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Circular RNA HIPK3 promotes glioma progression by binding to miR-124-3p
Accepted Manuscript Circular RNA HIPK3 promotes glioma progression by binding to miR-124-3p
Daling Hu, Yin Zhang PII: DOI: Reference:
S0378-1119(18)31216-2 https://doi.org/10.1016/j.gene.2018.11.073 GENE 43418
To appear in:
Gene
Received date: Revised date: Accepted date:
13 September 2018 13 November 2018 21 November 2018
Please cite this article as: Daling Hu, Yin Zhang , Circular RNA HIPK3 promotes glioma progression by binding to miR-124-3p. Gene (2018), https://doi.org/10.1016/ j.gene.2018.11.073
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ACCEPTED MANUSCRIPT Circular RNA HIPK3 promotes glioma progression by binding to miR-124-3p Daling Hu1, Yin Zhang2* 1 Department of Geriatrics, Sir Run Run Hospital of Nanjing Medical University 2 Department of Neurosurgery, Sir Run Run Hospital of Nanjing Medical University *Correspondence author: Yin Zhang, Department of Neurosurgery, The Affiliated Sir Run Run Hospital of Nanjing Medical University, Nanjing 210008, China. E-mail: [email protected]
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Abstract This study aims to investigate whether circ-HIPK3 could promote the proliferation and invasion of
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glioma cells by upregulating STAT3 after binding to miR-124-3p, thus participating in the development
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of glioma. Expression levels of circ-HIPK3, miR-124-3p and STAT3 in glioma cell lines were determined using qRT-PCR. The regulatory effects of circ-HIPK3, miR-124-3p and STAT3 on
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proliferative and invasive capacities of glioma cells were accessed using EdU assay, CCK-8 assay and invasion assay, respectively. Cell cycle assay and cell apoptosis assay were performed by flow
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cytometry. Dual-luciferase reporter gene assay was conducted to determine the binding condition among circ-HIPK3, miR-124-3p and STAT3. Rescue experiments were performed in co-transfected glioma cells. QRT-PCR data showed that circ-HIPK3 and STAT3 are highly expressed, whereas
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miR-124-3p is lowly expressed in glioma cells than those of negative control cell. Knockdown of
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circ-HIPK3 in U87 and U251 cells inhibited their proliferative and invasive capacities. On the contrary, miR-124-3p knockdown improved proliferative and migratory capacities. Dual-luciferase reporter gene assay exerted that circ-HIPK3 could bind to miR-124-3p and STAT3 is the target gene of miR-124-3p.
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Western blot results elucidated that circ-HIPK3 stabilizes STAT3 expression, whereas miR-124-3p
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degrades STAT3 expression. Rescue experiments demonstrated that overexpression of circ-HIPK3 could partially reverse the inhibited proliferative and migratory capacities induced by miR-124-3p in U87 and U251 cells. In summary，we found that overexpression of circ-HIPK3 promotes proliferative and invasive capacities of glioma cells by sponging miR-124-3p to upregulate STAT3 expression.
Key words: circ-HIPK3; miR-124-3p; proliferation; invasion; STAT3
1. Introduction Gliomas, known as the most prevalent primary brain tumor of central nervous system, account for significant mortality worldwide annually(Chen et al., 2012; Yu et al., 2017). Based on the degree of
ACCEPTED MANUSCRIPT malignancy and histopathological features, gliomas could be classified into I to IV levels(Fuller and Scheithauer, 2007; Louis et al., 2007). Although great efforts such as novel surgical treatments, radiation and conventional chemotherapies made in recent decades toward improvement for gliomas, higher-grade gliomas are still significant severe and associated with poor prognosis(Zhou et al., 2010). Therefore, exploring the molecular mechanism associated with the pathogenesis of glioma is essential,
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and might lead to the development of novel targeted therapies. Non-coding RNAs (ncRNAs) are a group of functional RNA having little protein-coding
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ability(Anastasiadou et al., 2018). Based on the structure and length, ncRNAs could be further classified into various groups, including microRNAs (miRNAs), long noncoding RNAs (lncRNAs) and
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circular RNAs (circRNAs)(Ransohoff et al., 2018; Wu et al., 2018). CircRNAs is referred to as a new class of functional molecule with a covalenty closed loop structure linking the 3’ and 5’
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terminals(Enuka et al., 2016). Emerging evidence suggests that circRNAs are conserved and stable, and most of them play their role in post-transcriptional level(Veno et al., 2015). Considering that circRNAs
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contains many miRNAs binding sites, in mainly function as miRNA sponges to regulate gene expression(Hansen et al., 2013). It’s reported that circRNAs have multiple functions in many aspects of
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physiological or pathological processes, including cell proliferation, migration, invasion, apoptosis, and
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cell cycle(Chen et al., 2017; Dai et al., 2018). Recent studies show that circRNAs have been identified to be dysregulated and involved in various of cancers. For examples, downregulated circ-LARP4 is detected in gastric cancer which represents poor prognosis(Zhang et al., 2017), upregulated
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circ-CCDC66 fosters the growth and metastasis of colon cancer(Hsiao et al., 2017), circ-ITCH functions as a tumor inhibitor via a novel circ-ITCH/miR-17, miR-224/p21, PTEN axis in bladder
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cancer(Yang et al., 2018a).
CircHIPK3(hsa_circ_0000284) is derived from HIPK3 gene and consisted of the head-to-tail splicing of exon 2 (1099bp)(Zheng et al., 2016). It is demonstrated that circHIPK3 could facilitate cell proliferation and invasion by binding and inhibiting many tumor-suppressive miRNAs(Xiao-Long et al., 2018). Ding-kai et al. found that the expression of circHIPK3 in gallbladder cancer is increased, which promotes cancer cell growth via spongingmiR-124(Kai et al., 2018). Similarly, Chen et al. revealed circHIPK3 promotes cell proliferation and migration and upregulates AQP3 expression in hepatocellular carcinoma(Chen et al., 2018). Although Jin and co-workers have confirmed that circ-HIPK3 is up-regulated in glioma tissues and may serve as a prognostic biomarker(Jin et al., 2018),
ACCEPTED MANUSCRIPT the underlying mechanism of circ-HIPK3 in glioma remains to be investigated. Our study discovered that circHIPK3 may boost cell proliferation and invasion via binding to miR-124-3p from STAT3.
2. Materials and methods 2.1. Cell culture and transfection Normal brain glial cell lines (HEB) and human glioma cells (U87, U251, LN229 and LN308)
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were provided by American Type Culture Collection (ATCC, Manassas VA, USA). All the cells were cultured in RPMI-1640 containing 10% FBS (Life Technologies, USA) and maintained at 37°C, 5%
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CO2. Cells were seeded into 6-well plates and cell transfection was performed until 60-70% of
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confluence using Lipofectamine 2000 (Invitrogen, CA, USA) following the manufacturer’s instructions. After incubation for 48 h, cells were used for further assay. Circ-HIPK3 siRNA, pcDNA-circHIPK3,
Genepharma Company (Shanghai, China).
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2.2. Quantitative RT-PCR
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miR-124-3p mimics, miR-124-3p inhibitor and the negative controls were all synthesized by
TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was utilized to extract total RNA in cell lines. For detection of mRNA,RNA was reversely transcribed into complimentary DNA (cDNA) with
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Reverse Transcription Kit (Takara, Tokyo, Japan). For detection of miR-124-3p-3p, total RNA was
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reverse-transcribed to cDNA using the TaqMan miRNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA). Then miR-124-3p-3p was quantified using the TaqMan miRNA kit
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(Applied Biosystems). QRT-PCR was carried out utilizing SYBR® Premix Ex TaqTM (Takara) on the ABI 7500HT (Applied Biosystems). GAPDH and U6 were used as endogenous control, respectively.
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The relative expression levels were calculated utilizing the 2−△△Ct method. The experiments were repeated in triplicate independently and all primers involved were listed in Table 1. 2.3. RNase R digestion For RNase R digestion, total RNA (5 ug) was incubated for 15 min at 37°C using 3U/ug of RNase R (Epicentre Biotechnologies). Based on previously published procedures(Shan et al., 2017), the RNase R digestion reaction was done twice. 2.4. EdU proliferation assay EdU assay kit (Ribobio, Guangzhou, China) was used for cell proliferation assay according to the the manufacturer’s instructions. In brief, 100 μmol/L of EdU were added to the transfected U87 and
ACCEPTED MANUSCRIPT U251 cells for 2 hours at 37°C, after which the cultured cells were fixed using 4% paraformaldehyde for 25 minutes. Then the cells were stained with 1 × Apollo reaction cocktail (deionized water, Apollo reaction buffer, Apollo catalyst solution, Apollo fluorescent dye solution and Apollo buffer
additive) for 30 minutes and then incubated with 100 μL of Hoechst33342 at 5 μg/mL for 30 minutes. Fluorescent microscope was utilized to examine the percentage of EdU-positive cells. All assays were
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independently carried out in triplicate. 2.5. CCK-8 assay
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After treatment, cells were plated at a dose of 3000 cells/well into 96-well plates. After cell cultured for 24 h, 48 h, 72 h and 96 h, 10 μl of Cell-Counting Kit 8 (CCK8; Dojindo Laboratories,
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Kumamoto, Japan) reagent was added in each well according to the manufacturer’s instructions. Two hours later, the OD value at the wavelength of 450 nm was measured utilizing a microplate reader.
