Circular RNA circRHOBTB3 is downregulated in hepatocellular carcinoma and suppresses cell proliferation by inhibiting miR-18a maturation

Background Circular RNA circRHOBTB3 has been characterized as a tumor suppressor in gastric cancer, while its role in hepatocellular carcinoma (HCC) is unknown. This study was carried out to analyze the role of circRHOBTB3 in HCC. Methods In this study, circRHOBTB3, mature miR-18a, and miR-18a precursor in HCC and paired non-cancer tissues were detected by RT-qPCR. The role of circRHOBTB3 in the production of mature miR-18a was explored by transfecting circRHOBTB3 expression vector into HCC cells, followed by RT-qPCR to determine the expression of mature miR-18a and miR-18a precursor. The role of circRHOBTB3 and miR-18a in HCC cell proliferation was studied using CCK-8 assay. Results CircRHOBTB3 was under-expressed in HCC compared to normal tissues. In HCC cells, circRHOBTB3 overexpression decreased mature miR-18a level but not miR-18a precursor. Cell proliferation analysis showed that circRHOBTB3 overexpression decreased cell proliferation while miR-18a overexpression increased cell proliferation. Moreover, circRHOBTB3 suppressed the role of miR-18a in cell proliferation. Conclusions CircRHOBTB3 is downregulated in HCC and may suppress cell proliferation by reducing miR-18a production. Supplementary Information The online version contains supplementary material available at 10.1186/s13027-021-00384-1.


Introduction
Hepatocellular carcinoma (HCC) is a major malignancy that originated from the liver and accounted for more than 85% of all liver cancer cases [1]. The incidence of HCC varies greatly worldwide, with the highest incidence rate observed in African countries and eastern Asia [2,3], mainly owing to the high prevalence of infections of hepatitis B virus (HBV) and hepatitis C virus (HCV) in these regions [4]. Especially in China, HCV and HBV affect about 30 million and 93 million people, accounting for about 9% of the Chinese population. As a result, about 53% HCC cases are believed to come from China [5,6]. Therefore, preventative and treatment approaches for HCC are urgently needed.
Although HCV and HBV infections are the main risk factors for HCC, they are not sufficient for its occurrence and development [5,6]. HCC is characterized by its hypervascularity and hepatocarcinogenesis. Receptor tyrosine kinases (RTKs) signaling pathways, such as Ras/ Raf/MEK/ERK, Jak/Stat, and PI3K/AKT/mTOR, could activate many angiogenic signaling pathways, resulting in tumor cell proliferation, invasion, and metastasis [7,8]. These data provide rational targets for innovative HCC therapies. mTOR inhibitors appear to be the most promising agents in this category [9][10][11]. Although many efforts have been made in molecularly targeted therapy in HCC, the effective targets for HCC remain lacking. As covalently closed RNAs with no or limited protein-coding capacity, circular RNAs (circRNAs) are emerging novel targets for targeted cancer therapy, mainly owing to their roles in regulating gene expression [12,13]. In a recent study, Deng et al. reported a novel circRNA, circRHOBTB3 originated from exon 6 and exon 7 of RHOBTB3 gene as a tumor suppressor in gastric cancer [14]. Our preliminary microarray analysis revealed that circRHOBTB3 expression is altered in HCC (Fig.S1), and circRHOBTB3 is inversely correlated with miR-18a (data not shown).
miR-18a is one of the most conserved and multifunctional miRNAs in the polycistronic miR-17-92 cluster and is frequently overexpressed in malignant tumors [15]. Studies have shown that miR-18a is overexpressed in non-small-cell lung cancer (NSCLC) [16], cervical cancer (CC) [17], and gastric cancer (GC) [18]. Savitsky D et al. have verified that the 3′-UTR of IRF2 is a target of miR-18a, which can promote NSCLC development via downregulating IRF2 [19]. Mezache L et al. confirmed that miR-18a indirectly upregulates PD-L1 expression by activating the PI3K/AKT, MEK/ ERK, and Wnt/β-catenin pathways, resulting in CC progression [20]. In addition, another study showed that miR-18a is an oncogene and plays a vital role in GC development by negatively regulating the protein inhibitor of activated STAT (PIAS)-3 and thereby modulating STAT3 target genes [18]. Liu L et al. showed that miR-18a is upregulated in human HCC tissues and cell lines and promotes HCC cell proliferation and migration by targeting KLF4 and its p21 [21]. Despite emerging evidence about the role of miRNAs in cancer progression, fewer studies have explored the functions of miR-18a than other miR-NAs in the miR-17-92 cluster in cancer development [15]. In this study, we focused on analyzing the crosstalk between circRHOBTB3 and miR-18a in HCC.

