Circular RNA circ-PAN3 (hsa_circ_0100181) Promotes Hepatocellular Carcinoma Growth Through Sponging miR-153 to Elevate Cyclin D1 Expression

Background: Circular RNAs (circRNAs) are engaged in hepatocellular carcinoma (HCC) progression, but the mechanisms remain to be elucidated. This study aimed to unveil the expression pattern and potential biological mechanisms of a newly indentied circRNA, circ-PAN3, in HCC. Methods: Cell Counting Kit-8 (CCK ‐ 8) assay and colony formation assay were used to assess cell proliferation. Transcription-quantitative PCR (RT-qPCR) analysis and western blot analysis were used to determine the relative expression level of mRNA and protein, respectively. Cell apoptosis assay was used to evaluate the apoptosis rate of transfected cells. CircInteractome and Targetscan were utilized to predict the possible targets of circRNAs and miRNAs, respectively. Luciferase reporter assay and RNA pull-down assay were used to assess the direct interaction of RNAs. HCC cancer xenograft model was used to evaluate the biological process of circ-PAN3 in vivo. Student’s t test, χ 2 test or one-way ANOVA was adopted appropriately. Results: Circ-PAN3 was elevated in HCC tissues, and patients with high Circ-PAN3 expression had a poor survival outcome. Knockdown of circ-PAN3 expression suppressed cell viability, colony formation and cell proliferation in vitro and in vivo. Circ-PAN3 elevates cyclin D1 expression to promote HCC progression. Subsequently, using miR-153 was downregulated miR-153 downregulated by miR-153. miR-153 signicantly and proliferation induced by of miR-153 Conclusion: study axis regulatory axis that promoted HCC progression.


Introduction
Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world [1,2]. Due to limited diagnostic methodologies, most patients are diagnosed at an advanced stage and are ineligible for liver transplantation or surgical resection [3,4]. Besides, this malignancy often exhibited high aggressiveness, invasiveness and frequent recurrence after resection [3]. As a result, the prognosis of HCC remains unsatisfactory [1]. It is urgent to investigate the detailed mechanism contributing to the initiation and progression of HCC in order to discover novel therapeutic targets for HCC patients.
Accumulating evidences showed that circular RNAs (circRNAs) are normally produced by scrambling of exons at the splicing process and are recognized as a novel class of endogenous noncoding RNA [5,6].
CircRNAs are highly conserved and are characterized as covalently closed loop structures with neither a 5′ to 3′ polarity nor a polyadenylated tail; they are RNA transcripts generated by the back-splicing of a single pre-mRNA and have gene-regulatory potential. Recently, studies have revealed that several circRNAs were abnormally expressed in HCC tissues and correlated with disease progression and prognosis [7][8][9].
However, the actual role of circRNA in HCC remains controversial. While several studies demonstrated an inhibitory role of circRNA in HCC [8], various studies also indicated a promotive role of circRNA [7].
Previous studies demonstrated that circ_0091570 could sponge miR-1307 to suppress the progression of HCC [8]. CircMTO1 could suppress HCC progression through miR-9/p-21 axis [10]. Recent studies elucidated that circSLC3A2 promoted HCC by sponging miR-490-3p and regulating PPM1F expression [11]. Recently, a newly circ-PAN3 was shown to be involved in drug resistance in acute myeloid leukemia and self-renewal of intestinal stem cells [12,13]. However, its role in HCC remains unknown. Therefore, the speci c roles and potential mechanisms of circRNA in HCC remains elucidated further.
Cyclin D1 is a key cell cycle regulator, which plays a key role in completion of the G1/S transition in mammalian cells [14]. Multiple studies have demonstrated that aberrant expression of cyclin D1 induced various oncogenic responses and was associated with shorter survival outcome [15]. Previous studies reported that cyclin D1 could be positively regulated by X-linked inhibitor of apoptosis protein (XIAP) through E3 ligase-mediated phosphorylation [16]. Interestingly, circ-PAN3 could regulate positively XIAP to induce chemo-resistance in acute myeloid leukemia [17]. Although both circ-PAN3 and cyclin D1 are upregulated in several malignancies, the possible role of circ-PAN3 in the regulation of cyclin D1 remains largely unknow.