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Each assay was repeated in triplicate. 2.6. Cell invasion assay
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Transwell chambers (8 um pore size, Millipore Corporation, Billerica, MA) coated with with 0.5 mg/ml collagen type I (BD Bioscience) and a 1:15 dilution of Matrigel (BD Bioscience) were used to
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measure the invasiveness of cancer cells. In brief, transfected cells (1×105) were seeded in the Matrigel
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invasion chambers () in RPMI-1640 without serum, while 600 μl of medium containing 10% FBS were added to the lower chamber. After 48 hours of cell culture, we carefully removed the noninvasive cells in the top chambers. The cells that invaded to the lower chamber were fixed with 4% paraformaldehyde
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for 30 minutes, and then stained with crystal violet for 20 min. Penetrating cells were captured in 5 randomly selected fields of each sample. All the assays were conducted three times independently.
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2.7. Cell cycle assay
U87 and U251 cells were harvested 48 h after the transfection and then washed with ice-cold phosphate buffered saline (PBS). The cells were fixed in 70% ethanol for 24 h, before being re-suspended in 500 μl of PBS which contains 50 μg/mL of propidium iodide (Sigma), 10 μg/mL RNase A, 0.1% sodium citrate and also 0.1% Triton X-100, and incubated for 15 min at room temperature in the dark. Cell cycle was immediately analyzed utilizing the flow cytometer (Millipore Guava). 2.8. Cell apoptosis assay The cells were harvested 48 h after the transfection and then incubated with Annexin V-FITC
ACCEPTED MANUSCRIPT Apoptosis Detection Kit (Beyotime Biotech, Haimen, China) following the manufacturer’s instructions. Annexin V-FITC was utilized to stain the cells which were then resuspended in binding buffer (190 μL), before adding 10 μL of PI (20 μg/mL), and also incubated for no shorter than 15 min in the dark at room temperature. Flow cytometry (Millipore Guava) was applied to detect and quantify the apoptotic cells based on the manufacturer’s instructions.
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2.9. Subcellular fractionation location Nuclear and cytoplasmic RNA was isolated with the PARIS Kit (Life Technologies, USA) as described
were considered as cytoplasmic and nuclear markers, respectively.
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2.10. Dual-luciferase reporter gene assay
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in directions. Total RNA isolated from each fraction was determined by qRT-PCR. GAPDH and U6
STAT3-WT 3'UTR, STAT3-MUT 3'UTR, circ-HIPK3-WT 3'UTR, or circ-HIPK3-MUT 3'UTR
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was inserted into pGL3. Cells were then seeded into the 24-well plates respectively with 5×105 cells per well. They were co-transfected with 0.12 μg vector and 40 nM miR-124-3p mimics or negative
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control for 48 h. Dual-Luciferase reporter assay system (Promega, Madison, WI, USA) was utilized to measure the luciferase activity.
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2.11. RNA immunoprecipitation
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The Imprint RNA Immunoprecipitation Kit (Sigma, St. Louis) together with the AGO2 antibody (Cell Signaling, Rockford) were used to perform RNA immunoprecipitation (RIP) assays. Then protein A/G beads were utilized to recover the AGO2 antibody. qRT-PCR was performed to measure
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circ-HIPK3 and miR-124-3p RNA levels in the immunoprecipitates. 2.12. Western blot
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Total protein was extracted utilizing a cell lysate (RIPA). The protein concentration of each cell lysate was quantified using BCA Protein Assay Kit Protein (Beyotime, Nantong, China). Equal amounts of samples were separated in the SDS-PAGE gel before being transferred to the PVDF membrane. Next, the membranes were blocked with the blocking solution for 1 hour And incubated overnight at 4°C using primary antibody. After being washed with TBST, corresponding secondary antibody was used for incubation for 2 hours at room temperature. At last, a Tanon detection system using ECL reagent (Thermo) was utilized to capture the images of protein bands. 2.13. Statistical analysis The data analyses were carried out using SPSS 22.0 and Graphpad Prism 6.0. Data were shown as
ACCEPTED MANUSCRIPT the mean ± one standard deviation (SD) from no less than three separate experiments. The Student t test was applied to analyze the comparisons between groups. And P < 0.05 was considered to be statistically significant.
3. Results 3.1. Circ-HIPK3 was highly expressed in glioma cell lines and-promoted cell proliferation and
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invasion The expression levels of circ-HIPK3 in normal cell lines (HEB) and glioma cell lines (U87, U251,
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LN229, LN308) were detected by qRT-PCR. The results revealed that circ-HIPK3 was upregulated in glioma cell lines (Figure 1A). Among them, the expression difference was pronounced in U251 and
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U87 cell lines, which were selected for subsequent experiments. Subsequently, to investigate the circular nature of circ-HIPK3, total RNA was treated with RNaseR, which could deplete linear RNAs
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featuring a free 3’terminus while having no impact on circRNAs. The results unveiled that circular RNAs were insusceptible of RNaseR digestion, but the liner RNAs were not (Figure 1B). Then, two
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circ-HIPK3 siRNAs (si- circ-HIPK3-1 and si- circ-HIPK3-2) were transfected into U87 and U251 cells, respectively. Two siRNAs all markedly downregulated circ-HIPK3 expression (Figure S1A).
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Particularly, si- circ-HIPK3-1 showed the highest interference efficiency, and was selected for
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subsequent experiments. We also found that circ-HIPK3 siRNA could decrease the expression levels of circ-HIPK3 whilemake no effect on
HIPK3 mRNA (Figure 1C). Morever, the proliferation of
si-circHIPK3 transfected U87 and U251 cell lines was evaluated by EdU assays and CCK8 assays. As
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shown in Figure 1D and 1E, we found that cell proliferation was significantly suppressed in si-circHIPK3 transfected cells, but no difference was observed between the si-HIPK3 and siRNA
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negative control transfected cells. The effect of circ-HIPK3 on cell cycle and apoptosis was analyzed using flow cytometry. The results indicated that cells in si-circHIPK3 groups were arrested chiefly in G0/G1 phase compared with siRNA negative control groups (Figure 1F). What’s more, the apoptosis ratio was higher in si-circHIPK3 group compared with the control group (Figure 1G). Here, we subsequently investigated the effect of circ-HIPK3 on invasion of U87 and U251 cells. As displayed in Figure 2A, si-circHIPK3 transfected cells showed a strongly suppressed invasive ability. These results suggested that circ-HIPK3 might act as a promoter in glioma cell progress. 3.2. Circ-HIPK3 was targeted by miR-124-3p We then searched for the subcellular location of circ-HIPK3 in order to look into the role of
ACCEPTED MANUSCRIPT circ-HIPK3 in glioma progression. The results indicated that circ-HIPK3 was localized mostly in the cytoplasm of U87 and U251 cells (Figure 2B), which meant that circ-HIPK3 might regulate cell progress at the post-transcriptional level. Previous study proved that circ-HIPK3 could function as miRNAs sponges and bind to miR-124-3p directly and inhibit the activity of miR-124-3p(Zheng et al., 2016). Considering that miR-124-3p had been reported to participated in glioma, we hypothesized that
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circ-HIPK3 might promote glioma cell proliferation and invasion by binging to miR-124-3p. Bioinformatics analysis showed that circ-HIPK3 comprises mutual miRNA response elements to
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miR-124-3p (Figure 2C). We transfected U251 and U87 cells with miR-124-3p mimics to achieve miR-124-3p overexpressed and verified the transfection efficiency using qPCR (Figure 2D). Then
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luciferase reporter assay was performed to explore the correlation between circ-HIPK3 and miR-124-3p in U251 and U87 cells. As presented in Figure 2E, we found that the luciferase activity in
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circHIPK3-WT group decreased dramatically with the presence of miR-124-3p mimics compared with that of NC. However, no significant change was seen in luciferase activity of the circ-HIPK3-MUT
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3'UTR group. Furthermore, RIP assay for AGO2 was conducted on 293T cells and both circ-HIPK3 and miR-124-3p were detected with approximately several-fold enrichment compared with IgG (Figure
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2F), showing that circ-HIPK3 and miR-124-3p-3p existed in RNA-induced silencing complex (RISC)
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and miR-124-3p directly binds to circ-HIPK3. We also found that the expression level of miR-124-3p in glioma cell lines was lowerthan that in normal cell lines (Figure 2G). Hence, we wondered whether circ-HIPK3 could suppress the expression of miR-124-3p. Then we detected the expression of
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miR-124-3p by qRT-PCR after U87 and U251 cells were transfected with circ-HIPK3 siRNA or pcDNA-circHIPK3. The results indicated that circ-HIPK3 might be able to bind to miR-124-3p and
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inhibit its expression (Figure S1B-C). 3.3. Circ-HIPK3 upregulated STAT3 expression by sponging miR-124-3p in glioma cells We predicted genes that bind to miR-124-3p through bioinformatics and performed functional analysis. Finally, STAT3 was screened out (Figure 3A). Dual-luciferase reporter gene assay revealed decreased luciferase activity in U87 and U251 cells of STAT3-WT 3'UTR group, but no significant change in luciferase activity was seen in STAT3-MUT 3'UTR group (Figure 3B), indicating that STAT3 binds to miR-124-3p. Subsequently, qRT-PCR was utilized to examine the expression level of STAT3 in glioma cell lines. The data showed that STAT3 is up-expressed in glioma cells (Figure 3C). After interference with circ-HIPK3 overexpressed plasmid in U87 and U251 cells, circ-HIPK3 expression
ACCEPTED MANUSCRIPT increased (Figure 3D). Similarly, cells were transfected with miR-124-3p inhibitor, which had high transfection efficiency (Figure 3E). We further assessed the effects of circ-HIPK3 and miR-124-3p on STAT3 by qRT-PCR and Western blot analysis. As illustrated in Figure 2F, the mRNA level of STAT3 was significantly decreased in circ-HIPK3 knockdown or miR-124-3p mimics glioma cells, while co-transfected with miR-124-3p inhibitor or circ-HIPK3 overexpressed plasmid could reverse the
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inhibited effect (Figure 3F). What’s more, we also found that overexpression of circ-HIPK3 or knockdown of miR-124-3p individually in U87 and U251 cells could increase the expression of STAT3
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(Figure S1D-E). Accordant with qRT-PCR results, the protein level of STAT3 presented similar trends in U87 and U251 cells following circ-HIPK3 or miR-124-3p interference (Figure 3G).
