Circular RNA hsa_circ_0001564 facilitates tumorigenesis of osteosarcoma via sponging miR-29c-3p

Abstract

Circular RNAs are a novel type of non-coding RNAs generated from back splicing, which has been verified to mediate multiple tumorigenesis. However, the role of circular RNA in osteosarcoma is still unclear. In this study, we preliminarily screened the circular RNAs expression profiles in osteosarcoma and investigated the potential regulation mechanism. The circular RNAs expression profiles in osteosarcoma were screened using circular RNA microarray analysis, and results showed that there were 1152 circular RNAs upregulated and 915 circular RNAs downregulated in tumor tissue compared to adjacent tissue. Hsa_circ_0001564, located at 5q35.3 and its associated gene symbol is CANX, was one of the significantly overexpressed circular RNAs in osteosarcoma tissue, as well as in osteosarcoma cell lines. In functional experiments, hsa_circ_001564 knockdown significantly suppressed the proliferation activity, induced cell-cycle arrest in G0/G1 phase, and promoted apoptosis in HOS and MG-63 cells. Subsequently, we explored the probable mechanism of hsa_circ_001564, and fortunately, bioinformatics analysis revealed that miR-29c-3p contained the complementary binding region with hsa_circ_0001564, which was confirmed by dual-luciferase reporter assay. Moreover, rescue experiments illustrated that miR-29c-3p could reverse the oncogenesis effect of hsa_circ_001564. Our study discovers that hsa_circ_0001564 acts as miR-29c-3p sponge to mediate the tumorigenicity, which could act as a potential biomarker for the osteosarcoma and provide a novel insight for competing endogenous RNA mechanism in osteosarcoma.

Introduction

Osteosarcoma is widely regarded as the most common primary bone tumor and the third most common tumor in children and adolescents, which is characterized by rapid progression, high metastatic potential, and poor clinical prognosis.^1,2 Overall survival rate of osteosarcoma has not been effectively increased with the existing therapeutic methods and the morbidity is still increasing by 1.4% per year, which is second in frequency to multiple myeloma. ³ The poor 5-year survival rate for osteosarcoma patients indicates that molecular mechanisms involving osteosarcoma formation and development need in-depth study. Molecular targeted therapy and immunotherapy have been tried to apply to the treatment of osteosarcoma. Unfortunately, there are still no effective treatments of osteosarcoma to improve the long-term survival of osteosarcoma patients.

Circular RNAs (circRNAs) are a large class of endogenous non-coding RNAs and generated through “backsplicing” without a free 3′- or 5′- end compared with linear RNAs, which are terminated with 5′ caps and 3′ tails. ⁴ In 1991, Nigro et al. ⁵ first found circRNA by identifying spliced transcripts of a candidate tumor suppressor gene. In the beginning, the novel type of non-coding RNA products was regarded as splicing errors and overlooked by researchers for decades. Recently, due to the high-throughput sequencing and bioinformatics, more and more circRNAs and their properties are being discovered and reported. CircRNAs are more stable than corresponding linear mRNAs to resist to RNase activity in vivo. ⁶ Abundant reports revealed that circRNAs specifically express in tissues or developmental stage and participate in a wide range of biological metabolic process. ⁷

The circRNA hsa_circ_0001564 (ID in circBase; www.circbase.org/) locates at 5q35.3 (chr5: 179132679-179137066) and its associated gene symbol is CANX. In this study, we applied microarray analysis to detect the expression profiles of circRNAs and microRNAs in osteosarcoma samples and paired adjacent noncancerous samples using. Our results revealed that the circRNA and microRNA expression profiles differ significantly between normal bone tissue and osteosarcoma.

Materials and methods

Patient samples and ethical statement

Human primary osteosarcoma samples (n = 11) and their paired adjacent noncancerous tissue samples were obtained from surgery excision in Huaihe Hospital of Henan University. None of the patients received radiotherapy or chemotherapy prior to surgery. All samples were immediately frozen in liquid nitrogen after resection and stored at −80°C until use. This study was approved by the Institutional Review Boards of Huaihe Hospital of Henan University. The written informed consents were obtained from all patients.

