circRASGRF2 functions as an oncogenic gene in hepatocellular carcinoma by acting as a miR-1224 sponge

Di Wu; Anliang Xia; Tianlong Fan; Guoqiang Li

doi:10.1016/j.omtn.2020.10.035

This article has been retracted.

Retraction in: Mol Ther Nucleic Acids. 2024 Apr 17;35(2):102188 See also: PMC Retraction Policy

. 2020 Oct 27;23:13–26. doi: 10.1016/j.omtn.2020.10.035

circRASGRF2 functions as an oncogenic gene in hepatocellular carcinoma by acting as a miR-1224 sponge

Di Wu ^1,⁴, Anliang Xia ^2,^3,⁴, Tianlong Fan ^1,⁴, Guoqiang Li ^2,^3,^∗

PMCID: PMC7711184 PMID: 33312757

Abstract

Circular RNAs (circRNAs) are a class of non-coding RNAs broadly expressed in cells of various species. However, the contributions and molecular mechanisms of circRNAs to hepatocellular carcinoma (HCC) remain largely unknown. In the present study, we compared the expression of circRNAs between five paired HCC and adjacent noncancerous liver (ANL) tissues by using RNA-sequencing (RNA-seq). circRASGRF2 (a circRNA located on chromosome 5 and derived from RASGRF2, hsa_circ_0073181) was identified and validated by quantitative reverse transcriptase PCR. The role of circRASGRF2 in HCC progression was assessed both in vitro and in vivo. Mechanistically, RNA immunoprecipitation and luciferase reporter assays were performed to confirm the interaction between circRASGRF2 and miR-1224 in HCC. circRASGRF2 was found to be significantly upregulated in HCC tissues and HCC cell lines compared with paired ANL tissues and normal cells. Our in vivo and in vitro data indicated that knockdown of circRASGRF2 inhibits the proliferation and migration of HCC cells. Mechanistically, we found that circRASGRF2 could promote the expression of focal adhesion kinase (FAK) by sponging miR-1224. Our data showed that circRASGRF2 is a central component linking circRNAs to progression of HCC, making it a potential therapeutic target.

Keywords: circRNAs, circRASGRF2, ceRNA, hepatocellular carcinoma

Graphical Abstract

Wu and colleagues found that circular RNA (circRNA) circRASGRF2 is a central component linking circRNAs to progression of HCC, making it a potential therapeutic target. circRASGRF2 promotes the expression of FAK by sponging miR-1224.

Introduction

Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide and the second most lethal human cancer.¹ Due to the lack of symptoms in the early disease stages, most patients were diagnosed at an advanced stage, thus losing the opportunity of curative resection.² The 5-year overall survival rate of HCC patients was less than 25% due to the late-stage detection and lack of effective therapies. At present, the precise mechanisms underlying HCC progression remain to be investigated.

Circular RNAs (circRNAs) are a class of RNA transcripts generated by back-splicing that involve transcriptional and post-transcriptional gene regulation.³ Although circRNAs were initially considered byproducts of splicing, high-throughput sequencing analysis has identified many circRNAs widely expressed in a tissue-specific or cell type-specific manner.⁴ Emerging evidence indicates that circRNAs play important roles in multiple biological functions of different types of cancers.⁵ Existing reports find that circRNAs can function as competing endogenous RNAs (ceRNAs) or protein-coding RNAs or interact with RNA-binding proteins to regulate the expression of genes involved in tumorigenesis and progression.³^,⁷ In addition, aberrant circRNAs exert effects on cancer detection, and circRNAs have been reported as potential biomarkers for diagnosis and prognosis of different cancers.⁸^,⁹ Even though the activities of various circRNAs in HCC have been studied well, there is little doubt that investigation of additional circRNAs may offer novel and valuable insights and thereby facilitate drug research and development.

In this study, we identified circRASGRF2 (hsa_circ_0073181) as a specific circRNA upregulated in HCC patients using RNA sequencing (RNA-seq). circRASGRF2 promotes HCC progression by functioning as a miR-1224 sponge to upregulate focal adhesion kinase (FAK) expression. Therefore, circRASGRF2 may serve as a promising biomarker and as a potential therapeutic target for HCC patients.

Results

circRNA profiling in human HCC tissues and circRASGRF2 characterization

To identify and characterize differentially expressed circRNAs in HCC, RNA-seq was implemented in five pairs of HCC and adjacent noncancerous liver (ANL) tissues. When we set the filter criteria as a fold-change ≥ 2 and p value < 0.01, we found 784 differentially expressed circRNAs, of which 443 were upregulated and 341 were downregulated in HCC tissues. Among them, the top 10 dysregulated circRNAs are shown in Figure 1A. Among the 784 differentially expressed circRNAs, 311, including 166 upregulated ones and 145 downregulated ones, were verified as novel circRNAs; 473 circRNAs, including 277 upregulated ones and 196 downregulated ones, had been identified beforehand and listed in the circRNA database (circBase; http://www.circbase.org) (Figure 1B). The 784 identified circRNAs were divided into five different categories on the basis of the way they were produced. Exonic circRNAs consisting of the protein-encoding exons accounted for 70.92% (556/784), intronic circRNAs from intron lariats comprised 6.89% (54/784), sense overlapping circRNAs that originated from exon and other sequence circRNAs comprised 13.01% (102/784), and intergenic circRNAs composed of unannotated sequences of the gene and antisense circRNAs originating from antisense regions equally comprised 9.18% (72/784) (Figure 1C). Expression levels of the top 10 dysregulated circRNAs were measured by quantitative real-time PCR in HCC tissues and ANL tissues (Figure 1D). In the top five upregulated circRNAs, we focused on a novel circRNA, circRASGRF2 (hsa_circ_0073181), which is located on chromosome 5 and derived from RASGRF2 gene by back-splicing (2,182 bp) on the basis of the annotation of circBase (http://www.circbase.org/). There was an increasing trend in circRASGRF2 levels from ANL tissues to HCC tumor tissues, with a more than 4-fold change from RNA-seq analysis (Figure 1E).

