The circular RNA 0010117 promotes the aggressive behavior of glioblastoma by regulating miRNA-6779-5p/SPEN axis

doi:10.21203/rs.3.rs-1502464/v1

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The circular RNA 0010117 promotes the aggressive behavior of glioblastoma by regulating miRNA-6779-5p/SPEN axis

https://doi.org/10.21203/rs.3.rs-1502464/v1

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Background: Noncoding RNAs (ncRNAs) play important roles in cancer biology, providing potential targets for cancer intervention. As a new class of endogenous noncoding RNAs, circular RNAs (circRNAs) have been recently identified in cell development and function, and certain types of pathological responses, to contribute to cancer progression including glioblastoma. However, the potential mechanisms underlying the relationship between circRNAs and glioblastoma progression are still largely unknow. Methods: The expression and roles of circular RNA 0010117 (circ-0010117) were examined in vitro and in vivo. Quantitative RT-PCR and western blotting were used to measure the expression of circ RNA, miRNA, each gene, or relative proteins. Cell biology experiments were performed to detect the biological function of circ-0010117 in glioblastoma cell lines. Moreover, bioinformatics analysis, luciferase reporter assay, and functional complementation analysis were carried out to investigate the target gene. Tumorigenesis was also evaluated by xenografting cells into nude mice.

Results: In this study, we find that circ-0010117 is down-regulated in glioblastoma compared with corresponding paratumoral tissues. Subsequently, we observe that circ-0010117 can regulate aggressiveness in glioblastoma cells through miR-6779-5p. Furthermore, SPEN is verified as a direct target of miR-6779-5p and also contributes to circ-0010117 regulatory network. In addition, we identify that overexpression of circ-0010117 can suppresses tumorigenesis in nude mice.

Conclusion: Our findings indicate that the circular RNA 0010117 promotes the aggressive behavior of glioblastoma by regulating miRNA-6779-5p/SPEN axis. And our results provide the rationale for the use of circ-0010117 as a novel potential therapeutic target in glioblastoma.

glioblastoma

circ-0010117

miR-6779-5p

SPEN

Glioblastoma is the most common primary brain cancer of glial origin in worldwide and is the top leading cause of neurologic cancer-related death worldwide[1]. High-grade glioblastoma in particular has a very poor prognosis and poses a serious threat to human health. In recent years, although advances have been made in surgery, chemotherapy and molecular targeted therapies, glioblastoma still has poor morbidity and mortality. Given the poor strategies for diagnosis and treatment, a better understanding of glioblastoma pathogenesis is critical for advancing and providing potential biomarker-driven treatments.

Noncoding RNAs (ncRNAs) are a class of non-protein translation RNA, that includes: long non-coding RNA (lncRNA) more than 200 nucleotides in length, microRNA (miRNA) 20–22 nucleotides in length, and circular RNA (circRNA), which are characterized by covalent closed loops. Like other ncRNA, circRNA are expressed in multiple tissues with great variability. CircRNAs are a special class of noncoding RNAs which originates from exons, introns, intergenic or untranslational regions by a non-canonical splicing process termed back splicing[2]. Importantly, circRNA participates in the occurrence of many kinds of tumors and regulates the tumor development. Circular RNA hsa_circ_0001785 has been reported to inhibit the proliferation, migration and invasion of breast cancer cells in vitro and in vivo by sponging miR-942 to upregulate SOCS3[3]. A circular RNA hsa_circ_0079929 has also been validated to inhibit tumor growth in hepatocellular carcinoma[4]. Another novel circular RNA circENTPD7 has been confirmed to contribute to glioblastoma progression by targeting ROS1 as well[5]. However, it is still unclear about the potentially important functional role of circ-0010117 in the progression of glioblastoma.

In this study, we first investigated the expression of circ-0010117 in human glioblastoma tissues. We subsequently investigated the effects of circ-0010117 on aggressiveness of human glioblastoma cells. Furthermore, we also explored the molecular mechanisms underlying these effects.

2.1 Human osteosarcoma clinical specimens

A total of 6 paired osteosarcoma and corresponding paratumoral tissues were collected at the First Affiliated Hospital of Nanchang University from November 2021 to January 2022. All tissues specimens were resected during surgery and directly snap frozen in liquid nitrogen. The study protocol was approved by the ethics committee of the First Affiliated Hospital of Nanchang University. All written informed consents had received from patient who underwent surgery resection.