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3.4. Over-expression of circHIPK3 reverses miR-124-3p-induced inhibition of cell migration, invasion in glioma cells
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To further verify the regulatory relationship among circ-HIPK3, miR-124-3p and STAT3, we tested proliferative and migratory capacities after U87 and U251 cells were interfered with both
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circ-HIPK3 and miR-124-3p. The results disclosed that the knockdown of circ-HIPK3 in U87 and U251 cells suppressed cell proliferation and migration, while co-transfection with miR-124-3p
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inhibitor reserved the inhibited proliferation and migration. Similarly, up-regulation of circ-HIPK3
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could reverse the inhibition induced by miR-124-3p mimics in glioma cells (Figure 4A and 4B). We also performed flow cytometry assays to investigate the effects of circ-HIPK3/miR-124-3p/STAT3 on glioma cell proliferation associated with cell cycle and cell apoptosis. As shown in Figure 4C and 4D,
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U251 and U87 cells transfected with circ-HIPK3 siRNA or miR-124-3p mimics could make cell cycle arrested in G0/G1 and induce cell apoptosis, while cells co-transfected with miR-124-3p inhibitor or
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pcDNA-circHIPK3 reversed the effect. These results provided evidence
that circ-HIPK3 may
promote cell proliferation and migration in glioma cells by upregulating STAT3 expression after binding to miR-124-3p.
4. Discussion Recently, researchers have identified many exonic transcripts have the ability to forming circRNAs by gene arrangement or non-liner reverse splicing(Zheng et al., 2017). Circ-RNAs are conserved and widely expressed in tissues and cells(Song et al., 2016). CircRNAs also play a vital role in regulating cellular progresses and tumorigenesis. Many recent researches show that circRNAs play a significant role in glioma. Li et al., showed that circ-0046701 can increase the expression level of
ACCEPTED MANUSCRIPT miR-142-3p target ITGB8 in glioma and therefore promote carcinogenesis(Li et al., 2018a), Wang et al., have identified that circ-0001649 has oncogenic features in glioma(Wang et al., 2018), Bian et al., found that circ-CFH could promote glioma progression by directly sponging miR-149 and regulating AKT1(Bian et al., 2018). In our study, we unveiled that circ-HIPK3 is upregulated in glioma cell lines. Subsequent
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experiments suggested that circ-HIPK3 silencing by targeted siRNAs significantly inhibited cell proliferation, invasion and arrested cell cycle in G0/G1, while inducing cell apoptosis. Then we
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conducted the experiment and found that circ-HIPK3 located mostly in cytoplasm fraction of cells, which meant it may affect cell function via acting as miRNAs sponge. Bioinformatics prediction
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revealed that several miRNAs including miR-124-3p proved to dysregulation in glioma could bind to circ-HIPK3. Hence, we selected miR-124-3p for further research.
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Recent studies have demonstrated that miRNAs could serve as vital roles in biological progresses for virous of human diseases, such as cardiovascular diseases, inflammatory disease and
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cancers(Deiuliis, 2016). Among them, some miRNAs are recognized as critical epigenetic regulators in many cancers. miR-124-3p has been identified to be decreased in various of malignant tumors, such as
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non-small cell lung cancer(Li et al., 2018b), osteosarcoma(Cong et al., 2018), ovarian cancer(Yuan et
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al., 2017). Moreover, miR-124-3p is a brain-enriched miRNA and is down-regulated in glioma tissues according to some studies, indicating that it may be involved in brain tumor progression(Xiao et al., 2018; Yang et al., 2018b; Zhaohui et al., 2018). We observed that miR-124-3p was decreased in glioma
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cell lines compared with normal cells. We further found that miR-124-3p could bind to circ-HIPK3 and cell proliferation and invasion were significantly suppressed by miR-124-3p mimics. Similarly, the
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effect of circ-HIPK3 siRNA on cell proliferation and invasion could be partly reversed by miR-124-3p inhibitor transfection.
Furthermore, we revealed that miR-124-3p could directly bind to 3’UTR of STAT3 and inhibit U87 and U251 cells growth and invasion. STAT3 (Signal transducer and activator of transcription 3) is a multifunctional signaling pathway belonging to the STAT3s family(Nicolas et al., 2013; Lu et al., 2017). It has been reported that STAT3 is greatly involved in the occurrence, development and metastasis of cancers(Don-Doncow et al., 2014; Gaykalova et al., 2015; Zhang et al., 2018) (25857630 24755219 29572071). Researchers also proved that STAT3 is dysregulated in glioma and has positive effects on glioma cell growth and metastasis(Peng et al., 2016; Wu et al., 2016; Li et al., 2017). In our
ACCEPTED MANUSCRIPT study, we found that STAT3 was up-regulated in glioma cell lines. By base sequence analysis, miR-124-3p was found to be able to complementarily pair with both the bases of circ-HIPK3 and STAT3 3'UTR. We speculated that circ-HIPK3 may indirectly affect the expression of STAT3 by sponging miR-124-3p. Our studied showed that highly expressed circ-HIPK3 can bind to miR-124-3p and abolished miR-124-3p-induced STAT3 degradation. Furthermore, we proved that circ-HIPK3 could
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promote cell proliferation and invasion in glioma cells, while miR-124-3p inhibited cell proliferation and invasion. It is concluded that circ-HIPK3 may indirectly foster STAT3 expression by inhibiting the
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expression of miR-124-3p, thereby affecting the proliferative and invasive capacities of glioma cells. Besides, we also identified that circ-HIPK3 could promote cell cycle and suppress cell apoptosis, while
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miR-124-3p had the opposite effect on glioma cells. All the findings in our study might provide novel insight for the occurrence of glioma, which need further validation in clinical trials and bare mouse
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model.
In summary, we reported circ-HIPK3 was down-regulated in glioma cell lines. Circ-HIPK3 may
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promote the progression of glioma by circ-HIPK3/miR-124-3p/STAT3 regulatory network. This study proposed that circ-HIPK3 may exert diagnostic and therapeutic values on glioma.
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Acknowledgements
Confilct of interest
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None.
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The authors declare no conflict of interest.