circRASGRF2 was upregulated in HCC tissues and cell lines

We measured circRASGRF2 expression levels in our cohort of 68 HCC patients and their corresponding ANL tissues by quantitative real-time PCR. As expected, circRASGRF2 was expressed at a higher level in HCC samples compared to ANL tissues (Figure 1F, p < 0.001). We next sought to determine whether circRASGRF2 expression level in HCC was associated with specific clinicopathological characteristics. We used the receiver operating characteristic (ROC) curve to examine the diagnostic value of circRASGRF2 in HCC tissues compared with ANL tissues, and found the area under the ROC curve (AUC) to be 0.882 (95% confidence interval [CI], 0.814–0.950, p < 0.0001; Figure 1G). Using the median expression level of circRASGRF2 as a cutoff value, we divided the 68 HCC patients into low and high expression groups. We found that a higher circRASGRF2 expression level was significantly correlated not only with poorer tumor differentiation and more advanced tumor stage, but also with tumor size and the presence of microvascular invasion (Table 1). Furthermore, HCC patients with high expression of circRASGRF2 displayed obviously shorter overall survival times than those with low expression of circRASGRF2 according to Kaplan-Meier survival curve analysis (p = 0.0036) (Figure 1H).

Table 1.

Clinical characteristics of 68 patients with HCC according to circRASGRF2 expression level

Variable	circRASGRF2
Variable	Low	High	p Value
All cases	34	34
Age, years, ≥50:<50	16:18	18:16	0.808
Sex, male:female	19:15	22:12	0.620
HBsAg, positive:negative	16:18	19:15	0.628
Liver cirrhosis, with:without	12:22	15:19	0.621
AFP, μg/L, >20:≤20	14:20	16:18	0.807
Pathological satellite, present:absent	17:17	19:15	0.808
No. tumors, multiple:solitary	6:28	8:26	0.765
Edmondson’s grade, III+IV:I+II	11:23	22:12	0.014∗
Tumor size, cm, >5:≤5	11:23	24:10	0.003∗
Microvascular invasion, present:absent	4:30	12:22	0.043∗
Encapsulation, incomplete:complete	18:16	20:14	0.808
TNM stage, II+III:I	10:24	20:14	0.027∗
BCLC stage, B+C:A	9:25	24:10	0.0006∗

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Α χ² test was used to test the association between two categorical variables. AFP, alpha-fetoprotein; BCLC, Barcelona Clinic Liver Cancer; HCC, hepatocellular carcinoma; HBsAg, hepatitis B surface antigen; TNM, tumor-node-metastasis. ∗Statistically significant.

To demonstrate the universal expression of circRASGRF2 in HCC cell lines, we selected normal liver cell line L02, as well as AFP-positive, AFP-negative, high metastatic potential, and low metastatic potential HCC cell lines, including HLE, Huh7, MHCC97H, BEL7402, SMMC7721, MHCC97L, Hep3B, HCCLM3, and HCCLM6 cells. Notably, MHCC97H and Hep3B cells were characterized as having relatively high and low metastatic potential, in which the circRASGRF2 was highly and lowly expressed, respectively (Figure 1I). On the basis of this result, MHCC97H cells were selected for a loss-of-function assay, whereas Hep3B cells were selected for a gain-of-function assay. These data indicated that circRASGRF2 might be a participant in tumorigenesis of HCC.

Confirmation of subcellular localization of circRASGRF2

To further evaluate the stability of circRASGRF2, actinomycin D was used to inhibit the synthesis of new RNA. The results showed that circRASGRF2 was more stable than linear RASGRF2 after treatment with actinomycin D (Figures 2A and 2B). RNase R is an exoribonuclease that can degrade RNA from its 3′ to 5′ end, but it does not act on circRNA. In contrast to the linear RASGRF2 mRNA, circRASGRF2 was resistant to RNase R (Figures 2C and 2D). Subsequently, the subcellular localization of circRASGRF2 was detected in HCC cells by nuclear-cytoplasmic fractionation assays. It was found that circRASGRF2 was mainly present in the cytoplasm of MHCC97H and Hep3B cells (Figures 2E and 2F). Our results implied that circRASGRF2 harbored a loop structure and was predominantly localized in the cytoplasm.

circRASGRF2 loss of function dramatically impairs the HCC cell malignant phenotypes *in vitro*

(A) Quantitative real-time PCR for the abundance of circRASGRF2 and RASGRF2 in MHCC97H cells treated with actinomycin D at the indicated time point. (B) Quantitative real-time PCR for the abundance of circRASGRF2 and RASGRF2 in Hep3B cells treated with actinomycin D at the indicated time point. (C) Quantitative real-time PCR for the expression of circRASGRF2 and RASGRF2 mRNA in MHCC97H cells treated with or without RNase R. (D) Quantitative real-time PCR for the expression of circRASGRF2 and RASGRF2 mRNA in Hep3B cells treated with or without RNase R. (E) Levels of circRASGRF2 in the nuclear and cytoplasmic fractions of MHCC97H cells. (F) Levels of circRASGRF2 in the nuclear and cytoplasmic fractions of Hep3B cells. (G) The expression of circRASGRF2 was only downregulated by si-circRASGRF2 but was not affected by si-RASGRF2. (H) The circRASGRF2 expression vector markedly increased the expression of circRASGRF2 compared with the empty vector. (I) CCK-8 assays showed circRASGRF2 knockdown significantly decreased the cell viability of MHCC97H cells. (J) Overexpression of circRASGRF2 promoted the proliferative ability of Hep3B cells. (K) Colony formation assays revealed that circRASGRF2 knockdown greatly attenuated the numbers of visible colonies of MHCC97H cells. (L) Colony formation assays revealed that overexpression of circRASGRF2 greatly increased the numbers of visible colonies of Hep3B cells. All tests were performed at least three times. Data are expressed as mean ± SD. ∗∗p < 0.01, ∗∗∗p < 0.001.