2.2 Cell culture

The U251 and U87 cell lines (ATCC, Manassas, VA, USA) are commonly used as experimental models of glioblastoma. Cells were cultured in Dulbecco’s Modified Eagle’s medium (Gibco, Waltham, MA, USA), supplemented with 10% fetal bovine serum (FBS, Gibco, Waltham, MA, USA), pyruvate (1 mM; Sigma-Aldrich, Shanghai, China), non-essential amino acids (0.1 mM; Sigma-Aldrich, Shanghai, China), and a mix of antibiotics (1 mM; Sigma-Aldrich, Shanghai, China) in a humidified atmosphere with 5% CO2 at 37°C.

2.3 Quantitative RT-PCR

Total RNA from tissue samples and cells were isolated using the mirVana™ miRNA isolation kit (Ambion, Austin, TX, USA) according to the manufacturer’s instruction. RT-qPCR analysis was performed with SYBR green in a 7500 Fast Real-Time PCR system (Applied Biosystems, Carlsbad, CA, USA). Circ-0010117, microRNA-6779-5p (miR-669-5p) and SPEN were detected by qRT-PCR. U6 was used as internal references for circ-0010117 and miR-669-5p, as previously described[6]. β-Actin was used as an internal control for SPEN. All primers were commercially available from Sangon Biotech (Shanghai, China). The amount of circ RNA, miRNA or each gene relative to the internal control was calculated as previously described[7].

2.4 RNA interference of circ-0010117

Circ-0010117 siRNAs (siRNA-#1, siRNA-#2 and siRNA-#3) targeting the junction region of the circRNA-0010117 sequence were commercially available from GeneCopoeia (Rockville, MD, USA). Transfection of siRNA was performed using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The shcirc-0010117 is also commercially available from GeneCopoeia (Rockville, MD).

For circ-0010117 over-expression plasmid (circ-0010117), the sequence was cloned into GV248. The backbone of pLCDH-ciR (Geneseed, Guangzhou, China) was used to construct the circ-0010117 overexpression plasmid. The linear sequence of circ-0010117 together with the flanking circ-0010117-inducing sequence was amplified and subcloned into EcoRI and BamHI sites of pLCDH-ciR.

2.5 MiR-6779-5p/SPEN overexpression and inhibition

For SPEN overexpression in vitro, we used SPEN vector construct (OriGene Technologies, Inc., Rockville, MD). U251 and U87 cells were seeded in a 6-well plate (Corning, Cambridge, MA, USA) (2X10⁵ cells/well) with the complete media. The cells were transfected with 2ug SPEN vector or the vector control (OriGene Technologies) using lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) with Opti-MEN (Gibco) for 4h. Then, we replaced the Opti-MEN with the complete medium. After 48h, the cells were collected to the next determination. MiR-6779-5p mimics were commercially available from life technologies. Transfection of miRNA mimics was performed using the RNAiMAX Reagent (Invitrogen, Carlsbad, CA, USA) followed the manufacturer’s instruction. miRNA vector control (NC mimic) was used as a vector control. The shSPEN and miR-6779-5p inhibitor are also commercially available from GeneCopoeia (Rockville, MD).

2.6 Cell Counting Kit-8 (CCK8) assay

CCK-8 assay were performed to assess the proliferation of U251 and U87 cells. For CCK-8 assay, the mock and infected cells (1×10⁵ cells/well) were seeded into 96-well plates (Corning, Cambridge, MA, USA) after 24h transfection and cultured for 24h, 48h and 72h. The absorbance at 450 nm was measured using CCK-8 (Dojindo Laboratories, Kumamoto, Japan) at indicated time according to the manufacturer’s protocols.

2.7 Colony formation assay

For colony formation assay, we melt 1.4% (v/v) agarose (Sigma-Aldrich, Shanghai, China) in a microwave and cool to room temperature. Then, we mixed equal volumes of the 1.4% melted agarose with the complete cell culture medium. After that, 2ml of 0.7% (v/v) low melt agar was added into each well of the 6-well plate (Corning, Cambridge, MA, USA) and set aside to allow agarose to solidify. Cells (5×10³ cells/well) were mixed with 1.4% agarose in complete culture medium, plated on top of the solidified layer to form colonies in1-3 weeks. Cells were fed with complete culture medium every 3 d.