In the present study, we aimed to explore the role of circ-PAN3 in HCC. The data from the present study revealed that circ-PAN3 promoted HCC cell proliferation by regulating cyclin D1 through modulating miR-153. The current study is the rst to suggest that circ-PAN3 could promote HCC progression through circ-PAN3/miR-153/ cyclin D1 regulatory axis. Circ-PAN3 may be utilized as a promising diagnostic and therapeutic target in HCC patients.
RNA from HCC cell lines or normal liver cell was isolated using TRIzol® reagent (Invitrogen; Thermo Fisher Scienti c, Inc.). Then, PrimeScript RT master mix (cat. no. RR036A; Takara Bio, Inc.) was used to reverse-transcribe RNA (1000 ng) into cDNA according to manufacturer's protocol. qPCR was conducted Cell proliferation assay.
Cell proliferation was assessed with Cell Counting Kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Inc.). After transfection, cells were seeded in 96-well plates with a density of 2x10 3 cells/well. After cultured for 24, 48 and 72 h at 37˚C, CCK-8 solution (10 μl) was supplemented to the culture medium.
Subsequently, the mixture was incubated for 90 min at 37˚C, and the absorbance was detected at 450 nm using a spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA). Relative cell viability (%) = (absorbance at 450 nm of the treated group-absorbance at 450 nm of the blank) / (absorbance at 450 nm of the control group-absorbance at 450 nm of the blank) x 100%.
Colony formation assay.
Transfected cells were planted into 6-well plates and maintained for 14 days at 37˚C. Subsequently, cells were washed with PBS and xed with 4% paraformaldehyde. The cells were then stained with 0.1 % crystal violet (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). The stained colonies were viewed and calculated using a uorescence microscope.

Apoptosis analysis.
For apoptosis analysis, transfected cells were seeded and maintained for 48 h and then collected and washed twice with PBS and resuspended in buffer. Cell apoptosis assay was conducted by using annexin V-uorescein isothiocyanate apoptosis detection kit (cat. no. 556547; BD Biosciences) in accordance to the manufacturer's protocol.
Cells were cultured to 60-70% con uence for transfection. Circ-PAN3-WT vector or Circ-PAN3-mutant (MUT) vector were created with or without a 3′-untranslated region binding site for miR-153 using pmirGLO vector (Promega Corporation) and CCDN1-WT vector or CCDN1-MUT vector were constructed similarly. Cells were co-transfected with miRNA mimics or NC mimics along with luciferase reporter vector using Lipofectamine 3000 (cat. no. L3000008; Invitrogen; Thermo Fisher Scienti c, Inc.). Following transfection for 48 h, luciferase activity was detected with a Dual-Luciferase Reporter Assay System (cat. no. E1910; Promega Corporation).
RNA pull-down assay HCC cells were transfected, harvested, lysed and sonicated. The circ-PAN3 probe was used for incubation with C-1 magnetic beads (Life Technologies) at 25 °C for 2 h to generate probe-coated beads. Cell lysate with circ-PAN3 probe or oligo probe was incubated at 4 °C for one night. After washing with wash buffer, the RNA mix bound to the beads was eluted and extracted with a RNeasy Mini Kit (QIAGEN) for subsequent RT-qPCR.
Protein extraction and western blot analysis.
Proteins were isolated using RIPA buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) supplemented with EDTA-free protease inhibitor cocktail (cat. no. 04693159001; Roche Diagnostics GmbH) as described previously. The extracted proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then transferred to PVDF membranes. Then the PVDF membranes were blocked with 5% skimmed milk. Subsequently, the membranes were incubated with primary antibodies at a dilution ratio of 1:1000 with PBST containing 5% BSA overnight at 4˚C. Next, the membranes were incubated with horseradish peroxidase-labeled secondary antibodies at dilution ratio of 1:5000 for 1 h at room temperature. Enhanced chemiluminescence (cat. no. 407207; EMD Millipore; Merck KGaA) was used to visualize the target proteins using the Tanon 4600 imaging system (Tanon Science and Technology Co., Ltd. University (Nanjing, China). Transfected Huh7 cells (3x10^6) in serum-free DMEM medium were injected into the right ank of the nude mice. After 4 weeks, tumor volume (V) was calculated using the following formula: V = length x width 2 /2 and tumor weight was measured.