HCC patients
A total of 62 HCC patients (38 to 66 years; median age: 52 years), including 42 males and 20 females, at Nanfang Hospital, Southern Medical University from May 2018 to May 2020 were enrolled in the study. These patients included 28 cases at AJCC stage I or II and 34 cases at III or IV. HBV infection was observed in 22 cases, and HCV infection was observed in 30 cases. To exclude other factors that may affect the expression of target genes, patients with initiated therapy or other severe clinical disorders, such as diabetes, heart diseases, severe infections, and other malignancies were excluded. The study was approved by the Ethics Committee of Nanfang Hospital. All patients signed the informed consent.

HCC tissue collections and HCC cell lines
HCC and adjacent non-cancer tissues were collected by aspirating 3 times in different directions using fine needles from all patients prior to therapies. Tissue samples were confirmed histopathologically using hematoxylineosin (H&E) staining and stored in liquid nitrogen prior to the subsequent assays.
SNU-449 and SNU-387 (ATCC) cell lines were used as HCC cell models in this study. Normal liver THLE-2 cell line (ATCC) was used as the control. Cells were cultured in RPMI-1640 medium with 10% FBS at 37°C and 5% CO 2 .

RNA preparations
RNAs were extracted from SNU-449 and SNU-387 cells and tissues collected from the 62 HCC patients using Ribozol reagent (VWR Life Science) and treated with DNase I (Invitrogen) for 1 h at 37°C to completely remove genomic DNAs. RNA integrity was examined by electrophoresis on 6% urea-PAGE gel, and RNA purity was reflected by OD260/280 ratio.

Cell proliferation assay
At 48 h of post-transfection, cell proliferation was analyzed using CCK-8 kits (Sigma-Aldrich). In brief, 5000 cells were placed in each well of 96-well plates. Cell proliferation was measured every 24 h for 4 times at 2 h after the addition of 10% CCK-8 by measuring the optical density (OD) at 450 nm.

Statistical analysis
CircRHOBTB3 and miR-18a expression in tissues and cell lines fulfilled the normal distribution, which was analyzed by the Kolmogorov-Smirnov test in SPSS software. Thus, circRHOBTB3 and miR-18a expression in tissues from HCC patients (n = 62) were compared by paired t test. Comparisons between two independent groups were performed by unpaired t test. ANOVA Tukey's test was used to compare differences among multiple transfection groups. P < 0.05 was statistically significant.