Cell lines and culture

Human osteosarcoma cell lines (U2OS, Saos-2, HOS, and MG-63) were purchased from the American Type Culture Collection (ATCC, USA). Cells were cultured in RPMI 1640 medium (Gibco, USA) added with 10% fetal bovine serum (FBS). The normal human osteoplastic cell line (NHOst) and HEK-293 were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and were cultured in Dulbecco’s modified Eagle’s medium (DMEM)-contained 10% FBS (Gibco), 100 U/mL penicillin, and 100 ng/mL streptomycin.

Microarray analysis

After the total RNA was extracted from samples using NanoDrop ND-1000, the preparation and microarray hybridization were operated according to the Arraystar’s standard protocols. First, the extracted total RNAs were digested with RNase R (Epicentre, Inc., WI, Madison, USA) to remove linear RNAs and increase the abundance of circRNAs. The enriched circRNA was transfected into fluorescent cRNA using Arraystar Super RNA Labeling Kit (Arraystar, USA). The labeled cRNAs were hybridized onto the Arraystar Human circRNA Array (8 × 15K; Arraystar). Then, the images were analyzed by Agilent Feature Extraction software.

Transfection

For knockdown, specific small interfering RNAs (siRNAs) were designed and synthesized by GenePharma Co., Ltd. (China). The sequences were provided by GenePharma and shown as follows: si-hsa_circ_0001564-1, 5′-GUCAACAGACCCGAAAUGCAAUU-3′; si-hsa_circ_0001564-2, 5′-GACCGGAACACAAGAACGAUGUU-3′; miR-29c-3p inhibitor, 5′-GGGCUCAUAAAGAGCGCACAGU-3′. Cell transfection and co-transfection with si-hsa_circ_0001564 or miR-29c-3p inhibitor were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.

Quantitative real-time polymerase chain reaction

Total RNA was extracted from samples and cell lines using TRIzol reagent (Invitrogen) as mentioned above. Then, complementary DNA (cDNA) was synthesized with the Reverse Transcription System (Promega, USA). Real-time polymerase chain reaction (RT-PCR) was performed using the SYBR Green Master Mixture (Roche, USA) reagent in ABI 7500 Real-time PCR instrument. The involved primers were designed using primer 5.0 and shown as follows: hsa_circ_0001564: forward, 5′-CATCCTTTGCGCTCAGAGGA-3′; reverse, 5′-GATTGGCCTGACCACAGTCTA-3′. MiR-29c-3p: forward, 5′-CCTGTTTCCTGCCTCTGAAG-3′; reverse, 5′-CCTGGGGAAGTACTGTTCA-3′. β-actin: forward, 5′-CTGGAGAAGAGCTATGAGCTG-3′; reverse, 5′-AATCTCCTTCTGATCCTGTC-3′. Relative levels of gene expression were expressed relative to β-actin and calculated using the 2^−ΔΔCt method.

Colony formation assay

Cells were seeded in a fresh six-well plate and maintained in RPMI 1640 medium supplemented with 10% FBS. After 2 weeks, cells were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet (Beyotime, China). After being washed mildly with phosphate-buffered saline (PBS) and air-dried, the visible colonies were counted for three times. The average value was regarded as the final value. The independent experiments were performed for three replicates.

Cell apoptosis assay

Cells were added into Eppendorf (EP) tube and centrifuged at 1000 r/min for 10 min and then resuspended in 100 µL binding buffer (pH 7.4). The cells were added with slight pancreatin and digested in incubator at 37°C for 10 min. Cells were stained with 10 µL fluorescein isothiocyanate (FITC)–Annexin V and propidium iodide (Becton Dickinson, Germany) and incubated at room temperature for 15 min. With 400 µL buffer being added, flow cytometry (FACScan; BD Biosciences, USA) was performed according to the manufacturer’s instructions. The independent experiments were performed for three replicates.

Cell-cycle analysis

After transfection, cells were washed in PBS and fixed in 70% ethanol, and the cell-cycle analysis was performed by Cell-Cycle Analysis Kit (Lianke, China). Cells (4 × 10⁵ per well) were seeded in six-well plates for 24 h and starved in FBS-free medium for 12 h. Then, cells were fixed with 70% cold ethanol for 2 h. The cells were added with 100 µL of RNase and incubated at 37°C for 30 min. Then, 400 µL of propidium iodide (PI) was added and the cells were incubated at 4°C for 30 min away from light. Finally, flow cytometry (FACScan) was performed. The independent experiments were performed for three replicates.