circRASGRF2 enhances proliferation of HCC cells

Given that circRASGRF2 exhibited the highest expression in MHCC97H cells and the lowest expression in Hep3B cells, we knocked down circRASGRF2 in MHCC97H cells by designing small interfering RNA (siRNA) oligonucleotides that would target the unique backsplice junction and overexpressed circRASGRF2 in Hep3B cell lines. The backsplice junction-specific siRNAs successfully decreased circRASGRF2 expression but did not affect the linear RASGRF2 mRNA level in MHCC97H cells. Compared with the negative control siRNA, the expression of circRASGRF2 was only downregulated by si-circRASGRF2 but was not affected by si-RASGRF2 (Figure 2G). Also, the expression vector markedly increased the expression of circRASGRF2 compared with the empty vector (Figure 2H). Functionally, cell counting kit-8 (CCK-8) assays showed that circRASGRF2 knockdown significantly decreased the cell viability of MHCC97H cells (Figure 2I), whereas overexpression of circRASGRF2 promoted the proliferative ability of Hep3B cells (Figure 2J). Similarly, colony formation assays revealed that circRASGRF2 knockdown greatly attenuated the numbers of visible colonies of MHCC97H cells (Figure 2K), whereas ectopic expression of circRASGRF2 in Hep3B cells led to an opposite result (Figure 2L).

Downregulation of circRASGRF2 induces cell cycle arrest and apoptosis in HCC cells

To further explore the role of circRASGRF2 in HCC progression, cell cycle, apoptosis, and autophagy were investigated after circRASGRF2 knockdown or overexpression in vitro. The cell cycle analysis showed that more MHCC97H cells were distributed in the G₁ phase and less in the S phase after silencing circRASGRF2, which suggested that MHCC97H cells were arrested at the G₁ phase by si-circRASGRF2 (Figure 3A). However, fewer Hep3B cells were distributed in the G₁ phase and more in the S phase after overexpression of circRASGRF2 (Figure 3B). Next, we assessed the effect of circRASGRF2 knockdown or overexpression on apoptosis of HCC cells. Flow cytometry analysis with annexin V/propidium iodide (PI) double staining showed that the apoptotic rates of MHCC97H cells in the si-circRASGRF2 group were significantly higher than those of the control group cells (Figure 3C). Conversely, apoptosis of transfected Hep3B cells was significantly reduced compared to the normal control group (Figure 3D). Furthermore, a TUNEL (terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling) assay indicated that downregulation of circRASGRF2 significantly increased the number of TUNEL-positive cells compared with control groups (Figure 3E). However, overexpression of circRASGRF2 decreased the number of TUNEL-positive cells of Hep3B cells (Figure 3D). These data collectively demonstrated that circRASGRF2 regulates cell cycle and apoptosis of HCC cells.

Knockdown of circRASGRF2 inhibits HCC growth in vivo

To identify the effect of circRASGRF2 on tumor growth in vivo, the impact of circRASGRF2 knockdown upon tumor growth in vivo was investigated. A xenograft tumor model of MHCC97H cells was developed and then treated with intratumoral injection of cholesterol-conjugated si-circRASGRF2 or negative control siRNA (si-NC). We found that treatment with si-circRASGRF2 significantly inhibited tumor volumes and weights of MHCC97H in vivo (Figures 4A and 4B). After harvesting the subcutaneous tumor tissues, immunohistochemistry (IHC) was performed. Results of IHC revealed that xenograft tumors derived from MHCC97H cells with circRASGRF2 knockdown had lower expression of Ki67 and higher expression of TUNEL compared with control groups (Figure 4C). In addition, we established a nude mice xenograft model by implanting Hep3B cells with vector or circRASGRF2. The tumor volumes were monitored from the 14 days after Hep3B cell injection. We found that overexpression of circRASGRF2 drastically increased tumor growth of Hep3B cells. The tumor volumes and weights were significantly accelerated by circRASGRF2 (Figures 4D and 4E). Results of IHC revealed that xenograft tumors derived from Hep3B cells with overexpression of circRASGRF2 had lower expression of TUNEL and higher expression of Ki67 than did the vector group (Figure 4F). Taken together, these findings suggest that circRASGRF2 may play an oncogenic role in HCC in vivo.

circRASGRF2 promotes migration and invasion of HCC cells in vitro and enhances the metastasis of xenograft tumors of HCC cells in vivo

The mobility of HCC cells was prominently decreased by downregulation of circRASGRF2, and the effect was also confirmed by transwell migration and invasion assays in MHCC97H cells (Figure 5A). Nevertheless, overexpression of circRASGRF2 promoted the migration and invasion of Hep3B cells (Figure 5B). These data collectively demonstrated that circRASGRF2 promoted cell migration and invasion in HCC in vitro.

To measure the effect of silencing circRASGRF2 expression on epithelial-to-mesenchymal transition (EMT) of HCC cells, western blot was performed to examine the expression of EMT-related markers in HCC cells after inhibition of circRASGRF2. As expected, circRASGRF2 knockdown remarkably increased the expression of E-cadherin and meanwhile greatly decreased the expression of N-cadherin and Vimentin in MHCC97H cells (Figure 5C). Correspondingly, overexpression of circRASGRF2 decreased the expression of E-cadherin and meanwhile greatly increased the expression of N-cadherin and vimentin in Hep3B cells, indicating that downregulation of circRASGRF2 obviously blocked the EMT process (Figure 5D).

To further confirm the above findings in vivo, a lung metastasis model was introduced by inoculating MHCC97H cells with modified circRASGRF2 expression directly into the tail veins of nude mice. Luciferase signals were monitored to observe location and growth of tumor xenografts in the lung. As a result, at 35 days, luciferase signals were lower in the circRASGRF2 knockdown group compared with those in the control group, and circRASGRF2 depletion significantly reduced the lung metastasis burden of MHCC97H cells (Figures 5E and 5F). All of these findings suggest a crucial role of circRASGRF2 in promoting HCC cell invasion and metastasis.