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Figure legends：
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gene in human glioma and the association with tumor malignancy. J Exp Clin Cancer
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Figure 1. Circ-HIPK3 knockdown inhibited proliferation and invasion of glioma cells. A. Circ-HIPK3 was highly expressed in glioma cells. B. Circ-HIPK3 was resistant to RNase R digestion in U87 and U251 cell lines. C. Konckdown efficacy of circ-HIPK3 and HIPK3 mRNA in U87 and U251 cell lines was determined by qRT-PCR. D. The effect of circ-HIPK3 negative control and circ-HIPK3 siRNA on cell proliferation was examined by EdU incorporation assay. Pictures were captured under a light microscopewith the magnification, ×10. E. The effect of negative control, circ-HIPK3 siRNA and HIPK3 siRNA on cell proliferation was examined by CCK8 assay. F and G. The effect of negative control, circ-HIPK3 siRNA and HIPK3 siRNA on cell cycle and apoptosis was examined by Flow cytometry. *P<0.05, **P<0.01, ***p<0.0001, ns, no significantly difference, data represent the mean ± SD Figure 2. Circ-HIPK3 was targeted by miR-124-3p A. Downregulating circ-HIPK3 reduced the invasion ability of U87 and U251 cells in
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vitro, magnification, ×100. B. The expression levels were assessed by qRT-PCR in cytoplasmic and nuclear fractions, GAPDH and U6 were used as cytoplasmic and nuclear markers, respectively. C. The binding site between circ-HIPK3 and miR-124-3p predicted by bioinformatics. B. Dual-luciferase reporter gene assay verified that miR-124-3p could bind to circ-HIPK3 3’-UTR in U87 and U251 cells. D. Overexpress efficacy of miR-124-3p in U87 and U251 cell lines was determined by qRT-PCR, respectively. E. Dual-luciferase reporter gene assay verified that miR-124-3p could bind to STAT3 3’-UTR. F. RNA immunoprecipitation (RIP) experiments were performed using extracts of 293T cells. Ago2 and IgG antibody were used to immunoprecipitate and qRT-PCR was applied to detect relative RNA levels of circ-HIPK3 and miR-124-3p. G. MiR-124-3p was lowly expressed in glioma cells. *P<0.05, **P<0.01, ***p<0.0001, data represent the mean ± SD
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Figure 3. Circ-HIPK3 upregulated STAT3 expression by sponging miR-124-3p A. The binding site between miR-124-3p and STAT3 predicted by bioinformatics. B. Dual-luciferase reporter gene assay verified that miR-124-3p could bind to STAT3 3’-UTR in U87 and U251 cells. C. STAT3 was highly expressed in glioma cells. D. QRT–PCR analysis of the effect on overexpression of circHIPK3 by vector transfection in the U87 and U251 cell lines. E. Knockdown efficacy of miR-124-3p in U87 and U251 cell lines was determined by qRT-PCR. F and G. Downregulation of circ-HIPK3 or overexpressed miR-124-3p in U87 and U251 cells could downregulate STAT3 mRNA and protein expression, while co-transfected with miR-124-3p inhibitor or pcDNA-circHIPK3 could reverse STAT3 expression. *P<0.05, **P<0.01, data represent the mean ± SD
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Figure 4. Circ-HIPK3 promoted proliferative and invasive capacities of glioma cells by sponging miR-124-3p A. EdU assays showed that knockdown of circ-HIPK3 or overexpression of miR-124-3p inhibits cellular proliferation. Co-transfecting the miR-124-3p inhibitor or circ-HIPK3 plasmid in U87 and U251 abolished the decreased proliferation. Pictures were captured under a light microscope with the magnification, ×10. B. Cell invasion assays showed that knockdown of circ-HIPK3 or overexpression of miR-124-3p inhibits cell invasion. Co-transfecting the miR-124-3p inhibitor or circ-HIPK3 plasmid in U87 and U251 promoted cell invasion, magnification, ×100. C. Cell cycle assays showed that knockdown of circ-HIPK3 or overexpression of miR-124-3p induced cell cycle arrested. Co-transfecting the miR-124-3p inhibitor or circ-HIPK3 plasmid in U87 and U251 promoted cell cycle. D. Cell apoptosis assays showed that knockdown of circ-HIPK3 or overexpression of miR-124-3p induced cell apoptosis. Co-transfecting the miR-124-3p inhibitor or circ-HIPK3 plasmid in U87 and U251 suppressed cell apoptosis. *P<0.05, **P<0.01, data represent the mean ± SD
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miR-124-3p miR-124-3p mimics miR-124-3p inhibitor Circ-HIPK3 siRNA-1
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Circ-HIPK3 siRNA-2
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U6
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GAPDH
Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Sense Antisense Sense Sense Antisense Sense Antisense
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STAT3
Sequence 5’-TATGTTGGTGGATCCTGTTCGGCA-3’ 5’-TGGTGGGTAGACCAAGACTTGTGA-3’ 5’-GAAGAATCCAACAACGGC-3’ 5’-TCACAATCAGGGAAGCAT-3’ 5’-GCACCGTCAAGGCTGAGAAC-3’ 5’-GGATCTCGCTCCTGGAAGATG-3’ 5’-CTCGCTTCGGCAGCACA-3’ 5’-AACGCTTCACGAATTTGCGT-3’ 5’-CGGTAAGGCACGCGGTGA-3’ 5’-AGTGCGAACTGTGGCGAT-3’ 5’-UAAGGCACGCGGUGAAUGCCAA-3’ 5’-GGCAUUCACCGCGUGCCUUAUU-3’ 5’-UUGGCAUUCACCGCGUGCCUUA -3’ 5’-GCCAUAGACAUGUGGUCAUTT-3’ 5’-AUGACCACAUGUCUAUGGCTT-3’ 5’-GCUUCAAGCAGUACUGCUATT-3’ 5’-UAGCAGUACUGCUUGAAGCTT-3’
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Name Circ-HIPK3
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Figure 1. Circ-HIPK3 knockdown inhibited proliferation and invasion of glioma cells. A. Circ-HIPK3 was highly expressed in glioma cells. B. Circ-HIPK3 was resistant to RNase R digestion in U87 and U251 cell lines. C. Konckdown efficacy of circ-HIPK3 and HIPK3 mRNA in U87 and U251 cell lines was determined by qRT-PCR. D. The effect of circ-HIPK3 negative control and circ-HIPK3 siRNA on cell proliferation was examined by EdU incorporation assay. Pictures were captured under a light microscopewith the magnification, ×10. E. The effect of negative control, circ-HIPK3 siRNA and HIPK3 siRNA on cell proliferation was examined by CCK8 assay. F and G. The effect of negative control, circ-HIPK3 siRNA and HIPK3 siRNA on cell cycle and apoptosis was examined by Flow cytometry. *P<0.05, **P<0.01, ***p<0.0001, ns, no significantly difference, data represent the mean ± SD
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Figure 2. Circ-HIPK3 was targeted by miR-124-3p A. Downregulating circ-HIPK3 reduced the invasion ability of U87 and U251 cells in vitro, magnification, ×100. B. The expression levels were assessed by qRT-PCR in cytoplasmic and nuclear fractions, GAPDH and U6 were used as cytoplasmic and nuclear markers, respectively. C. The binding site between circ-HIPK3 and miR-124-3p predicted by bioinformatics. B. Dual-luciferase reporter gene assay verified that miR-124-3p could bind to circ-HIPK3 3’-UTR in U87 and U251 cells. D. Overexpress efficacy of miR-124-3p in U87 and U251 cell lines was determined by qRT-PCR, respectively. E. Dual-luciferase reporter gene assay verified that miR-124-3p could bind to STAT3 3’-UTR. F. RNA immunoprecipitation (RIP) experiments were performed using extracts of 293T cells. Ago2 and IgG antibody were used to immunoprecipitate and qRT-PCR was applied to detect relative RNA levels of circ-HIPK3 and miR-124-3p. G. MiR-124-3p was lowly expressed in glioma cells. *P<0.05, **P<0.01, ***p<0.0001, data represent the mean ± SD
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Figure 3. Circ-HIPK3 upregulated STAT3 expression by sponging miR-124-3p A. The binding site between miR-124-3p and STAT3 predicted by bioinformatics. B. Dual-luciferase reporter gene assay verified that miR-124-3p could bind to STAT3 3’-UTR in U87 and U251 cells. C. STAT3 was highly expressed in glioma cells. D. QRT–PCR analysis of the effect on overexpression of circHIPK3 by vector transfection in the U87 and U251 cell lines. E. Knockdown efficacy of miR-124-3p in U87 and U251 cell lines was determined by qRT-PCR. F and G. Downregulation of circ-HIPK3 or overexpressed miR-124-3p in U87 and U251 cells could downregulate STAT3 mRNA and protein expression, while co-transfected with miR-124-3p inhibitor or pcDNA-circHIPK3 could reverse STAT3 expression. *P<0.05, **P<0.01, data represent the mean ± SD Figure 4. Circ-HIPK3 promoted proliferative and invasive capacities of glioma cells by sponging miR-124-3p A. EdU assays showed that knockdown of circ-HIPK3 or overexpression of miR-124-3p inhibits cellular proliferation. Co-transfecting the miR-124-3p inhibitor
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or circ-HIPK3 plasmid in U87 and U251 abolished the decreased proliferation. Pictures were captured under a light microscope with the magnification, ×10. B. Cell invasion assays showed that knockdown of circ-HIPK3 or overexpression of miR-124-3p inhibits cell invasion. Co-transfecting the miR-124-3p inhibitor or circ-HIPK3 plasmid in U87 and U251 promoted cell invasion, magnification, ×100. C. Cell cycle assays showed that knockdown of circ-HIPK3 or overexpression of miR-124-3p induced cell cycle arrested. Co-transfecting the miR-124-3p inhibitor or circ-HIPK3 plasmid in U87 and U251 promoted cell cycle. D. Cell apoptosis assays showed that knockdown of circ-HIPK3 or overexpression of miR-124-3p induced cell apoptosis. Co-transfecting the miR-124-3p inhibitor or circ-HIPK3 plasmid in U87 and U251 suppressed cell apoptosis. *P<0.05, **P<0.01, data represent the mean ± SD
ACCEPTED MANUSCRIPT Highlights Circular RNA HIPK3 is up-regulated in glioma cell lines. Circular RNA HIPK3 promotes cell proliferation, invasion and cell cycle, inhibits cell apoptosis.