Statistical analysis

Data are presented as the mean ± standard deviation (SD). Statistical analysis were analyzed by student’s t-test and one-way analysis of variance (ANOVA) and performed with the SPSS 19.0 software (SPSS, USA) and GraphPad Prism 6.0 (GraphPad Software, Inc., USA); p < 0.05 was considered to be statistically significant. The independent experiments were performed for three replicates.

Results

CircRNA profile in osteosarcoma tissues

To identify whether circRNAs are differentially expressed within osteosarcoma tissues and normal adjacent tissue, we performed circRNA microarray analysis to detect the circRNA profile in excised tumor tissue. Results showed that there were 1152 circRNAs upregulated and 915 circRNAs downregulated, and among them, 15 circRNAs upregulated and 21 circRNAs downregulated with fold changes >2.0 and p values <0.05 between the tumor and adjacent tissue, which was shown in heat maps (Figure 1(a)). Afterwards, the 10 upregulated circRNAs were selected and confirmed by quantitative RT-PCR (qRT-PCR) to verify the marked expression (fold changes >5.0 and p values <0.01; Figure 1(b)). Finally, we randomly select one of the markedly upregulated circRNAs, hsa_circ_0001564, as our research object. Our results first revealed the circRNA profile in osteosarcoma tissues and ascertained multi-target circRNAs, which provided research orientation for in-depth mechanism.

Figure 1.

CircRNA microarray analysis of excised tumor tissue from osteosarcoma patients. (a) Heat maps of circRNA expression fold change. Red indicates a higher fold change and green indicates a smaller fold change. (b) The selected 10 upregulated circRNAs (fold changes >5.0 and p values <0.01) on the basis of microarray analysis were confirmed by qRT-PCR. Data were expressed as mean ± SD.

Mediation of hsa_circ_0001564 on osteosarcoma cell lines

Our previous study had discovered the dysregulated circRNAs in osteosarcoma tissue. Next, we confirmed the expression of hsa_circ_0001564 in several common osteosarcoma cell lines, including U2OS, Saos-2, HOS, and MG-63, compared to normal human osteoplastic cell line NHOst. Results showed that hsa_circ_0001564 was significantly increased in these cell lines, especially HOS and MG-63 (Figure 2(a)). In order to evaluate the upregulated hsa_circ_0001564, we transfected interference oligonucleotide into HOS and MG-63 cell lines to achieve hsa_circ_0001564 knockdown. The interference oligonucleotide, si-hsa_circRNA-1 and si-hsa_circRNA-2, could effectively suppress the hsa_circ_0001564 expression (Figure 2(b)). Cell Counting Kit-8 (CCK-8) assay showed that the cell viability was evidently impeded after siRNA transfection (Figure 2(c) and (d)). Colony formation assay showed that hsa_circ_0001564 knockdown observably repressed the colony formation vitality of HOS and MG-63 cells (Figure 2(e)). Afterwards, we assessed the function of hsa_circ_0001564 knockdown on the cell cycle and apoptosis. Results showed that the lower expression could significantly induced cell-cycle arrest in G0/G1 phase (Figure 2(f)–(h)) and promoted apoptosis of HOS and MG-63 cells (Figure 2(i) and (j)). Generally, the functional experiments showed that hsa_circ_0001564 knockdown could suppress the tumorigenicity of osteosarcoma cell lines, which similarly illustrates the potential tumor-promoting effect of hsa_circ_0001564.

Figure 2.

Regulation of hsa_circ_0001564 on osteosarcoma cell lines. (a) The expression of hsa_circ_0001564 measured by qRT-PCR in several common osteosarcoma cell lines, including U2OS, Saos-2, HOS, and MG-63, compared to normal human osteoplastic cell line NHOst. (b) Knockdown of hsa_circ_0001564 transfected with si-hsa_circRNA-1/2 in HOS and MG-63 cell lines. (c and d) Cell viability assessed by CCK-8 assay was performed after transfection. (E) Clone number was counted in colony formation assay. (f–h) Cell cycle detected by flow cytometry showed the cycle arrest in G0/G1 phase. (i and j) Apoptotic cell rates of osteosarcoma cell lines were detected by flow cytometry. Data are presented as the mean ± SD (*p < 0.05 and **p < 0.01 compared to the si-blank control).