Statistical analysis.
All data generated by above procedures were statistically analyzed using the GraphPad Prism v8.0 (GraphPad Software, Inc.). Survival curves were produced using the Kaplan-Meier method, and a log-rank test was used for comparison. The correlation between the expression levels of two genes was analyzed using Pearson's correlation analysis. Student's t test, χ 2 test or one-way ANOVA was adopted appropriately. P<0.05 was considered to exhibit a signi cant difference statistically. Data are presented as mean ± SEM from three independent experiments.

Results
Circ-PAN3 is frequently overexpressed in HCC and associates with poor prognosis To investigate the expression pattern of circ-PAN3 in HCC, the levels of circ-PAN3 in 80 HCC tumor tissues and 80 adjacent normal tissues was detected. Consistant with the studies in other malignancy, the expression level of circ-PAN3 was signi cantly elevated in HCC tumor tissues compared with that in adjacent normal tissues ( Fig. 1A; p < 0.001). Then, the expression level of circ-PAN3 was measured in HCC cell lines (Huh7, HCCLM3, SK-HEP-1) and normal liver cell LO2 . The results showed that the level of circ-PAN3 in HCC cell lines was statistically higher than that in normal liver cell LO2 ( Fig. 1B; p < 0.001). Moreover, the association of circ-PAN3 level and clinical characteristics was analyzed. The expression of circ-PAN3 is statistically associated with tumor size, TNM staging, and lymph node metastasis, but the association between circ-PAN3 level and age, sex or tumor differentiation was marginal (Table 1). Next, the association between circ-PAN3 expression level and clinical outcomes of HCC patients was explored. The results showed that elevated expression of circ-PAN3 was signi cantly associated with poor overall survival time ( Fig. 1C; p < 0.001).

Knockdown of circ-PAN3 inhibitsHCC cell proliferation in vitro as well as tumor growth in vivo
We further elucidate the functional mechanisms of circ-PAN3 in HCC in vitro and in vivo. Two cell lines of high-level circ-PAN3 were selected to be transfected with si-circ-PAN3 to knockdown the expression level of circ-PAN3 ( Fig. 2A). Following transfection, the cell viability, colony formation and cell proliferation assays were conducted. The results exhibited that cell proliferation and colony formation were validated to be signi cantly inhibited after transfection with si-circ-PAN3 in SK-Hep-1 and Huh7 cells (Fig. 2B-C).
Further, cell apoptosis analysis was performed and indicated that downregulation of circ-PAN3 caused an apparent increase in apoptosis rate of HCC cells, when compared with that in the control groups (Fig. 2D). Together, these results manifested that downregulation of circ-PAN3 suppressed HCC cell growth through induction of cell apoptosis in vitro.
Next, we clari ed the function of circ-PAN3 using HCC tumor xenograft models. The results showed that injection with si-circ-PAN3 inhibited tumor growth compared to the si-nc groups signi cantly (Fig. 2E). Altogether, these data revealed that the downregulation of circ-PAN3 could signi cantly induce inhibit HCC proliferation in vivo.
Circ-PAN3 was positively correlated with cyclin D1 expression in HCC cells Since cyclin D1 was upregulated in several malignancies with high expression level of circ-PAN3, we explored the relationship of circ-PAN3 and cyclin D1. Our results showed that circ-PAN3 knockdown downregulated the expression of cyclin D1, but induced merely marginal alteration of other cell cycleassociated genes in SK-Hep-1 and Huh7 cells (Fig. 3A). Moreover, the expression levels of cyclin D1 was found to be upregulation in HCC cell lines with high expression of circ-PAN3 (Fig. 3B & 3C). The expression level of cyclin D1 was signi cantly higher in HCC tumor tissues compared with that in adjacent normal tissues (Fig. 3D). Pearson's correlation analysis indicated the positive correlation between the expression levels of circ-PAN3 and cyclin D1 (Fig. 3E). These results showed that circ-PAN3 expression was positively correlated with cyclin D1 in HCC, which indicated the possible regulatory role of circ-PAN3 on cyclin D1.