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Circ-HIPK3/miR-124/STAT3 regulatory axis plays a significant role
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in glioma progression.
ACCEPTED MANUSCRIPT Abbreviations circular RNAs competing endogenous RNAs microRNAs signal transducer and activator of transcription 3 quantitative real-time polymerase chain reaction small interfering RNAs Non-coding RNAs Long noncoding RNAs
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circRNAs ceRNAs miRNAs STAT3 qRT-PCR siRNAs ncRNAs lncRNAs
Daling Hu, Yin Zhang PII: DOI: Reference:
S0378-1119(18)31216-2 https://doi.org/10.1016/j.gene.2018.11.073 GENE 43418
To appear in:
Gene
Received date: Revised date: Accepted date:
13 September 2018 13 November 2018 21 November 2018
Please cite this article as: Daling Hu, Yin Zhang , Circular RNA HIPK3 promotes glioma progression by binding to miR-124-3p. Gene (2018), https://doi.org/10.1016/ j.gene.2018.11.073
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ACCEPTED MANUSCRIPT Circular RNA HIPK3 promotes glioma progression by binding to miR-124-3p Daling Hu1, Yin Zhang2* 1 Department of Geriatrics, Sir Run Run Hospital of Nanjing Medical University 2 Department of Neurosurgery, Sir Run Run Hospital of Nanjing Medical University *Correspondence author: Yin Zhang, Department of Neurosurgery, The Affiliated Sir Run Run Hospital of Nanjing Medical University, Nanjing 210008, China. E-mail: [email protected]
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Abstract This study aims to investigate whether circ-HIPK3 could promote the proliferation and invasion of
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glioma cells by upregulating STAT3 after binding to miR-124-3p, thus participating in the development
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of glioma. Expression levels of circ-HIPK3, miR-124-3p and STAT3 in glioma cell lines were determined using qRT-PCR. The regulatory effects of circ-HIPK3, miR-124-3p and STAT3 on
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proliferative and invasive capacities of glioma cells were accessed using EdU assay, CCK-8 assay and invasion assay, respectively. Cell cycle assay and cell apoptosis assay were performed by flow
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cytometry. Dual-luciferase reporter gene assay was conducted to determine the binding condition among circ-HIPK3, miR-124-3p and STAT3. Rescue experiments were performed in co-transfected glioma cells. QRT-PCR data showed that circ-HIPK3 and STAT3 are highly expressed, whereas
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miR-124-3p is lowly expressed in glioma cells than those of negative control cell. Knockdown of
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circ-HIPK3 in U87 and U251 cells inhibited their proliferative and invasive capacities. On the contrary, miR-124-3p knockdown improved proliferative and migratory capacities. Dual-luciferase reporter gene assay exerted that circ-HIPK3 could bind to miR-124-3p and STAT3 is the target gene of miR-124-3p.
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Western blot results elucidated that circ-HIPK3 stabilizes STAT3 expression, whereas miR-124-3p
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degrades STAT3 expression. Rescue experiments demonstrated that overexpression of circ-HIPK3 could partially reverse the inhibited proliferative and migratory capacities induced by miR-124-3p in U87 and U251 cells. In summary，we found that overexpression of circ-HIPK3 promotes proliferative and invasive capacities of glioma cells by sponging miR-124-3p to upregulate STAT3 expression.
Key words: circ-HIPK3; miR-124-3p; proliferation; invasion; STAT3
1. Introduction Gliomas, known as the most prevalent primary brain tumor of central nervous system, account for significant mortality worldwide annually(Chen et al., 2012; Yu et al., 2017). Based on the degree of
ACCEPTED MANUSCRIPT malignancy and histopathological features, gliomas could be classified into I to IV levels(Fuller and Scheithauer, 2007; Louis et al., 2007). Although great efforts such as novel surgical treatments, radiation and conventional chemotherapies made in recent decades toward improvement for gliomas, higher-grade gliomas are still significant severe and associated with poor prognosis(Zhou et al., 2010). Therefore, exploring the molecular mechanism associated with the pathogenesis of glioma is essential,
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and might lead to the development of novel targeted therapies. Non-coding RNAs (ncRNAs) are a group of functional RNA having little protein-coding
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ability(Anastasiadou et al., 2018). Based on the structure and length, ncRNAs could be further classified into various groups, including microRNAs (miRNAs), long noncoding RNAs (lncRNAs) and
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circular RNAs (circRNAs)(Ransohoff et al., 2018; Wu et al., 2018). CircRNAs is referred to as a new class of functional molecule with a covalenty closed loop structure linking the 3’ and 5’
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terminals(Enuka et al., 2016). Emerging evidence suggests that circRNAs are conserved and stable, and most of them play their role in post-transcriptional level(Veno et al., 2015). Considering that circRNAs
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contains many miRNAs binding sites, in mainly function as miRNA sponges to regulate gene expression(Hansen et al., 2013). It’s reported that circRNAs have multiple functions in many aspects of
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physiological or pathological processes, including cell proliferation, migration, invasion, apoptosis, and
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cell cycle(Chen et al., 2017; Dai et al., 2018). Recent studies show that circRNAs have been identified to be dysregulated and involved in various of cancers. For examples, downregulated circ-LARP4 is detected in gastric cancer which represents poor prognosis(Zhang et al., 2017), upregulated
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circ-CCDC66 fosters the growth and metastasis of colon cancer(Hsiao et al., 2017), circ-ITCH functions as a tumor inhibitor via a novel circ-ITCH/miR-17, miR-224/p21, PTEN axis in bladder
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cancer(Yang et al., 2018a).
CircHIPK3(hsa_circ_0000284) is derived from HIPK3 gene and consisted of the head-to-tail splicing of exon 2 (1099bp)(Zheng et al., 2016). It is demonstrated that circHIPK3 could facilitate cell proliferation and invasion by binding and inhibiting many tumor-suppressive miRNAs(Xiao-Long et al., 2018). Ding-kai et al. found that the expression of circHIPK3 in gallbladder cancer is increased, which promotes cancer cell growth via spongingmiR-124(Kai et al., 2018). Similarly, Chen et al. revealed circHIPK3 promotes cell proliferation and migration and upregulates AQP3 expression in hepatocellular carcinoma(Chen et al., 2018). Although Jin and co-workers have confirmed that circ-HIPK3 is up-regulated in glioma tissues and may serve as a prognostic biomarker(Jin et al., 2018),
ACCEPTED MANUSCRIPT the underlying mechanism of circ-HIPK3 in glioma remains to be investigated. Our study discovered that circHIPK3 may boost cell proliferation and invasion via binding to miR-124-3p from STAT3.
2. Materials and methods 2.1. Cell culture and transfection Normal brain glial cell lines (HEB) and human glioma cells (U87, U251, LN229 and LN308)
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were provided by American Type Culture Collection (ATCC, Manassas VA, USA). All the cells were cultured in RPMI-1640 containing 10% FBS (Life Technologies, USA) and maintained at 37°C, 5%
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CO2. Cells were seeded into 6-well plates and cell transfection was performed until 60-70% of
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confluence using Lipofectamine 2000 (Invitrogen, CA, USA) following the manufacturer’s instructions. After incubation for 48 h, cells were used for further assay. Circ-HIPK3 siRNA, pcDNA-circHIPK3,
Genepharma Company (Shanghai, China).
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2.2. Quantitative RT-PCR
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miR-124-3p mimics, miR-124-3p inhibitor and the negative controls were all synthesized by
TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was utilized to extract total RNA in cell lines. For detection of mRNA,RNA was reversely transcribed into complimentary DNA (cDNA) with
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Reverse Transcription Kit (Takara, Tokyo, Japan). For detection of miR-124-3p-3p, total RNA was
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reverse-transcribed to cDNA using the TaqMan miRNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA). Then miR-124-3p-3p was quantified using the TaqMan miRNA kit
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(Applied Biosystems). QRT-PCR was carried out utilizing SYBR® Premix Ex TaqTM (Takara) on the ABI 7500HT (Applied Biosystems). GAPDH and U6 were used as endogenous control, respectively.
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The relative expression levels were calculated utilizing the 2−△△Ct method. The experiments were repeated in triplicate independently and all primers involved were listed in Table 1. 2.3. RNase R digestion For RNase R digestion, total RNA (5 ug) was incubated for 15 min at 37°C using 3U/ug of RNase R (Epicentre Biotechnologies). Based on previously published procedures(Shan et al., 2017), the RNase R digestion reaction was done twice. 2.4. EdU proliferation assay EdU assay kit (Ribobio, Guangzhou, China) was used for cell proliferation assay according to the the manufacturer’s instructions. In brief, 100 μmol/L of EdU were added to the transfected U87 and
ACCEPTED MANUSCRIPT U251 cells for 2 hours at 37°C, after which the cultured cells were fixed using 4% paraformaldehyde for 25 minutes. Then the cells were stained with 1 × Apollo reaction cocktail (deionized water, Apollo reaction buffer, Apollo catalyst solution, Apollo fluorescent dye solution and Apollo buffer
additive) for 30 minutes and then incubated with 100 μL of Hoechst33342 at 5 μg/mL for 30 minutes. Fluorescent microscope was utilized to examine the percentage of EdU-positive cells. All assays were
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independently carried out in triplicate. 2.5. CCK-8 assay
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After treatment, cells were plated at a dose of 3000 cells/well into 96-well plates. After cell cultured for 24 h, 48 h, 72 h and 96 h, 10 μl of Cell-Counting Kit 8 (CCK8; Dojindo Laboratories,
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Kumamoto, Japan) reagent was added in each well according to the manufacturer’s instructions. Two hours later, the OD value at the wavelength of 450 nm was measured utilizing a microplate reader.
