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HIF1A in hepatocellular carcinoma under hypoxia
Biomedicine & Pharmacotherapy 125 (2020) 109703
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LncRNA HOTAIR knockdown inhibits glycolysis by regulating miR-130a-3p/ HIF1A in hepatocellular carcinoma under hypoxia
T
Mingxing Hua,b,c, Qiang Fua,b,c, Chan Jinga,b,c, Xu Zhanga,b,c, Tao Qina,b,c,*, Yanfeng Pand,** a
Department of Hepatobiliary and Pancreatic Surgery, the Henan Provincial People’s Hospital, Zhengzhou, Henan, China Department of Hepatobiliary and Pancreatic Surgery, the People's Hospital of Zhengzhou University, Zhengzhou, Henan, China c Department of Hepatobiliary and Pancreatic Surgery, the School of Clinical Medicine, Henan University, Zhengzhou, Henan, China d Department of Infectious Diseases, the First Affiliated Hospital of Zhengzhou University, No. Jianshe East Road, Zhengzhou 450000, Henan, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Hepatocellular carcinoma Glycolysis Hypoxia HOTAIR miR-130a-3p HIF1A
High rate of glycolysis supports hepatocellular carcinoma (HCC) cell growth even in a hypoxic environment. However, the mechanism underlying glycolysis under hypoxia remains largely unknown. Long noncoding RNAs (lncRNAs) play essential roles in regulating glucose metabolism in cancers. This study aimed to explore the role of lncRNA homeobox transcript antisense RNA (HOTAIR) in HCC glycolysis under hypoxia. Thirty-eight HCC patients were recruited. HepG2 and Huh7 cells were used for study in vitro. The expression levels of HOTAIR, microRNA-130a-3p (miR-130a-3p) and hypoxia inducible factor 1 alpha (HIF1A) were measured by quantitative real-time polymerase chain reaction and western blot, respectively. The glycolysis under hypoxia (1 % O2) condition was investigated by glucose consumption, lactate production and hexokinase 2 (HK2) level. The target interaction between miR-130a-3p and HOTIR or HIF1A was analyzed by bioinformatics analysis, luciferase assay, RNA pull-down and RNA immunoprecipitation. We found that HOTAIR expression was enhanced in HCC tissues and cells. Under hypoxia condition, HOTAIR expression was increased and its knockdown inhibited glycolysis in HCC cells. HOTAIR was validated as a decoy of miR-130a-3p and miR-130a-3p deficiency reversed the suppressive effect of HOTAIR silence on glycolysis under hypoxia. HIF1A was indicated as a target of miR130a-3p and miR-130a-3p overexpression repressed glycolysis under hypoxia by targeting HIF1A. Moreover, HIF1A expression was regulated by HOTAIR and miR-130a-3p. In conclusion, knockdown of HOTAIR suppressed glycolysis by regulating miR-130a-3p and HIF1A in HCC cells treated by hypoxia, elucidating a novel mechanism in HCC glycolysis.
1. Introduction Hypoxia is one of the most important features of the tumor microenvironment, which leads to the reprogramming of glycolytic metabolism, maintaining the survival of cancer cells under hypoxia condition [1]. Targeting glycolysis provides potential for therapeutic opportunities in human cancers [2]. Hepatocellular carcinoma (HCC) is the major type of liver cancer with high incidence and mortality worldwide [3]. The glycolysis under hypoxia stress contributes to malignancy of HCC [4,5]. However, the mechanism underlying glycolysis under hypoxia condition in HCC remains largely unknown. Noncoding RNAs have been regarded as essential targets for diagnosis and therapeutics of HCC [6]. Long noncoding RNAs (lncRNAs) are one class of noncoding RNAs with more than 200 nucleotides, which
are implicated in glucose metabolism by regulating glycolytic enzymes or related signaling pathways in cancers [7]. LncRNA homeobox transcript antisense RNA (HOTAIR) has been reported to be an oncogene in human cancers [8]. Previous studies revealed that HOTAIR acts as a carcinogenic lncRNA in HCC to contribute to HCC malignancy by promoting epithelial-mesenchymal transition, proliferation and cisplatin resistance [9–11]. However, little is known about the effect of HOTAIR on glycolysis under hypoxia in HCC. microRNAs (miRNAs) are also a class of small noncoding RNAs with ∼23 nucleotides, which play pivotal roles in prognosis and treatment of HCC [12]. Moreover, miRNAs could participate in HCC progression through regulating glucose metabolism [13,14]. Former works indicated miR-130a-3p as a tumor suppressor in many cancers, such as non-small-cell lung cancer, breast cancer and glioma [15–17]. In
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Corresponding author at: Department of Hepatobiliary and Pancreatic Surgery, the Henan Provincial People’s Hospital, No. 7 Weiwu Road, Zhengzhou 450003, Henan, China. ⁎⁎ Corresponding author. E-mail address: [email protected] (Y. Pan). https://doi.org/10.1016/j.biopha.2019.109703 Received 10 September 2019; Received in revised form 14 November 2019; Accepted 24 November 2019 0753-3322/ © 2019 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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empty vector (vector) were generated from Genepharma (Shanghai, China). miR-130a-3p mimic (miR-130a-3p) (cat. no. miR10000425-1-5) and miR-130a-3p inhibitor (anti-miR-130a-3p) (cat. no. miR200004251-5) along with their corresponding negative control (miR-NC, antimiR-NC) were purchased from Ribobio (Guangzhou, China). When reaching 60 % confluence, HepG2 and Huh7 cells in 6-well plates were transfected with 30 nM oligonucleotides or 500 ng vectors using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufactures’ instructions for 6 h. After transfection for 24 h, cells were harvested for subsequent experiments.
addition, previous studies demonstrated that miR-130a-3p is downregulated and suppresses HCC malignancy [18,19]. Hypoxia-inducible factor 1 (HIF1) is a key regulator of glucose metabolism under hypoxia, providing cancer cells a growth advantage in solid tumors [20]. HIF1A is a major subunit of HIF1, which plays as a poor prognostic factor in patients with HCC [21]. We assumed that miR-130a-3p and HIF1A were involved in HOTAIR-addressed mechanism in HCC due to the predicted binding sites of miR-130a-3p and HOTAIR or HIF1A by bioinformatics analysis. Here we first measured the changes of HOTAIR level in HCC in response to hypoxia. Moreover, we investigated the effect of HOTAIR knockdown on glycolysis by analyses of glucose consumption, lactate production and hexokinase 2 (HK2) protein level. Besides, we explored the potential mechanism by analyzing the association among HOTAIR, miR-130a-3p and HIF1A.
2.3. Detection of glucose consumption and lactate production HepG2 and Huh7 cells (2 × 105/well) were seeded into 6-well plates overnight and then cultured in hypoxia or normoxia condition for 48 h. The glucose consumption and lactate production were measured by using Glucose Assay Kit and Lactate Assay Kit (Sigma, St. Louis, MO, USA) following the manufacturer’s protocol. The relative levels of glucose consumption and lactate production were analyzed by normalizing to indicated control group.
2. Materials and methods 2.1. Patients and tissues A total of 38 HCC patients without history of chemotherapy, radiotherapy and other therapy were recruited from the Henan Provincial People’s Hospital. The tumor tissues and corresponding adjacent normal samples were collected from all participants who have provided the written informed consent. This study was approved by the ethics committee of the Henan Provincial People’s Hospital. The clinicopathologic features of patients were displayed in Table 1.
