Hsa_circ_0001806 promotes glycolysis and cell progression in hepatocellular carcinoma through miR‐125b/HK2

Abstract Objective Hepatocellular carcinoma (HCC) is one of the most common malignant tumours and a leading cause of cancer death. Circular RNA (circRNA) has been demonstrated to play an important role in regulating tumour development. The current study aims to explore the specific role of hsa_circ_0001806 during HCC progression. Methods The expression of hsa_circ_0001806 in HCC tissues and cells was measured through qRT‐PCR. Cell proliferation, apoptosis and migration were measured using CCK‐8 and Annexin V/PI staining kits, and Transwell assay. Bioinformatics prediction and dual‐luciferase reporter assay were adopted to explore the mechanism underlying the cell function of hsa_circ_0001806 in HCC cells. In addition, glycolysis was assessed by measuring the glucose uptake, lactate production and ATP level using a glucose assay kit, fluorometric lactate assay kit and ATP detection assay kit. Results Hsa_circ_0001806 was up‐regulated in HCC tissues and cells and positively associated with the advanced TNM stage, metastasis and poor overall survival. The overexpression of hsa_circ_0001806 promoted HCC cell proliferation, migration and glycolysis and inhibited cell apoptosis, while the silence of hsa_circ_0001806 showed an opposite effect. Furthermore, hsa_circ_0001806 acted as a sponge of miR‐125b to up‐regulate hexokinase II (HK2) expression. In addition, the inhibition of miR‐125b and HK2 overexpression partly reversed the inhibitory effect of hsa_circ_0001806 silencing on HCC cell proliferation, migration and glycolysis. Conclusion The inhibition of hsa_circ_0001806 suppressed HCC cell proliferation, migration and glycolysis through mediating miR‐125b/HK2 axis.


| INTRODUC TI ON
Hepatocellular carcinoma (HCC) is one of the most common malignant primary tumours occurred in liver and the third leading cause of cancer death. There were about 782,000 cases diagnosed and 746,000 deaths in 2012. 1 Most patients were diagnosed as HCC at an advanced stage with no obvious symptoms found in the early stages. 2 According to statistics, the 5 years survival rates of patients with early stage of HCC reached 40% ~70% following radical surgery, while the survival time for patients with advanced liver cancer was only 6-18 months. 3 In addition, a high recurrence rate was also presented in HCC patients. 4 Chronic hepatitis B and C, cirrhosis, non-alcoholic fatty liver disease, excessive alcohol intake and diabetes were reported to be crucial factors leading to HCC. [5][6][7] Besides, gene alteration was also involved in the tumorigenesis of HCC. 8 Hence, it is necessary to determine the underlying mechanism by which HCC tumorigenesis is regulated and identify new biomarkers and targets for HCC diagnosis and therapy.
First, circular RNAs (circRNAs) are discovered in 1976 and found to be more stable than long non-coding RNA. 9 A covalently closed continuous loop structure without 5′-caps or 3′-tails is formed within circRNAs by back-splicing at exon or intron regions. 10 Increasing evidence indicated that circRNAs played an important role in tumour evolution and progression. 11,12 The biological regulatory mechanisms of circRNAs include working as sponges of miRNAs, interacting with RNA-binding proteins (RBPs), serving as transcriptional and translational regulators, affecting the splicing of pre-mRNAs and being translated into peptides. [12][13][14] Based on that, large amount of circRNAs had been regarded as essential potential biomarkers of various tumour diseases. 15 In addition, growing evidence indicated that numerous circRNAs were dysregulated in HCC tissues and associated with HCC progression 13,16,17 and could act as competing endogenous RNAs (ceR-NAs) to participate tumour progression. 17 For example, circRNA-104718 functioned as a ceRNA of miR-218 to promote HCC progression by regulating thioredoxin domain-containing protein 5. 12 Hsa_circ_0001806 derives from centrosome and spindle pole associated protein 1 (CSPP1), located on chromosome 8 (68018139 ~ 68028357). It was also found that hsa_ circ_0001806 was dramatically up-regulated in colorectal cancer tissues and positively associated with tumour node metastasis (TNM) stage, invasion and lymphatic metastasis. 18 Meanwhile, hsa_circ_0001806 enhanced the stemness of colorectal cancer cells by activating the miR-193a-5p/ COL1A1 axis. 18 Recently, evidence discovered an increased expression of hsa_circ_0001806 in HCC tissues and cells. 16 However, the specific function and mechanism of hsa_circ_0001806 in HCC is still unclear.
In this study, we verified the alteration of hsa_circ_0001806 in HCC tissues and cells, and its relevance with the survival outcomes of HCC patients. Additionally, the role of hsa_circ_0001806 in HCC cell viability, apoptosis, migration and glycolysis was explored. A potential mechanism in which hsa_circ_0001806 exerted on HCC cells was investigated. Overall, our study indicated that hsa_circ_0001806 may act as a potential target for HCC prognosis and therapy.