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Each assay was repeated in triplicate. 2.6. Cell invasion assay
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Transwell chambers (8 um pore size, Millipore Corporation, Billerica, MA) coated with with 0.5 mg/ml collagen type I (BD Bioscience) and a 1:15 dilution of Matrigel (BD Bioscience) were used to
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invasion chambers () in RPMI-1640 without serum, while 600 μl of medium containing 10% FBS were added to the lower chamber. After 48 hours of cell culture, we carefully removed the noninvasive cells in the top chambers. The cells that invaded to the lower chamber were fixed with 4% paraformaldehyde
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for 30 minutes, and then stained with crystal violet for 20 min. Penetrating cells were captured in 5 randomly selected fields of each sample. All the assays were conducted three times independently.
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2.7. Cell cycle assay
U87 and U251 cells were harvested 48 h after the transfection and then washed with ice-cold phosphate buffered saline (PBS). The cells were fixed in 70% ethanol for 24 h, before being re-suspended in 500 μl of PBS which contains 50 μg/mL of propidium iodide (Sigma), 10 μg/mL RNase A, 0.1% sodium citrate and also 0.1% Triton X-100, and incubated for 15 min at room temperature in the dark. Cell cycle was immediately analyzed utilizing the flow cytometer (Millipore Guava). 2.8. Cell apoptosis assay The cells were harvested 48 h after the transfection and then incubated with Annexin V-FITC
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2.9. Subcellular fractionation location Nuclear and cytoplasmic RNA was isolated with the PARIS Kit (Life Technologies, USA) as described
were considered as cytoplasmic and nuclear markers, respectively.
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2.10. Dual-luciferase reporter gene assay
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in directions. Total RNA isolated from each fraction was determined by qRT-PCR. GAPDH and U6
STAT3-WT 3'UTR, STAT3-MUT 3'UTR, circ-HIPK3-WT 3'UTR, or circ-HIPK3-MUT 3'UTR
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was inserted into pGL3. Cells were then seeded into the 24-well plates respectively with 5×105 cells per well. They were co-transfected with 0.12 μg vector and 40 nM miR-124-3p mimics or negative
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control for 48 h. Dual-Luciferase reporter assay system (Promega, Madison, WI, USA) was utilized to measure the luciferase activity.
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2.11. RNA immunoprecipitation
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The Imprint RNA Immunoprecipitation Kit (Sigma, St. Louis) together with the AGO2 antibody (Cell Signaling, Rockford) were used to perform RNA immunoprecipitation (RIP) assays. Then protein A/G beads were utilized to recover the AGO2 antibody. qRT-PCR was performed to measure
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circ-HIPK3 and miR-124-3p RNA levels in the immunoprecipitates. 2.12. Western blot
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Total protein was extracted utilizing a cell lysate (RIPA). The protein concentration of each cell lysate was quantified using BCA Protein Assay Kit Protein (Beyotime, Nantong, China). Equal amounts of samples were separated in the SDS-PAGE gel before being transferred to the PVDF membrane. Next, the membranes were blocked with the blocking solution for 1 hour And incubated overnight at 4°C using primary antibody. After being washed with TBST, corresponding secondary antibody was used for incubation for 2 hours at room temperature. At last, a Tanon detection system using ECL reagent (Thermo) was utilized to capture the images of protein bands. 2.13. Statistical analysis The data analyses were carried out using SPSS 22.0 and Graphpad Prism 6.0. Data were shown as
ACCEPTED MANUSCRIPT the mean ± one standard deviation (SD) from no less than three separate experiments. The Student t test was applied to analyze the comparisons between groups. And P < 0.05 was considered to be statistically significant.
3. Results 3.1. Circ-HIPK3 was highly expressed in glioma cell lines and-promoted cell proliferation and
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invasion The expression levels of circ-HIPK3 in normal cell lines (HEB) and glioma cell lines (U87, U251,
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LN229, LN308) were detected by qRT-PCR. The results revealed that circ-HIPK3 was upregulated in glioma cell lines (Figure 1A). Among them, the expression difference was pronounced in U251 and
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U87 cell lines, which were selected for subsequent experiments. Subsequently, to investigate the circular nature of circ-HIPK3, total RNA was treated with RNaseR, which could deplete linear RNAs
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featuring a free 3’terminus while having no impact on circRNAs. The results unveiled that circular RNAs were insusceptible of RNaseR digestion, but the liner RNAs were not (Figure 1B). Then, two
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circ-HIPK3 siRNAs (si- circ-HIPK3-1 and si- circ-HIPK3-2) were transfected into U87 and U251 cells, respectively. Two siRNAs all markedly downregulated circ-HIPK3 expression (Figure S1A).
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Particularly, si- circ-HIPK3-1 showed the highest interference efficiency, and was selected for
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subsequent experiments. We also found that circ-HIPK3 siRNA could decrease the expression levels of circ-HIPK3 whilemake no effect on
HIPK3 mRNA (Figure 1C). Morever, the proliferation of
si-circHIPK3 transfected U87 and U251 cell lines was evaluated by EdU assays and CCK8 assays. As
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shown in Figure 1D and 1E, we found that cell proliferation was significantly suppressed in si-circHIPK3 transfected cells, but no difference was observed between the si-HIPK3 and siRNA
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negative control transfected cells. The effect of circ-HIPK3 on cell cycle and apoptosis was analyzed using flow cytometry. The results indicated that cells in si-circHIPK3 groups were arrested chiefly in G0/G1 phase compared with siRNA negative control groups (Figure 1F). What’s more, the apoptosis ratio was higher in si-circHIPK3 group compared with the control group (Figure 1G). Here, we subsequently investigated the effect of circ-HIPK3 on invasion of U87 and U251 cells. As displayed in Figure 2A, si-circHIPK3 transfected cells showed a strongly suppressed invasive ability. These results suggested that circ-HIPK3 might act as a promoter in glioma cell progress. 3.2. Circ-HIPK3 was targeted by miR-124-3p We then searched for the subcellular location of circ-HIPK3 in order to look into the role of
ACCEPTED MANUSCRIPT circ-HIPK3 in glioma progression. The results indicated that circ-HIPK3 was localized mostly in the cytoplasm of U87 and U251 cells (Figure 2B), which meant that circ-HIPK3 might regulate cell progress at the post-transcriptional level. Previous study proved that circ-HIPK3 could function as miRNAs sponges and bind to miR-124-3p directly and inhibit the activity of miR-124-3p(Zheng et al., 2016). Considering that miR-124-3p had been reported to participated in glioma, we hypothesized that
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circ-HIPK3 might promote glioma cell proliferation and invasion by binging to miR-124-3p. Bioinformatics analysis showed that circ-HIPK3 comprises mutual miRNA response elements to
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miR-124-3p (Figure 2C). We transfected U251 and U87 cells with miR-124-3p mimics to achieve miR-124-3p overexpressed and verified the transfection efficiency using qPCR (Figure 2D). Then
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luciferase reporter assay was performed to explore the correlation between circ-HIPK3 and miR-124-3p in U251 and U87 cells. As presented in Figure 2E, we found that the luciferase activity in
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circHIPK3-WT group decreased dramatically with the presence of miR-124-3p mimics compared with that of NC. However, no significant change was seen in luciferase activity of the circ-HIPK3-MUT
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3'UTR group. Furthermore, RIP assay for AGO2 was conducted on 293T cells and both circ-HIPK3 and miR-124-3p were detected with approximately several-fold enrichment compared with IgG (Figure
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2F), showing that circ-HIPK3 and miR-124-3p-3p existed in RNA-induced silencing complex (RISC)
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and miR-124-3p directly binds to circ-HIPK3. We also found that the expression level of miR-124-3p in glioma cell lines was lowerthan that in normal cell lines (Figure 2G). Hence, we wondered whether circ-HIPK3 could suppress the expression of miR-124-3p. Then we detected the expression of
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miR-124-3p by qRT-PCR after U87 and U251 cells were transfected with circ-HIPK3 siRNA or pcDNA-circHIPK3. The results indicated that circ-HIPK3 might be able to bind to miR-124-3p and
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inhibit its expression (Figure S1B-C). 3.3. Circ-HIPK3 upregulated STAT3 expression by sponging miR-124-3p in glioma cells We predicted genes that bind to miR-124-3p through bioinformatics and performed functional analysis. Finally, STAT3 was screened out (Figure 3A). Dual-luciferase reporter gene assay revealed decreased luciferase activity in U87 and U251 cells of STAT3-WT 3'UTR group, but no significant change in luciferase activity was seen in STAT3-MUT 3'UTR group (Figure 3B), indicating that STAT3 binds to miR-124-3p. Subsequently, qRT-PCR was utilized to examine the expression level of STAT3 in glioma cell lines. The data showed that STAT3 is up-expressed in glioma cells (Figure 3C). After interference with circ-HIPK3 overexpressed plasmid in U87 and U251 cells, circ-HIPK3 expression
ACCEPTED MANUSCRIPT increased (Figure 3D). Similarly, cells were transfected with miR-124-3p inhibitor, which had high transfection efficiency (Figure 3E). We further assessed the effects of circ-HIPK3 and miR-124-3p on STAT3 by qRT-PCR and Western blot analysis. As illustrated in Figure 2F, the mRNA level of STAT3 was significantly decreased in circ-HIPK3 knockdown or miR-124-3p mimics glioma cells, while co-transfected with miR-124-3p inhibitor or circ-HIPK3 overexpressed plasmid could reverse the
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inhibited effect (Figure 3F). What’s more, we also found that overexpression of circ-HIPK3 or knockdown of miR-124-3p individually in U87 and U251 cells could increase the expression of STAT3
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(Figure S1D-E). Accordant with qRT-PCR results, the protein level of STAT3 presented similar trends in U87 and U251 cells following circ-HIPK3 or miR-124-3p interference (Figure 3G).