2.4. Quantitative real-time polymerase chain reaction (qRT-PCR) RNA was isolated from HCC tissues and cells using Trizol reagent (Thermo Fisher Scientific, Waltham, MA USA) following the manufacturer’s protocols. The samples were reverse transcribed using TaqMan™ mRNA or microRNA Reverse Transcription Kit (Thermo Fisher Scientific). The qRT-PCR was performed with SYBR Green mix (Thermo Fisher Scientific) on the Roche LightCycler 480 system. All primers used in this study were generated by Sangon (Shanghai, China) as follows: HOTAIR (Forward, 5′-GGTAGAAAAAGCAACCACGAAGC-3′; Reverse, 5′- ACATAAACCTCTGTCTGTGAGTGCC-3′); HIF1A (Forward, 5′-TTCCCGACTAGGCCCATTC-3′; Reverse, 5′-CAGGTATTCAAGGTCCC ATTTCA-3′); GAPDH (Forward, 5′-AGAAGGCTGGGGCTCATTTG-3′; Reverse, 5′- AGGGGCCATCCACAGTCTTC-3′); miR-130a-3p (Forward, 5′- TTGCGATTCTGTTTTGTGCT-3′; Reverse, 5′-GTGGGGTCCTCAGT GGG-3′); U6 (Forward, 5′-CTCGCTTCGGCAGCACA-3′; Reverse, 5′AACGCTTCACGAATTTGCGT-3′). GAPDH and U6 were regarded as internal control. The relative expression levels of RNA were analyzed according to the 2−ΔΔCt method [22].
2.2. Cell culture, hypoxia treatment and transfection HCC cell lines (HepG2 and Huh7), normal human hepatic cell line LO2 and 293 T cells were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). All cells were cultured at 37 °C under 5 % CO2 in RPMI-1640 medium (Gibco, Grand Island, NY, USA) containing 10 % fetal bovine serum. For hypoxia stimulation, HepG2 and Huh7 cells were incubated in a hypoxia chamber with 1 % O2 for different times (0, 6, 12, 24 and 48 h). Small interfering RNA (siRNA) against HOTAIR (si-HOTAIR) (sense: 5′-UUCUAAAUCCGUUCCAUUCCACUGCGA-3′, antisense: 5′-GCAGUG GAAUGGAACGGAUUUAGAA-3′), siRNA negative control (si-NC) (sense: 5′-UCUCCGAACGUGUCACGUTT-3′, antisense: 5′-GUGACACGU UCGGAGAATT-3′), pcDNA-based HOTAIR overexpression vector (HOTAIR), pcDNA-based HIF1A overexpression vector (HIF1A), pcDNA
2.5. Western blot
Table 1 Relationship between HOTAIR level and clinicopathologic features of HCC patients. Clinicopathologic features
Age(years) ≥50 < 50 Gender Male Female AJCC stage I-II III-IV HBV infection Positive Negative Histological grade Well-Moderately Poor
N (%)
Relative HOTAIR level High (%)
Low (%)
23 (60.5) 15 (39.5)
13 (56.5) 9 (60.0)
10 (43.5) 6 (40.0)
30 (78.9) 8 (21.1)
16 (53.3) 6 (75.0)
14 (46.7) 2 (25.0)
18 (47.4) 20 (52.6)
6 (33.3) 16 (80.0)
12 (66.7) 4 (20.0)
25 (65.8) 13 (44.2)
17 (68.0) 5 (38.5)
8 (32.0) 8 (61.5)
21 (55.3) 17 (44.7)
8 (38.1) 14 (82.4)
13 (61.9) 3 (17.6)
Proteins were extracted from HepG2 and Huh7 cells using RadioImmunoprecipitation Assay (RIPA) buffer (Beyotime, Shanghai, China). For detecting HIF-1A level, after stimulation of 1 % O2 for 48 h, cells were washed with ice phosphate buffer saline (PBS) and scraped directly in lysis buffer containing protease and phosphatase inhibitors, together with dimethyloxalylglycine (Sigma), an inhibitor of prolyl-4hydroxylase domain enzymes involved in HIF1A degradation. The procedures were performed as quickly as possible. Samples were separated by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking with 5 % non-fat milk, the membranes were incubated with indicated primary antibodies overnight at 4 °C and then interacted with secondary antibodies for 2 h. The anti-HK2 (cat. no. ab227198, Abcam, Cambridge, MA, USA), antiHIF1A (cat. no. 20960-1-AP, ProteinTech, Chicago, IL, USA), anti-βactin (cat. no. ab227387, Abcam) and secondary antibody (cat. no. ab205718, Abcam) were used. The protein signals were developed using BeyoECL Plus (Beyotime) and relative expression levels of HK2 and HIF1A were analyzed with β-actin as a loading control according to the densitometry analysis.
P value
0.9014
0.4839
0.0098
0.2091
0.0156
Abbreviations: AJCC, American Joint Committee on Cancer; HBV, hepatitis B virus. 2
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age, gender and hepatitis B virus (HBV) infection (Table 1). Meanwhile, the level of HOTAIR was markedly increased in HepG2 and Huh7 cells compared with that in LO2 cells (Fig. 1B). Moreover, exposure of hypoxia progressively increased HOTAIR level in a time dependent manner in the two cell lines (Fig. 1C and D).
2.6. Bioinformatics analysis, luciferase assay, RNA pull-down and RNA immunoprecipitation (RIP) Bioinformatics analysis was performed using starBase, predicting the binding sites of miR-130a-3p and HOTAIR or HIF1A. The sequences of HOTAIR containing the putative binding sites of miR-130a-3p ( UUGCACU) were inserted into pmirGLO vectors (Promega, Madison, WI, USA) to generate wild-type (WT) luciferase reporter vector HOTAIR-WT. The mutant (HOTAIR-MUT) was generated via mutating the seed sites to GGAUCAG. The wild-type 3′ untranslated region (UTR) sequences of HIF1A (UGCACU) containing miR-130a-3p binding sites and the mutant (MUT) by changing to (CAACUG) were cloned into pmirGLO vectors to generate corresponding luciferase reporter vectors (HIF1A-WT or HIF1A-MUT). Luciferase assay was performed in 293 T cells co-transfected with 200 ng luciferase reporter vectors and 30 nM miR-130a-3p or miR-NC using Lipofectamine 2000. After 24 h of transfection, cells were harvested for luciferase activity assay with a luciferase reporter assay kit (Promega). For RNA pull-down assay, biotinylated HOTAIR probe (BioHOTAIR-probe-WT), biotinylated HOTAIR probe mutant (Bio-HOTAIRprobe-MUT), biotinylated HIF1A probe (Bio-HIF1A-probe-WT), biotinylated HIF1A probe mutant (Bio-HIF1A-probe-MUT), biotinylated miR-130a-3p (Bio-miR-130a-3p-WT), biotinylated miR-130a-3p mutant (Bio-miR-130a-3p-MUT) and biotinylated negative control (Bio-NC) were synthesized by Genepharma and incubated with streptavidin magnetic beads (Invitrogen) for 2 h. HepG2 and Huh7 cells were lysed and incubated with probe-coated beads, followed by detecting the levels of HOTAIR, miR-130a-3p, HIF1A by qRT-PCR. RIP assay was performed in HepG2 and Huh7 cells transfected with miR-130a-3p or miR-NC using the Magna RNA immunoprecipitation kit (Millipore). Collected cells were lysed in RIP lysis buffer and then incubated with magnetic beads pre-coated with Argonaute 2 (Ago2) antibody (cat. no. ab32381, Abcam) or IgG (cat. no. AP112, Sigma). qRTPCR was performed to measure the levels of HOTAIR and HIF1A enriched in complex.
3.2. Knockdown of HOTAIR suppresses glycolysis in HCC cells treated by hypoxia To explore the effect of HOTAIR on glycolysis under hypoxia, the abundance of HOTAIR was significantly decreased by using siRNA in hypoxia-challenged HepG2 and Huh7 cells (Fig. 2A and B). Moreover, in comparison to normoxia group, treatment of hypoxia led to increased glucose consumption and lactate production in HepG2 and Huh7 cells (Fig. 2C-F). However, these events were abrogated by knockdown of HOTAIR. In addition, the expression of HK2 protein was evidently elevated in HepG2 and Huh7 cells after treatment of hypoxia, which was weakened by knockdown of HOTAIR (Fig. 2G and H).