circRASGRF2 may function as a sponge for miR-1224

Given that circRNAs have been reported to function as sponges for microRNAs (miRNAs) and that circRASGRF2 is stable and located in the cytoplasm, we tried to explore whether circRASGRF2 could bind to miRNAs. First, we conducted RNA immunoprecipitation (RIP) with an antibody against Argonaute 2 (AGO2) in MHCC97H and Hep3B cells. RIP showed that circRASGRF2, but not circANRIL (a circular RNA reported not to bind to AGO2), was significantly enriched by the AGO2 antibody (Figure 6A). This result suggested that circRASGRF2 may act as a binding platform for AGO2 and miRNAs. We therefore analyzed the sequence of circRASGRF2 using miRanda, PITA, and RNAhybrid and identified five candidate miRNAs by overlapping the prediction results of the miRNA recognition elements in the circRASGRF2 sequence (miRNA-1224, miRNA-1204, miRNA-1208, miRNA-1231, and miRNA-1236). We purified the circRASGRF2-associated RNAs, by circRIP, using probes specifically against circRASGRF2, and analyzed the five candidate miRNAs in the complex. We found a specific enrichment of circRASGRF2/miR-1224 compared with the controls, while the other miRNAs had no enrichment, indicating that miR-1224 is the circRASGRF2-associated miRNA in HCC cells (Figure 6B).

circRASGRF2 acted as a miR-1224 sponge in HCC cells

(A) Endogenous circRASGRF2 was efficiently pulled down by anti-Ago2. (B) miRNA pull-down assay showed that circRASGRF2 was only efficiently enriched by miR-1224. (C) Binding sequence between miR-1224 and circRASGRF2. (D) The luciferase reporter systems showed that the miR-1224 mimic considerably reduced the luciferase activity of the WT-circRASGRF2 luciferase reporter vector compared with the negative control, while the miR-1224 mimic did not pose any impact on the luciferase activity of MUT-circRASGRF2-transfected MHCC97H cells. (E) circRASGRF2 and miR-1224 simultaneously existed in the production precipitated by anti-AGO2. (F) Silencing of circRASGRF2 did not affect the expression of miR-1224. (G) Transfection of miR-1224 mimics did not affect the expression of circRASGRF2. (H) The level of miR-1224 was significantly decreased in HCC cells compared to L02 cells. (I) The level of miR-1224 was significantly decreased in HCC tissues compared to ANL tissues. (J) miR-1224 could significantly reverse the circRASGRF2 overexpression-mediated promotion of proliferation. All tests were performed at least three times. Data are expressed as mean ± SD. ∗∗p < 0.01.

To validate the interaction between circRASGRF2 and miR-1224, we obtained the binding sequence between miR-1224 and circRASGRF2 (Figure 6C). Then, we performed a luciferase assay using these miR-1224 mimics that were co-transfected with the luciferase reporters into HEK293 T cells. Compared with the control RNA, miR-1224 reduced the luciferase reporter activity by at least 60% (Figure 6D, p < 0.01). We then mutated the target sites for miR-1224 from the luciferase reporter, and the luciferase activity did not significantly change after transfection of the corresponding miRNA into HEK293 T cells (Figure 6D). In addition, the RIP assay revealed that miR-1224 was efficiently pulled down by the anti-AGO2 antibody but not by the nonspecific anti-immunoglobulin G (IgG) antibody (Figure 6E). Furthermore, silencing of circRASGRF2 did not affect the expression of miR-1224, and transfection of miR-1224 mimics did not affect the expression of circRASGRF2 (Figures 6F and 6G), which indicated circRASGRF2 functions as a miRNA sponge without affecting the expression of sponged miRNAs.

Then, we detected markedly decreased expression of miR-1224 in HCC cells in comparison to L02 cells (Figure 6H). Consistently, tumor tissues in HCC samples also displayed lower miR-1224 expression than that in ANL tissues (Figure 6I). To functionally confirm that circRASGRF2 promotes HCC progression by sponging miR-1224, we transfected miR-1224 mimics into circRASGRF2-overexpressing cells to examine whether the tumor-promoting effect of circRASGRF2 overexpression could be reversed by miR-1224 mimics. The results showed that miR-1224 could significantly reverse the circRASGRF2 overexpression-mediated promotion of proliferation (Figure 6J, p < 0.01). These data suggested that circRASGRF2 might exert its functions by sponging miR-1224 in HCC.

FAK was positively regulated by circRASGRF2/miR-1224

To validate whether circRASGRF2 sponges miR-1224 and liberates the expression of its downstream target, we identified five target genes of miR-1224 by overlapping the prediction results of the four algorithms (miRanda, RNAhybrid, miRWalk, and TargetScan) prediction, and miR-1224 could target the 3′ UTRs of KLF3, Nfib, Sp1, FAK, and HGF (Figure 7A). To further verify the downstream targets of circRASGRF2, mRNA levels of five candidate target genes were detected after silencing circRASGRF2, and we found that only FAK was downregulated (Figure 7B). To verify whether FAK was the direct target of miR-1224, we first performed a miRNA biotin pull-down assay. We found that miR-1224 could significantly enrich the 3′ UTR of FAK mRNA. FAK is a focal adhesion-associated protein kinase involved in cellular adhesion and spreading processes. Gene set enrichment analysis (GSEA) showed that the gene set of KEGG_FOCAL ADHESION_PATHWAY was significantly enriched in the si-circRASGRF2 group compared to the si-NC group, indicating that this gene set could be significantly associated with circRASGRF2 expression in HCC (Figure 7C). To verify whether the 3′ UTR of FAK mRNA was a target of miR-1224, a luciferase reporter gene assay was used. The wild-type (WT) 3′ UTR sequence or mutant (MUT) 3′ UTR sequence of FAK was cloned into a luciferase reporter vector. The luciferase activity was significantly inhibited by the miR-1224 mimics in WT 3′ UTR sequence-transfected HEK293 T cells. Conversely, the luciferase activity was not inhibited by the miR-1224 mimics in MUT 3′ UTR sequence-transfected HEK293 T cells (Figures 7D and 7E). These results suggested that miR-1224 binds to the 3′ UTR of FAK and directly downregulates FAK expression.

