CircFAT1 sponges miR-375 to promote the expression of Yes-associated protein 1 in osteosarcoma cells
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There is an urgent need to identify new molecular targets for treatment of osteosarcoma. Circular RNAs are a class of endogenous RNAs that are extensively found in mammalian cells and exert critical functions in the regulation of gene expression, but in osteosarcoma the underlying molecular mechanism of circular RNAs remain poorly understood. Here we assessed the tumorigenesis properties of a circular RNA, circFAT1 in osteosarcoma.
The effects of circFAT1/miR-375/YAP1 was evaluated on human osteosarcoma cells growth, apoptosis, migration, invasion and tumorigenesis. Signaling pathways were analyzed by western blotting, qRT-PCR, fluorescence in situ hybridization, chromogenic in situ hybridization,RNA Binding Protein Immunoprecipitation and immunofluorescence. The consequence of circFAT1 short hairpin RNA combined or not with miR-375 sponge was evaluated in mice bearing 143B xenografts on tumor growth.
In this study, we observed significant upregulation of circFAT1 originating from exon 2 of the FAT1 gene in human osteosarcoma tissues and cell lines. Inhibition of circFAT1 effectively prevented the migration, invasion, and tumorigenesis of osteosarcoma cells in vitro and repressed osteosarcoma growth in vivo. Mechanistic studies revealed that circFAT1 contains a binding site for the microRNA-375 (miR-375) and can abundantly sponge miR-375 to upregulate the expression of Yes-associated protein 1. Moreover, inhibition of miR-375 reversed attenuation of cell proliferation, migration, and invasion, which was induced by circFAT1 knockdown, and therefore promoted tumorigenesis.
Our findings demonstrate a novel function of circFAT1 in tumorigenesis and suggest a new therapeutic target for the treatment of osteosarcoma.
KeywordsOsteosarcoma circFAT1 circular RNA YAP miR-375
3′- untranslated region
Competing endogenous RNA
small Small hairpin RNA
Yes-associated protein 1
Osteosarcoma (OS), a common malignant bone cancer, has the highest morbidity in adolescence and childhood [1, 2]. Even with multiple treatments including surgery with chemotherapy, the 5-year survival rate is less than 70% because of tumor recurrence caused by metastasis and drug resistance . Furthermore, efficient therapeutic targets or diagnostic markers for OS have not been identified, making it difficult to improve outcomes. Therefore, it is critical to develop better strategies and new therapeutic methods for avoiding resistance and distant metastasis, thus improving the prognosis of OS.
As a distinct group of noncoding transcripts, circular RNAs (circRNAs), form a closed continuous loop with the 3′RNA and 5′ RNA joined covalently [4, 5, 6]. In the past 40 years, circRNAs have been identified in eukaryotic cells by electron microscopy  and were previously considered as splicing error by-products. Through the application of high-throughput sequencing and bioinformatics, circRNAs have been successively identified in multiple cell lines and various species [7, 8, 9, 10, 11]. Most circRNAs are formed by exon or intron back-splicing. This process differs from the formation of linear RNAs. Two mechanisms exist for the formation of exonic or exon–intron circRNAs: exon skipping and back-splicing [12, 13, 14]. Previous studies reported circRNAs as “miRNA sponges” that play an inhibitory role in miRNA regulation [10, 14]. Known as “miR-7 sponges,” antisense to the cerebellar degeneration-related protein 1 (CDR1as), also known as ciRS-7, is one of the most widely known and effective circRNAs [15, 16]. Its function is to sponge miR-7 with approximately 74 canonical binding sites. Therefore, circRNAs may be critical biological markers in the identification of disease mechanisms and for developing new methods for precise diagnosis and effective treatment. However, the function of circRNAs as miRNA sponges for OS remains unknown.
In this study, we identified and characterized the circRNA circFAT1 (originating from exon 2 of FAT1 gene) . Importantly, we found that circFAT1 RNA, rather than FAT1 mRNA, was significantly upregulated in OS tissues and cell lines. Furthermore, we found that inhibiting circFAT1 expression significantly reduced the invasion, metastasis, and proliferation of OS cells via sponging of miR-375, which downregulated the expression of Yes-associated protein 1 (YAP1).
All animal experiments were carried out according to the Guide for the Care and Use of Laboratory Animals published by National Institutes of Health and approved by the Ethics Committee of Sir Run Run Shaw Hospital. All experiments strictly followed the panel’s specific guidelines regarding the care, treatment and euthanasia of animals used in the study.
Slices of formalin-fixed and paraffin-embedded primary osteosarcoma and chondroma tissues were obtained from biopsies in each 12 patients before administration of neo-adjuvant chemotherapy. Osteosarcoma, chondroma biopsies were histologically characterized by pathologists according to the criteria defined by the World Health Organization. Written informed consent was obtained from each patient before entering this study, and all study protocols were approved by the Ethics Committee of Sir Run Run Shaw Hospital.
