Circular RNA Circrna_0072387 Promotes Hepatoblastoma Progression by Regulating Hsa-Mir-490/Histone Deacetylase 2 Axis

Research Article

Thromb Haemost Res. 2022; 6(1): 1074.

Circular RNA Circrna_0072387 Promotes Hepatoblastoma Progression by Regulating Hsa-Mir-490/Histone Deacetylase 2 Axis

Gou H1#, Yu R2#, Luo J1, Liu L3, Sun X4 and Xu C1*

1Department of Clinical Laboratory Medicine, Shanghai Tenth People’s Hospital of Tongji University, Shanghai, China

2The Second School Affiliated to Fudan University, Shanghai, China

3Department of Laboratory Medicine, Shanghai Children’s Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai, China

4Shanghai World Foreign Language Middle School, Shanghai, China

#Contributed equally to this work

*Corresponding author: Chang Xu, Department of Clinical Laboratory Medicine, Shanghai Tenth People’s Hospital of Tongji University, 1238, Gonghexin Road, Jingan District, Shanghai, China; Tel: +8613122189907; Email: [email protected]

Received: February 21, 2022; Accepted: March 18, 2022; Published: March 25, 2022


Circular RNAs have been reported to be associated with the occurrence and progress of various cancers. However, its biological roles in hepatoblastoma (HB) remain undefined. This study intended to explain a possible mechanism of hsa_circ_0072387 (circ72387) in hepatoblastoma progression. The expression of circ72387, hsa-miR-490 (miR490) and histone deacetylase 2 (HDAC2) in HB tissues and cell lines (HepG2/Huh6) were determined by quantitative realtime polymerase chain reaction (qPCR) or Western blot. Cell Counting Kit-8 (CCK-8) and colony formation assay were used to detect the proliferation of HB cell lines. The interaction between miR490 with circ72387 was predicted by CircInteractome and was verified by Dual-Luciferase reporter assay while the interaction between miR490 with HDAC2 was predicted by Targetscan and was verified by western blotting assay. Results showed that circ72387 was upregulated in both HB tissues and cells, and miR490 was downregulated in HB cells. Furthermore, the silence of circ72387 suppressed the proliferation of hepatoblastoma cells. Circ72387 promoted the proliferation of HB cells through competitively binding to HDAC2 against miR490 acting as a Sponge of miR490 in HB. Circ72387 played an essential role in HB development through the circ72387-miR490-HDAC2 regulatory axis, which may pave the way for potential diagnostic markers and therapeutic targets research in HB.

Keywords: Circular RNAs; Hepatoblastoma; Hsa_circ_0072387; HsamiR-490; HDAC2


Hepatoblastoma (HB) is a kind of malignant liver tumor originated from hepatic precursor cells that almost occurred in infants and toddlers [1]. In Western countries, there are nearly 1.0- 1.5 cases in a million children below 15 year-old every year [2]. So far, the mainstream HB treatment is neoadjuvant chemotherapy combined with hepatectomy. The overall survival (OS) rate was 75% if patients could be treated with immediate hepatectomy in an early stage [3]. However, due to its nonspecific symptoms and incipient development, some patients may have already progressed to macrovascular tumor invasion and extrahepatic tumor extension when newly diagnosed [4]. Metastasis was observed in nearly 20% of newly diagnosed HB patients who lost the opportunity of surgery and might result in an unfavorable prognosis [5]. Therefore, more reliable diagnostic biomarkers are required to predict HB patients’ prognosis.

Circular RNAs (circRNAs) are RNAs with continuous covalently closed loops formed by alternative splicing event [6]. Without 5' caps and 3' tails at the two terminals, circRNAs are more stable and difficult to be degraded by RNA exonuclease than linear RNAs because of their closed loop structures [7,8]. It has been proved that circRNAs might play a role as competitive endogenous RNAs (ceRNAs) and regulator of MicroRNA (miRNA) activity by competing for miRNA-binding sites [9]. MiRNAs are short RNA molecules which can bind to target mRNAs, leading to translational suppression and gene silencing [10]. Therefore, circRNAs combined with miRNA can regulate the expression of target gene and build a circRNAs-miRNA-mRNA axis. Many research revealed that CircRNAs were closely relevant to various kinds of disease, including cancers [11,12]. Therefore, figuring out the association between circRNAs and HB may provide a brand-new way for the hepatoblastoma diagnosis and therapy.