To further confirm the effects of circRASGRF2 on FAK expression, MHCC97H cells were transfected with the circRASGRF2 siRNA, and the FAK mRNA levels were detected. The results showed that knockdown of circRASGRF2 expression significantly reduced the FAK mRNA and protein levels in MHCC97H cells (Figure 7F). Moreover, inhibition of the circRASGRF2-mediated decrease of FAK mRNA and protein expression was significantly recuperated following miR-1224 inhibitors (Figure 7F). To determine the expression levels of FAK in HCC, we analyzed the HCC dataset from The Cancer Genome Atlas (TCGA) database and found that the level of FAK was significantly upregulated in HCC tissue compared with normal liver tissue (Figure 7G). To explore the functional role of FAK in the control of HCC, we knocked down FAK in MHCC97H cells by two short hairpin RNAs (shRNAs) (Figure 7H). MHCC97H cells transduced with FAK shRNAs had a slower growth rate (Figure 7I). Moreover, as a counterweight to the increase of cells populations in the G₀/G₁ and S phases, the proportion of cells in the G₂/M phase was lower in shRNA (sh)FAK than in control HCC cells (Figure 7J) coherently with an increased early apoptosis (Figure 7K). All of these data made us draw a conclusion that circRASGRF2 positively regulated FAK expression by interacting with miR-1224 in HCC cells.

Discussion

Recently, the extensive distribution and functional regulation of circRNAs have been wildly validated in human cancers.10, 11, 12, 13 HCC is one of the most common human cancers, accounting for 70%–85% of primary liver malignancies, and it is the third leading cause of cancer-related death worldwide. To date, only a few circRNAs have been reported in HCC.¹⁴^,¹⁵ In this study, we screened the circRNAs that are differentially expressed between HCC and matched ANL tissues by RNA-seq, focusing on the expression of circRASGRF2, and we gradually confirmed the regulatory role of circRASGRF2 and its sponging effect for miRNAs in HCC through functional and molecular experiments. We found that circRASGRF2 was upregulated and positively correlated with poorer tumor differentiation, more advanced tumor stage, tumor size, and the presence of microvascular invasion (Table 1). High expression of circRASGRF2 was further identified in different HCC cells. After downregulation and overexpression of circRASGRF2, we further confirmed its oncogenic role in cellular functions. Functionally, circRASGRF2 promotes tumor growth of HCC cells in vitro and in vivo, implying that circRASGRF2 is a tumor promoter in HCC. Due to the stable loop structure, great resistance to exoribonuclease, and high abundance in the cytoplasm, circRASGRF2 may be an efficient diagnostic and therapeutic target and a promising biomarker for prognosis in HCC.

Several studies suggest that circRNAs regulate gene expression and participate in numerous cellular processes by sponging miRNAs.¹⁶^,¹⁷ circRNAs contain one or more miRNA response elements (MREs) that act as miRNA sponges to negatively modulate miRNA activity, attenuating the inhibitory effect on their target genes.¹⁸ In the present study, bioinformatics analysis showed that circRASGRF2 and FAK share a MRE of miR-1224, implying formation of the circRASGRF2/miR-1224/FAK axis. In the current study, in vitro precipitation, a luciferase reporter assay, a RIP assay, and biotin-coupled miRNA capture were undertaken to reveal the direct interaction between circRASGRF2 and miR-1224. These findings suggested that circRASGRF2 acts as a miRNA sponge, interacting with miR-1224 and suppressing the activation of miR-1224. Likewise, loss- and gain-of-function assays and luciferase reporter assays demonstrated that miR-1224 directly suppresses FAK expression through binding to the 3′ UTR of FAK mRNA. The circRNA-miRNA-mRNA axis has been reported to be associated with cancer progression.⁸^,²⁰ circRASGRF2 regulates FAK expression by sponging miR-1224 because miR-1224 inhibition releases the inhibitory effect of circRASGRF2 deficiency on FAK expression.

Recently, a few studies have implicated that FAK transmits extracellular stimuli to phosphatidylinositol 3-kinase (PI3K)/Akt, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and Rho-family small guanosine triphosphatase signaling, contributing to the fundamental cell biological processes, such as cell adhesion, migration, proliferation, and survival.²¹ FAK is a nonreceptor tyrosine kinase that participates in FA complex formation. Its dysregulation is found in various types of cancer in relation to tumor metastasis.²² Many kinases act downstream of integrins to promote metastasis of HCC, including FAK and SRC.²³ We found that FAK also exerts cancer-promoting actions in HCC.

Taking the results together, we showed that circRASGRF2 expression is elevated in HCC and revealed that the circRNA functions as an oncogene. circRASGRF2 mechanically sponges miR-1224 expression, resulting in the upregulation of FAK and HCC progression. Furthermore, our results advocate the circRASGRF2-miR-1224-FAK axis as a potential research avenue for new drug development strategies in HCC.

Materials and methods

Patient specimens

We obtained 68 paired HCC tissues and adjacent normal tissues from the Department of Hepatobiliary Surgery, The Affiliated Drum Tower Hospital of Nanjing University Medical School. Samples receiving chemotherapy or radiotherapy before collection were excluded. The tissue samples were confirmed by two histopathologists. All samples were immediately snap-frozen in liquid nitrogen and subsequently stored at −80°C until RNA extraction and protein isolation. Written consent approving the usage of HCC tissues and serum in our study was obtained from each patient. This study was approved by the Ethics Committee of The First Affiliated Hospital of Nanjing Medical University.

Cell culture

The human liver cell line of L02 (normal), human HCC cell lines of Hep3B with low invasiveness, and highly invasive HLE, Huh7, BEL7402, SMCC7721, MHCC97L, MHCC97H, HCCLM3, and HCCLM6 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). All cell lines were grown routinely in Roswell Park Memorial Institute (RPMI) 1640 medium (Invitrogen, CA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen). Cells were cultured in a humidified incubator at 37°C with 5% CO₂. The culture medium was replaced every 3 days. Cells were passaged as soon as the cell attachment rate reached 80%–90%.

RNA sequencing of circRNA extracted from human HCC tissues

The total RNA was extracted from five pairs of fresh-frozen HCC tissues and ANL tissues using TRIzol reagent (Takara, Dalian, China). Next, RNA was purified by rRNA depletion, followed by cDNA synthesis and RNA amplification. The RNA-seq libraries were constructed and sequenced utilizing the Illumina HiSeq2500 platform (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol.