HCC exhibited downregulated circRHOBTB3 expression
CircRHOBTB3 expression in paired tissues from HCC patients (n = 62) was analyzed by RT-qPCR. Cir-cRHOBTB3 was significantly under-expressed in HCC tissues (Fig. 1A, p < 0.01). CircRHOBTB3 expression in HCC tissues was not significantly different between AJCC stages (I or II vs. III or IV; Fig. 1B, p < 0.01). Among the HCC patients, HBV infection was observed in 22 cases, HCV infection was observed in 30 cases, and non-infection was observed in 10 cases. No significant differences in circRHOBTB3 expression were found among these three groups (Fig. 1C). CircRHOBTB3 levels in SNU-448, SNU-387, and normal liver cell line THLE-2 were measured by RT-qPCR. The results showed that circRHOBTB3 was also downregulated in SNU-448 and SNU-387 cell lines compared to normal THLE-2 cell line (Fig. 1D). It is worth noting that RHOBTB3 mRNA was also downregulated in HCC cell lines (Fig. S2). Both mature miR-18a and miR-18a precursor were upregulated in HCC, but circRHOBTB3 was only inversely correlated with mature miR-18a Mature miR-18a and miR-18a precursor expressions in paired tissues from HCC patients (n = 62) were also analyzed by RT-qPCRs. Mature miR-18a ( Fig. 2A) and miR-18a precursor (Fig. 2B) were significantly overexpressed in HCC tissues than in non-tumor tissues (p < 0.01). Correlation analysis performed by Pearson's correlation coefficient showed thatcircRHOBTB3 was inversely correlated with mature miR-18a (Fig. 2C), but not miR-18a precursor (Fig. 2D) across HCC tissues. Therefore, we hypothesized that circRHOBTB3 is likely involved in miR-18a maturation.

CircRHOBTB3 overexpression decreased HCC cell proliferation
The role of circRHOBTB3 and miR-18a in the proliferation of SNU-449 and SNU-387 cells was analyzed by CCK-8 assay. CircRHOBTB3 overexpression decreased cell proliferation, while miR-18a overexpression increased cell proliferation. Moreover, circRHOBTB3 suppressed the role of miR-18a in cell proliferation (Fig. 4, p < 0.05). However, CircRHOBTB3 and miR-18a overexpression have no effect on the proliferation of THLE-2 cell lines (Fig. S2).

Discussion
We analyzed the differential expression of cir-cRHOBTB3 in HCC and studied its crosstalk with miR-18a. The results showed that circRHOBTB3 was underexpressed in HCC and inhibited HCC cell proliferation by suppressing miR-18a maturation. Fig. 2 Mature miR-18a and miR-18a precursor were both upregulated in HCC, but circRHOBTB3 was only inversely correlated with mature miR-18a. Mature miR-18a (A) and miR-18a precursor (B) expressions in paired tissues from HCC patients (n = 62) were analyzed by qRT-PCRs. Expression levels of mature miR-18a and miR-18a precursor in tissue samples from HCC patients (n = 62) were expressed as the average of three technical qRT-PCR replicates. U6 was used for normalization. ** p < 0.01. Correlations between circRHOBTB3 and mature miR-18a (C) or miR-18a precursor (D) were analyzed by Pearson's correlation coefficient To study the role of circRHOBTB3 in regulating miR-18a maturation, SNU-449, SNU-387, and THLE-2 cells were transfected with circRHOBTB3 expression vector or miR-18a mimic, followed by qRT-PCRs to confirm the overexpression of circRHOBTB3 and mature miR-18a (A). The effects of circRHOBTB3 overexpression on mature miR-18a (B) and miR-18a precursor (C) were also analyzed by qRT-PCR. Expressions of circRHOBTB3, mature miR-18a, and miR-18a precursor were expressed as the average of three technical qRT-PCR replicates. GAPDH was used for circRHOBTB3 normalization, and U6 was used for mature miR-18a and miR-18a precursor normalization. * p < 0.05 CircRHOBTB3 is significantly downregulated in gastric cancer [14]. In addition, circRHOBTB3 overexpression sponges miR-654-3p to suppress the growth of gastric tumor, suggesting the role of circRHOBTB3 as a tumor suppressor in this malignancy [14]. This study, for the first time, reported circRHOBTB3 downregulation in HCC. Interestingly, circRHOBTB3 expression was not significantly affected by clinical stags, suggesting that circRHOBTB3 may participate in the whole process of HCC development but not specific stages. Moreover, circRHOBTB3 expression was also not significantly affected by HBV and HCV infections, the major risk factors for HCC [5,6]. Therefore, circRHOBTB3 may participate in HCC through HBV-and HCVindependent pathways.
MiR-18a has been characterized as an oncogenic miRNA in many cancers, including HCC [21]. MiR-18a is overexpressed in HCC and targets KLF4 to promote tumor metastasis and growth in HCC [21]. Consistently, our study confirmed the upregulation of both miR-18a precursor and mature miR-18a in HCC. Therefore, the miR-18a may be upregulated at the transcription level from the process from pri-miRNA to miRNA precursor.
Besides regulating gene expression at transcriptional and translational levels, circRNAs may also sponge miR-NAs to participate in human cancers, including HCC [22]. For instance, circRNA circMTO1 may sponge miR-9 to inhibit the progression of HCC [22]. In this study, we reported the interaction between circRHOBTB3 and miR-18a. Interestingly, circRHOBTB3 is unlikely a sponge of miR-18a. Memczak S et al. found that CDR1as can function as a miR-7 sponge to reduce midbrain sizes in zebrafish, and this biological activity of CDR1as could be partially rescued by injecting miR-7 precursor [23]. Our results showed that circRHOBTB3 overexpression decreased mature miR-18a level, leading to reduced HCC cell proliferation while had no effect on miR-18a precursor level. These results suggested that the effect of circRHOBTB3 overexpression on HCC cell proliferation cannot be rescued by the miR-18a precursor. However, our results showed that the effect of circRHOBTB3 on HCC cell proliferation could be rescued by mature miR-18a overexpression. Therefore, we believe that cir-cRHOBTB3 overexpression-reduced HCC cell proliferation may be due to its role to suppress miR-18a maturation rather than its role as a molecular sponge of miR-18. Our findings revealed another type of interaction between circRHOBTB3 and miR-18a and suggested that circRHOBTB3 overexpression hinders the formation of mature miR-18a instead of miR-18a precursor, thereby reducing HCC cell proliferation. Because the formation of mature miRNAs requires transportation of miRNA precursors from the nucleus to the cytoplasm, we speculate that circRHOBTB3 may be involved in forming mature miRNAs from miRNA precursors. Our future studies will explore this possibility. Interestingly, we did not observe the increased level of miR-18a precursor along with decreased mature miR-18a in cells with circRHOBTB3 overexpression. It possibly indicated that circRHOBTB3 could also downregulate total miR-18a. In addition, we found that RHOBTB3 mRNA level was downregulated in HCC line cells. MiRNAs regulate physiological and pathological processes via inhibiting target mRNA translation or promoting mRNA degradation [24]. In this study, we only revealed the interaction between circRHOBTB3 and miR-18a. The underlying mechanism for this interaction needs to be further explored.