3.3. HOTAIR is a decoy of miR-130a-3p in HCC cells To elucidate the mechanism of HOTAIR in HCC, its target miRNAs were explored by starBase. Among those targets, we selected five miRNAs which have been reported to participate in HCC development and be in response to hypoxia, in which miR-130a-3p exhibited lowest expression in cells after treatment of hypoxia (Supplementary Fig. 2). Hence, miR-130a-3p was chosen for further experiments. The bioinformatics analysis displayed the predicted binding sites of HOTAIR and miR-130a-3p (Fig. 3A). As shown in Fig. 3B, overexpression of miR130a-3p led to a 60 % reduction of luciferase activity in HOTAIR-WT group, while it did not affect the activity when mutating the binding sites of miR-130a-3p and HOTAIR in HOTAIR-MUT group. Furthermore, the results of RNA pull-down showed the obvious enrichment between HOTAIR and miR-130a-3p (Fig. 3C and D). Moreover, the level of HOTAIR enriched by Ago2 RIP was obviously enhanced in HepG2 and Huh7 cells with miR-130a-3p overexpression compared with that in miR-NC group, while IgG RIP showed little enrichment (Fig. 3E and F). In addition, the data of qRT-PCR demonstrated that the expression level of miR-130a-3p in the two cells was greatly decreased by overexpression of HOTAIR and increased by silence of HOTAIR (Fig. 3G and H).
2.7. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) HepG2 and Huh7 cells (5 × 103 cells per well) were seeded into 96well plates. After incubation for 0, 24, 48 and 72 h, 10 μL MTT solution (Beyotime) was added and cells were incubated for 4 h. Then, 100 μL dimethyl sulfoxide (Beyotime) was applied to dissolve the formazan. The optical density (OD) value at 490 nm was examined through a microplate reader (Bio-Rad, Hercules, CA, USA).
3.4. HOTAIR regulates glycolysis by sponging miR-130a-3p in HCC cells treated by hypoxia
2.8. Statistical analysis Statistical analyses were performed by using GraphPad Prism 7 software (GraphPad Inc., La Jolla, CA, USA). Data of results were expressed as mean ± standard deviation (S.D.) from three independent experiments. Student’s t test (for two groups) or ANOVA (for more than two groups) with Tukey’s post hoc test were performed for difference analyses. P < 0.05 was considered significant (*P < 0.05, **P < 0.01, ***P < 0.001).
As described in Fig. 4A and B, the expression of miR-130a-3p was significantly reduced in HepG2 and Huh7 cells after treatment of hypoxia in a time dependent manner. Moreover, the data of starBase v3.0 showed that miR-130a-3p expression was decreased in liver cancer tissues (Supplementary Fig. 1B). Similarly, this study also found that miR-130a-3p expression was declined in HCC tumor tissues compared with that in adjacent normal samples (Supplementary Fig. 1C). Meanwhile, there was a negative correlation between the levels of HOTAIR and miR-130a-3p in HCC tissues (Supplementary Fig. 1D). To explore whether miR-130a-3p was involved in the regulatory network of HOTAIR, HepG2 and Huh7 cells were transfected with si-NC, si-HOTAIR, si-HOTAIR and anti-miR-NC or anti-miR-130a-3p before treatment of hypoxia. As shown in Fig. 4C-F, deficiency of miR-130a-3p reversed silencing HOTAIR-induced suppression of glucose conscription and lactate production in hypoxia-treated HepG2 and Huh7 cells. Furthermore, the HK2 protein level inhibited by HOTAIR interference was restored by knockdown of miR-130a-3p in the two cell lines treated by hypoxia (Fig. 4G and H).
3. Results 3.1. HOTAIR expression is increased in HCC To explore the role of HOTAIR in HCC, its expression was measured in HCC. As shown in Fig. 1A, the expression of HOTAIR was significantly enhanced in HCC tumor tissues compared with that in adjacent normal samples (n = 38), which was similar to the data predicted by starBase v3.0 (Supplementary Fig. 1A). Furthermore, high expression of HOTAIR was associated with American Joint Committee on Cancer (AJCC) stage and Histological grade of patients but not with 3
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Fig. 1. HOTAIR expression is enhanced in HCC. (A) The expression of HOTAIR was measured in HCC tissues and normal samples by qRT-PCR. n = 38. (B) The level of HOTAIR was detected in HCC cells (HepG2 and Huh7) and normal LO2 cells by qRT-PCR. (C and D) The expression of HOTAIR was measured in HepG2 and Huh7 cells after treatment of hypoxia for different exposure times (0, 6, 12, 24 and 48 h) by qRT-PCR. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 2. Knockdown of HOTAIR inhibits glycolysis in HCC cells treated by hypoxia. (A and B) The expression of HOTAIR was measured in HepG2 and Huh7 cells transfected with si-HOTAIR or si-NC after treatment of hypoxia by qRT-PCR. The glucose consumption (C and D) and lactate production (E and F) were analyzed in HepG2 and Huh7 cells transfected with si-HOTAIR or si-NC after treatment of hypoxia. (G and H) The expression of HK2 protein was detected in HepG2 and Huh7 cells transfected with si-HOTAIR or si-NC after treatment of hypoxia by western blot. **P < 0.01, ***P < 0.001.
miR-130a-3p overexpression in HIF1A-WT group (Fig. 5B). Additionally, there was a great enrichment between miR-130a-3p and HIF1A in HepG2 and Huh7 cells after RNA pull-down assay (Fig. 5C and D). Furthermore, overexpression of miR-130a-3p resulted in higher level of HIF1A enriched by Ago2 RIP in HepG2 and Huh7 cells (Fig. 5E and F). Besides, the results of western blot described that addition of miR-130a-3p induced great decrease of HIF1A protein level in the two
3.5. HIF1A is a target of miR-130a-3p in HCC cells To further explore the mechanism in this study, the potential target of miR-130a-3p was explored by bioinformatics analysis, which displayed the binding sites of miR-130a-3p and HIF1A (Fig. 5A). Luciferase assay was performed to validate the targeting association between them with the results of a 70 % reduction of luciferase activity by 4
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Fig. 3. miR-130a-3p is bound to HOTAIR in HCC cells. (A) The predicted binding sites of HOTAIR and miR-130a-3p was provided by starBase. (B) Luciferase activity was measured in 293 T cells co-transfected with HOTAIR-WT or HOTAIR-MUT luciferase reporter vector and miR-130-3p or miR-NC. (C and D) RNA pulldown assay was performed in HepG2 and Huh7 cells by using Bio-NC, Bio-miR-130a-3p-WT, Bio-miR-130a-3p-MUT, Bio-HOTAIR-probe-WT or Bio-HOTAIR-probeMUT, followed by detection of level of HOTAIR or miR-130a-3p. (E and F) The level of HOTAIR enriched by Ago2 RIP was detected in HepG2 and Huh7 cells transfected with miR-130a-3p or miR-NC. (G and H) The expression of miR-130a-3p was detected in HepG2 and Huh7 cells transfected with vector, HOTAIR, si-NC or si-JOTAIR by qRT-PCR. ***P < 0.001, NS: not significant.
production in HepG2 and Huh7 cells under hypoxia condition. Moreover, overexpression of miR-130a-3p led to great decrease of HK2 protein level in HepG2 and Huh7 cells after insult of hypoxia (Fig. 6E and F). Besides, to explore whether miR-130a-3p-mediated glycolysis suppression was associated with HIF1A, HepG2 and Huh7 cells were cotransfected with miR-130a-3p and vector or HIF1A. Results described that introduction of HIF1A notably attenuated the inhibitive effect of miR-130a-3p on glucose consumption, lactate production and HK2 protein expression in HepG2 and Huh7 cells suffered from hypoxia treatment (Fig. 6A-F).
cell lines, while miR-130a-3p knockdown caused an opposite effect (Fig. 5G and H).