However, the roles of circRNAs in HB have rarely been reported, and the functions and regulatory mechanism of hsa_circ_0072387 (circ72387) remain unknown. In 2018, Liu et al. identified 869 differentially expressed circRNAs in hepatoblastoma samples by circular RNA microarray. They confirmed that circRNAs plays a role in the pathogenesis of hepatoblastoma by bioinformatics analysis [13]. A recent study also pointed out that CDR1as, a highly expressed circRNA in HB cell lines, might promote HB proliferation and stemness as an oncogene [14]. In conclusion, exploring the molecular mechanisms of HB occurrence and progress from the perspective of circRNAs may provide a new sight to the precise treatment in HB.

Our research mainly focused on a down-stream target miRNA, hsa-miR-490 (miR490), which down-regulated in cancers revealed by many other studies. The targets of miR-490 includes a range of cancerrelated genes, which involve in proliferation, migration, and invasion, apoptotic cell death, angiogenesis, and so on [15]. More importantly, down-regulation of miR490 is closely related to the development of cancers including Hepatocellular carcinoma (HCC) [16-26]. It has been proved that miR490 can suppress tumor by repressing the translation of a range of cancer-related genes like SOX2 [16], HDAC2 [18], POU3F2 [19], E2F2 [20], PPM1F [22], AURKA [25], BUB1 [27], CDK1 [28], TNKS2 [29]. These evidences strongly support that miR490 is a potential biomarker in the diagnosis and prognosis of HB.

In this research, we analyzed the function of highly-expressed hsa_circ_0072387 in HB promotion and its mechanism as ceRNAs. hsa_circ_0072387 regulates down-stream mRNA of Histone deacetylase 2 (HDAC2) by competitively binding with miR490, which finally promotes proliferation of HB cells. We clarified that hsa_circ_0072387 might play an essential role in HB development by circ72387-miR490-HDAC2 regulatory axis, which might become a novel diagnostic marker and potential therapeutic target in HB.

Materials and Methods

Clinical specimens

HB Patients from the Department of Hepatobiliary Surgery of Shanghai Children’s Medical Center (Shanghai, China) were included in this research excluding patients who had received chemotherapy, radiotherapy, or targeted therapy. After hepatectomy surgery, 22 pairs of HB tissue and corresponding para-cancerous liver tissues were resected and immediately stored in liquid nitrogen, which were later kept at -80°C until use. Approval of investigation by the Ethics Committee of Shanghai Children’s Medical Center was acquired. Each patient was informed with this investigation, and they consented to this research.

HB cell lines

HB cell lines HepG2 and Huh6 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and were cultured in Minimum Eagle’s Medium (MEM) and Dulbecco’s Modified Eagle’s Medium (DMEM), respectively, supplemented with high glucose, 10% fetal bovine serum (FBS), 100U/ml streptomycin, and 100U/ml penicillin in 5% CO2/95%O2 at 37°C.

Oligonucleotide transfection

SiRNAs for hsa_circ_0072387, siRNA-NC, miR-490 mimics, miR-NC mimics, miR-490 inhibitor, and miR-NC inhibitor were chemically synthesized by GenePharma. The sequences of which mentioned above are provided in the supplemental material (Table S1). HepG2 and Huh6 cells were transfected with the oligonucleotides using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.

RNA extraction and real time-quantitative reversetranscription polymerase chain reaction (qRT-PCR)

To extract total RNA from HB specimens and cell lines, Trizol reagent (Ambion, Texas, America) was used to according to the manual provided by the manufacturer. cDNA was synthesized by 1μg total RNA with the PrimeScriptTM RT Reagent Kit No. RR037A (TaKaRa, Dalian, Liaoning, China). RT-qPCR was performed with TaKaRa TB GreenTM Premix Ex TaqTM II No. RR820A (TaKaRa, Dalian, Liaoning, China) on an ABI7900 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). All data were analyzed by the 2-ΔΔCT method. Primers used were provided in Table S1. Amplification curve and melting curve were confirmed after finishing all the cycles.