Conclusion
In conclusion, circRHOBTB3 is under-expressed in HCC. Moreover, circRHOBTB3 may suppress the maturation of miR-18a to promote HCC cell proliferation.
Additional file 1 Fig. S1 The microarray results of circRHOBTB3 and miR-18a Additional file 2 Fig. S2 RHOBTB3 mRNA expression in cell lines.

Acknowledgments
Not Applicable.

Authors' contributions
Gang Hu, Shusen Zhai, Ran Gao: study concepts, literature research, clinical studies, data analysis, experimental studies, manuscript writing and review; Sheng Yu: study design, literature research, experimental studies and manuscript editing; Zhen Huang: definition of intellectual content, clinical studies, data acquisition and statistical analysis. The author(s) read and approved the final manuscript.

Funding
Not Applicable.

Availability of data and materials
The data that support the findings of this study are available on request from the corresponding author: Sheng Yu, Division of Hepatobiliopancreatic Surgery, Department of General Surgery, Nanfang Hospital, Southern Medical University, No. 1838 Guangzhou Avenue North, Guangzhou City, Guangdong Province, 510,515, P. R. China. The data are not publicly available due to their containing information that could compromise the privacy of research participants.

Declarations
Ethical approval and consent to participate The study was approved by the Ethics Committee of Nanfang Hospital, Southern Medical University. Informed consent was obtained from all individual participants included in this study. Procedures operated in this research were completed in keeping with the standards set out in the Announcement of Helsinki and the Laboratory Guidelines of Research in China.

Consent for publication
Not applicable.