Hsa_circ_0001564 acts as a sponge for miR-29c-3p

In our early study about osteosarcoma, we had explored the discrepant miRNA expression profiles in osteosarcoma surgery excision tissue. Part of the aberrant expression miRNAs were listed as follows (Figure 3(a)). Because increasing evidence had indicated that circRNAs could act as miRNA sponges to mediate relevant physiology, ⁸ we performed bioinformatics analysis to uncover the potential interconnection within hsa_circ_0001564 and miRNAs. Fortunately, among these aberrant expression miRNAs, miR-29c-3p was downregulated (Figure 3(b)) and predicted to have binding sites with hsa_circ_0001564 using starBase V2.0 (http://starbase.sysu.edu.cn/). ⁹ A putative complementary region of hsa_circ_0001564 and miR-29c-3p was shown below (Figure 3(c)), and the complementary binding was confirmed by dual-luciferase reporter assay (Figure 3(d)). Pearson’s correlation indicated that hsa_circ_0001564 was negatively correlated to miR-29c-3p expression in osteosarcoma. A series of data indicated that hsa_circ_0001564 directly target miR-29c-3p and acted as a sponge to it.

Figure 3.

Hsa_circ_0001564 directly targeted miR-29c-3p and acted as a sponge. (a) MiRNA expression profiles in osteosarcoma surgery excision tissue compared to adjacent normal tissue measured by qRT-PCR. (b) Relative expression of miR-29c-3p was detected again by qRT-PCR in osteosarcoma tissue compared to control normal. (c) A putative complementary sites of miR-29c-3p and hsa_circ_0001564, including original and mutation in the hsa_circ_0001564 sequence to create the wild-type and mutant luciferase reporter constructs. (d) Dual-luciferase reporter assay showed the putative complementary sites within miR-29c-3p with hsa_circ_0001564. (e) The correlations between hsa_circ_0001564 and miR-29c-3p expression were shown by Pearson’s correlation (R ²  = 0.3108, p < 0.01) in 11 samples. All data were expressed as mean ± SD (**p < 0.01 and *p < 0.05 represented for statistical differences).

Regulation of hsa_circ_0001564 and miR-29c-3p on tumorigenicity of osteosarcoma

Because previous experiments had verified that hsa_circ_0001564 knockdown suppressed the tumorigenicity of osteosarcoma cell lines and hsa_circ_0001564 acted as miR-29c-3p sponge, we performed rescue experiments to assess whether hsa_circ_0001564 knockdown mediated the tumorigenesis inhibition on osteosarcoma through targeting miR-29c-3p. The expression of miR-29c-3p was increased when hsa_circ_0001564 was downregulated by oligonucleotides, which was reversed by miR-29c-3p inhibitor (Figure 4(a)). Afterwards, hsa_circ_0001564 knockdown suppressed the proliferation and colony formation ability, which was rescued by miR-29c-3p inhibitor (Figure 4(b) and (c)). Analogously, miR-29c-3p inhibitor co-transfection significantly reversed the cell-cycle arrest in G0/G1 phase and apoptosis induced by si-hsa_circ_001564 (Figure 4(d)–(g)). Rescue experiments revealed that the oncogenesis effect of hsa_circ_001564 was carried by targeting miR-29c-3p, which also illustrated the important role of competing endogenous RNA mechanism of hsa_circ_001564 and miR-29c-3p.

Figure 4.

Rescue experiments were performed to assess the regulation of hsa_circ_0001564 and miR-29c-3p on proliferation, cell cycle, and apoptosis of HOS cell lines. (a) The miR-29c-3p expression of HOS cell lines with co-transfection of miR-29c-3p inhibitor and si-hsa_circ_0001564. (b) Cell viability was detected by CCK-8 assay. (c) Colony formation assay. (d and e) Cell cycle detected by flow cytometry. (f and g) Apoptosis was detected by flow cytometry. Data are presented as the mean ± SD (**p < 0.01 and *p < 0.05 represented for statistical differences).

Discussion

With the evolution of high-throughput sequencing or next-generation sequencing (NGS) and bioinformatics analysis, more and more functional circRNAs have been discovered and identified in multiple diseases, especially tumorigenesis. ¹⁰ Our study uncovers and verifies a novel aberrant expression circRNA, hsa_circ_0001564, in osteosarcoma and explores the potential regulatory mechanism using a series of functional experiments.