The colony is denied to consist of at least 50 cells [8]. Clones were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet (Millipore, Billerica, MA, USA). Visible colonies were manually counted.

2.8 Apoptosis assay

Flow cytometry was performed for the apoptosis analysis. U251 and U87 cells were digested with trypsin (Invitrogen, Carlsbad, CA, USA) and washed with PBS. After blocking with 10% FBS, cells were stained with 5 µl FITC annexin-V and 1 µl propidiumiodide (Becton Dickinson, Heidelberg, Germany) and incubated at room temperature for 15 mins in the dark. Then, flow cytometric was performed (Life Technologies, Darmstadt, Germany). The cells were then analyzed with CellQuest software (BD Biosciences, San Jose, CA). The dead cells and the relative ratio of apoptotic cells was compared in each condition.

2.9 Wound scratch assay analysis

U251 and U87 cells were first transfected with shcirc-0010117 or circ-0010117 or their related control for 24hrs. Wound scratch assay was performed as previous description[9–12]. After then, cells were seeded into 12-well plates (5X10⁵ cells/well) with medium and cultured for 24h to reach ~ 90% confluence. A 10µl pipette tip was used to make a scratch line. Cells were washed with PBS for 3 times to remove the scratched cells. After then, cells were cultured in medium containing 0.5% FBS. The cells were incubated at 37°C and images were captured at 0 and 48h using a light microscope (Olympus Corporation, Tokyo, Japan).

2.10 Cellular Invasion

Boyden chamber assay with 24-well collagen-based cell invasion assay kit (Millipore, Bedford, MA, USA) was used to perform transwell assay. U251 and U87 cells were first transfected with shcirc-0010117 or circ-0010117 or their related control for 24hrs. After then, 200ul of serum-free medium containing 5X10⁵ cells were added to the filter. 750µl complete cell culture medium was added into the bottom chamber in which the FBS as a chemoattractant. After incubated for indicated time, the non-invasive cells were cleaned by scrubbing with a cotton swab. The cells that adhered to the outside of the membrane were fixed and dyed with Cristal Violet solution (Millipore, Billerica, MA, USA). An Olympus optical microscope (Olympus Corporation, Tokyo, Japan) was used to observe invasive cells.

2.11 Dual-luciferase reporter assay

The binding sites of circ-0010117 for miR-6779-5p of its 3’-UTR and mutant were commercially available at GeneCopoeia (Rockville, MD). U251 and U87 cells were seeded in a 6-well plate (Corning, Cambridge, MA, USA) at a density of 2.5 X 10⁵ cells/ well with the complete media. The cells were co-transfected with 1ug circ-0010117 3’-UTR (circ0010117-WT) or mutant luciferase reporter construct (circ0010117-Mut) with 20nM miRNA mimic or NC mimic using lipofectamine 2000 (Carlsbad, CA, USA) with Opti-MEN (Gibco, Waltham, MA, USA) for 48h. Luciferase assays were performed using the dual-luciferase reporter assay reagent from GeneCopoeia. Data are expressed as the ratio of firefly luciferase activity by Renilla luciferase activity.

2.12 Western blotting

The total protein was extracted from cell with and without circ-0010117 with T-PER Tissue Protein Extraction Reagent (Thermo Pierce, Rockford, IL, USA) with 1% Halt Protease and Phosphatase Inhibitor (Thermo Pierce, Rockford, IL, USA). After then, the extracted protein was loaded and ran on odium dodecyl sulfate-polyacrylamide gels (8–12% separating gel, 5% concentrated gel). The protein in the gel was transferred onto a PVDF membrane (Millipore, Bedford, MA, USA) for antibody detection. The PVDF membrane was blocked for 1 h at room temperature in 5% non-fat milk in Tris-buffered saline with 0.1% Tween- 20 detergent (TBST). The membrane was washed by TBST for 3 times for 10 min each in room temperature and followed by incubation with primary antibodies (anti-truncated-cas-3, full-cas-3, cas-8, cas-9, BCL-2, BAX, P53, and JNK1) overnight at 4℃. β-actin was served as internal control (Abcam, Cambrigde, MA, USA). After then, the membrane was washed by TBST for 3 times for 10min each in room temperature, and incubated with horseradish peroxidase (HRP) labeled goat anti-mouse IgG or goat anti-rabbit IgG secondary antibody, respectively (Thermo Pierce, Rockford, IL, USA) for 1h in room temperature. Proteins were visualized by using ECL western blotting detection reagents (SuperSignal® West Dura Extended Duration Substrate, Thermo Pierce, Rockford, IL, USA) after the membrane was washed by TBST for 3 times for 10 min each in room temperature. The relative expression of the target protein was quantitatively analyzed by ImageJ (NIH, Bethesda, MD, USA).

