Has_circ_0008583 modulates hepatocellular carcinoma progression through the miR-1301-3p/METTL3 pathway.

Has_circ_0008583 is reported to be involved in the progression of hepatocellular carcinoma (HCC), while its biological role in HCC remains unclear. Here, the qRT-PCR was used to detect the expression of has_circ_0008583. The CCK-8 kit was performed to measure cell proliferation. The cell migration and invasion were evaluated by Transwell. A dual-luciferase reporter assay was performed to confirm the target combination between the genes in has_circ_0008583/miR-1301-3p/METTL3 axis. The in vivo role of has_circ_0008583 was verified by murine xenograft assay. Our data showed that hsa_circ_0008583 was upregulated in HCC tissues and cells. Hsa_circ_0008583 overexpression promoted Hep3B cell proliferation, migration and invasion, but hsa_circ_0008583 silencing had an opposing influence. MiR-1301-3p is directly bound to hsa_circ_0008583 and METTL3. MiR-1301-3p overexpression or METTL3 knockdown could partially counteract hsa_circ_0008583 overexpression-mediated influence on HCC cell behaviors. In addition, hsa_circ_0008583 depletion inhibits HCC tumor growth in vivo. In conclusion, hsa_circ_0008583 promotes HCC progression through the miR-1301-3p/METTL3 axis.

Introduction

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world [1,2]. About 30–40% of patients with HCC have received a potentially radical resection. However, the prognosis of these patients is still poor due to the high frequency of metastasis and recurrence [3,4]. The numerous published literature reported that the changes in cell function, tumor microenvironment, and abnormal activation of signaling pathways could promote the development of HCC [5,6], but there is still not enough information to understand the progress of HCC. Therefore, new target molecules are urgently needed for the application of diagnostic biomarkers and therapeutic drugs.

CircRNAs are endogenous RNAs with covalently closed-loop structures, which can be used as transcription and splicing regulators, protein scaffolds, and miRNA sponges to exert their biological functions [7,8]. A large number of studies have shown that the disorder of circRNA is related to the occurrence and development of cancer. For example, Zhu et al. [9] have found a significantly increased circ-0004277 in HCC tissues, cells, and plasma exosomes compared to that in normal controls. Further experiments proved that circ-0004277 could promote EMT of HCC. Wang et al. [10] identified hsa_circ_0009910, hsa_circ_0049783, and hsa_circ_0089172 were significantly upregulated in HCC tissues, which revealed that circRNAs was a potentially effective target for the treatment of HCC. Recently, Sun et al.’s study [11] showed that hsa_circ_0008583 is highly expressed in HCC tissues, but its role and mechanism in the development of HCC has not been reported. Here, we focused on the underlying working mechanism of hsa_circ_0008583 in HCC.

MiRNAs could regulate the progression of cancers by degrading or restraining the translation of target genes. MiR-1301-3p has been reported to act as a tumor suppressor in HCC [12-15]. Utilizing circinteractome databases, we found that miR-1301-3p is a potential target of hsa_circ_0008583. Therefore, we speculated that hsa_circ_0008583 may promote HCC progression through miR-1301-3p.

N6 methyladenosine (m6A) is one of the most common chemical modifications of eukaryotic messenger RNA (mRNA), which can regulate the stability, splicing, and translation of mRNA. Methyltransferase like 3 (METTL3) is a major RNA N6 adenosine methyltransferase, which is significantly increased in tumor tissues and contributes to tumor progression [16]. In particular, METTL3 was significantly increased in HCC and contributes to HCC progression [17]. Moreover, METTL3 was forecasted as a target of miR-1301-3p via starBase. Thus, we analyze the role of METTL3 in the hsa_circ_0008583-mediated HCC development.

In this study, we speculate that the expression of hsa_circ_0008583 was significantly upregulated in HCC tissues and cells. Additionally, gain-of-function experiments and loss-of-function experiments were carried out to explore the regulatory mechanism of hsa_circ_0008583 on the malignancy of HCC cells.

Material and methods

Clinical tissue samples

The specimens of HCC tissue (tumor) and paracancerous tissue (non-tumor) were collected from 12 HCC patients who underwent surgical resection in our hospital. The study was approved by the Medical Ethics Committee of the Second Affiliated Hospital of Nanchang University.