3.6. miR-130a-3p inhibits glycolysis by targeting HIF1A in HCC cells treated by hypoxia To explore the effect of miR-130a-3p on glycolysis under hypoxia, HepG2 and Huh7 cells were transfected with miR-130a-3p or miR-NC and then suffered from hypoxia. As shown in Fig. 6A-D, overexpression of miR-130a-3p significantly repressed glucose consumption and lactate
Fig. 4. miR-130a-3p knockdown reverses the effect of HOTAIR silence on glycolysis in HCC cells treated by hypoxia. (A and B) The expression of miR-130a3p was measured in HepG2 and Huh7 cells after exposure of hypoxia for different times (0, 6, 12, 24 and 48 h) by qRT-PCR. The glucose consumption (C and D), lactate production (E and F) and HK2 protein level (G and H) were detected in HepG2 and Huh7 cells transfected with si-NC, si-HOTAIR, si-HOTAIR and anti-miR-NC or anti-miR-130a-3p after treatment of hypoxia. *P < 0.05, **P < 0.01, ***P < 0.001. 5
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Fig. 5. HIF1A is a target of miR-130a-3p in HCC cells. (A) The binding sites of miR-130a-3p and HIF1A were predicted by starBase. (B) Luciferase activity was analyzed in 293 T cells co-transfected with HIF1A-WT or HIF1A-MUT and miR-130a-3p or miR-NC. (C and D) RNA pull-down assay was performed in HepG2 and Huh7 cells by using bio-NC, Bio-miR-130a-3p-WT, Bio-miR-130a-3p-MUT, Bio-HIF1A-probe-WT or Bio-HIF1A-probe-MUT, followed by detection of level of HIF1A or miR-130a-3p. (E and F) The level of HIF1A was measured in HepG2 and Huh7 cells transfected with miR-130a-3p or miR-NC after Ago2 RIP. (G and H) The protein level of HK2 was detected in HepG2 and Huh7 cells transfected with miR-NC, miR-130a-3p, anti-miR-NC or anti-miR-130a-3p by western blot. ***P < 0.001, NS: not significant.
ceRNA for miR-1, miR-23b-3p and miR-122 to mediate HCC malignancy [9,30,31]. To figure out whether HOTAIR addressed glycolysis by the ceRNA network, we first indicated HOTAIR as a sponge of miR130a-3p, which was confirmed by luciferase assay, RNA pull-down and RIP. The previous studies suggested that miR-130a-3p could play a suppressive role in the development of HCC [18,19]. In this study, we found that miR-130a-3p expression was decreased in hypoxia-treated HCC cells and miR-130a-3p overexpression suppressed glycolysis in HCC cells. Moreover, miR-130a-3p deficiency reversed the suppressive role of HOTAIR knockdown in glycolysis, uncovering that HOTAIR regulated glycolysis under hypoxia in HCC by sponging miR-130a-3p. The function of miRNA is realized by regulating its target mRNA. This study first validated HIF1A as a functional target of miR-130a-3p in HCC. Hypoxia could induce activation of HIF1A and then regulate reprogramming of glucose metabolism in cancers [32,33]. Additionally, previous report demonstrated that HIF1A facilitated HCC glycolysis under hypoxia [13]. Here we found that HIF1A restoration weakened miR-130a-3p-mediated glycolysis inhibition, suggesting that miR-130a3p blocked glycolysis by targeting HIF1A. Moreover, former study showed that HOTAIR could increase HIF1A level by acting as a ceRNA for miR-217 in renal cell carcinoma [34]. In the present study, we confirmed that HOTAIR could derepress HIF1A expression by competitively sponging miR-130a-3p in HCC cells. Furthermore, we confirmed that knockdown of HOTAIR could decrease HCC cell viability under hypoxic condition by regulating miR-130a-3p and HIF1A (Supplementary Fig. 4A-D), indicating that the network of HOTAIR/miR-130a-3p/ HIF1A could mediate HCC progression by regulating metabolic reprogramming under hypoxic condition. Besides, the transcriptional regulation mediated by HOTAIR might be also responsible for understanding the potential mechanism which should be explored in future work [35,36]. HK2, a key enzyme contributing to glycolysis is a downstream of HIF1A. HIF1A is a key factor in response to hypoxia, which could bind to hypoxia response element in transcription-regulatory regions (promoters or enhancers) of downstream glycolytic genes, including HK2 [37]. In this study, we confirmed the ceRNA
3.7. HOTAIR regulates HIF1A expression by competitively sponging miR130a-3p To implicate the competing endogenous RNA (ceRNA) network of HOTAIR, the association among HOTAIR, miR-130a-3p and HIF1A was analyzed. In HIF1A-WT-transfected 293 T cells, overexpression of miR130a-3p markedly decreased the luciferase activity, while this event was alleviated by introduction of HOTAIR (Fig. 7A). Moreover, overexpression of HOTAIR significantly enhanced the protein level of HIF1A in HepG2 cells, which was counteracted by miR-130a-3p overexpression (Fig. 7B). Meanwhile, the expression of HIF1A protein was greatly inhibited by interference of HOTAIR, which was mitigated by deficiency of miR-130a-3p (Fig. 7C). 4. Discussion HCC cells have a high dependence of glycolysis especially under hypoxia condition [23]. HOTAIR is a lncRNA associated with hypoxia stress by interacting with HIF proteins [24,25]. Furthermore, HOTAIR was highly expressed and promoted HCC progression [9–11]. In this study, we were the first to demonstrate that HOTAIR promoted glycolysis under hypoxia in HCC and elucidate the regulatory mechanism of HOTAIR/miR-130a-3p/HIF1A (Supplementary Fig. 3). This study displayed that HOTAIR was highly expressed in HCC and associated with hypoxia stress, indicating that HOTAIR might contribute to HCC malignancy. Glycolysis, characterized by glucose consumption and lactate production, maintains cancer cell survival under hypoxia. HK2 is a pivotal player in glycolysis and utilized as a metabolic target for treatment of HCC [26,27]. The loss-of-function experiments showed that knockdown of HOTAIR inhibited glycolysis under hypoxia, revealed by reduction of glucose consumption, lactate production and HK2 protein abundance in HCC cells, which is also in agreement with former works [28,29]. However, the mechanism underlying HOTAIR regulating glycolysis remains elusive. Previous studies have indicated that HOTAIR could serve as a 6
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Fig. 6. HIF1A attenuates miR-130a-3p-mediated glycolysis inhibition in HCC cells treated by hypoxia. HepG2 and Huh7 cells were transfected with miR-NC, miR-130a-3p, miR-130a-3p and vector or HIF1A and then exposed to hypoxia. The glucose consumption (A and B), lactate production (C and D) and HK2 protein level (E and F) were detected in the treated HepG2 and Huh7 cells. **P < 0.01, ***P < 0.001.
Declaration of Competing Interest
network of HOTAIR/miR-130a-3p/HIF1A, indicating the glycolysis mediated by HOTAIR is regulated in an HIF1A-dependent manner. We hypothesized that might be realized by binding the sites in promoter regions of HK2, which is needed to be further confirmed in future. In conclusion, here we indicated a novel mechanism underlying the glycolysis progression under hypoxia in HCC. HOTAIR expression was increased in HCC cells after hypoxia treatment. Knockdown of HOTAIR inhibited glycolysis in HCC cells stimulated with hypoxia, possibly by acting as a ceRNA network of HOTAIR/miR-130a-3p/HIF1A. This study may provide a new therapeutic target for treatment of HCC.
The authors declare that there are no conflicts of interest.
Acknowledgement This work was supported by National Natural Science Foundation of China (No. 931671440), the Science and Technology Self-help Project of Department of Henan Science and Technology (No.172102310099), and the National Natural Fund Joint Fund (No. U1404309).
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Fig. 7. HIF1A expression is regulated by HOTAIR and miR-130a-3p. (A) Luciferase activity was measured in 293 T cells transfected with HIF1A-WT, HIF1A-WT + miR130a-3p, HIF1A-WT + miR-130a-3p + vector and HIF1A-WT + miR-130a-3p+HOTAIR. (B) The protein level of HIF1A was measured in HepG2 cells transfected with vector, HOTAIR, HOTAIR and miR-NC or miR-130a-3p. (C) The expression of HIF1A protein was detected in Huh7 cells transfected with si-NC, si-HOTAIR, si-HOTAIR and anti-miR-NC or anti-miR-130a3p. **P < 0.01, ***P < 0.001.