2.13 Mouse xenograft assays

Female nude mice (BALB/c, SPF grade, weighing 16–20 g and 4-week-old) were purchased from Shanghai Lingchang Biotechnology Co., Ltd (Shanghai, China). Transfected cells were digested with trypsin and washed twice with PBS. Cells were resuspended in 200 µL solution containing 100 µL PBS and 100 µL Matrigel substrate at a final density of 2×10⁷ cells/0.2 mL. Four-week-old female BALB/c nude mice were subcutaneously injected with 2×10⁷ tumor cells. Mice were sacrificed at day 35 since tumor cells have been injected and tumor volume was measured.

2.14 Statistical analysis

Statistical analyses were performed using GraphPad Prism and all data were presented as mean ± standard deviation (SD) from triplicate experiments. Differences between groups were analyzed by unpaired t-test. A p-value < 0.05 is considered significant.

3.1 Circ-0010117 is down-regulated in glioblastoma

In this study, we first determined the mRNA expression levels of circ-0010117 in 6 paired glioblastoma and corresponding paratumoral tissues with qRT-PCR. Circ-0010117 mRNA levels was significant down-regulated compared with the normal tissues (P < 0.05, Fig. 1A). Subsequently, we tested circ-0010117 expression in U251 and U87 cells. Synthesized interfering oligonucleotides targeting circ-0010117 significantly decreased the expression of circ-0010117 in U251 and U87 cell lines (Fig. 1B and C). Circ-0010117 overexpression strongly increased the expression of circ-0010117 in U251 and U87 cell lines (Fig. 1D).

3.2 Circ-0010117 regulates aggressiveness in glioblastoma

The silencing of circ-0010117 in U251 and U87 cell lines remarkably enhanced cell proliferation compared with control (Fig. 2A and B). While, the overexpression of circ-0010117 in U251 and U87 cell lines significantly suppressed cell proliferation (Fig. 2C and D).We next sought to establish whether alternation of circ-0010117 could affect U251 and U87 clonogenicity. As shown in Fig. 2E, F, G and H, circ-0010117 silencing significantly enhanced the anchorage-dependent growth of MSTO-211H (Fig. 2E and F). In the meantime, circ-0010117 overexpression strongly decreased the anchorage-dependent growth of U251 and U87 (Fig. 2G and H). Thus, circ-0010117 can regulate growth activities in glioblastoma cells.

Enhanced migration and invasion are another key feature across the metastatic cascade. To assess the effect of circ-0010117 on the migration and invasion of MPM cells, wound scratch and transwell assays were performed, respectively. As shown in Fig. 2I, the rate of gap closure was greatly enhanced after circ-0010117 silencing in the U251 and U87 cells compared with control. While, the rate of gap closure was significantly dropped after circ-0010117 overexpression in the U251 and U87 cells compared with control (Fig. 2J).

Similarly, a remarkably increased cell invasion in U251 and U87 cells silencing circ-0010117 compared with control by transwell assay (Fig. 2K). In contrast, we observed that a significantly decreased cell invasion in U251 and U87 cells overexpression circ-0010117compared with control (Fig. 2L). We also assessed apoptosis alternation by circ-0010117 regulation through flow cytometry. U251 and U87 cells was observed to have reduced percentage of cells exhibiting apoptosis after circ-0010117 silence (Fig. 2M). For U251 and U87 cells with circ-0010117 overexpression, the percentage of cells exhibiting apoptosis were significantly increased (Fig. 2N). These results suggest that circ-0010117 regulates aggressiveness in glioblastoma.