Antibodies, mice, and other materials. Information regarding the materials used in this study is included in the Additional file 1: Materials and Methods.
The human cell line hFOB1.19, human osteosarcoma cell lines MG-63, u2os, SJSA-1, HOS and 143B were purchased from FuHeng Cell Center (Shanghai, China). The OS cell lines were authenticated at the by the ShangHai Biowing Applied Biotechnology Co. Ltd., performing a STR profiling analysis, as described by Capes-Davis and according to the ANSI Standard (ASN-0002) set forth by the ATCC Standards Development Organization. Mycoplasma testing was performed using the Venor GeM Mycoplasma Detection Kit (Minerva Biolabs, Berlin, Germany). Cells were cultured as described in detail in the Additional file 1: Materials section.
Transfection, and viral infection
All the cell lines Cell culture procedures are described in the Additional file 1: Materials and Methods.
Xenograft tumorigenesis model
Nude mice (nu/nu, male 3- to 4-week-old) were injected subcutaneously with 5 × 106 143B stable cells. Tumor volumes were calculated from the length (a) and the width (b) by using the following formula: volume (ml3) = ab2/2. Five weeks after injection, the animals were sacrificed, and tumors were harvested (measured and weighed) and fixed in 4% paraformaldehyde. Wet tumor weight was calculated as mean weight ± standard deviation (SD) in each group.
Ago2-binding sites from CLIP data sets
The evidence for Ago2-binding sites was obtained from published online cross-linking immunoprecipitation (CLIP) data sets, which are available from doRiNA (a database of RNA interactions in post-transcriptional regulation, http://dorina.mdc-berlin.de). These data sets include Ago2 HITS-CLIP and PAR-CLIP data from HEK-293 cells and several lymphoma cells. We downloaded the available data sets (http://dorina.mdc-berlin.de) and acquired the Ago2-binding sites of circFAT1 genomic region.
RIP experiments were performed by using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA). The Ago-RIP assay was conducted in HOS cells stably expressing vector or shcircFAT1. We first constructed the lentivirus vectors harbouring vector or shcircFAT1, and established two stable cell lines of HOS. Approximately 1×107 cells were pelleted and re-suspended with an equal pellet volume of RIP Lysis Buffer (about 100 ml) plus protease inhibitors cocktail and RNase inhibitors. The cell lysates (200 μl) were incubated with 5 μg of control rabbit IgG or antibody against Ago2 (Millipore) coated beads with rotation at 4 °C overnight, respectively. After treating with proteinase K buffer, the immunoprecipitated RNAs were extracted by RNeasy MinElute Cleanup Kit (Qiagen) and reversely transcripted using Prime- Script RT Master Mix (TaKaRa). The abundance of circFAT1 level was detected by qRT–PCR assay.
Luciferase reporter assay
Cells were seeded in 96-well plates at a density of 5×103 cells per well 24 h before transfection. The cells were co-transfected with a mixture of 50 ng firefly luciferase (FL) reporter vectors, 5 ng Renilla luciferase (RL) reporter vectors (pRL-TK), and miRNA mimics at the indicated concentration. The miRNA mimics were obtained from Life Technologies. After 48 h, the luciferase activity was measured with a dual luciferase reporter assay system (Promega, Madison, WI). Each miRNA or NC RNA was co-transfected with RL reporter and FL reporter with or without the circFAT1 3’-UTR. For comparison, the FL activity was first normalized with RL activity. The effect of each miRNA on luciferase reporter with circFAT1 3’-UTR was then normalized with that on luciferase reporter without circFAT1 3’-UTR. Finally, the fold-change was calculated by each miRNA compared with NC.
RNA in situ hybridization
Cy3-labeled locked nucleic acid miR-375 probes and Alexa Fluor 488-labeled circFAT1 probes were designed and synthesized by RiboBio (Guangzhou, China), and the probes sequences were available upon request. The signals of the probes were detected by Fluorescent In Situ Hybridization Kit (RiboBio, Guangzhou, China) according to the manufacturer’s instructions. The images were acquired on Nikon A1Si Laser Scanning Confocal Microscope (Nikon Instruments Inc, Japan).
Pull-down assay with biotinylated circFAT1 probe
Pull-down assay was performed as indicated: In brief, 1 × 107 osteosarcoma cells were harvested, lysed, and sonicated. The circFAT1 probe was incubated with C-1 magnetic beads (Life Technologies) at 25°C for 2 h to generate probe-coated beads. The cell lysates were incubated with circFAT1 probe or oligo probe at 4°C overnight. After washing with the wash buffer, the RNA complexes bound to the beads were eluted and extracted with RNeasy Mini Kit (QIAGEN) for RT–PCR or real-time PCR. Biotinylated-circFAT1 probe was designed and synthesized by RiboBio (Guangzhou, China).