Bioinformatics analysis

Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images. Data processing were performed using the R software package. Differentially expressed circRNAs with statistical significance between two groups were identified through volcano plot filtering. Differentially expressed circRNAs between two samples were identified through fold change filtering. The circRNA/miRNA interaction was predicted with Arraystar’s home-made software based on miRanda, PITA, and RNAhybrid.

RNA extraction, nuclear-cytoplasmic fractionation, RNase R and actinomycin D treatment, and quantitative real-time PCR assays

Total RNA was extracted from tissues and cultured cells using the RNAiso Plus (Takara, Japan) according to the manufacturer’s protocol. RNA isolation of nuclear and cytoplasmic fractions was performed with NE-PER nuclear and cytoplasmic extraction reagents (Thermo Scientific, USA) according to the manufacturer’s protocol. RNase R treatment was executed at 37°C with 4 U/μg of RNase R (Epicenter Biotechnologies, Madison, WI, USA) for 10, 20, 30, and 40 min, respectively. In addition, total RNA from HCC cells was treated with 100 ng/mL actinomycin D (Cell Signaling Technology, Beverly, MA, USA) against new RNA synthesis for 12 and 24 h. RNA was reversed transcribed into cDNAs with the PrimeScript RT reagent kit (Takara, Dalian, China) according to the manufacturer’s instructions. Reverse transcription was performed with PrimeScript RT master mix (Takara, Japan) for circRNA and mRNA. For miRNA, cDNA was synthesized using a miRNA first-strand cDNA synthesis kit (Sangon Biotech, China). Subsequently, the cDNA was subjected to real-time PCR on a QuantStudio Dx system (Applied Biosystems, Singapore). GAPDH and small nuclear U6 were used as internal controls. The 2^-ΔΔCt method was used to calculate relative expression. The sequences of primers for detection of circRASGRF2 are as follows: forward, 5′-CACGCGTGGATCTGTGTAAC-3′; reverse, 5′-TGAATGGCCTCCATCCACTC-3′.

Oligonucleotides, vectors, and transfection

siRNAs that were used to knock down endogenous circRASGRF2 expression (si-circRASGRF2) and si-NC were synthesized by RiboBio (Guangzhou, China). A miR-1224 mimic, miRNA negative control (miR-NC), miR-1224 inhibitor (in-miR-1224), and negative control inhibitor (inmiR- NC) were acquired from GenePharma (Shanghai, China). For the overexpression of circRASGRF2, the sequences of circRASGRF2 were inserted into vector pcDNA3.1 to construct pcDNA3.1/circRASGRF2 (termed circRASGRF2), and empty vector was considered as a negative control in this study. HCC cells were seeded in six-well plates, and cell transfection was conducted using the Lipofectamine 2000 reagent (Invitrogen). Cells transfected with the aforementioned oligonucleotide(s) or plasmid(s) were harvested and used in subsequent experiments.

Cell growth assay

Cell growth was assessed using CCK-8 (CK04; Dojindo, Kumamoto, Japan). A total of 1,500 cells/well were seeded in 96-well plates. After cells had adhered to the wells, CCK-8 reagent was added to 100 μL of RPMI 1640 medium with 10% FBS in each well and cells were incubated at 37°C for 3 h. Absorbance was then detected at 450 nm (A₄₅₀) using a microplate reader (Tecan, Mechelen, Belgium).

Colony formation assay

Cells were cultured in six-well plates (2,000–3,000 cells per well) in complete medium for 7–12 days, depending on the sizes of the colonies. The cells were then fixed with methanol for 15 min and stained using 0.1% crystal violet for 1 h.

Cell cycle and apoptosis assays

For cell cycle analysis, cells were harvested and fixed in pre-cold 70% ethanol at 4°C overnight, then stained with PI and measured by flow cytometry (BD FACSCalibur, BD Biosciences, NY, USA). The cells were stained with annexin V-fluorescein isothiocyanate (FITC) and PI, and subsequently the ratio of apoptotic cells was tested by flow cytometry. In addition, cell apoptosis was further detected with TUNEL (Beyotime, Shanghai, China) according to the manufacturer’s protocols. The images were observed under a fluorescence microscope (Leica, Wetzlar, Germany). All experiments were performed independently in triplicate.

Cell migration and invasion assay

Transwell migration and invasion abilities were assessed using 8-μm pore transwell chambers either without Matrigel (for migration assays) or with Matrigel (for invasion assays). Each well of the upper chamber was filled with 300 μL of serum-free RPMI 1640 medium containing 1 × 10⁵ infected cells/mL. The lower chambers contained 700 μL of complete medium. The cells were fixed in 4% paraformaldehyde and stained with crystal violet after a 48-h incubation. The cells remaining in the top chamber were removed with cotton swabs and imaged by light microscopy (Olympus, China). All assays were performed three times independently using triplicate wells.

Western blotting analysis

Cells were washed twice with phosphate-buffered saline and solubilized in lysis buffer. Aliquots of approximately 30 μg of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes; the membranes were blocked with 5% bovine serum albumin in Tris-buffered saline with Tween 20, then incubated with primary antibodies at 4°C overnight. Subsequently, the membranes were hybridized with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1.5 h, then washed in Tris-buffered saline with Tween 20. The signal density was visualized on FluorChem E (ProteinSimple, San Jose, CA, USA).

Xenograft model

For the xenograft model of MHCC97H cells, 2 × 10⁶ MHCC97H cells were subcutaneously injected into a single flank of each mouse (12 mice in total). Two weeks later, mice with palpable tumors were randomly divided into two groups (six mice per group), and 50 nmol of cholesterol-conjugated si-NC or si-circRASGRF2 was intratumorally injected into the two groups three times per week for 2 weeks. Tumor growth was examined every 4 days. After mice were sacrificed, tumors were weighed and processed for further histological analysis. Tumor volume was calculated as follows: V (volume) = (length × width2)/2. All animal experiments were performed under approval by the Experimental Animal Care Commission of The Affiliated Drum Tower Hospital of Nanjing University Medical School.

For the xenograft model of Hep3B cells, 6-week-old male BALB/c nude mice were housed under standard conditions and cared for according to protocols. 2 × 10⁶ Hep3B cells with circRASGRF2 overexpressed vector or control vector were suspended in 200 μL of serum-free RPMI 1640 and subcutaneously injected into the right flank of each mouse. The volumes of tumors were measured from 14 days after injections. After 31 days the mice were sacrificed.