Cell culture and transfection

The human normal hepatocytes (LO2) and two hepatoma cell lines (Hep3B and Huh-7) were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China), cultured in DMEM medium containing 10% FBS, 100 µg/mL streptomycin, and 100 U/mL penicillin, and incubated in 5% CO2 at 37°C. Cell transfection was performed in Hep3B or Huh-7 cells using has_circ_0008583 expressing plasmid, si-has_circ_0008583, miR-1301-3p mimics, si-METTL3 or the controls (vector, si-NC, miR-NC, scramble), which were obtained by chemical synthesis (GenePharma, Shanghai, China).

Relative hsa_circ_0008583 expression analysis

Cells were digested and lysed, and total RNA isolation was extracted using TRIzol reagent (Thermo Fisher Scientific). Amplification reactions were conducted using the SYBR Green PCR Master Mix (Vazyme, Nanjing, China). Hsa_circ_0008583 primers were designed by the CircPrimer [18], as follows: 5ʹ-GGAGCAGATGATGAATAGT −3ʹ(F), 5ʹ- AAGAAGCTGACATAGAGGG−3ʹ(R); GAPDH primers as 5ʹ-CGGACCAATACGACCAAATCCG-3ʹ (F),5ʹ-AGCCACATCGCTCAGACACC-3ʹ (R). Relative expression was figured using the 2−ΔΔCt method and normalized to GAPDH.

Cell proliferation assay

After transfection for 24–48 h, the cells were collected and seeded into 96-well plates and cultured for 4 days. On each day, 10 µL of CCK-8 solution was added to each well and followed by incubation for another 2 h in a humidified incubator. The OD value was determined at 450 nm.

Cell apoptosis assay

The cells were collected, washed and then suspended in a binding buffer. Annexin V-Fluorescein isothiocyanate (FITC) and Propidium iodide (PI, Sigma-Aldrich, Corp. St. Louis, MO, USA) was then added to the binding buffer and incubated in the dark at room temperature for 15 minutes. Finally, the apoptosis rate of cells was analyzed by flow cytometry and cell quest software (BD Biosciences, Mountain View, CA).

Bioinformatics analysis and luciferase reporter assay

The binding sites between hsa_circ_0008583/METTL3 3ʹUTR and miR-1301-3p were, respectively, predicted by circinteractome databases and Starbase 2.0. hsa_circ_0008583 sequence or METTL3 3ʹUTR containing the miR-1301-3p binding sites were constructed into psiCHECK-2 vector (Promega), as wild type (WT) hsa_circ_0008583 or METTL3 3ʹUTR. Meanwhile, the miR-1301-3p binding site of hsa_circ_0008583 sequence/METTL3 3ʹUTR was mutated and introduced into psiCHECK-2 vector to construct mutant report vector, mutant (Mut) hsa_circ_0008583 or METTL3 3ʹUTR. Then, cells were co-transfected with miR-1301-3p mimic/mimic NC and these reporter vectors using Lipofectamine 2000 reagent. The luciferase activities were determined at 48 h post-transfection with the luciferase reporter assay kit (Promega).

Transwell migration and invasion assay

About 1 × 105 Hep3B and Huh-7 cells were cultured in the transwell upper chamber with a serum-free medium. For cell invasion assay, 2 mg/mL Matrigel (BD Biosciences) was used to pre-coat the insert. 600 µL of DMEM containing 10% FBS was added to the upper chamber. After 24 h of incubation, cells in the upper chamber were discarded with a cotton swab, and the cells left (invaded or migrated cells) were fixed in methanol and stained with crystal violet. Cells were then imaged, and the number of invaded or migrated cells was counted under an optical microscope.

Establishment of stably transfected Huh-7 cell line

For hsa_circ_0008583 knockdown, the lentivirus expressing siRNA for hsa_circ_0008583 (si-hsa_circ_0008583) was purchased from Shanghai GenePharma (China), the si-NC was used as the negative control. After cell infection in Huh-7 cells, using puromycin (SigmaAldrich) to screen cells, and the interference effect of hsa_circ_0008583 was detected by qRT-PCR.

