Circular RNA hsa_circ_0061395 accelerates hepatocellular carcinoma progression via regulation of the miR-877-5p/PIK3R3 axis

Circular RNA hsa_circ_0061395 (circ_0061395) has been reported to accelerate the advancement of hepatocellular carcinoma (HCC). However, the regulatory mechanism by which circ_0061395 modulates the progression of HCC is unclear. The morphology and size of exosomes were analyzed by transmission electron microscope (TEM) and nanoparticle-tracking analysis (NTA). Protein levels were detected by western blotting. Expression levels of circ_0061395, microRNA (miR)-877-5p, and phosphoinositide-3-kinase regulatory subunit 3 (PIK3R3) mRNA were assessed by quantitative real time polymerase chain reaction (qRT-PCR). The proliferation, invasion, migration, cell cycle progression, and apoptosis were analyzed by cell counting kit-8 (CCK-8), plate clone, transwell, or flow cytometry assays. The targeting relationship between circ_0061395 or PIK3R3 and miR-877-5p was verified using the dual-luciferase reporter and/or RNA immunoprecipitation (RIP) assays. Xenograft assay was performed to confirm the biological function of circ_0061395 in HCC. Circ_0061395 was upregulated in HCC tissues, serum, cells, and serum-derived exosomes. Circ_0061395 silencing decreased tumor growth in vivo, and induced cell cycle arrest, apoptosis, repressed proliferation, invasion, and migration of HCC cells in vitro. MiR-877-5p was downregulated while PIK3R3 was upregulated in HCC. Circ_0061395 regulated PIK3R3 expression via competitively binding to miR-877-5p. MiR-877-5p inhibitor overturned circ_0061395 knockdown-mediated influence on malignant behaviors of HCC cells. PIK3R3 overexpression reversed the suppressive influence of miR-877-5p mimic on malignant behaviors of HCC cells. Circ_0061395 facilitated HCC progression via regulating the miR-877-5p/PIK3R3 axis, providing a new perspective on the advancement of HCC.


Introduction
Liver cancer ranks fourth among cancer-related death causes globally [1]. Hepatocellular carcinoma (HCC) is one of the most common pathological types of primary liver cancer, accounting for about 85% of all liver cancer cases [2]. Although new breakthroughs have been made in the treatment of HCC, the 5-year survival rate is still low [3,4]. Hence, studying the underlying molecular mechanisms of HCC advancement can provide potential strategies for the treatment of HCC.
Circular RNAs (CircRNAs) are a type of endogenous RNA molecules with the characteristics of covalently closed loops [5]. Unlike liner transcripts, circRNAs are more resistant to RNase R due to their lack of 5′ cap and 3′ poly(A) tail [6]. Some circRNAs can act as competing endogenous RNAs (ceRNAs) and regulate gene expression by interacting with microRNAs (miRs) [7]. Moreover, circRNAs have been revealed to be implicated in the advancement of diverse tumors [8]. For instance, pancreatic cancer cells-secreted exosomal circRNA_IARS accelerated tumor metastasis via regulation of the permeability of the endothelial cell monolayer [9]. CircRNA hsa_circ_0061395 (circ_0061395), located at chromosome 21: 30698379-3070201, is formed by head-to-tail splicing of exons 3 and 4 of the BTB domain and CNC homolog 1 (BACH1) gene (http://www.circb ank.cn/searc h.html?selec tValu e=hsa_circ_00613 95). It was reported that circ_0061395 facilitated HCC progression via inhibiting p27 expression [10]. However, the molecular mechanism that circ_0061395 promotes HCC progression has not been fully explained.
In the present study, circ_0061395 was upregulated in HCC tissues, serum, cells, and serum-derived exosomes.
Mechanically, circ_0061395 facilitated HCC progression via elevating PIK3R3 expression via competitively binding to miR-877-5p, which provided a new insight into for the advancement of HCC.

Human blood and tissue samples
Human blood and/or tissue samples (HCC tissues and adjacent non-tumor tissues) were collected from 30 HCC patients and 30 healthy controls that were administrated at the Second Hospital of Jilin University. These blood samples were centrifuged at 1200×g for 20 min to obtain serum samples. The research was approved by the Ethics Committee of the Second Hospital of Jilin University. All recruited participants (HCC patients and healthy controls) signed written informed consents.