Western blotting assay

Cells and tissues were lysed by RIPA lysis buffer (Beyotime, Shanghai, China) for extracting the total protein. A sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was applied to the separation of protein. The separated protein was then transferred onto nitrocellulose membranes (GE Healthcare, Natick, MA, USA). After blocked with 5% non-fat milk (Sangon Biotech), the membranes were incubated with primary antibody against histone deacetylase 2 (HDAC2) (1:2000, ab32117, Abcam, Cambridge, UK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1000, 97166, Abcam) for 12h at 4°C. After washed with PBS for 3 times, the membranes were incubated with a secondary antibody (1:4000, ab205718, Abcam).Finally, the membranes were scanned by an Odyssey instrument (LI-COR Biosciences, Lincoln, NE, USA) to detect the protein signals. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control.

Cell proliferation and colony formation assays

For colony formation assays, HB cells with a cell density of 500 cells per cells were inoculated into 12-well plates and were incubated for about 7-9 days in fresh medium. Then, the cells were washed with PBS. A 95% ethanol was used to fix the cells for the preparation of staining. Finally, the fixed cells were stained with crystal violet with a concentration of 0.1%, and the amount of cell colonies were analyzed by Image J, which represented the colony-forming ability.

For cell counting kit-8 (CCK8) assays, 1000 HB cells transfected in advance were inoculated in triplicate into 96-well plates. At the indicated time points, the medium of each well was replaced with 100μl fresh medium which was supplemented with 10μl CCK8 reagent (Beyotime, Shanghai, China), and the cells were incubated for 2-4 h at 37°C. Finally, the absorbance was determined by a multiplate reader (Bio Tek, Vermont, USA) at a wavelength of 450nm.

Measurement of Caspase3 activity

100μl Caspase-Glo® 3/7 reagent (Promega, G8090) was added into 96-well plates with 100μl blank control, negative control and HB cell lines. 96-well plates were vibrated at 300-500 rpm for 30s followed by incubation at room temperature (RT) for 3h. Fluorescence intensity was determined by Luminometer.

Dual-Luciferase reporter assay

In order to explore the interaction between circ72387 and miR490, a psiCHECKTM-2-circ72387 vector, a dual luciferase plasmid psiCHECKTM-2 Vector (Yeasen, Shanghai, China), was constructed and transfected into 293T cells. The whole sequence of circ72387 with miR490 binding site was planted downstream of the Renilla luciferase gene. Before transfection, 293T cells were seeded in 24- well plates and incubated for 24h. 0.8μg of psiCHECKTM-2-circ72387 vector or psiCHECKTM-2 vector was transfected into cells combined with 20pmol of miRNA mimics or negative control mimics using Lipofectamine 2000 (Invitrogen). 48h after transfection, the signal intensity of firefly and Renilla were determined by Luminometer.

Statistical analysis

At least three independent experiments were carried for all data which were shown as the mean ± standard deviation (SD). SPSS 23.0 and GraphPad Prism 8.0 were utilized to perform statistical data analysis. The differences between different groups were determined by student’s t-test or a one-way analysis of variance (ANOVA) analyze. Statistical significance was considered when P <0.05.


Circ72387 was upregulated in HB tissues and cells

Based on the previous HB tissue sequencing data, we focused on circ72387 which up-regulated in HB. To further confirm the level of circ72387 in HB tissues and cells, qRT-PCR was performed. The result indicated that circ72387 was remarkably upregulated in HB tissues (n=22) compared with normal tissues (n=22) (Figure 1a). Equally, circ72387 level was significantly increased in HB cell lines (HepG2 and Huh6) compared to normal human hepatocyte (7701) (Figure 1b). We analyzed circ72387 by an online database CircInteractome ( that circ72387 with a genomic length of 3111bp was generated from exon 5-8 of HMGCS1 gene which located on chromosome 5 (Figure 1c). To sum up, the observation supplies a basis for circ72387 to be a circular RNA and a potential prognosis marker.