Xenograft assay

Nine female BALB/c nude mice (4–6 weeks) were randomly divided into three groups (n = 3). These two groups of mice were, respectively, injected with Huh-7 cells stably expressing si-hsa_circ_0008583 or si-NC. The mice that were injected with no treatment Huh-7 cell as control. The Huh7 cells (1 × 107) were injected into the left anterior armpit of BALB/C nude mice. Tumor volume was measured every 5 days. The tumor volume formula: Volume = (length × width2)/2. Thirty days after injection, the mice were anesthetized with 50 mg/kg Nembutal and then killed by cervical dislocation. The tumor tissues of mice were excised and weighed. The experiment had been authorized by the Animal Ethics Committee of the Second Affiliated Hospital of Nanchang University.

Statistical analysis

The experiment was carried out three times, expressed as mean ± standard deviation (SD). Student's t-test or one-way ANOVA was used for comparison between the two groups or among multiple groups. P < 0.05 was classified as significant.

Results

Upregulation of Hsa_circ_0008583 expression in HCC

To investigate the role of hsa_circ_0008583 in HCC, we detected the expression of hsa_circ_0008583 in HCC tissues and cells. A conspicuous elevation in the abundance of hsa_circ_0008583 was found in HCC tissues (n = 12) compared with adjacent nontumor tissues (n = 12) (Figure 1(a)), implying that hsa_circ_0008583 might exert an important role in HCC. To disclose the functions of hsa_circ_0008583 at the cellular level, the enrichment of hsa_circ_0008583 in HCC cells (Hep3B and Huh-7) and normal hepatocytes (LO2) was detected by RT-PCR. Consistent with the tissue-level data, the hsa_circ_0008583 expression in HCC cells was extremely enhanced compared with that in normal hepatocytes (Figure 1(b)). The data suggested that hsa_circ_0008583 was involved in the development of HCC.

Figure 1.

Hsa_circ_0008583 was up-regulated in HCC tissues and cells. A. Relative hsa_circ_0008583 expression level in HCC tumor and adjacent tissues (n = 12). B. Relative hsa_circ_0008583 expression level in human normal hepatocytes (LO2) and two hepatoma cell lines (Hep3B and Huh-7). *p < 0.05,**p < 0.01 vs Non-tumor, #p < 0.05,##p < 0.01 vs LO2 cell.

Effects of altered hsa_circ_0008583 expression on the progression of HCC cell in vitro

To explore the effect of hsa_circ_0008583 on HCC cell proliferation, migration, and invasion, hsa_circ_0008583 was overexpressed in Hep3B cells and silenced in Huh-7 cells. Figure 2(a) shows that hsa_circ_0008583 was significantly upregulated by hsa_circ_0008583 overexpressing plasmid in Hep3B cells. Subsequently, we found that the proliferation of hsa_circ_0008583 overexpressing Hep3B cells increased significantly (Figure 2(b)). Hep3B cell migration and invasion capacity were enhanced by hsa_circ_0008583 overexpressing plasmid (Figure 2(c-d)). The apoptosis of hsa_circ_0008583 overexpressing Hep3B cells decreased significantly (Figure 2(e)). The data revealed that overexpressed hsa_circ_0008583 promoted the progression of HCC cells in vitro.

Figure 2.

Hsa_circ_0008583 overexpression increased the proliferation, migration and invasion of Hep3B cell. A. RT-PCR measured the overexpression efficiency of hsa_circ_0008583 in Hep3B cell. B. The cell proliferation was measured by CCK8. C and D. Transwell assay was used to evaluate the cell migration/invasion. E. The cell apoptosis was analyzed by Flow cytometry. **P < 0.01 vs vector.

Moreover, HCC cells were infected with si-hsa_circ_0008583 to observe the effect of hsa_circ_0008583 inhibition on the progression of HCC cells. Figure 3(a) shows that hsa_circ_0008583 was dramatically decreased by si-hsa_circ_0008583 in Huh-7 cells. Similarly, we found that the proliferation of hsa_circ_0008583 knockdown Huh-7 cells decreased significantly (Figure 3(b)). Transwell assays demonstrated that Huh-7 cell migration and invasion capacity were disturbed by si-hsa_circ_0008583 (Figure 3(c-d)). Meanwhile, the apoptosis of hsa_circ_0008583 knockdown Huh-7 cells increased significantly (Figure 3(e)). The data indicated that inhibition of hsa_circ_0008583 inhibited the growth of HCC cells in vitro.

Figure 3.