Exosome isolation
Exosomes were isolated from the serum of HCC patients and healthy controls using the ExoQuick exosome precipitation solution (System Biosciences, Mountain View, CA, USA). In short, the mixture of serum and ExoQuick exosome precipitation solution was stored at 4 °C for 24 h. Thereafter, the sample was centrifuged at 1500×g for 30 min to obtain exosome pellets. Then, these exosome pellets were suspended in phosphate-buffered saline for subsequent analysis. For nanoparticle-tracking analysis (NTA), the number and size of exosomes were tracked using NanoSight NS 300 system (NanoSight, Malvern, UK) and particle tracking software (NanoSight).

Transmission electron microscopy (TEM)
The exosome suspension was placed on a special copper mesh of the H-7650 electron microscope (Hitachi, Tokyo, Japan), followed by negative staining with 2% phosphotungstic acid (20 μL) for 10 min. The morphology of exosomes was observed by the H-7650 electron microscope (Hitachi) at 100 kV.

Quantitative real-time polymerase chain reaction (qRT-PCR)
Nuclear and cytoplasmic RNA of HCC cells were extracted with the PARIS kit (Life Technologies). RNAiso Plus (TaKaRa, Tokyo, Japan) or miRNeasy Serum/ Plasma Kit (Qiagen, Valencia, CA, USA) was utilized to isolate total RNA from sera, tissue samples, and cells.

Cell viability assay
After a specific transfection, HCC cells (1 × 10 3 cells/ well) were seeded to a 96-well plate. After culturing for 48 h, the cell counting kit-8 (CCK-8) solution (10 μL, Dojindo, Kumamoto, Japan) was added to each well and incubated for 1 h. The absorbance at 450 nm was measured by the Microplate Reader (Bio-Rad).

Plate clone assay
Transfected HCC cells (1 × 10 2 cells/well) were seeded into 6-well plates. After culture for 2 weeks, the cells were washed with phosphate buffer solution and then fixed with paraformaldehyde (4%, Solarbio). Thereafter, the cells were stained with crystal violet (0.5%, Solarbio). Finally, the number of cell colonies (> 50 cells) were counted and photographed with an inverted microscope (Nikon, Tokyo, Japan).

Transwell assay
The invasion and migration abilities of HCC cells were analyzed by transwell chambers with an 8 μm pore membrane (Costar, Cambridge, MA, USA). For the invasion assay, the transwell chamber was pre-covered with Matrigel (Sigma). In short, the upper chamber was added with serum-free DMEM or RPMI-1640 medium (200 μL) containing HCC cells (1 × 10 5 cells), and the lower chamber was added with DMEM or RPMI-1640 medium (600 μL) containing FBS (10%). After 24 h incubation, the migrating and invading cells were fixed with paraformaldehyde (4%, Solarbio) and then stained with crystal violet (0.5%, Solarbio). The number of the migrating and invading cells was figured by the inverted microscope (Nikon) (magnification, 100 ×).

Flow cytometry assay
Transfected HCC cells were harvested and treated with trypsin, followed by washing with phosphate buffer solution. For the cell cycle assay, these cells were fixed with ethanol (70%) and then stained with propidium iodide (PI)/RNase solution (Sigma). For the cell apoptosis analysis, these cells were stained with the Annexin V-fluorescein isothiocyanate (FITC)/PI Apoptosis Detection Kit (BD Biosciences, San Jose, CA, USA). The cell distribution was analyzed by using a FACScan flow cytometry (BD Biosciences) with a FACS Diva Software (BD Biosciences). The data of the cell cycle experiment and the cell apoptosis experiment were presented in Additional file 1 and Additional file 2, respectively.

Xenograft assay
The protocols of xenograft assay were authorized by the Animal Ethics Committee of the Second Hospital of Jilin University. For xenograft assay, Huh7 cells (1 × 10 6 cells/200 μL) with sh-circ_0061395 or sh-NC were subcutaneously injected into the right flank of BALB/c nude mice (6 weeks old, Huafukang Bioscience, Beijing, China) (5 mice/group). Tumor volume was measured once a week with a digital caliper. Until day 28, these mice were sacrificed by cervical dislocation under isoflurane (5%) for subsequent analysis. Tumor volume was calculated in accordance with the following equation: Volume = (length × width 2 )/2. All mice were kept under specific pathogen-free conditions. For stable knockdown of circ_0061395, Huh7 cells were transduced with lentiviral particles carrying sh-circ_0061395.