| Cell culture
Human normal liver cells, LO2 and HCC cells lines (HepG2, Hep3B and Huh7) were acquired from the Institute of Biochemistry and Cell Biology at the Chinese Academy of Sciences. Cells were cultured in DMEM (Hyclone, Salt Lake City, UT) supplemented with 10% foetal bovine serum (Invitrogen) and 1% penicillin/streptomycin (Gibco). Cells were incubated in a cell culture incubator at 37°C in 5% CO 2 atmosphere.

| Plasmid construction and stable transfection
Overexpression vector (ov) of hsa_circ_0001806 and HK2, small interfering RNA (siRNA) of hsa_circ_0001806, miR-125b mimic, miR-125b inhibitor and negative controls (NC) were synthesized and constructed by GenePharma. Cell transfection was performed by using Lipofectamine 3000 (Invitrogen) according to the manufacturer's protocol. Transfection efficiency was confirmed by qRT-PCR and Western blot at 48 h after transfection.

| RNA preparation and qRT-PCR
Total RNA was isolated using TRIzol reagent (Invitrogen). The obtained RNA samples were quantified on a Nanodrop platform (Thermo Scientific Fisher) and purified using miRNEasy spin columns (QIAGEN).
To detect hsa_circ_0001806 expression, the RNA extract was incubated with RNase R (Epicenter, Madison) before RNA purification. Then, RNA was reverse-transcribed using HiScript II Q RT SuperMix for qPCR K E Y W O R D S glycolysis, hepatocellular carcinoma, HK2, hsa_circ_0001806, miR-125b (Vazyme). qRT-PCR was performed using the AceQ SYBR Green PCR Master Mix (Vazyme, Nanjing, China) on the ABI 7900 HT sequence detection system (Applied Biosystems). The circRNA and mRNA levels were normalized to U6 and GAPDH levels. Triplicates of each experiment were performed. The primer sequences used in this study are

| Dual-luciferase reporter assay
The wild type (WT) and mutant type (MUT) of hsa_circ_0001806 luciferase reporter plasmids were constructed and co-transfected with miRNA mimics into Hep3B cells using Lipofectamine 3000 (Thermo Fisher Scientific). The NC mimic was adopted as a negative

control. After being cultured for 48 h, a Dual-Luciferase Reporter
System was used to measure the Firefly and Renilla luciferase activities following the manufacturer's instructions (Promega).

| Cell proliferation assay
Hep3B cells (3000 cells/well) were planted into 96-microwell plates and cultured overnight. After being transfected with overexpression vectors or siRNAs for 48 h, 10 μL of CCK-8 assay reagent (Dojindo Corp) was added and incubated for another 2 h. The absorbance value was measured at 450 nm in a microplate reader (Thermo Fisher Scientific, Inc.). This experiment was repeated in triplicate.

| Cell apoptosis assay
Transfected Hep3B cells (1 × 10 5 cells/ml) were planted into six-well plates. After being cultured for 48 h, cells were harvested using a cell scraper and collected into an EP tube. Following washed by PBS twice, cells were double-stained with Annexin V and propidium iodide (PI) (BD Biosciences Pharmingen) and analysed on an EPICS XL-MCL flow cytometer (Beckman Coulter, Brea). Each experiment was independently repeated three times.

| Cell migration test
Cell migration was evaluated through the Transwell assay. The cultured cells (5 × 10 4 cells) in 300 ml serum free medium were planted onto the upper chamber (8 μm pore size, Corning). The lower chamber was supplemented with 600 ml medium and 10% FBS. After being cultured for 48 h, cells migrated to the lower chamber were fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet. The representative images were obtained under an upright microscope, and the migrated cells were counted in 5 randomly selected fields per chamber. This experiment was repeated in triplicate.

| Western blot analysis
Cells were harvested and lysed by the treatment with RAPI lysis buffer at 4°C for 30 min. After being centrifuged at 4°C with a speed of 25,764 g for 10 min, the supernatant was obtained and the protein concentration was analysed

| Glucose uptake, lactate production, and ATP levels
The glucose uptake, lactate production and ATP levels were quantified by using the glucose assay kit (Sigma-Aldrich), the fluorometric lactate assay kit (Abcam) and Luminescent ATP Detection Assay (Abcam) following the manufacturers' instructions.

| Statistical analysis
Statistical analyses were carried out using GraphPad Prism (GraphPad). Student's t test was used to compare the statistical significance of differences between two groups. Survival curves for HCC patients were analysed using the Kaplan-Meier method and logrank test. Data were presented as mean ± standard deviation (SD). P < 0.05 indicated that the difference was statistically significant.

| Hsa_circ_0001806 was up-regulated in HCC tissues and cells and correlated with the poor prognosis of patients
As shown in Figure 1A (Table 1). Meanwhile, prognosis analysis calculation suggested that the high level of hsa_circ_0001806 expression was positively associated with lower survival rate (p = 0.0291) ( Figure 1C). In addition, we found that hsa_circ_0001806 was notably up-regulated in HCC cells (HepG2, Hep3B and Huh7), compared with LO2 cells ( Figure 1D).

| Effect of hsa_circ_0001806 on cell viability, apoptosis, migration, and glycolysis in Hep3B cells
To detect the functional effects of hsa_circ_0001806 on the cell processes of HCC, hsa_circ_0001806 expression was significantly depressed by siRNA (si-hsa_circ_0001806) and up-regulated by  The significance level (p) was determined by chi-square, *p < 0.05.

