LncRNA NR2F1-AS1 regulates hepatocellular carcinoma oxaliplatin resistance by targeting ABCC1 via miR-363

Abstract

Emerging evidence has validated the vital role of long non-coding RNA (lncRNA) in the chemoresistance of cancer treatment. In the present study, we investigate the function of lncRNA NR2F1-AS1 on oxaliplatin (OXA) resistance of hepatocellular carcinoma (HCC) and discover the underlying molecular mechanism. Results revealed that lncRNA NR2F1-AS1 was up-regulated in oxaliplatin-resistant HCC tissue and cells using microarray analysis and RT-PCR. Meanwhile, ABCC1 protein was overexpressed in OXA-resistant HCC cells (Huh7/OXA and HepG2/OXA). In vitro, NR2F1-AS1 knockdown reduced the invasion, migration, drug-resistant gene (MDR1, MRP5, LRP1) and IC50 value in Huh7/OXA and HepG2/OXA cells. In vivo, NR2F1-AS1 knockdown decreased the tumour weight of HCC cells. Bioinformatics tools and luciferase reporter assay confirmed miR-363 targeted the 3'-UTR of NR2F1-AS1 and ABCC1 mRNA, presenting that NR2F1-AS1 promoted ABCC1 expression through endogenous sponging miR-363. In summary, results conclude that NR2F1-AS1 regulates HCC OXA resistance through targeting miR-363-ABCC1 pathway, providing a vital theoretic mechanism and therapeutic target for HCC chemoresistance.

Keywords: ABCC1; NR2F1-AS1; hepatocellular carcinoma; miR-363; oxaliplatin resistant.

Figure 1

NR2F1‐AS1 was up‐regulated in oxaliplatin‐resistant hepatocellular carcinoma (HCC) tissue and cell lines. A, Heat map shows the representative dysregulated lncRNA with more than fourfold changes. Red symbol presented the high‐expression RNAs. Green symbol presents the low‐expression RNAs. B, NR2F1‐AS1 was up‐regulated in 47 cases of oxaliplatin‐resistant HCC tissue compared with oxaliplatin‐sensitive tissue samples. C, RT‐PCR showed that NR2F1‐AS1 was up‐regulated in human oxaliplatin‐resistant HCC cell lines (Huh7/OXA, HepG2/OXA) compared with parental cell lines. Lo‐2 was the normal liver cells. Data are presented as the mean ± SD. *P < .05, **P < .01 compared to control group

Figure 2

ABCC1 was up‐regulated in oxaliplatin‐resistant cells and NR2F1‐AS1 knockdown suppressed the oxaliplatin resistance of hepatocellular carcinoma (HCC) cells. A, The survival percentage of oxaliplatin‐resistant HCC cells (Huh7/OXA, HepG2/OXA) and their parental cells when treated with increasing concentration of oxaliplatin (OXA). B, C, Western blot showed the ABCC1 protein expression in oxaliplatin‐resistant HCC cells (Huh7/OXA, HepG2/OXA) and their parental cells. D, Interfering oligonucleotides were synthesized and transfected into Huh7/OXA and HepG2/OXA cells. E, RT‐PCR showed the mRNA expression levels of drug resistance‐related genes, including MDR1, MRP5, LRP1. F, G, The 50% inhibitory concentration (IC50) value was measure in Huh7/OXA and HepG2/OXA cells. si‐1#, 2#, 3# present “siRNA‐targeting NR2F1‐AS1.” Data were expressed as mean ± SD. *P < .05, **P < .01 represents statistically difference

Figure 3

NR2F1‐AS1 knockdown inhibited the migration, invasion and tumour growth of oxaliplatin‐resistant hepatocellular carcinoma (HCC) cells in vitro and in vivo. A, Transwell assay showed the invasive cell number in Huh7/OXA and HepG2/OXA cells transfected with NR2F1‐AS1 siRNA compared to empty vector‐transfected cells. B, Quantitative value of invasive cell number. C, Transwell assay showed the migrative cell number in Huh7/OXA and HepG2/OXA cells transfected with NR2F1‐AS1 siRNA compared to empty vector‐transfected cells. D, Quantitative value of migrative cell number. E, Photographs of xenograft mice and neoplasm. F, Tumour weight of neoplasm in mice injected with sh‐NR2F1‐AS1 or empty vector. Data were expressed as mean ± SD. *P < .05, **P < .01 represent the statistical difference

Figure 4

NR2F1‐AS1 sponged miR‐363 with complementary binding at 3′‐UTR. A, Schematic diagram demonstrated the complementary bound within miR‐363 and NR2F1‐AS1 3′‐UTR with binding sites predicted by bioinformatics programs (StarBase V 2.0, http://starbase.sysu.edu.cn/mirLncRNA). B, Luciferase reporter gene assay was performed in HEK‐293T cells transfected with NR2F1‐AS1 wild/mutant type and miR‐363 mimics/control. C, miR‐363 expression levels were measured in oxaliplatin‐resistant cell lines and parental cell lines using RT‐PCR. D, RT‐PCR showed the miR‐363 expression levels in Huh7/OXA and HepG2/OXA cells transfected with NR2F1‐AS1 siRNA or empty vector. E, miR‐363 was measured in 15 pairs of oxaliplatin‐resistant hepatocellular carcinoma (HCC) samples and oxaliplatin‐sensitive samples. Data were expressed as mean ± SD. *P < .05 represents the statistical difference

Figure 5

NR2F1‐AS1 modulated ABCC1 protein expression through targeting miR‐363. A, Schematic diagram shows the binding sites within miR‐363 and ABCC1 mRNA 3′‐UTR. B, Luciferase reporter assay shows the molecular bond within miR‐363 and ABCC1 mRNA 3′‐UTR. C, RT‐PCR shows the ABCC1 mRNA expression levels in Huh7/OXA cells transfected with miR‐363 inhibitor. D, Western blot images of ABCC1. E, Quantitative ABCC1 protein expression in Huh7/OXA cells transfected with blank vector, miR‐363 inhibitor and/or si‐NR2F1‐AS1. Data were expressed as mean ± SD. *P < .05, **P < .01 represent the statistically difference