Statistical analysis
The experiments in vitro were repeated at last 3 times and each experiment was carried out in triplicate. The original data of this study was displayed in Additional file 3. Statistical analysis was implemented with SPSS 20.0 software (SPSS, Chicago, IL, USA). Data were displayed as the mean ± standard deviation. These data followed a normal distribution using the Kolmogorov-Smirnov test. The homogeneity of variance between 2 groups was assessed by F-test. The difference between 2 groups was evaluated with paired or unpaired Student's t test. The differences among 3 or more groups were evaluated by one-way variance analysis (ANOVA) with Turkey's post hoc test. Correlation between circ_0061395 expression and clinicopathological parameters of HCC patients were evaluated by Chi-square test. P < 0.05 was considered statistically significant.

Circ_0061395 was upregulated in serum exosomes of HCC patients
At the outset, we extracted exosomes from the serum of HCC patients and healthy controls. TEM presented that the serum exosomes of HCC patients and healthy controls were round (Fig. 1a). NTA analysis displayed that the size of exosomes was about 100 nm (Fig. 1b). Western blotting showed the presence of CD63 and CD81 (exosome markers) present in exosomes derived from the serum of HCC patients and healthy controls (Fig. 1c). QRT-PCR exhibited that circ_0061395 expression was observably increased in exosomes derived from serum of HCC patients compared to exosomes derived from healthy control serum (Fig. 1d). These results indicated that high circ_0061395 expression in serum-derived exosomes might be involved in HCC development.

Circ_0061395 expression was apparently elevated in HCC
To investigate the biological function of circ_0061395 in HCC, we detected circ_0061395 expression in the serum of 30 HCC patients and 30 healthy controls. QRT-PCR presented that circ_0061395 was higher expression in Fig. 1 Circ_0061395 was upregulated in serum exosomes of HCC patients. a The morphology of exosomes from the serum of HCC patients and healthy controls were observed under TEM. b NTA analysis revealed the size of exosomes from the serum of HCC patients and healthy controls. c Western blotting was executed to detect the exosomal markers CD63 and CD81 in serum exosomes of HCC patients and healthy controls. d The expression of circ_0061395 in serum exosomes of HCC patients and healthy controls was analyzed by qRT-PCR. *P < 0.05 Fig. 2 Circ_0061395 was highly expressed in HCC. a-c QRT-PCR was performed to assess the expression of circ_0061395 in HCC serum, tissues, and cells. d The levels of circ_0061395, U6, and GAPDH in the cytoplasm and nucleus of SNU-387 and Huh7 cells were analyzed by qRT-PCR. *P < 0.05 the serum of HCC patients than that the healthy controls (Fig. 2a). Also, circ_0061395 expression was elevated in HCC tissues when compared to the adjacent non-tumor tissues (Fig. 2b). Furthermore, circ_0061395 expression was associated with the tumor size, TNM stages, and lymphatic metastasis of HCC patients (Table 1). We also observed that circ_0061395 expression was elevated in HCC cells (SNU-387 and Huh7) than that in THLE-2 cells (Fig. 2c). Nucleus and cytoplasm separation experiment and qRT-PCR exhibited that circ_0061395 was mainly located in the cytoplasm of SNU-387 and Huh7 cells (Fig. 2d). These data indicated that high circ_0061395 expression might be associated with HCC advancement.

Circ_0061395 knockdown constrained malignant behaviors of HCC cells
Subsequently, we investigated the biological role of circ_0061395 in HCC through the loss-of-function experiments. The knockdown efficiency of si-circ_0061395 in SNU-387 and Huh7 cells was exhibited in Fig. 3a. The transfection of si-circ_0061395 had no overt influence on the expression of BACH1 mRNA (Fig. 3b). CCK-8 and plate clone assays indicated that circ_0061395 silencing reduced the proliferation of SNU-387 and Huh7 cells (Fig. 3c, d). Transwell assay exhibited that circ_0061395 knockdown impeded the invasion and migration of SNU-387 and Huh7 cells (Fig. 3e). Flow cytometry assay displayed that decreased circ_0061395 expression induced cell cycle arrest and apoptosis in SNU-387 and Huh7 cells (Fig. 3f, g). Taken together, these data indicated that circ_0061395 knockdown could reduce the malignancy of HCC cells.