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3.4. Over-expression of circHIPK3 reverses miR-124-3p-induced inhibition of cell migration, invasion in glioma cells
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To further verify the regulatory relationship among circ-HIPK3, miR-124-3p and STAT3, we tested proliferative and migratory capacities after U87 and U251 cells were interfered with both
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circ-HIPK3 and miR-124-3p. The results disclosed that the knockdown of circ-HIPK3 in U87 and U251 cells suppressed cell proliferation and migration, while co-transfection with miR-124-3p
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inhibitor reserved the inhibited proliferation and migration. Similarly, up-regulation of circ-HIPK3
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could reverse the inhibition induced by miR-124-3p mimics in glioma cells (Figure 4A and 4B). We also performed flow cytometry assays to investigate the effects of circ-HIPK3/miR-124-3p/STAT3 on glioma cell proliferation associated with cell cycle and cell apoptosis. As shown in Figure 4C and 4D,
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U251 and U87 cells transfected with circ-HIPK3 siRNA or miR-124-3p mimics could make cell cycle arrested in G0/G1 and induce cell apoptosis, while cells co-transfected with miR-124-3p inhibitor or
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pcDNA-circHIPK3 reversed the effect. These results provided evidence
that circ-HIPK3 may
promote cell proliferation and migration in glioma cells by upregulating STAT3 expression after binding to miR-124-3p.
4. Discussion Recently, researchers have identified many exonic transcripts have the ability to forming circRNAs by gene arrangement or non-liner reverse splicing(Zheng et al., 2017). Circ-RNAs are conserved and widely expressed in tissues and cells(Song et al., 2016). CircRNAs also play a vital role in regulating cellular progresses and tumorigenesis. Many recent researches show that circRNAs play a significant role in glioma. Li et al., showed that circ-0046701 can increase the expression level of
ACCEPTED MANUSCRIPT miR-142-3p target ITGB8 in glioma and therefore promote carcinogenesis(Li et al., 2018a), Wang et al., have identified that circ-0001649 has oncogenic features in glioma(Wang et al., 2018), Bian et al., found that circ-CFH could promote glioma progression by directly sponging miR-149 and regulating AKT1(Bian et al., 2018). In our study, we unveiled that circ-HIPK3 is upregulated in glioma cell lines. Subsequent
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experiments suggested that circ-HIPK3 silencing by targeted siRNAs significantly inhibited cell proliferation, invasion and arrested cell cycle in G0/G1, while inducing cell apoptosis. Then we
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conducted the experiment and found that circ-HIPK3 located mostly in cytoplasm fraction of cells, which meant it may affect cell function via acting as miRNAs sponge. Bioinformatics prediction
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revealed that several miRNAs including miR-124-3p proved to dysregulation in glioma could bind to circ-HIPK3. Hence, we selected miR-124-3p for further research.
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Recent studies have demonstrated that miRNAs could serve as vital roles in biological progresses for virous of human diseases, such as cardiovascular diseases, inflammatory disease and
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cancers(Deiuliis, 2016). Among them, some miRNAs are recognized as critical epigenetic regulators in many cancers. miR-124-3p has been identified to be decreased in various of malignant tumors, such as
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non-small cell lung cancer(Li et al., 2018b), osteosarcoma(Cong et al., 2018), ovarian cancer(Yuan et
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al., 2017). Moreover, miR-124-3p is a brain-enriched miRNA and is down-regulated in glioma tissues according to some studies, indicating that it may be involved in brain tumor progression(Xiao et al., 2018; Yang et al., 2018b; Zhaohui et al., 2018). We observed that miR-124-3p was decreased in glioma
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cell lines compared with normal cells. We further found that miR-124-3p could bind to circ-HIPK3 and cell proliferation and invasion were significantly suppressed by miR-124-3p mimics. Similarly, the
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effect of circ-HIPK3 siRNA on cell proliferation and invasion could be partly reversed by miR-124-3p inhibitor transfection.
Furthermore, we revealed that miR-124-3p could directly bind to 3’UTR of STAT3 and inhibit U87 and U251 cells growth and invasion. STAT3 (Signal transducer and activator of transcription 3) is a multifunctional signaling pathway belonging to the STAT3s family(Nicolas et al., 2013; Lu et al., 2017). It has been reported that STAT3 is greatly involved in the occurrence, development and metastasis of cancers(Don-Doncow et al., 2014; Gaykalova et al., 2015; Zhang et al., 2018) (25857630 24755219 29572071). Researchers also proved that STAT3 is dysregulated in glioma and has positive effects on glioma cell growth and metastasis(Peng et al., 2016; Wu et al., 2016; Li et al., 2017). In our
ACCEPTED MANUSCRIPT study, we found that STAT3 was up-regulated in glioma cell lines. By base sequence analysis, miR-124-3p was found to be able to complementarily pair with both the bases of circ-HIPK3 and STAT3 3'UTR. We speculated that circ-HIPK3 may indirectly affect the expression of STAT3 by sponging miR-124-3p. Our studied showed that highly expressed circ-HIPK3 can bind to miR-124-3p and abolished miR-124-3p-induced STAT3 degradation. Furthermore, we proved that circ-HIPK3 could
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promote cell proliferation and invasion in glioma cells, while miR-124-3p inhibited cell proliferation and invasion. It is concluded that circ-HIPK3 may indirectly foster STAT3 expression by inhibiting the
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expression of miR-124-3p, thereby affecting the proliferative and invasive capacities of glioma cells. Besides, we also identified that circ-HIPK3 could promote cell cycle and suppress cell apoptosis, while
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miR-124-3p had the opposite effect on glioma cells. All the findings in our study might provide novel insight for the occurrence of glioma, which need further validation in clinical trials and bare mouse
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model.
In summary, we reported circ-HIPK3 was down-regulated in glioma cell lines. Circ-HIPK3 may
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promote the progression of glioma by circ-HIPK3/miR-124-3p/STAT3 regulatory network. This study proposed that circ-HIPK3 may exert diagnostic and therapeutic values on glioma.
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Acknowledgements
Confilct of interest
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None.
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The authors declare no conflict of interest.