Hsa_circ_0008583 silencing decreased the proliferation, migration and invasion of Huh-7 cell. A. RT-PCR measured the efficiency of hsa_circ_0008583 silencing in Huh-7 cell. B. The cell proliferation was measured by CCK8. C and D. Transwell assay was used to evaluate the cell migration/invasion. E. The cell apoptosis was analyzed by Flow cytometry. **P < 0.01 vs si-NC.

Has_circ_0008583 accelerates the HCC cells progression through miR-1301-3p/METTL3 pathway

As the Circinteractome databases predict, hsa_circ_0008583 was a potential target of miR-1301-3p (Figure 4(a)). The transfection of miR-1301-3p mimics instead of mimic NC caused a dramatic downregulation in the luciferase activity of hsa_circ_0008583 WT transfected cells, but there was no obvious change in the luciferase activity of Hep3B cell in hsa_circ_0008583 Mut group with the co-transfection of mimic NC or miR-1301-3p mimic (Figure 4(b)), demonstrated that miR-1301-3p bound to hsa_circ_0008583 via the putative binding sites. Then, the METTL3 3ʹUTR possessed the complementary sites with miR-1301-3p (Figure 4(c)). As shown in Figure 4(d), the luciferase activity of miR-1301-3p mimics and METTL3 WT transfected cells were notably decreased relative to mimic NC and METTL3 WT transfected cells, suggesting the METTL3 was a target of miR-1301-3p.

Figure 4.

The relationship between hsa_circ_0008583/METTL3 and miR-1301-3p. A. The Circinteractome database showed the binding sites of miR-1301-3p on hsa_circ_0008583. B. The binding of miR-1301-3p and hsa_circ_0008583 was verified by dual-luciferase reporter assay. mC. The starBase showed the binding sites of miR-1301-3p and METTL3 3ʹUTR. D. The binding of miR-1301-3p and METTL3 3ʹUTR was verified by dual-luciferase reporter assay. **P < 0.01 vs. mimic NC.

Next, to prove that miR-1301-3p and METTL3 are involved in hsa_circ_0008583 mediated HCC cell progression, a series of rescue experiments were carried out. Figure 5(a) and Figure 7(a) show that the increased OD value at 450 nm mediated by hsa_circ_0008583 overexpression was ameliorated by miR-1301-3p mimic and si-METTL3, respectively. More importantly, hsa_circ_0008583 overexpression-mediated the increased migration and invasion ability (Figures 5(b-c), Figures 7(b-c)) are all alleviated by miR-1301-3p mimic and si-METTL3. And the decreased apoptosis mediated by hsa_circ_0008583 overexpression (Figures 5(d) and 7(d)) is increased by miR-1301-3p mimic and si-METTL3. In addition, the miR-1301-3p inhibitor could reverse the inhibitory effect of si-hsa_circ_0008583 on the proliferation, migration and invasion of Huh-7 cells (Figure 6). In a word, hsa_circ_0008583 modulates the progression of HCC cells by regulating the miR-1301-3p/METTL3 pathway.

Figure 5.

MiR-1301-3p mimic reverses the promoting effect of hsa_circ_0008583 on the proliferation, migration and invasion of hepatoma cells. Hep3B cells were transfected with vector+mimic NC, hsa_circ_0008583+ mimic NC, vector+miR-1301-3p mimic, or hsa_circ_0008583+ miR-1301-3p mimic. A. The proliferation of Hep3B cells was measured by CCK8. B and C. Transwell assay was used to evaluate the cell migration/invasion. D. The cell apoptosis was analyzed by Flow cytometry. **P < 0.01 vs vector+mimic NC; ##p < 0.01 vs hsa_circ_0008583+ mimic NC.

Figure 7.

Hsa_circ_0008583 regulated HCC cell proliferation, migration and invasion via METTL3. Hep3B cells were co-transfected with vector+Scramble, hsa_circ_0008583+ Scramble, vector+si-METTL3, or hsa_circ_0008583+ si-METTL3. A. The proliferation of Hep3B cells was measured by CCK8. B and C. Transwell assay was used to evaluate the cell migration/invasion. D. The cell apoptosis was analyzed by Flow cytometry. **p < 0.01 vs vector+ Scramble; ##p < 0.01 vs hsa_circ_0008583+ Scramble.

Figure 6.

