blot and Immunofluorescence were used to detect target-relationship between miR-539-5p and POU2F1. qRT-PCR was used to detect the expression of miR-539-5p and POU2F1 in clinical tissues. Rescue experiments was used to evaluate the association among lncRNA CRNDE, miR-539-5p and POU2F1. Finally, we used Western blot to detect the effects of lncRNA CRNDE, miR-539-5p and POU2F1 on NF-κB and AKT pathway.

Results: lncRNA CRNDE was highly expressed in HCC cells and HCC tissues compared with normal cells and the corresponding adjacent normal tissues. lncRNA CRNDE promoted the cell viability, migration and invasion of HCC cells, while inhibited the apoptosis and promoted the EMT process of HCC cells. lncRNA CRNDE adsorbed miR-539-5p acts as a competitive endogenous RNA to regulate POU2F1 expression indirectly. In HCC clinical tissues, miR-539-5p expression decreased and POU2F1 increased compared with the corresponding adjacent normal tissues. lncRNA CRNDE/miR-539-5p/POU2-F1 participated the NF-κB and AKT pathway in HCC.

Conclusion: lncRNA CRNDE promotes the expression of POU2F1 by adsorbing miR-539-5p, thus promoting the progression of HCC.

Keywords: HCC; POU2F1; ceRNA; lncRNA CRNDE; miR-539-5p.

Fig. 1

lncRNA CRNDE expression was upregulated in HCC cells and clinical tissues. a lncRNA CRNDE relative expression levels were determined in 18 paired HCC clinical tissues and their corresponding normal samples. The level of lncRNA CRNDE expression was normalized to GAPDH. b qRT-PCR analysis of lncRNA CRNDE expression in three HCC cells and one cultured human liver epithelial cells. c Correlation analysis was used to detect the expression of lncRNA CRNDE in HCC and clinicopathological indicators. All bars indicate the means±SD. All experiments were performed independently at least three times

Fig. 2

Knockdown of lncRNA CRNDE inhibits proliferation and metastasis in HCC cells. We divided the cells into four groups: pcDNA3, pcDNA3/lncRNA CRNDE, si-NC, and si-lncRNA CRNDE, transfected for 48 h, and then performed subsequent experiments. a MTT assay was performed to detect the proliferation ability of HCC cells when lncRNA CRNDE was overexpressed or knocked down. b FCM of apoptosis in HepG2 cells transfected with silncRNA CRNDE and siNC. c qRT-PCR and Western blot was used to detect the effects of lncRNA CRNDE on apoptosis associated genes. d, e Transwell migration and invasion assays were performed to detect the migration and invasion ability of QGY-7703 cells when lncRNA CRNDE was overexpressed or knocked down. f qRT-PCR and Western blot was used to detect the effects of lncRNA CRNDE on EMT associated genes. g, h Transwell migration and invasion assays were performed to detect the migration and invasion ability of HepG2 cells when lncRNA CRNDE was overexpressed or knocked down. i Tumor tissues in nude mice. *p < 0.05, **p < 0.01. All bars indicate the means±SD. All experiments were performed independently at least three times

Fig. 3

lncRNA CRNDE acted as a ceRNA by sponging miR-539-5p and regulated POU2F1 expression indirectly. We divided the cells into four groups: mimics NC, miR-539-5p mimics, inhibitor NC, and miR-539-5p inhibitor, transfected for 48 h, and then performed subsequent experiments. a The putative target sites between lncRNA CRNDE and miR-539-5p were shown. b Dual-luciferase analysis was proformed when HCC cells were co-transfected with lncRNA CRNDE-wt and miR-539-5p mimic or lncRNA CRNDE-mut and miR-539-5p mimic. The activity of luciferase was detected. c miR-539-5p expression level was detected by qRT-PCR when lncRNA CRNDE was overexpressed or knocked down. d RNA pulldown was used to presented the binding between lncRNA CRNDE and miR-539-5p as fold enrichment. e Prediction program was used to find that there is a putative target of miR-539-5p in the POU2F1 sequences. f Dual-luciferase analysis showed that effects of miR-539-5p on the luciferase activity of constructs of the type binding site. qRT-PCR (g) and Western blot (j) were used to detect the POU2F1 level when the miR-539-5p was overexpressed or knocked down. miR-539-5p. k Immunofluorescence was used to detect the expression of POU2F1 in HepG2 cells. qRT-PCR (l) and Western blot (m) were used to detect the level of miR-539-5p and POU2F1 in nude tissues. o and POU2F1 (p) relative expression levels were determined in 18 paired HCC clinical tissues and their corresponding normal samples.*p < 0.05, **p < 0.01. All bars indicate the means±SD. All experiments were performed independently at least three times

Fig. 4

The promoting effects of lncRNA CRNDE can be reversed by miR-539-5p and POU2F1. a qRT-PCR analysis of miR-539-5p expression in three HCC cells and one cultured human liver epithelial cells. b qRT-PCR analysis of POU2F1 expression in three HCC cells and one cultured human liver epithelial cells. Western Blot analysis of POU2F1 expression in three HCC cells and one cultured human liver epithelial cells. c We divided the cells into groups: pcDNA3/lncRNA CRNDE, miR-539-5p mimics, pcDNA3, transfected for 48 h. MTT assays were performed to detect the proliferation ability of QGY-7703 and HepG2 cells. d We divided the cells into groups: miR-539-5p mimics, pcDNA3/POU2F1, pcDNA3, transfected for 48 h. MTT assays were performed to detect the proliferation ability of QGY-7703 and HepG2 cells. e, f We divided the cells into three groups: pcDNA3/lncRNA CRNDE, miR-539-5p mimics, pcDNA3, transfected for 48 h. Transwell migration and invasion assays were performed to detect the migration and invasion ability of QGY-7703 and HepG2 cells. *p < 0.05, **p < 0.01. All bars indicate the means±SD. All experiments were performed independently at least three times. g, h We divided the cells into groups: miR-539-5p mimics, pcDNA3/POU2F1, pcDNA3, transfected for 48 h. Transwell migration and invasion assays were performed to detect the migration and invasion ability of QGY-7703 and HepG2 cells. *p < 0.05, **p < 0.01. All bars indicate the means±SD. All experiments were performed independently at least three times

Fig. 5

lncRNA CRNDE/miR-539-5p/POU2F1 involved in NF-κB and AKT pathway activity. We divided the cells into groups: pcDNA3 + siPOU2F1, pcDNA3 + siNC, pcDNA3/ lncRNA CRNDE+siPOU2F1, lncRNA CRNDE+siNC, transfected for 48 h. Western blot was performed to detect the effects of lncRNA CRNDE/miR-539-5p/POU2F1 on IKBa(a), AKT(b), ERK(c) phosphorylation level. d The NF-κB(p65) level in nuclear was detected when cells were treated with lncRNA CRNDE/miR-539-5p/POU2F1. *p < 0.05, **p < 0.01. All bars indicate the means±SD. All experiments were performed independently at least three times

Fig. 6

The model of lncRNA CRNDE/miR-539-5p/POU2F1 in HCC. The overview of this study