3.3 Circ-0010117 regulates aggressiveness via miRNA-6779-5p

To unravel the relevant mechanism underlying circ-0010117 contributed to the malignant phenotype of glioblastoma. We searched for potential regulation mechanism of circ-0010117. We next asked whether miRNAs play important roles in the regulation of circ-0010117 related to the malignant phenotype of glioblastoma. We found miR-6779-5p can significantly reduce luciferase activities of circ-0010117 WT but not Mut in U251 and U87 cells (Fig. 3A). We next investigated the effect of inhibiting or overexpressing miR-6779-5p in U251 and U87 cells with circ-0010117 silencing or circ-0010117 overexpression. The miR-6779-5p expression level has been confirmed after inhibiting or overexpression miR-677-5p (Fig. 3B).

Shcirc-0010117 and miR-6779-5p inhibitor were transfected into U251 and U87 cells to knockdown the circ-0010117 and miR-6779-5p expression. The effect of miR-6779-5p inhibitor on the U251 and U87 cells obligated the effect caused by circ-0010117 in cell proliferation detected with CCK-8 (Fig. 3C). In contrast, circ-0010117 and miR-6779-5p mimic were transfected into U251 and U87 cells to overexpress the circ-0010117 and miR-6779-5p. We found that miR-6779-5p overexpression enhanced U251 and U87 cells proliferation, which reversed the effect of circ-0010117 overexpression on the cell proliferation (Fig. 3D).

Similarity was found in the invasion and apoptosis. The effect of miR-6779-5p inhibitor on the U251 and U87 cells obligated the effect caused by circ-0010117 in cell invasion and apoptosis detected with transwell assay and flow cytometry (Fig. 3E and G). In the meantime, we found that miR-6779-5p overexpression enhanced U251 and U87 cells invasion and apoptosis, which reversed the effect of circ-0010117 overexpression on the cell invasion and apoptosis in the cells overexpressing circ-0010117 and miR-6779-5p (Fig. 3F and H).

3.4 SPEN contributed to miRNA-6779-5p regulation

To explore the regulatory mechanism underlying circ-0010117 and miR-6779-5p for the malignant phenotype of glioblastoma. We subsequently identified potential target of miR-6779-5p. We found miR-6779-5p can significantly reduce luciferase activities of SPEN WT but not Mut in U251 and U87 cells (Fig. 4A). SPEN mRNA level was also found to be significantly decreased after miR-6779-5p inhibition in U251 and U87 cells (Fig. 4B), but remarkably increased after miR-6779-5p overexpression (Fig. 4C).

MiR-6779-5p mimic and SPEN overexpression plasmid were transfected into U251 and U87 cells to increase the miR-6779-5p and SPEN expression. The SPEN overexpression on the U251 and U87 cells reversed the effect caused by miR-6779-5p overexpression on cell proliferation detected with CCK-8 (Fig. 4D). We also transfected U251 and U87 cells with miR-6779-5p inhibitor and shSPEN to knock down miR-6779-5p and SPEN. As shown in Fig. 4D, SPEN knock down blocked the effect of miR-6779-5p inhibition caused suppression on cell proliferation.

In consistent with cell proliferation, SPEN knock down reversed the effect of miR-6997-5p silencing on invasion and apoptosis in the U251 and U87 cells (Fig. 4E and G). But SPEN overexpression repressed U251 and U87 cells invasion and apoptosis caused by miR-6779-5p overexpression (Fig. 4F and H).

3.5 Circ-0010117 up-regulation inhibited tumor growth in vivo

To further assess whether overexpression of circ-0010117 could suppress tumor formation in vivo, we inoculated stable overexpressed circ-0010117 with U251 cell line (Fig. 5A) subcutaneously into 4-week-old nude mice. The results showed that tumor size and weight were remarkably reduced when transplanted with tumor cells overexpressed U251 (Fig. 5B, C and D).

To investigate the molecular mechanism by which circ-0010117 overexpression influences the caspase pathway, we determined each component involved in the pathway by Western blotting (Fig. 5E). The truncated-cas-3, full-cas-3, cas-8, cas-9, BAX, P53, and JNK1were enhanced by circ-0010117 overexpression (Fig. 5E). But BCL-2 was suppressed by circ-0010117 overexpression (Fig. 5E). Therefore, we concluded that overexpression of circ-0010117 also suppressed tumorigenesis through caspase pathway in vivo.