MiR-1301-3p inhibitor reverses the inhibitory effect of si-hsa_circ_0008583 on the proliferation, migration and invasion of hepatoma cells Huh-7 cells were transfected with si-NC+inhibitor NC, si-hsa_circ_0008583+ inhibitor NC, si-NC+miR-1301-3p inhibitor, or si-hsa_circ_0008583+ miR-1301-3p inhibitor. A. The proliferation of Huh-7 cells was measured by CCK8. B and C. Transwell assay was used to evaluate the cell migration/invasion. D. The cell apoptosis was analyzed by Flow cytometry. **P < 0.01 vs si-NC+inhibitor NC; ##p < 0.01 vs si-hsa_circ_0008583+ inhibitor NC.

Hsa_circ_0008583 depletion prevents the growth of HCC tumors in vivo

To verify whether hsa_circ_0008583 exerted a similar role in vivo, we monitored the volume and weight of HCC tumors in nude mice. No treatment Huh-7 cell as control. As exhibited in Figure 8(a-b), HCC tumors were smaller in nude mice injected with Huh-7 cells stably expressing LV-si-hsa_circ_0008583 other than LV-NC. In addition, LV-si-hsa_circ_0008583 greatly inhibited tumor weight (Figure 8(c)). These results suggested that inhibition of hsa_circ_0008583 repressed HCC progression in vivo.

Figure 8.

Silencing hsa_circ_0008583 inhibited the growth of HCC in vivo. A. 30 days after implantation, the tumor image of mice in each group were taken. B and C. Comparison of the volume and weight of subcutaneous transplanted tumor in each group. **p < 0.01 vs LV-NC.

Discussion

There is growing evidence supporting that circRNAs are involved in the growth of HCC [19,20]. Hsa_circ_0008583 has been found highly expressed in HCC tissues [11]. In our experiments, we found hsa_circ_0008583 was greatly elevated in HCC tumor tissues, which was consistent with those reported in the literature. In addition, our experiments also found that hsa_circ_0008583 was greatly elevated in HCC cell lines, and demonstrated that hsa_circ_0008583 knockout can inhibit the progression of HCC in vitro and in vivo.

CircRNAs can play their role as ceRNAs in regulating the development of HCC. In this study, miR-1301-3p was predicted to be a target of hsa_circ_0008583, which was confirmed by the luciferase reporter assay. Studies have shown that miR-1301-3p plays a dual role in tumor progression and can be used as a prognostic biomarker and therapeutic target for some malignant tumors [21-24]. In addition, miR-1301-3p was sponged by Circ_0004370, CircRNA MYLK and participated in cancer progression [25,26]. Here, our results showed that miR-1301-3p mimic could partially reverse the effect of hsa_circ_0008583 overexpression on HCC cell progression, which indicated that hsa_circ_0008583 played a carcinogenic role in HCC by sponging miR-1301-3p.

Previous studies have confirmed that the miR-1301 expression was significantly downregulated in HCC tissues/cell lines. And miR-1301 mimic inhibits HCC progression by targeting STAT3 [15] or BCL9 [14]. Similarly, our study also confirmed the anti-hepatoma activity of miR-1301-3p. Using Starbase 2.0, METTL3 was found to be one of the downstream target genes of miR-1301-3p. Then, the relationship between miR-1301-3p and METTL3 was confirmed by luciferase reporter assay.

At present, a lot of literature has reported the function of METTL3 in malignant tumors [27,28]. METTL3 is frequently upregulated in HCC and is involved in the progression of HCC [17]. Our results demonstrated that METTL3 knockout significantly reversed the effect of hsa_circ_0008583 overexpression on HCC proliferation, migration, and invasion, indicating that METTL3 is related to the HCC progression along the hsa_circ_0008583/miR-1301-3p/METTL3 axis. The study had some shortcomings: First, a larger number of clinical samples are required to evaluate the potential of hsa_circ_0008583 as a prognostic biomarker in HCC. Besides, the upstream regulation mechanism of hsa_circ_0008583 is needed to be clarified in the future, which will further explain their roles in HCC development.

Conclusion

The hsa_circ_0008583 was up-regulated in HCC tissues and cells. Moreover, we also confirmed that hsa_circ_0008583 plays a carcinogenic role in HCC partially by regulating the miR-1301-3p/METTL3 pathway.

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