Up to now, increasing researches and achievements involving circRNAs have been reported on literatures, which provide in-depth understanding of the various physiological functions.^4,11 However, the homologous studies on osteosarcoma are still deficient, which deserve our deepgoing exploration to excavate new fields. In this study, the expression profiles of circRNAs were detected in osteosarcoma tissues using circRNA microarray analysis. Results showed that there are 1152 circRNAs upregulated and 915 circRNAs downregulated within the tumor and adjacent control tissue, and among them, 15 circRNAs upregulated and 21 circRNAs downregulated with fold changes >2.0 and p values <0.05. We randomly select one of the markedly upregulated circRNAs, hsa_circ_0001564, as our research object, which is located at 5q35.3 and its associated gene symbol is CANX. The function of hsa_circ_0001564 in cells was identified by loss-of-function methods. The hsa_circ_0001564 knockdown transfected with siRNAs could effectively suppress the cell proliferation and colony formation vitality of HOS and MG-63 cell. Afterwards, hsa_circ_0001564 knockdown induces the cell-cycle arrest in G0/G1 phase and promotes apoptosis. There are increasing functional circRNAs being reported; for example, the silencing of circHIPK3 significantly inhibits growth of six normal tissues and seven cancer cells by directly binds to miR-124 and inhibits miR-124 activity. ¹² Furthermore, in colorectal cancer, circ_001569 acts as a positive regulator in cell proliferation and invasion as a sponge of miR-145. ¹³

The characteristics and critical role of circRNA in transcription and post-transcriptional regulation were well recognized due to the “microRNA sponge” function of circRNA. ¹⁴ Being similar with lncRNAs, circRNAs could bind with miRNAs and consequently repress their function and regulate miRNA activity. In our study, bioinformatics analysis (starBase V2.0; http://starbase.sysu.edu.cn/) predicts a putative complementary region of hsa_circ_0001564 and miR-29c-3p, which is confirmed by dual-luciferase reporter assay. Rescue experiments revealed that hsa_circ_0001564 exerts the oncogenesis effect by targeting miR-29c-3p, which illustrates the ceRNA mechanism of hsa_circ_0001564. In human glioma cells, upregulated circ-TTBK2 promoted cell proliferation, migration, and invasion and inhibited apoptosis through decreasing miR-217 expression. ¹⁵ In myocardial infarction, CiRS-7 functions as a powerful miR-7a sponge and repress the protective role of miR-7a by targeting PARP and SP1. ¹⁶

Compared to miRNA and long non-coding RNAs, circRNAs is a type of re-discovered endogenous non-coding RNA and become a hotspot in RNA research.^11,17 So far, with the rapid blossom of NGS, enormous quantity of circRNAs has been discovered; however, there is not a unitive systematic nomenclature for the thousands of new-found circRNAs in human or mouse. Most of the classical small ncRNA genes have now been awarded unique nomenclature by HUGO Gene Nomenclature Committee (HGNC), as well as the partial lncRNAs. ¹⁸ The new reported circRNA, hsa_circ_0001564, is located at 5q35.3 and the associated gene symbol is CANX, and it might be named circ-CANX according to the current default rules. Presently, the homologous naming pattern contains cir-ZNF609, ¹⁹ circHIPK3, ¹² and circ-TTBK2. ¹⁵ In order to improve the academic communication, unitive systematic nomenclature is urgently needed.

The characteristics, including high stability, abundance, and tissue-specific expression patterns, make circRNA been valuable for clinical research and could function as novel biomarker. ²⁰ Studies have revealed the biomarker function of circRNAs in human diseases, for example, hsa_circ_0001649 might serve as a biomarker in hepatocellular carcinoma ²¹ and hsa_circ_0000190 acts as a diagnosis biomarker for gastric cancer. ²² Generally, our study illustrates that hsa_circ_0001564 is closely related to tumorigenicity of osteosarcoma cell lines and could act as a potential biomarker for the osteosarcoma.

In summary, our study reveals that hsa_circ_001564 is upregulated in human osteosarcoma tissues and aggravates the malignant process via negatively targeting miR-29c-3p, which could be considered as an independent biomarker and provide an effective therapeutic target for osteosarcoma patients.