IHC

The sections were deparaffinized, rehydrated, and incubated in 3% hydrogen peroxide at room temperature for 10 min. Antigens were retrieved in 0.01 mol/L citric buffer (pH 6.0) at 95°C–98°C for 25 min. Slides were cooled down for 1 h before blocking with a preferred blocking solution for 30 min at room temperature. Staining with primary antibodies was performed at 4°C overnight. Each sample was measured by the intensity (0, negative; 1, weak; 2, moderate; and 3, strong) and the percentage of positive cells (0, 0%; 1, 1%–25%; 2, 26%–50%; 3, 51%–75%; and 4, 76%–100%). The final IHC score = intensity score × percentage score. The following antibody was used for IHC: Ki67 (A2094; ABclonal).

RIP

The EZMagna RIP kit (Millipore) was used following the manufacturer’s protocol. Cells were lysed in complete RIP lysis buffer, and the cell extract was incubated with magnetic beads conjugated with anti-Argonaute 2 (AGO2) or control anti-IgG antibody (Millipore) for 6 h at 4°C. The beads were washed and incubated with proteinase K to remove proteins. Finally, purified RNA was subjected to quantitative real-time PCR analysis.

Biotin-coupled miRNA capture

Briefly, the 30-end biotinylated miR-RNA mimic or control biotin-RNA (RiboBio) was transfected into cells at a final concentration of 20 nmol/L for 1 day. The biotin-coupled RNA complex was pulled down by incubating the cell lysate with streptavidin-coated magnetic beads (Ambion, Life Technologies). The abundance of circRNA in bound fractions was evaluated by quantitative real-time PCR analysis.

Luciferase reporter assay

circRASGRF2 sequences harboring the WT or MUT miR-1224-binding site were amplified by GenePharma and inserted (separately) into the psi-CHECK2 luciferase reporter vector (Promega, Madison, WI, USA). The generated luciferase reporter vectors are referred to as circRASGRF2-WT and circRASGRF2-MUT, respectively. For the reporter assay, Lipofectamine 2000 reagent was used to co-transfect HEK293 T cells with either the miR-1224 mimic or miR-NC and either circRASGRF2-WT or circRASGRF2-MUT. Luciferase activities were detected with the Dual-Luciferase reporter assay (Promega Corporation) at 48 h post-transfection. Renilla luciferase activity was utilized as an internal control for firefly luciferase activity.

TCGA dataset analysis

The data and the corresponding clinical information for patients were collected from TCGA database (https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga). We used the edgeR package of R packages to perform the difference analysis (http://www.bioconductor.org/packages/release/bioc/html/edgeR.html) and used the pheatmap package of R packages to perform the cluster analysis (https://cran.r-project.org/web/packages/pheatmap/index.html). The sva R package was used to remove the batch effect. Genes with adjusted p values <0.05 and absolute fold changes (FC) >1.5 were considered differentially expressed genes. Kaplan-Meier survival curves were drawn to analyze the relationships between genes and overall survival in the survival package. The corresponding statistical analysis and graphics were performed in R software (R version 3.3.2).

GSEA

GSEA was also performed using the Broad Institute GSEA version 3.0 software. The gene sets used for the enrichment analysis were downloaded from the Molecular Signatures Database (MsigDB, https://software.broadinstitute.org/gsea/index.jsp).

Statistical analysis

All statistical analyses were performed with SPSS 20.0 and GraphPad Prism 7.0 software. Associations of the expression of circRASGRF2 with clinicopathological parameters were evaluated by a chi-square test. Other statistical significance between groups was determined by a Student’s t test or a one-way ANOVA test. The ROC and AUC were used to estimate the diagnostic value of each index for HCC. A combined ROC was calculated based on the logistic regression model. A p value <0.05 was regarded as statistically significant.

Acknowledgments

The study is supported by the National Natural Science Foundation of Jiangsu Province of China (BK20131445), the Scientific Research Program of Ministry of Health (201302009), Jiangsu Province’s Key Provincial Talents Program (RC201152).

Author contributions

Conception and design, G.L.; in vitro and in vivo experiments, D.W.; analysis and interpretation of data, A.X.; writing, review, and revision, T.F.

Declaration of interests

The authors declare no competing interests.

References

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[bib3] 3.Chen L.L. The biogenesis and emerging roles of circular RNAs. Nat. Rev. Mol. Cell Biol. 2016;17:205–211. doi: 10.1038/nrm.2015.32. [DOI] [PubMed] [Google Scholar]

[bib4] 4.Jeck W.R., Sharpless N.E. Detecting and characterizing circular RNAs. Nat. Biotechnol. 2014;32:453–461. doi: 10.1038/nbt.2890. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib5] 5.Han B., Chao J., Yao H. Circular RNA and its mechanisms in disease: from the bench to the clinic. Pharmacol. Ther. 2018;187:31–44. doi: 10.1016/j.pharmthera.2018.01.010. [DOI] [PubMed] [Google Scholar]

[bib7] 7.Jeck W.R., Sorrentino J.A., Wang K., Slevin M.K., Burd C.E., Liu J., Marzluff W.F., Sharpless N.E. Circular RNAs are abundant, conserved, and associated with ALU repeats. RNA. 2013;19:141–157. doi: 10.1261/rna.035667.112. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib8] 8.Memczak S., Jens M., Elefsinioti A., Torti F., Krueger J., Rybak A., Maier L., Mackowiak S.D., Gregersen L.H., Munschauer M. Circular RNAs are a large class of animal RNAs with regulatory potency. Nature. 2013;495:333–338. doi: 10.1038/nature11928. [DOI] [PubMed] [Google Scholar]

[bib9] 9.Conn S.J., Pillman K.A., Toubia J., Conn V.M., Salmanidis M., Phillips C.A., Roslan S., Schreiber A.W., Gregory P.A., Goodall G.J. The RNA binding protein quaking regulates formation of circRNAs. Cell. 2015;160:1125–1134. doi: 10.1016/j.cell.2015.02.014. [DOI] [PubMed] [Google Scholar]