Appendix A. Supplementary data
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LncRNA HOTAIR knockdown inhibits glycolysis by regulating miR-130a-3p/ HIF1A in hepatocellular carcinoma under hypoxia
T
Mingxing Hua,b,c, Qiang Fua,b,c, Chan Jinga,b,c, Xu Zhanga,b,c, Tao Qina,b,c,*, Yanfeng Pand,** a
Department of Hepatobiliary and Pancreatic Surgery, the Henan Provincial People’s Hospital, Zhengzhou, Henan, China Department of Hepatobiliary and Pancreatic Surgery, the People's Hospital of Zhengzhou University, Zhengzhou, Henan, China c Department of Hepatobiliary and Pancreatic Surgery, the School of Clinical Medicine, Henan University, Zhengzhou, Henan, China d Department of Infectious Diseases, the First Affiliated Hospital of Zhengzhou University, No. Jianshe East Road, Zhengzhou 450000, Henan, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Hepatocellular carcinoma Glycolysis Hypoxia HOTAIR miR-130a-3p HIF1A
High rate of glycolysis supports hepatocellular carcinoma (HCC) cell growth even in a hypoxic environment. However, the mechanism underlying glycolysis under hypoxia remains largely unknown. Long noncoding RNAs (lncRNAs) play essential roles in regulating glucose metabolism in cancers. This study aimed to explore the role of lncRNA homeobox transcript antisense RNA (HOTAIR) in HCC glycolysis under hypoxia. Thirty-eight HCC patients were recruited. HepG2 and Huh7 cells were used for study in vitro. The expression levels of HOTAIR, microRNA-130a-3p (miR-130a-3p) and hypoxia inducible factor 1 alpha (HIF1A) were measured by quantitative real-time polymerase chain reaction and western blot, respectively. The glycolysis under hypoxia (1 % O2) condition was investigated by glucose consumption, lactate production and hexokinase 2 (HK2) level. The target interaction between miR-130a-3p and HOTIR or HIF1A was analyzed by bioinformatics analysis, luciferase assay, RNA pull-down and RNA immunoprecipitation. We found that HOTAIR expression was enhanced in HCC tissues and cells. Under hypoxia condition, HOTAIR expression was increased and its knockdown inhibited glycolysis in HCC cells. HOTAIR was validated as a decoy of miR-130a-3p and miR-130a-3p deficiency reversed the suppressive effect of HOTAIR silence on glycolysis under hypoxia. HIF1A was indicated as a target of miR130a-3p and miR-130a-3p overexpression repressed glycolysis under hypoxia by targeting HIF1A. Moreover, HIF1A expression was regulated by HOTAIR and miR-130a-3p. In conclusion, knockdown of HOTAIR suppressed glycolysis by regulating miR-130a-3p and HIF1A in HCC cells treated by hypoxia, elucidating a novel mechanism in HCC glycolysis.
1. Introduction Hypoxia is one of the most important features of the tumor microenvironment, which leads to the reprogramming of glycolytic metabolism, maintaining the survival of cancer cells under hypoxia condition [1]. Targeting glycolysis provides potential for therapeutic opportunities in human cancers [2]. Hepatocellular carcinoma (HCC) is the major type of liver cancer with high incidence and mortality worldwide [3]. The glycolysis under hypoxia stress contributes to malignancy of HCC [4,5]. However, the mechanism underlying glycolysis under hypoxia condition in HCC remains largely unknown. Noncoding RNAs have been regarded as essential targets for diagnosis and therapeutics of HCC [6]. Long noncoding RNAs (lncRNAs) are one class of noncoding RNAs with more than 200 nucleotides, which
are implicated in glucose metabolism by regulating glycolytic enzymes or related signaling pathways in cancers [7]. LncRNA homeobox transcript antisense RNA (HOTAIR) has been reported to be an oncogene in human cancers [8]. Previous studies revealed that HOTAIR acts as a carcinogenic lncRNA in HCC to contribute to HCC malignancy by promoting epithelial-mesenchymal transition, proliferation and cisplatin resistance [9–11]. However, little is known about the effect of HOTAIR on glycolysis under hypoxia in HCC. microRNAs (miRNAs) are also a class of small noncoding RNAs with ∼23 nucleotides, which play pivotal roles in prognosis and treatment of HCC [12]. Moreover, miRNAs could participate in HCC progression through regulating glucose metabolism [13,14]. Former works indicated miR-130a-3p as a tumor suppressor in many cancers, such as non-small-cell lung cancer, breast cancer and glioma [15–17]. In
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Corresponding author at: Department of Hepatobiliary and Pancreatic Surgery, the Henan Provincial People’s Hospital, No. 7 Weiwu Road, Zhengzhou 450003, Henan, China. ⁎⁎ Corresponding author. E-mail address: [email protected] (Y. Pan). https://doi.org/10.1016/j.biopha.2019.109703 Received 10 September 2019; Received in revised form 14 November 2019; Accepted 24 November 2019 0753-3322/ © 2019 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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empty vector (vector) were generated from Genepharma (Shanghai, China). miR-130a-3p mimic (miR-130a-3p) (cat. no. miR10000425-1-5) and miR-130a-3p inhibitor (anti-miR-130a-3p) (cat. no. miR200004251-5) along with their corresponding negative control (miR-NC, antimiR-NC) were purchased from Ribobio (Guangzhou, China). When reaching 60 % confluence, HepG2 and Huh7 cells in 6-well plates were transfected with 30 nM oligonucleotides or 500 ng vectors using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufactures’ instructions for 6 h. After transfection for 24 h, cells were harvested for subsequent experiments.
addition, previous studies demonstrated that miR-130a-3p is downregulated and suppresses HCC malignancy [18,19]. Hypoxia-inducible factor 1 (HIF1) is a key regulator of glucose metabolism under hypoxia, providing cancer cells a growth advantage in solid tumors [20]. HIF1A is a major subunit of HIF1, which plays as a poor prognostic factor in patients with HCC [21]. We assumed that miR-130a-3p and HIF1A were involved in HOTAIR-addressed mechanism in HCC due to the predicted binding sites of miR-130a-3p and HOTAIR or HIF1A by bioinformatics analysis. Here we first measured the changes of HOTAIR level in HCC in response to hypoxia. Moreover, we investigated the effect of HOTAIR knockdown on glycolysis by analyses of glucose consumption, lactate production and hexokinase 2 (HK2) protein level. Besides, we explored the potential mechanism by analyzing the association among HOTAIR, miR-130a-3p and HIF1A.
2.3. Detection of glucose consumption and lactate production HepG2 and Huh7 cells (2 × 105/well) were seeded into 6-well plates overnight and then cultured in hypoxia or normoxia condition for 48 h. The glucose consumption and lactate production were measured by using Glucose Assay Kit and Lactate Assay Kit (Sigma, St. Louis, MO, USA) following the manufacturer’s protocol. The relative levels of glucose consumption and lactate production were analyzed by normalizing to indicated control group.
2. Materials and methods 2.1. Patients and tissues A total of 38 HCC patients without history of chemotherapy, radiotherapy and other therapy were recruited from the Henan Provincial People’s Hospital. The tumor tissues and corresponding adjacent normal samples were collected from all participants who have provided the written informed consent. This study was approved by the ethics committee of the Henan Provincial People’s Hospital. The clinicopathologic features of patients were displayed in Table 1.