Circ-PAN3 sponged miR-153 to regulate cyclin D1 in HCC cells
To further investigate the regulatory mechanisms of circ-PAN3 on cyclin D1, the following experiments were performed. Since circ-RNA function could be largely depended on subcellular localization, we analyzed subcellular RNA level to identify the predominant location of circ-PAN3. This analysis showed that circ-PAN3 was mostly located in cytoplasm in SK-Hep-1 and Huh7 cells (Fig. 4A). Previous studies have been reported that circRNAs could act as miRNA sponges. Thus, CircInteractome (https://circinteractome.nia.nih.gov/) and Targetscan (http://targetscan.org/vert_72) were used to identify the possible interaction between circ-PAN3 and cyclin D1. According to the prediction, circ-PAN3 could bind and sponge miR-153, which then in turn targeted cyclin D1 to accelerate the growth of HCC cells (Fig. 4B). To verify the prediction, luciferase reporter assays were conducted to con rm the predictive interaction among circ-PAN3, miR-153, and cyclin D1. In accordance to the prediction, the results indicated that miR-153 signi cantly reduced the luciferase activity in circ-PAN3-WT vector but not in circ-PAN3-MUT vector in SK-Hep-1 and Huh7 cells (Fig. 4C). In addition, miR-153 lso reduced the luciferase activity in CCND1-WT vector but not in CCND1-MUT vector signi cantly in SK-Hep-1 and Huh7 cells (Fig.  4D). Moreover, RNA pull-down with biotinylated-circ-PAN3 probe con rmed direct interaction between circ-PAN3 and miR-153 in SK-Hep-1 and Huh7 cells (Fig. 4E). RNA pull-down with biotinylated-CNND1 probe also validated direct interaction between CNND1 and miR-153 in SK-Hep-1 and Huh7 cells (Fig. 4E). Next, we detect the expression level of miR-153 following circ-PAN3 knock-down. We found knock-down of circ-PAN3 signi cantly elevated the expression of miR-153 (Fig. 4F). We then evaluated the expression level of cyclin D1 with downregulation of circ-PAN3. The results unveiled that downregulation of circ-PAN3 reduced the expression of cyclin D1 in SK-Hep-1 and Huh7 cells (Fig. 4F). Since cyclin D1 was a direct target of miR-153, we evaluated the expression level of cyclin D1 with upregulation of miR-153. The results showed that overexpression of miR-153 also reduced the expression of cyclin D1 in RNA and protein level (Fig. 4G). Together, these results indicated that circ-PAN3 positively regulated expression of cyclin D1 via sponging miR-153.
Silencing of miR-153 or overexpression of cyclin D1 rescues the decreased proliferative abilities of HCC cells caused by circ-PAN3 knockdown To further clarify the relationship between circ-PAN3, miR-153 and cyclin D1, rescue analysis was conducted. The results showed that the inhibitory expression of cyclin D1 following circ-PAN3 knockdown was reversed with the addition of miR-153 inhibitor or cyclin D1 plasmid (Fig. 5A & 5B). We then evaluated the cell phenotypes with rescue experiments. The results showed circ-PAN3 knockdown impaired cell proliferation and cell colony formation (Fig. 5C & 5D). Notably, the inhibition of cell proliferation and colony formation were recovered following the combination with miR-153 inhibitor, (Fig.   5C & 5D). Moreover, the suppression of cell proliferation and colony formation could be reversed following the combination with cyclin D1 plasmid (Fig. 5C & 5D). Combined with the results above, these results demonstrated the circ-PAN3/miR-153/cyclin D1 modulated the growth of HCC cells.

Discussion
CircRNAs are wildly reported to be involved in the tumorigenesis and progression of various types of cancers, but the role of circRNAs in HCC remains to be investigated further. In this study, it was revealed that circ-PAN3, was statistically increased in HCC clinical tissues and associated with poor survival rate. The experimental data revealed that circ-PAN3 served as a competitive endogenous RNA to deteriorate miR-153 activity. Impairment of miR-153 activity would then upregulate cyclin D1 and promote the proliferation of HCC cell. Interestingly, it was also demonstrated that overexpression of cyclin D1 reversed the inhibition of HCC proliferation induced by reducing circ-PAN3 expression. Taken together, it has been established that circ-PAN3 might be used as a promising diagnostic and therapeutic target in HCC.