Circ_0061395 was identified as a sponge for miR-877-5p in HCC cells
To investigate the underlying molecular mechanism of circ_0061395 in HCC, we predicted miRs which could bind to circ_0061395 using the CircInteractome database. As exhibited in Fig. 4a, circ_0061395 might be a sponge of miR-877-5p. Dual-luciferase reporter assay exhibited that miR-877-5p mimic could reduce the luciferase activity of the luciferase plasmid with WT-circ_0061395 in SNU-387 and Huh7 cells, while the luciferase activity of the luciferase plasmid containing MUT-circ_0061395 did not change (Fig. 4b). RIP assay presented that circ_0061395 and miR-877-5p were enriched in the anti-Ago2 group compared to the Anti-IgG group (Fig. 4b).

Circ_0061395 regulated malignant behaviors of HCC cells by sponging miR-877-5p
In consideration of the above findings, we explored whether circ_0061395 facilitated malignant behaviors of HCC cells through adsorbing miR-877-5p. The results exhibited that miR-877-5p inhibitor overturned the repressive influence of circ_0061395 silencing on proliferation, invasion, and migration of SNU-387 and Huh7 cells (Fig. 5a-c). Flow cytometry assay exhibited that miR-877-5p silencing offset the promoting influence of circ_0061395 inhibition on cell cycle arrest and apoptosis in SNU-387 and Huh7 cells (Fig. 5d, e). In sum, these findings suggested that circ_0061395 regulated the malignancy of HCC cells via sponging miR-877-5p in HCC cells.

Circ_0061395 knockdown decreased tumor growth in vivo
To verify the function of circ_0061395 in HCC, we performed xenograft assay through injecting Huh7 cells carrying sh-circ_0061395 or sh-NC into nude mice. We observed that tumor volume and weight were reduced in mice of the sh-circ_0061395 group relative to the sh-NC group (Fig. 7a, b). Moreover, circ_0061395 was downregulated while miR-877-5p was upregulated in mice tumor tissues of the sh-circ_0061395 group when compared with the sh-NC group (Fig. 7c, d). Also, the level of PIK3R3 protein was reduced in mice tumor tissues of the sh-circ_0061395 group (Fig. 7e). IHC staining displayed that the expression of Ki67 was decreased in mice tumor tissues of the sh-circ_0061395 group in contrast to the sh-NC group (Fig. 7f ). These data suggested that circ_0061395 inhibition could decrease tumor growth in vivo.