2.4. Quantitative real-time polymerase chain reaction (qRT-PCR) RNA was isolated from HCC tissues and cells using Trizol reagent (Thermo Fisher Scientific, Waltham, MA USA) following the manufacturer’s protocols. The samples were reverse transcribed using TaqMan™ mRNA or microRNA Reverse Transcription Kit (Thermo Fisher Scientific). The qRT-PCR was performed with SYBR Green mix (Thermo Fisher Scientific) on the Roche LightCycler 480 system. All primers used in this study were generated by Sangon (Shanghai, China) as follows: HOTAIR (Forward, 5′-GGTAGAAAAAGCAACCACGAAGC-3′; Reverse, 5′- ACATAAACCTCTGTCTGTGAGTGCC-3′); HIF1A (Forward, 5′-TTCCCGACTAGGCCCATTC-3′; Reverse, 5′-CAGGTATTCAAGGTCCC ATTTCA-3′); GAPDH (Forward, 5′-AGAAGGCTGGGGCTCATTTG-3′; Reverse, 5′- AGGGGCCATCCACAGTCTTC-3′); miR-130a-3p (Forward, 5′- TTGCGATTCTGTTTTGTGCT-3′; Reverse, 5′-GTGGGGTCCTCAGT GGG-3′); U6 (Forward, 5′-CTCGCTTCGGCAGCACA-3′; Reverse, 5′AACGCTTCACGAATTTGCGT-3′). GAPDH and U6 were regarded as internal control. The relative expression levels of RNA were analyzed according to the 2−ΔΔCt method [22].
2.2. Cell culture, hypoxia treatment and transfection HCC cell lines (HepG2 and Huh7), normal human hepatic cell line LO2 and 293 T cells were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). All cells were cultured at 37 °C under 5 % CO2 in RPMI-1640 medium (Gibco, Grand Island, NY, USA) containing 10 % fetal bovine serum. For hypoxia stimulation, HepG2 and Huh7 cells were incubated in a hypoxia chamber with 1 % O2 for different times (0, 6, 12, 24 and 48 h). Small interfering RNA (siRNA) against HOTAIR (si-HOTAIR) (sense: 5′-UUCUAAAUCCGUUCCAUUCCACUGCGA-3′, antisense: 5′-GCAGUG GAAUGGAACGGAUUUAGAA-3′), siRNA negative control (si-NC) (sense: 5′-UCUCCGAACGUGUCACGUTT-3′, antisense: 5′-GUGACACGU UCGGAGAATT-3′), pcDNA-based HOTAIR overexpression vector (HOTAIR), pcDNA-based HIF1A overexpression vector (HIF1A), pcDNA
2.5. Western blot
Table 1 Relationship between HOTAIR level and clinicopathologic features of HCC patients. Clinicopathologic features
Age(years) ≥50 < 50 Gender Male Female AJCC stage I-II III-IV HBV infection Positive Negative Histological grade Well-Moderately Poor
N (%)
Relative HOTAIR level High (%)
Low (%)
23 (60.5) 15 (39.5)
13 (56.5) 9 (60.0)
10 (43.5) 6 (40.0)
30 (78.9) 8 (21.1)
16 (53.3) 6 (75.0)
14 (46.7) 2 (25.0)
18 (47.4) 20 (52.6)
6 (33.3) 16 (80.0)
12 (66.7) 4 (20.0)
25 (65.8) 13 (44.2)
17 (68.0) 5 (38.5)
8 (32.0) 8 (61.5)
21 (55.3) 17 (44.7)
8 (38.1) 14 (82.4)
13 (61.9) 3 (17.6)
Proteins were extracted from HepG2 and Huh7 cells using RadioImmunoprecipitation Assay (RIPA) buffer (Beyotime, Shanghai, China). For detecting HIF-1A level, after stimulation of 1 % O2 for 48 h, cells were washed with ice phosphate buffer saline (PBS) and scraped directly in lysis buffer containing protease and phosphatase inhibitors, together with dimethyloxalylglycine (Sigma), an inhibitor of prolyl-4hydroxylase domain enzymes involved in HIF1A degradation. The procedures were performed as quickly as possible. Samples were separated by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking with 5 % non-fat milk, the membranes were incubated with indicated primary antibodies overnight at 4 °C and then interacted with secondary antibodies for 2 h. The anti-HK2 (cat. no. ab227198, Abcam, Cambridge, MA, USA), antiHIF1A (cat. no. 20960-1-AP, ProteinTech, Chicago, IL, USA), anti-βactin (cat. no. ab227387, Abcam) and secondary antibody (cat. no. ab205718, Abcam) were used. The protein signals were developed using BeyoECL Plus (Beyotime) and relative expression levels of HK2 and HIF1A were analyzed with β-actin as a loading control according to the densitometry analysis.
P value
0.9014
0.4839
0.0098
0.2091
0.0156
Abbreviations: AJCC, American Joint Committee on Cancer; HBV, hepatitis B virus. 2
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age, gender and hepatitis B virus (HBV) infection (Table 1). Meanwhile, the level of HOTAIR was markedly increased in HepG2 and Huh7 cells compared with that in LO2 cells (Fig. 1B). Moreover, exposure of hypoxia progressively increased HOTAIR level in a time dependent manner in the two cell lines (Fig. 1C and D).
2.6. Bioinformatics analysis, luciferase assay, RNA pull-down and RNA immunoprecipitation (RIP) Bioinformatics analysis was performed using starBase, predicting the binding sites of miR-130a-3p and HOTAIR or HIF1A. The sequences of HOTAIR containing the putative binding sites of miR-130a-3p ( UUGCACU) were inserted into pmirGLO vectors (Promega, Madison, WI, USA) to generate wild-type (WT) luciferase reporter vector HOTAIR-WT. The mutant (HOTAIR-MUT) was generated via mutating the seed sites to GGAUCAG. The wild-type 3′ untranslated region (UTR) sequences of HIF1A (UGCACU) containing miR-130a-3p binding sites and the mutant (MUT) by changing to (CAACUG) were cloned into pmirGLO vectors to generate corresponding luciferase reporter vectors (HIF1A-WT or HIF1A-MUT). Luciferase assay was performed in 293 T cells co-transfected with 200 ng luciferase reporter vectors and 30 nM miR-130a-3p or miR-NC using Lipofectamine 2000. After 24 h of transfection, cells were harvested for luciferase activity assay with a luciferase reporter assay kit (Promega). For RNA pull-down assay, biotinylated HOTAIR probe (BioHOTAIR-probe-WT), biotinylated HOTAIR probe mutant (Bio-HOTAIRprobe-MUT), biotinylated HIF1A probe (Bio-HIF1A-probe-WT), biotinylated HIF1A probe mutant (Bio-HIF1A-probe-MUT), biotinylated miR-130a-3p (Bio-miR-130a-3p-WT), biotinylated miR-130a-3p mutant (Bio-miR-130a-3p-MUT) and biotinylated negative control (Bio-NC) were synthesized by Genepharma and incubated with streptavidin magnetic beads (Invitrogen) for 2 h. HepG2 and Huh7 cells were lysed and incubated with probe-coated beads, followed by detecting the levels of HOTAIR, miR-130a-3p, HIF1A by qRT-PCR. RIP assay was performed in HepG2 and Huh7 cells transfected with miR-130a-3p or miR-NC using the Magna RNA immunoprecipitation kit (Millipore). Collected cells were lysed in RIP lysis buffer and then incubated with magnetic beads pre-coated with Argonaute 2 (Ago2) antibody (cat. no. ab32381, Abcam) or IgG (cat. no. AP112, Sigma). qRTPCR was performed to measure the levels of HOTAIR and HIF1A enriched in complex.
3.2. Knockdown of HOTAIR suppresses glycolysis in HCC cells treated by hypoxia To explore the effect of HOTAIR on glycolysis under hypoxia, the abundance of HOTAIR was significantly decreased by using siRNA in hypoxia-challenged HepG2 and Huh7 cells (Fig. 2A and B). Moreover, in comparison to normoxia group, treatment of hypoxia led to increased glucose consumption and lactate production in HepG2 and Huh7 cells (Fig. 2C-F). However, these events were abrogated by knockdown of HOTAIR. In addition, the expression of HK2 protein was evidently elevated in HepG2 and Huh7 cells after treatment of hypoxia, which was weakened by knockdown of HOTAIR (Fig. 2G and H).