TA B L E 1 Correlation between hsa_ circ_0001806 levels in HCC patients and their clinicopathologic characteristics
that hsa_circ_0001806 overexpression promoted cell viability of Hep3B cells, while the knockdown of hsa_circ_0001806 suppressed the cell viability of Hep3B cells ( Figure 2B). Subsequently, we found that hsa_circ_0001806 inhibition significantly induced cell apoptosis in Hep3B cells ( Figure 2C). In addition, the result of the Transwell assay showed that the overexpression of hsa_ circ_0001806 facilitated Hep3B cell migration, while the silencing of hsa_circ_0001806 suppressed cell migration ( Figure 2D,E).
What is more, glucose uptake, lactate production and ATP level were significantly elevated by hsa_circ_0001806 overexpression and inhibited by hsa_circ_0001806 inhibition in Hep3B cells ( Figure 2F-H).

Mimic and inhibitor of miR-125b were constructed and transfected into
Hep3B cells to promote or suppress miR-125b expression ( Figure 3B).
Furthermore, the dual-luciferase reporter assay verified the direct binding between miR-125b and hsa_circ_0001806 ( Figure 3C,D). inhibition in Hep3B cells ( Figure 3E). Previous studies have reported that HK2 was a key target of miR-125b and was involved in tumour progression. 19,20 Our study verified that HK2 expression was increased in HCC cells, compared with LO2 cells (Figure 3F,G). Also, HK2 expression was promoted by hsa_circ_0001806 overexpression and the transfection of miR-125b inhibitor and suppressed by hsa_circ_0001806 inhibition and miR-125b overexpression in Hep3B cells ( Figure 3H-K).

| DISCUSS ION
In this study, we found that hsa_circ_0001806 was highly expressed in HCC tissues and cells, and the increased expression of hsa_circ_0001806 was related to advanced TNM stage, lymph gland and organ metastasis, as well as poor survival, of HCC patients. This discovery was consistent with previous reports that hsa_circ_0001806 was up-regulated in colorectal carcinoma and HCC tissues. 16,18 This suggested that hsa_circ_0001806 may serve as a molecular marker for HCC. In addition, our study indicated that Another study presented that circ_0004913 was dysregulated during HCC progression and the alteration of circ_0004913 could be used as an evaluation factor for HCC severity and prognosis. 22 29 Our study identified that HK2 served as a direct target of miR-125b in HCC cells and participated in hsa_ circ_0001806-mediated proliferation, apoptosis and migration in HCC cells.
Glycolysis provides the major energy for HCC tumour progression and is involved in cell proliferation, migration and invasion. 30,31 It is known that cancer cells exhibit aberrant metabolism and are in preference to glycolysis rather than mitochondrial oxidative phosphorylation to produce glycolytic intermediates and glucosedependent ATP for macromolecular biosynthesis, even under an oxygen abundant environment. 31 HK2 is the first and initial ratelimiting enzyme in the glycolytic pathway 27 and catalyses glucose transforming to glucose-6-phosphate. 32 The inhibition of HK2 suppressed glycolysis in HCC tumours. In addition, miR-125b targeted to HK2 and suppressed glycolysis in laryngeal squamous cell carcinoma. 20 The overexpression of miR-125b promoted the dysregulation of cellular glucose metabolism by directly targeting to HK2 in human retinal pigment epithelium cells. 33 A previous study found that astragalin suppressed HK2 through up-regulating miR-125b to inhibit HCC cell proliferation in vitro and in vivo. 34 In this study, oncogenic hsa_circ_0001806 promoted glycolysis in HCC cells, while the overexpression of HK2 and inhibition of miR-125b suppressed the inhibitory effect of hsa_circ_0001806 silencing on the glycolysis process in HCC cells.

| CON CLUS ION
In summary, this study investigated the role and mechanism of hsa_circ_0001806 in HCC tumours. The results verified that hsa_ circ_0001806 was significantly up-regulated in HCC tissues and cells and associated with an advanced TNM stage, lymph gland and organ metastasis and the poor prognosis of HCC patients. Moreover, hsa_circ_0001806 promoted cellular malignant behaviour and glycolysis by regulating the miR-125b/HK2 axis, which may provide a potential therapeutic target for HCC.

CO N FLI C T S O F I NTE R E S T
The authors declare no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are available from the corresponding author upon request.