Circ-PAN3 is a newly identi ed circRNA, which is located on 13q12.2 [17]. In recent years, several studies have investigated the role of circ-PAN3 in various diseases [12,13]. In acute myeloid leukemia, circ-PAN3 promoted drug resistance through regulation of autophagy as an autophagy inducer via the AMPK/mTOR pathway [12]. In another study of acute myeloid leukemia, circ-PAN3 was demonstrated to be a key mediator for chemo-resistance of AML cells through circ-PAN3-miR-153-5p/miR-183-5p-XIAP regulatory axis [17]. In cardiac brosis, circ-PAN3 exhibited pro brotic effects via miR-221/FoxO3/ATG7 axis [18]. However, the role and potential mechanisms of circ-PAN3 in HCC remains largely unknown. In this study, two cell lines (SK-Hep-1 and Huh7) were selected with signi cantly high expression of circ-PAN3 from four common HCC cell lines to investigate the possible function of circ-PAN3. It was showed that knockdown of circ-PAN3 signi cantly impaired HCC cell proliferation and colony formation. It was further revealed that circ-PAN3 knockdown induced HCC cell apoptosis signi cantly. These results suggest that circ-PAN3 functioned as a promoter in HCC cell proliferation.
CircRNAs can act through diverse mechanisms, including genomic targeting, regulation in cis or trans, and antisense interference [5]. In recent years, the role of circRNA as competitive endogenous RNA in multiple types of cancer has gained much attention and has been broadly reported [8,10]. The competitive endogenous RNA can decrease the stability of target miRNA, thereby upregulating the expression of the miRNA target gene. In the present study, the potential miRNA that circ-PAN3 might regulate in HCC was investigated. It was showed that circ-PAN3 contained the binding site of miR-153 using bioinformatic analysis. The interaction between circ-PAN3 and miR-153 was further demonstrated using luciferase assay and RIP. Moreover, circ-PAN3 knockdown increased the expression of miR-153. In addition, cell apoptosis induced by circ-PAN knockdown could be reversed by miR-153 inhibitor. These results indicate that circ-PAN3 might exert oncogenic function by inhibiting the expression of miR-153.
Multiple studies have reported that cyclin D1 plays a pro-tumorigenic role in HCC and downregulation of cyclin D1 was correlated with reduced cell proliferation and invasion [19,20]. The expression of cyclin D1 could be regulated by various mechanisms including autophagy degradation system and miRNA inhibition [21]. Previous studies reported that cyclin D1 could be positively regulated by X-linked inhibitor of apoptosis protein (XIAP) through E3 ligase-mediated phosphorylation [16]. Notably, circ-PAN3 could regulate positively XIAP to induce chemo-resistance in acute myeloid leukemia. This indicated a potential positive association between circ-PAN3 and cyclin D1 [17]. In the present study, we investigated the regulatory role of circ-PAN3 in cyclin D1. We found that circ-PAN3 could modulate the expression of cyclin D1 via miRNA sponge. Targetscan database was used to explore potential targets that could bind with miR-153. It was found that cyclin D1 directly interacted with miR-153 and this was validated using a luciferase assay and cyclin D1 expression was downregulated by miR-153. Subsequently, it was found that downregulation of circ-PAN3 could decrease cyclin D1 expression and this effect could be reversed by the addition of miR-153 inhibitor. Finally, it was con rmed that the inhibition of HCC cell proliferation induced by circ-PAN3 knockdown could be rescued by the overexpression of cyclin D1. In total, it was demonstrated that the novel circRNA, circ-PAN3, could promote the progression of HCC through the circ-PAN3/miR-153/cyclin D1 axis.

Conclusions
In conclusion, it was revealed that circ-PAN3 was increased in HCC tumor tissues and might be used as a predictive marker of prognosis. Notably, to the best of our knowledge this is the rst study to establish that the role of circ-PAN3 could accelerate the progression of HCC through the miR-153/cyclin D1 pathway. Thus, circ-PAN3 could be adopted as a novel diagnostic and therapeutic target in the management of HCC.