3.3. HOTAIR is a decoy of miR-130a-3p in HCC cells To elucidate the mechanism of HOTAIR in HCC, its target miRNAs were explored by starBase. Among those targets, we selected five miRNAs which have been reported to participate in HCC development and be in response to hypoxia, in which miR-130a-3p exhibited lowest expression in cells after treatment of hypoxia (Supplementary Fig. 2). Hence, miR-130a-3p was chosen for further experiments. The bioinformatics analysis displayed the predicted binding sites of HOTAIR and miR-130a-3p (Fig. 3A). As shown in Fig. 3B, overexpression of miR130a-3p led to a 60 % reduction of luciferase activity in HOTAIR-WT group, while it did not affect the activity when mutating the binding sites of miR-130a-3p and HOTAIR in HOTAIR-MUT group. Furthermore, the results of RNA pull-down showed the obvious enrichment between HOTAIR and miR-130a-3p (Fig. 3C and D). Moreover, the level of HOTAIR enriched by Ago2 RIP was obviously enhanced in HepG2 and Huh7 cells with miR-130a-3p overexpression compared with that in miR-NC group, while IgG RIP showed little enrichment (Fig. 3E and F). In addition, the data of qRT-PCR demonstrated that the expression level of miR-130a-3p in the two cells was greatly decreased by overexpression of HOTAIR and increased by silence of HOTAIR (Fig. 3G and H).
2.7. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) HepG2 and Huh7 cells (5 × 103 cells per well) were seeded into 96well plates. After incubation for 0, 24, 48 and 72 h, 10 μL MTT solution (Beyotime) was added and cells were incubated for 4 h. Then, 100 μL dimethyl sulfoxide (Beyotime) was applied to dissolve the formazan. The optical density (OD) value at 490 nm was examined through a microplate reader (Bio-Rad, Hercules, CA, USA).
3.4. HOTAIR regulates glycolysis by sponging miR-130a-3p in HCC cells treated by hypoxia
2.8. Statistical analysis Statistical analyses were performed by using GraphPad Prism 7 software (GraphPad Inc., La Jolla, CA, USA). Data of results were expressed as mean ± standard deviation (S.D.) from three independent experiments. Student’s t test (for two groups) or ANOVA (for more than two groups) with Tukey’s post hoc test were performed for difference analyses. P < 0.05 was considered significant (*P < 0.05, **P < 0.01, ***P < 0.001).
As described in Fig. 4A and B, the expression of miR-130a-3p was significantly reduced in HepG2 and Huh7 cells after treatment of hypoxia in a time dependent manner. Moreover, the data of starBase v3.0 showed that miR-130a-3p expression was decreased in liver cancer tissues (Supplementary Fig. 1B). Similarly, this study also found that miR-130a-3p expression was declined in HCC tumor tissues compared with that in adjacent normal samples (Supplementary Fig. 1C). Meanwhile, there was a negative correlation between the levels of HOTAIR and miR-130a-3p in HCC tissues (Supplementary Fig. 1D). To explore whether miR-130a-3p was involved in the regulatory network of HOTAIR, HepG2 and Huh7 cells were transfected with si-NC, si-HOTAIR, si-HOTAIR and anti-miR-NC or anti-miR-130a-3p before treatment of hypoxia. As shown in Fig. 4C-F, deficiency of miR-130a-3p reversed silencing HOTAIR-induced suppression of glucose conscription and lactate production in hypoxia-treated HepG2 and Huh7 cells. Furthermore, the HK2 protein level inhibited by HOTAIR interference was restored by knockdown of miR-130a-3p in the two cell lines treated by hypoxia (Fig. 4G and H).
3. Results 3.1. HOTAIR expression is increased in HCC To explore the role of HOTAIR in HCC, its expression was measured in HCC. As shown in Fig. 1A, the expression of HOTAIR was significantly enhanced in HCC tumor tissues compared with that in adjacent normal samples (n = 38), which was similar to the data predicted by starBase v3.0 (Supplementary Fig. 1A). Furthermore, high expression of HOTAIR was associated with American Joint Committee on Cancer (AJCC) stage and Histological grade of patients but not with 3
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Fig. 1. HOTAIR expression is enhanced in HCC. (A) The expression of HOTAIR was measured in HCC tissues and normal samples by qRT-PCR. n = 38. (B) The level of HOTAIR was detected in HCC cells (HepG2 and Huh7) and normal LO2 cells by qRT-PCR. (C and D) The expression of HOTAIR was measured in HepG2 and Huh7 cells after treatment of hypoxia for different exposure times (0, 6, 12, 24 and 48 h) by qRT-PCR. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 2. Knockdown of HOTAIR inhibits glycolysis in HCC cells treated by hypoxia. (A and B) The expression of HOTAIR was measured in HepG2 and Huh7 cells transfected with si-HOTAIR or si-NC after treatment of hypoxia by qRT-PCR. The glucose consumption (C and D) and lactate production (E and F) were analyzed in HepG2 and Huh7 cells transfected with si-HOTAIR or si-NC after treatment of hypoxia. (G and H) The expression of HK2 protein was detected in HepG2 and Huh7 cells transfected with si-HOTAIR or si-NC after treatment of hypoxia by western blot. **P < 0.01, ***P < 0.001.
miR-130a-3p overexpression in HIF1A-WT group (Fig. 5B). Additionally, there was a great enrichment between miR-130a-3p and HIF1A in HepG2 and Huh7 cells after RNA pull-down assay (Fig. 5C and D). Furthermore, overexpression of miR-130a-3p resulted in higher level of HIF1A enriched by Ago2 RIP in HepG2 and Huh7 cells (Fig. 5E and F). Besides, the results of western blot described that addition of miR-130a-3p induced great decrease of HIF1A protein level in the two
3.5. HIF1A is a target of miR-130a-3p in HCC cells To further explore the mechanism in this study, the potential target of miR-130a-3p was explored by bioinformatics analysis, which displayed the binding sites of miR-130a-3p and HIF1A (Fig. 5A). Luciferase assay was performed to validate the targeting association between them with the results of a 70 % reduction of luciferase activity by 4
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Fig. 3. miR-130a-3p is bound to HOTAIR in HCC cells. (A) The predicted binding sites of HOTAIR and miR-130a-3p was provided by starBase. (B) Luciferase activity was measured in 293 T cells co-transfected with HOTAIR-WT or HOTAIR-MUT luciferase reporter vector and miR-130-3p or miR-NC. (C and D) RNA pulldown assay was performed in HepG2 and Huh7 cells by using Bio-NC, Bio-miR-130a-3p-WT, Bio-miR-130a-3p-MUT, Bio-HOTAIR-probe-WT or Bio-HOTAIR-probeMUT, followed by detection of level of HOTAIR or miR-130a-3p. (E and F) The level of HOTAIR enriched by Ago2 RIP was detected in HepG2 and Huh7 cells transfected with miR-130a-3p or miR-NC. (G and H) The expression of miR-130a-3p was detected in HepG2 and Huh7 cells transfected with vector, HOTAIR, si-NC or si-JOTAIR by qRT-PCR. ***P < 0.001, NS: not significant.
production in HepG2 and Huh7 cells under hypoxia condition. Moreover, overexpression of miR-130a-3p led to great decrease of HK2 protein level in HepG2 and Huh7 cells after insult of hypoxia (Fig. 6E and F). Besides, to explore whether miR-130a-3p-mediated glycolysis suppression was associated with HIF1A, HepG2 and Huh7 cells were cotransfected with miR-130a-3p and vector or HIF1A. Results described that introduction of HIF1A notably attenuated the inhibitive effect of miR-130a-3p on glucose consumption, lactate production and HK2 protein expression in HepG2 and Huh7 cells suffered from hypoxia treatment (Fig. 6A-F).
cell lines, while miR-130a-3p knockdown caused an opposite effect (Fig. 5G and H).