Currently, the roles of circRNAs in cancer progression have attracted a lot of attention due to their key roles involved in human cancers. In this study, our goal was to explore the potential important roles of circ-0010117 during the glioblastoma progress. CircRNAs represent a new class of endogenous noncoding RNAs that have recently been recognized as important regulators of glioblastoma. MiRNAs are a group of are 19–22 nucleotides non-coding, single-stranded RNA molecules that participate in sequence-specific post-transcriptional regulation of gene expression [13]. Recently, emerging evidence confirms that circRNAs can regulate the expression of tumor-suppressive or oncogenic genes through circRNA-miRNA-mRNA axis. For instance, circular RNA circ-0001946 has been validated to act as a competing endogenous RNA to inhibit glioblastoma progression by modulating miR-671-5p and CDR1[14]. Circular RNA circMTO1 inhibits proliferation of glioblastoma cells via miR-92/WWOX signaling pathway[15]. Because their expression and function in glioblastoma development are still largely elusive, we screened the differentially expressed circRNAs between glioblastoma and matched nontumor liver tissues and focused on the role and underlying mechanism of the decreased circ-0010117 in glioblastoma progression. In this study, we found that circ-0010117 is down-regulated in glioblastoma. In addition, circ-0010117 can regulate aggressiveness in glioblastoma. Furthermore, circ-0010117 regulates aggressiveness is through miR-6779-5p. Moreover, circ-0010117 up-regulation inhibited tumor growth in vivo.

Split ends (SPEN) is an ERα corepressor that we have identified as a tumor suppressor protein in ERα-positive breast cancer cells[16]. It has been reported to play important roles in cancer progression. For instance, SPEN has been found to induce miR-4652-3p to target HIPK2 in nasopharyngeal carcinoma[17]. SPEN has also been identified as a crucial factor for Xist function through forward genetic screening in haploid embryonic stem cells[18]. However, it is still unclear whether SPEN contributes to the circ-0010117 and miR-6779-5p regulatory network. In this study, we found SPEN is a direct target of miR-6779-5p and SPEN contributed to miRNA-6779-5p regulatory network.

In summary, we determined that circ-0010117 is linked to aggressiveness in glioblastoma through miRNA-6779-5p and SPEN. Our results provide the rationale for the use of circ-0001946 as a novel potential therapeutic target in glioblastoma.

Author Contributions: SX and JX conceived and corresponded to this study together. XY performed the experiments, analyzed the data and drafted the manuscript. YL and XZ contributed to data analysis. KC and JX discussed the results. SY and SX revised the manuscript.

Funding: This work received no funding.

Data availability: The data used to support the findings of this study are available from the corresponding author upon request.

Ethics approval and consent to participate: The experimental protocols were approved by the Ethics Committee of the First Affiliated Hospital of Nanchang University (Reference NCUH20219). This paper has not been published elsewhere in whole or in part. All authors have read and approved the content, and agree to submit it for consideration for publication in your journal. There are no ethical/legal conflicts involved in the article. Informed consent was obtained from all individual participants included in the study

Competing interests: The authors have declared no conflicts of interest in this work

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No competing interests reported.

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The circular RNA 0010117 promotes the aggressive behavior of glioblastoma by regulating miRNA-6779-5p/SPEN axis

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Abstract

Figures

1 Introduction

2 Materials And Methods

2.1 Human osteosarcoma clinical specimens

2.2 Cell culture

2.3 Quantitative RT-PCR

2.4 RNA interference of circ-0010117

2.5 MiR-6779-5p/SPEN overexpression and inhibition

2.6 Cell Counting Kit-8 (CCK8) assay

2.7 Colony formation assay

2.8 Apoptosis assay

2.9 Wound scratch assay analysis

2.10 Cellular Invasion

3 Results

3.1 Circ-0010117 is down-regulated in glioblastoma

3.2 Circ-0010117 regulates aggressiveness in glioblastoma

3.3 Circ-0010117 regulates aggressiveness via miRNA-6779-5p

3.4 SPEN contributed to miRNA-6779-5p regulation

3.5 Circ-0010117 up-regulation inhibited tumor growth in vivo

4 Discussion

Declarations

References

Additional Declarations

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