[bib10] 10.Gan X., Zhu H., Jiang X., Obiegbusi S.C., Yong M., Long X., Hu J. circMUC16 promotes autophagy of epithelial ovarian cancer via interaction with ATG13 and miR-199a. Mol. Cancer. 2020;19:45. doi: 10.1186/s12943-020-01163-z. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib11] 11.Liu Y.P., Wan J., Long F., Tian J., Zhang C. circPVT1 facilitates invasion and metastasis by regulating miR-205-5p/c-FLIP axis in osteosarcoma. Cancer Manag. Res. 2020;12:1229–1240. doi: 10.2147/CMAR.S231872. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib12] 12.Wei J., Xu H., Wei W., Wang Z., Zhang Q., De W., Shu Y. circHIPK3 promotes cell proliferation and migration of gastric cancer by sponging miR-107 and regulating BDNF expression. OncoTargets Ther. 2020;13:1613–1624. doi: 10.2147/OTT.S226300. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]

[bib13] 13.Cao L., Wang M., Dong Y., Xu B., Chen J., Ding Y., Qiu S., Li L., Karamfilova Zaharieva E., Zhou X., Xu Y. Circular RNA circRNF20 promotes breast cancer tumorigenesis and Warburg effect through miR-487a/HIF-1α/HK2. Cell Death Dis. 2020;11:145. doi: 10.1038/s41419-020-2336-0. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib14] 14.Wei X., Zheng W., Tian P., He Y., Liu H., Peng M., Li X., Liu X. Oncogenic hsa_circ_0091581 promotes the malignancy of HCC cell through blocking miR-526b from degrading c-MYC mRNA. Cell Cycle. 2020;19:817–824. doi: 10.1080/15384101.2020.1731945. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib15] 15.Li Z., Liu Y., Yan J., Zeng Q., Hu Y., Wang H., Li H., Li J., Yu Z. Circular RNA hsa_circ_0056836 functions an oncogenic gene in hepatocellular carcinoma through modulating miR-766-3p/FOSL2 axis. Aging (Albany NY) 2020;12:2485–2497. doi: 10.18632/aging.102756. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]

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[bib17] 17.Piwecka M., Glazar P., Hernandez-Miranda L.R., Memczak S., Wolf S.A., Filipchyk A., Klironomos F., Cerda Jara C.A., Fenske P., Trimbuch T. Loss of a mammalian circular RNA locus causes miRNA deregulation and affects brain function. Science. 2017;357 doi: 10.1126/science.aam8526. eaam8526. [DOI] [PubMed] [Google Scholar]

[bib18] 18.Zheng Q., Bao C., Guo W., Li S., Chen J., Chen B., Luo Y., Lyu D., Li Y., Shi G. Circular RNA profiling reveals an abundant circHIPK3 that regulates cell growth by sponging multiple miRNAs. Nat. Commun. 2016;7:11215. doi: 10.1038/ncomms11215. [DOI] [PMC free article] [PubMed] [Google Scholar]

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[bib21] 21.Chen Y., Li Q., Tu K., Wang Y., Wang X., Liu D., Chen C., Liu D., Yang R., Qiu W., Kang N. Focal adhesion kinase promotes hepatic stellate cell activation by regulating plasma membrane localization of TGFβ receptor 2. Hepatol. Commun. 2019;4:268–283. doi: 10.1002/hep4.1452. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib22] 22.Yu H.G., Nam J.O., Miller N.L., Tanjoni I., Walsh C., Shi L., Kim L., Chen X.L., Tomar A., Lim S.T., Schlaepfer D.D. p190RhoGEF (Rgnef) promotes colon carcinoma tumor progression via interaction with focal adhesion kinase. Cancer Res. 2011;71:360–370. doi: 10.1158/0008-5472.CAN-10-2894. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib23] 23.Leng C., Zhang Z.G., Chen W.X., Luo H.P., Song J., Dong W., Zhu X.R., Chen X.P., Liang H.F., Zhang B.X. An integrin beta4-EGFR unit promotes hepatocellular carcinoma lung metastases by enhancing anchorage independence through activation of FAK-AKT pathway. Cancer Lett. 2016;376:188–196. doi: 10.1016/j.canlet.2016.03.023. [DOI] [PubMed] [Google Scholar]

PERMALINK

This article has been retracted.

circRASGRF2 functions as an oncogenic gene in hepatocellular carcinoma by acting as a miR-1224 sponge

Di Wu

Anliang Xia

Tianlong Fan

Guoqiang Li

Abstract

Graphical Abstract

Introduction

Results

circRNA profiling in human HCC tissues and circRASGRF2 characterization

Figure 1.

circRASGRF2 was upregulated in HCC tissues and cell lines

Table 1.

Confirmation of subcellular localization of circRASGRF2

Figure 2.

circRASGRF2 enhances proliferation of HCC cells

Downregulation of circRASGRF2 induces cell cycle arrest and apoptosis in HCC cells

Figure 3.

Knockdown of circRASGRF2 inhibits HCC growth in vivo

Figure 4.

circRASGRF2 promotes migration and invasion of HCC cells in vitro and enhances the metastasis of xenograft tumors of HCC cells in vivo

Figure 5.

circRASGRF2 may function as a sponge for miR-1224

Figure 6.

FAK was positively regulated by circRASGRF2/miR-1224

Figure 7.

Discussion

Materials and methods

Patient specimens

Cell culture

RNA sequencing of circRNA extracted from human HCC tissues

Bioinformatics analysis

RNA extraction, nuclear-cytoplasmic fractionation, RNase R and actinomycin D treatment, and quantitative real-time PCR assays

Oligonucleotides, vectors, and transfection

Cell growth assay

Colony formation assay

Cell cycle and apoptosis assays

Cell migration and invasion assay

Western blotting analysis

Xenograft model

IHC

RIP

Biotin-coupled miRNA capture

Luciferase reporter assay

TCGA dataset analysis

GSEA

Statistical analysis

Acknowledgments

Author contributions

Declaration of interests

References

ACTIONS

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