Discussion
Increasing researches have proved that circRNAs exert a vital role in tumor growth [23]. More and more circR-NAs have been revealed to exert carcinogenic or tumorsuppressive influence on HCC based on the development of sequencing technology. Studies have reported that circRNA circ_104718 [24], circRNA circ_CDR1AS [25], and circRNA circ_RHOT1 [26] play a cancerigenic role in HCC, but circRNA circ_5692 [27] and circRNA circ_ TRIM33-12 [28] exert anti-tumor role in HCC. In this study, circ_0061395 expression was elevated in HCC tissues, serum, and cells. Moreover, circ_0061395 inhibition reduced tumor growth in vivo and impeded cell viability, colony formation, invasion, migration, and induced cell cycle arrest and apoptosis in HCC cells in vitro. Liu et al. discovered that circ_0061395 facilitated HCC development via inhibiting P27 expression via binding to HuR [10]. These results manifested that circ_0061395 could accelerate the advancement of HCC. Studies have proved that the abnormal expression of miRs is related to the development of a series of Fig. 4 Circ_0061395 served as a sponge for miR-877-5p in HCC cells. a The complementary binding sites between circ_0061395 and miR-877-5p were predicted by the CircInteractome database. b Dual luciferase reporter assay was performed to assess the luciferase activity in SNU-387 and Huh7 cells cotransfected with luciferase plasmids containing MUT-circ_0061395 or WT-circ_0061395 and miRNA NC or miR-877-5p mimic. c After RIP assay, the levels of circ_0061395 and miR-877-5p were examined by qRT-PCR. d-f The expression of miR-877-5p in HCC serum, tissues, and cells was detected by qRT-PCR. g After inhibitor NC or miR-877-5p inhibitor transfection, the expression of miR-877-5p in SNU-387 and Huh7 was examined by qRT-PCR. (H) The expression of miR-877-5p in SNU-387 and Huh7 cells transfected with si-NC, si-circ_0061395, si-circ_0061395 + inhibitor NC, or si-circ_0061395 + miR-877-5p inhibitor was analyzed by qRT-PCR. *P < 0.05 cancers, including HCC [29,30]. Also, circRNAs take part in the advancement of a range of tumors by sponging miRs [31]. MiR-877-5p had been proved to exert a repressive role in several cancers. Liang et al. uncovered that lncRNA DSCAM-AS1 inhibition elevated the expression of miR-877-5p, thereby repressing invasion, migration, and proliferation of cervical cancer cells [13]. Report of Xie et al. revealed that lncRNA TRG-AS1 accelerated proliferation of glioblastoma through elevating SUZ12 expression by adsorbing miR-877-5p [14]. In HCC, miR-877-5p could decrease cancer cell invasion, migration, and growth via targeting CDK14 [15]. Herein, we discovered that circ_0061395 acted as a sponge for miR-877-5p. Downregulation of miR-877-5p overturned circ_0061395 silencing-mediated the malignancy of HCC cells. Therefore, we inferred that circ_0061395 accelerated the malignancy of HCC cells via sponging miR-877-5p.
Exosomes, a type of microvesicle (diameter about 40-100 nm), have been revealed to exert a vital role in HCC advancement [33]. Exosomes contain a variety of circRNAs, miRs, lipids, and proteins, which act as information transmitters in intercellular communication [34,35]. Previous study revealed that that the exosomal cir-cRNA circ_100383 derived from highly metastatic HCC cells contributed to HCC metastasis through elevating angiogenesis and invasiveness [36]. Zhang et al. indicated that adipocytes-derived exosomal circRNA circ_DB accelerated HCC progression via activation of the USP7/ cyclin A2 pathway via sponging miR-34a [37]. In the current study, we discovered that circ_0061395 was elevated Fig. 6 Circ_0061395 regulated PIK3R3 expression through sponging miR-877-5p in HCC cells. a The binding sites of PIK3R3 in miR-877-5p were predicted by the starBase database. b Dual-luciferase reporter assay was executed to determine the binding sites between PIK3R3 and miR-877-5p in SNU-387 and Huh7 cells. c-g QRT-PCR and western blotting were employed to assess the levels of PIK3R3 mRNA and/or protein in HCC serum, tissues, and cells. h After pc-NC or pc-PIK3R3 transfection, the level of PIK3R3 protein in SNU-387 and Huh7 cells was detected with western blotting. (I-N) SNU-387 and Huh7 cells were transfected with miRNA NC, miR-877-5p mimic, miR-877-5p mimic + pc-NC, or miR-877-5p mimic + pc-PIK3R3. i The level of PIK3R3 protein in SNU-387 and Huh7 cells was analyzed by western blotting. j-n The viability, colony formation, invasion, migration, cell cycle progression, and apoptosis of SNU-387 and Huh7 cells were analyzed by CCK-8, plate clone, transwell, or flow cytometry assays. o The level of PIK3R3 protein in SNU-387 and Huh7 cells transfected with si-NC, si-circ_0061395, si-circ_0061395 + inhibitor NC, or si-circ_0061395 + miR-877-5p inhibitor was measured by western blotting. *P < 0.05 in serum exosomes of HCC patients. Unfortunately, we did not explore whether the exosomal circ_0061395 derived from the serum of HCC patients affected the tumorigenesis and advancement of HCC, which can be investigated in the future (Additional files 1, 2 and 3).

Conclusion
In conclusion, circ_0061395 promoted the malignancy of HCC cells via elevating PIK3R3 expression through competitively binding to miR-877-5p, which offered that circ_0061395 might be a target for HCC treatment. c-e QRT-PCR and western blotting were performed to assess the levels of circ_0061395, miR-877-5p, and PIK3R3 protein in mice tumor tissues of the sh-circ_0061395 and sh-NC groups. f IHC analysis of Ki67 expression in mice tumor tissues of the sh-circ_0061395 and sh-NC groups. *P < 0.05