3.6. miR-130a-3p inhibits glycolysis by targeting HIF1A in HCC cells treated by hypoxia To explore the effect of miR-130a-3p on glycolysis under hypoxia, HepG2 and Huh7 cells were transfected with miR-130a-3p or miR-NC and then suffered from hypoxia. As shown in Fig. 6A-D, overexpression of miR-130a-3p significantly repressed glucose consumption and lactate
Fig. 4. miR-130a-3p knockdown reverses the effect of HOTAIR silence on glycolysis in HCC cells treated by hypoxia. (A and B) The expression of miR-130a3p was measured in HepG2 and Huh7 cells after exposure of hypoxia for different times (0, 6, 12, 24 and 48 h) by qRT-PCR. The glucose consumption (C and D), lactate production (E and F) and HK2 protein level (G and H) were detected in HepG2 and Huh7 cells transfected with si-NC, si-HOTAIR, si-HOTAIR and anti-miR-NC or anti-miR-130a-3p after treatment of hypoxia. *P < 0.05, **P < 0.01, ***P < 0.001. 5
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Fig. 5. HIF1A is a target of miR-130a-3p in HCC cells. (A) The binding sites of miR-130a-3p and HIF1A were predicted by starBase. (B) Luciferase activity was analyzed in 293 T cells co-transfected with HIF1A-WT or HIF1A-MUT and miR-130a-3p or miR-NC. (C and D) RNA pull-down assay was performed in HepG2 and Huh7 cells by using bio-NC, Bio-miR-130a-3p-WT, Bio-miR-130a-3p-MUT, Bio-HIF1A-probe-WT or Bio-HIF1A-probe-MUT, followed by detection of level of HIF1A or miR-130a-3p. (E and F) The level of HIF1A was measured in HepG2 and Huh7 cells transfected with miR-130a-3p or miR-NC after Ago2 RIP. (G and H) The protein level of HK2 was detected in HepG2 and Huh7 cells transfected with miR-NC, miR-130a-3p, anti-miR-NC or anti-miR-130a-3p by western blot. ***P < 0.001, NS: not significant.
ceRNA for miR-1, miR-23b-3p and miR-122 to mediate HCC malignancy [9,30,31]. To figure out whether HOTAIR addressed glycolysis by the ceRNA network, we first indicated HOTAIR as a sponge of miR130a-3p, which was confirmed by luciferase assay, RNA pull-down and RIP. The previous studies suggested that miR-130a-3p could play a suppressive role in the development of HCC [18,19]. In this study, we found that miR-130a-3p expression was decreased in hypoxia-treated HCC cells and miR-130a-3p overexpression suppressed glycolysis in HCC cells. Moreover, miR-130a-3p deficiency reversed the suppressive role of HOTAIR knockdown in glycolysis, uncovering that HOTAIR regulated glycolysis under hypoxia in HCC by sponging miR-130a-3p. The function of miRNA is realized by regulating its target mRNA. This study first validated HIF1A as a functional target of miR-130a-3p in HCC. Hypoxia could induce activation of HIF1A and then regulate reprogramming of glucose metabolism in cancers [32,33]. Additionally, previous report demonstrated that HIF1A facilitated HCC glycolysis under hypoxia [13]. Here we found that HIF1A restoration weakened miR-130a-3p-mediated glycolysis inhibition, suggesting that miR-130a3p blocked glycolysis by targeting HIF1A. Moreover, former study showed that HOTAIR could increase HIF1A level by acting as a ceRNA for miR-217 in renal cell carcinoma [34]. In the present study, we confirmed that HOTAIR could derepress HIF1A expression by competitively sponging miR-130a-3p in HCC cells. Furthermore, we confirmed that knockdown of HOTAIR could decrease HCC cell viability under hypoxic condition by regulating miR-130a-3p and HIF1A (Supplementary Fig. 4A-D), indicating that the network of HOTAIR/miR-130a-3p/ HIF1A could mediate HCC progression by regulating metabolic reprogramming under hypoxic condition. Besides, the transcriptional regulation mediated by HOTAIR might be also responsible for understanding the potential mechanism which should be explored in future work [35,36]. HK2, a key enzyme contributing to glycolysis is a downstream of HIF1A. HIF1A is a key factor in response to hypoxia, which could bind to hypoxia response element in transcription-regulatory regions (promoters or enhancers) of downstream glycolytic genes, including HK2 [37]. In this study, we confirmed the ceRNA
3.7. HOTAIR regulates HIF1A expression by competitively sponging miR130a-3p To implicate the competing endogenous RNA (ceRNA) network of HOTAIR, the association among HOTAIR, miR-130a-3p and HIF1A was analyzed. In HIF1A-WT-transfected 293 T cells, overexpression of miR130a-3p markedly decreased the luciferase activity, while this event was alleviated by introduction of HOTAIR (Fig. 7A). Moreover, overexpression of HOTAIR significantly enhanced the protein level of HIF1A in HepG2 cells, which was counteracted by miR-130a-3p overexpression (Fig. 7B). Meanwhile, the expression of HIF1A protein was greatly inhibited by interference of HOTAIR, which was mitigated by deficiency of miR-130a-3p (Fig. 7C). 4. Discussion HCC cells have a high dependence of glycolysis especially under hypoxia condition [23]. HOTAIR is a lncRNA associated with hypoxia stress by interacting with HIF proteins [24,25]. Furthermore, HOTAIR was highly expressed and promoted HCC progression [9–11]. In this study, we were the first to demonstrate that HOTAIR promoted glycolysis under hypoxia in HCC and elucidate the regulatory mechanism of HOTAIR/miR-130a-3p/HIF1A (Supplementary Fig. 3). This study displayed that HOTAIR was highly expressed in HCC and associated with hypoxia stress, indicating that HOTAIR might contribute to HCC malignancy. Glycolysis, characterized by glucose consumption and lactate production, maintains cancer cell survival under hypoxia. HK2 is a pivotal player in glycolysis and utilized as a metabolic target for treatment of HCC [26,27]. The loss-of-function experiments showed that knockdown of HOTAIR inhibited glycolysis under hypoxia, revealed by reduction of glucose consumption, lactate production and HK2 protein abundance in HCC cells, which is also in agreement with former works [28,29]. However, the mechanism underlying HOTAIR regulating glycolysis remains elusive. Previous studies have indicated that HOTAIR could serve as a 6
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Fig. 6. HIF1A attenuates miR-130a-3p-mediated glycolysis inhibition in HCC cells treated by hypoxia. HepG2 and Huh7 cells were transfected with miR-NC, miR-130a-3p, miR-130a-3p and vector or HIF1A and then exposed to hypoxia. The glucose consumption (A and B), lactate production (C and D) and HK2 protein level (E and F) were detected in the treated HepG2 and Huh7 cells. **P < 0.01, ***P < 0.001.
Declaration of Competing Interest
network of HOTAIR/miR-130a-3p/HIF1A, indicating the glycolysis mediated by HOTAIR is regulated in an HIF1A-dependent manner. We hypothesized that might be realized by binding the sites in promoter regions of HK2, which is needed to be further confirmed in future. In conclusion, here we indicated a novel mechanism underlying the glycolysis progression under hypoxia in HCC. HOTAIR expression was increased in HCC cells after hypoxia treatment. Knockdown of HOTAIR inhibited glycolysis in HCC cells stimulated with hypoxia, possibly by acting as a ceRNA network of HOTAIR/miR-130a-3p/HIF1A. This study may provide a new therapeutic target for treatment of HCC.
The authors declare that there are no conflicts of interest.
Acknowledgement This work was supported by National Natural Science Foundation of China (No. 931671440), the Science and Technology Self-help Project of Department of Henan Science and Technology (No.172102310099), and the National Natural Fund Joint Fund (No. U1404309).
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Fig. 7. HIF1A expression is regulated by HOTAIR and miR-130a-3p. (A) Luciferase activity was measured in 293 T cells transfected with HIF1A-WT, HIF1A-WT + miR130a-3p, HIF1A-WT + miR-130a-3p + vector and HIF1A-WT + miR-130a-3p+HOTAIR. (B) The protein level of HIF1A was measured in HepG2 cells transfected with vector, HOTAIR, HOTAIR and miR-NC or miR-130a-3p. (C) The expression of HIF1A protein was detected in Huh7 cells transfected with si-NC, si-HOTAIR, si-HOTAIR and anti-miR-NC or anti-miR-130a3p. **P < 0.01, ***P < 0.001.
Appendix A. Supplementary data
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