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Figure 1. Circ-HIPK3 knockdown inhibited proliferation and invasion of glioma cells. A. Circ-HIPK3 was highly expressed in glioma cells. B. Circ-HIPK3 was resistant to RNase R digestion in U87 and U251 cell lines. C. Konckdown efficacy of circ-HIPK3 and HIPK3 mRNA in U87 and U251 cell lines was determined by qRT-PCR. D. The effect of circ-HIPK3 negative control and circ-HIPK3 siRNA on cell proliferation was examined by EdU incorporation assay. Pictures were captured under a light microscopewith the magnification, ×10. E. The effect of negative control, circ-HIPK3 siRNA and HIPK3 siRNA on cell proliferation was examined by CCK8 assay. F and G. The effect of negative control, circ-HIPK3 siRNA and HIPK3 siRNA on cell cycle and apoptosis was examined by Flow cytometry. *P<0.05, **P<0.01, ***p<0.0001, ns, no significantly difference, data represent the mean ± SD Figure 2. Circ-HIPK3 was targeted by miR-124-3p A. Downregulating circ-HIPK3 reduced the invasion ability of U87 and U251 cells in
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vitro, magnification, ×100. B. The expression levels were assessed by qRT-PCR in cytoplasmic and nuclear fractions, GAPDH and U6 were used as cytoplasmic and nuclear markers, respectively. C. The binding site between circ-HIPK3 and miR-124-3p predicted by bioinformatics. B. Dual-luciferase reporter gene assay verified that miR-124-3p could bind to circ-HIPK3 3’-UTR in U87 and U251 cells. D. Overexpress efficacy of miR-124-3p in U87 and U251 cell lines was determined by qRT-PCR, respectively. E. Dual-luciferase reporter gene assay verified that miR-124-3p could bind to STAT3 3’-UTR. F. RNA immunoprecipitation (RIP) experiments were performed using extracts of 293T cells. Ago2 and IgG antibody were used to immunoprecipitate and qRT-PCR was applied to detect relative RNA levels of circ-HIPK3 and miR-124-3p. G. MiR-124-3p was lowly expressed in glioma cells. *P<0.05, **P<0.01, ***p<0.0001, data represent the mean ± SD
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Figure 3. Circ-HIPK3 upregulated STAT3 expression by sponging miR-124-3p A. The binding site between miR-124-3p and STAT3 predicted by bioinformatics. B. Dual-luciferase reporter gene assay verified that miR-124-3p could bind to STAT3 3’-UTR in U87 and U251 cells. C. STAT3 was highly expressed in glioma cells. D. QRT–PCR analysis of the effect on overexpression of circHIPK3 by vector transfection in the U87 and U251 cell lines. E. Knockdown efficacy of miR-124-3p in U87 and U251 cell lines was determined by qRT-PCR. F and G. Downregulation of circ-HIPK3 or overexpressed miR-124-3p in U87 and U251 cells could downregulate STAT3 mRNA and protein expression, while co-transfected with miR-124-3p inhibitor or pcDNA-circHIPK3 could reverse STAT3 expression. *P<0.05, **P<0.01, data represent the mean ± SD
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Figure 4. Circ-HIPK3 promoted proliferative and invasive capacities of glioma cells by sponging miR-124-3p A. EdU assays showed that knockdown of circ-HIPK3 or overexpression of miR-124-3p inhibits cellular proliferation. Co-transfecting the miR-124-3p inhibitor or circ-HIPK3 plasmid in U87 and U251 abolished the decreased proliferation. Pictures were captured under a light microscope with the magnification, ×10. B. Cell invasion assays showed that knockdown of circ-HIPK3 or overexpression of miR-124-3p inhibits cell invasion. Co-transfecting the miR-124-3p inhibitor or circ-HIPK3 plasmid in U87 and U251 promoted cell invasion, magnification, ×100. C. Cell cycle assays showed that knockdown of circ-HIPK3 or overexpression of miR-124-3p induced cell cycle arrested. Co-transfecting the miR-124-3p inhibitor or circ-HIPK3 plasmid in U87 and U251 promoted cell cycle. D. Cell apoptosis assays showed that knockdown of circ-HIPK3 or overexpression of miR-124-3p induced cell apoptosis. Co-transfecting the miR-124-3p inhibitor or circ-HIPK3 plasmid in U87 and U251 suppressed cell apoptosis. *P<0.05, **P<0.01, data represent the mean ± SD
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miR-124-3p miR-124-3p mimics miR-124-3p inhibitor Circ-HIPK3 siRNA-1
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Circ-HIPK3 siRNA-2
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GAPDH
Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Sense Antisense Sense Sense Antisense Sense Antisense
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Sequence 5’-TATGTTGGTGGATCCTGTTCGGCA-3’ 5’-TGGTGGGTAGACCAAGACTTGTGA-3’ 5’-GAAGAATCCAACAACGGC-3’ 5’-TCACAATCAGGGAAGCAT-3’ 5’-GCACCGTCAAGGCTGAGAAC-3’ 5’-GGATCTCGCTCCTGGAAGATG-3’ 5’-CTCGCTTCGGCAGCACA-3’ 5’-AACGCTTCACGAATTTGCGT-3’ 5’-CGGTAAGGCACGCGGTGA-3’ 5’-AGTGCGAACTGTGGCGAT-3’ 5’-UAAGGCACGCGGUGAAUGCCAA-3’ 5’-GGCAUUCACCGCGUGCCUUAUU-3’ 5’-UUGGCAUUCACCGCGUGCCUUA -3’ 5’-GCCAUAGACAUGUGGUCAUTT-3’ 5’-AUGACCACAUGUCUAUGGCTT-3’ 5’-GCUUCAAGCAGUACUGCUATT-3’ 5’-UAGCAGUACUGCUUGAAGCTT-3’
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Figure 1. Circ-HIPK3 knockdown inhibited proliferation and invasion of glioma cells. A. Circ-HIPK3 was highly expressed in glioma cells. B. Circ-HIPK3 was resistant to RNase R digestion in U87 and U251 cell lines. C. Konckdown efficacy of circ-HIPK3 and HIPK3 mRNA in U87 and U251 cell lines was determined by qRT-PCR. D. The effect of circ-HIPK3 negative control and circ-HIPK3 siRNA on cell proliferation was examined by EdU incorporation assay. Pictures were captured under a light microscopewith the magnification, ×10. E. The effect of negative control, circ-HIPK3 siRNA and HIPK3 siRNA on cell proliferation was examined by CCK8 assay. F and G. The effect of negative control, circ-HIPK3 siRNA and HIPK3 siRNA on cell cycle and apoptosis was examined by Flow cytometry. *P<0.05, **P<0.01, ***p<0.0001, ns, no significantly difference, data represent the mean ± SD
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Figure 2. Circ-HIPK3 was targeted by miR-124-3p A. Downregulating circ-HIPK3 reduced the invasion ability of U87 and U251 cells in vitro, magnification, ×100. B. The expression levels were assessed by qRT-PCR in cytoplasmic and nuclear fractions, GAPDH and U6 were used as cytoplasmic and nuclear markers, respectively. C. The binding site between circ-HIPK3 and miR-124-3p predicted by bioinformatics. B. Dual-luciferase reporter gene assay verified that miR-124-3p could bind to circ-HIPK3 3’-UTR in U87 and U251 cells. D. Overexpress efficacy of miR-124-3p in U87 and U251 cell lines was determined by qRT-PCR, respectively. E. Dual-luciferase reporter gene assay verified that miR-124-3p could bind to STAT3 3’-UTR. F. RNA immunoprecipitation (RIP) experiments were performed using extracts of 293T cells. Ago2 and IgG antibody were used to immunoprecipitate and qRT-PCR was applied to detect relative RNA levels of circ-HIPK3 and miR-124-3p. G. MiR-124-3p was lowly expressed in glioma cells. *P<0.05, **P<0.01, ***p<0.0001, data represent the mean ± SD
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Figure 3. Circ-HIPK3 upregulated STAT3 expression by sponging miR-124-3p A. The binding site between miR-124-3p and STAT3 predicted by bioinformatics. B. Dual-luciferase reporter gene assay verified that miR-124-3p could bind to STAT3 3’-UTR in U87 and U251 cells. C. STAT3 was highly expressed in glioma cells. D. QRT–PCR analysis of the effect on overexpression of circHIPK3 by vector transfection in the U87 and U251 cell lines. E. Knockdown efficacy of miR-124-3p in U87 and U251 cell lines was determined by qRT-PCR. F and G. Downregulation of circ-HIPK3 or overexpressed miR-124-3p in U87 and U251 cells could downregulate STAT3 mRNA and protein expression, while co-transfected with miR-124-3p inhibitor or pcDNA-circHIPK3 could reverse STAT3 expression. *P<0.05, **P<0.01, data represent the mean ± SD Figure 4. Circ-HIPK3 promoted proliferative and invasive capacities of glioma cells by sponging miR-124-3p A. EdU assays showed that knockdown of circ-HIPK3 or overexpression of miR-124-3p inhibits cellular proliferation. Co-transfecting the miR-124-3p inhibitor
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or circ-HIPK3 plasmid in U87 and U251 abolished the decreased proliferation. Pictures were captured under a light microscope with the magnification, ×10. B. Cell invasion assays showed that knockdown of circ-HIPK3 or overexpression of miR-124-3p inhibits cell invasion. Co-transfecting the miR-124-3p inhibitor or circ-HIPK3 plasmid in U87 and U251 promoted cell invasion, magnification, ×100. C. Cell cycle assays showed that knockdown of circ-HIPK3 or overexpression of miR-124-3p induced cell cycle arrested. Co-transfecting the miR-124-3p inhibitor or circ-HIPK3 plasmid in U87 and U251 promoted cell cycle. D. Cell apoptosis assays showed that knockdown of circ-HIPK3 or overexpression of miR-124-3p induced cell apoptosis. Co-transfecting the miR-124-3p inhibitor or circ-HIPK3 plasmid in U87 and U251 suppressed cell apoptosis. *P<0.05, **P<0.01, data represent the mean ± SD
ACCEPTED MANUSCRIPT Highlights Circular RNA HIPK3 is up-regulated in glioma cell lines. Circular RNA HIPK3 promotes cell proliferation, invasion and cell cycle, inhibits cell apoptosis.
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Circ-HIPK3/miR-124/STAT3 regulatory axis plays a significant role
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in glioma progression.
ACCEPTED MANUSCRIPT Abbreviations circular RNAs competing endogenous RNAs microRNAs signal transducer and activator of transcription 3 quantitative real-time polymerase chain reaction small interfering RNAs Non-coding RNAs Long noncoding RNAs
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circRNAs ceRNAs miRNAs STAT3 qRT-PCR siRNAs ncRNAs lncRNAs