Wound healing assay
HOS and 143B cells were cultured in six-well plates and scraped with the tip of 200 μl pipette tips (time 0 h). Cell migration was photographed using 10 high-power fields at 0 and 24 h after injury. Remodeling was measured as diminishing distance across the induced injury, normalized to the 0 h control, and expressed as relative migration.
Transwell migration and matrigel invasion assays
The migration and matrigel invasion assays were conducted by using transwell chamber (for migration assay) or transwell pre-coated matrigel chamber (for invasion assay) according to the manufacturer’s protocol (BD Science, Bedford, MA, USA). The homogeneous single cell suspensions (5 × 104 cells/well for migration, 1 × 10 /well for invasion) were added to the upper chambers and incubated for 24 h. The migration and invasion rates were quantified by counting the migratory and invasive cells at least three random fields.
For anoikis analysis, cells were cultured in suspension for 48 h. Cells were then trypsinized and stained with Annexin V- FITC –PI and analyzed by flow cytometry using the Annexin V-FITC/PI Apoptosis Detection kit (BD Biosciences) following the manufacturer’s instructions. Data were collected on a BD FACSCanto and analyzed using FlowJo software.
Other in vitro experiments
We performed in vitro experiments as described, including CCK-8 assay, cell transfection and colony formation , western blotting , flow cytometry, IHC and immunofluorescence microscopy [18, 20], real-time PCR using U6 as an internal control , and northern blotting [10, 14]. Given the different sizes of circFAT1 and linear FAT1 mRNA, the samples were separated on a 1.5% formaldehyde agarose gel for circFAT1 and a 0.6% gel for FAT1 mRNA by using voltages of 50 and 90 V, respectively. Tumor-formation assay was described previously . Further details are provided in the Additional file 1: Materials and Methods.
Statistical analyses were performed with 22.0 SPSS. Data are represented as means with SDs, and statistical significance was determined with unpaired Student’s t tests, unless indicated otherwise. P values less than 0.05 were considered statistically significant. Tumor sizes were measured daily to observe dynamic changes in tumor growth and calculated by a standard formula:
CircFAT1 is abundantly expressed in OS tissues and cell lines and predominantly localized in the cytoplasm.
Knockdown of circFAT1 inhibits migration and invasion of OS cells in vitro
CircFAT1 abundantly sponges miR-375 in OS cells
miR-375 is downregulated in OS tissues and cell lines and inhibits cell migration, invasion, and proliferation by targeting YAP1 in vitro
MiR-375 targets YAP1 in OS
To verify whether YAP1 is a direct target of miR-375, we constructed 3′-UTR sensors and co-transfected 293T cells with pre-miR-375. We observed reduced luciferase activity for of the YAP1 3′-UTR in the presence of miR-375 (Fig. 5d). To verify target specificity, we generated mutated forms of the YAP1 3′-UTR, in which the miR-375-binding site was abolished. Co-transfection of pre-miR-375 with the mutant construct abrogated the decrease in wild-type 3′-UTR luciferase activity, indicating that miR-375 specifically regulated YAP1 expression (Fig. 5d). Additionally, transfection of miR-375 mimics reduced endogenous YAP1 protein and mRNA levels, as well as the YAP1-targeted genes c-Myc and Birc5 (Fig. 5e, f and g). However, reducing endogenous miR-375 levels (using miR-375 inhibitor) had the opposite effect in both cell lines (Fig. 5e, f and g). These results indicate that miR-375 targets YAP1 in OS. Consistent with our previous studies , YAP1 is important for OS cell apoptosis during anoikis and cell detachment. Overexpression of YAP1 decreased anoikis in both HOS and 143B osteosarcoma cells (Fig. 5h).
Knockdown of miR-375 reverses shcircFAT1-induced attenuation of cell proliferation, migration, and invasion in OS cells
CircFAT1 functions as miR-375 sponge to promote tumorigenesis in vivo
We next evaluated the relationship between circFAT1 and YAP1 in vivo. circFAT1 knockdown reduced the mean area immunopositive for YAP1, c-Myc, and Birc5 in tumor tissues, as determined by immunohistochemistry (Fig. 8e), this corresponded to a decrease in the levels of YAP1, c-Myc, and Birc5 protein and mRNA (Fig. 8f and g). Inhibition of miR-375 reversed this effect. Interestingly, immunofluorescence (Additional file 4: Figure S5a) and western blot (Additional file 4: Figure S5b) results indicated that the upregulation of c-Myc expression induced by circFAT1 overexpression is independent on Wnt signal pathway. Besides, Kaplan-Meier survival curves of the TCGA sarcoma dataset showed that the patients with high expression of YAP and target genes had a low 10-year overall survival rate, while the patients with high expression of miR-375 had a high 10-year overall survival rate (Additional file 5: Figure S8). These results suggest that circFAT1 sponges miR-375 in OS and miR-375 mediates the tumor-suppressive function of circFAT1 in vivo (Fig. 8h).
In recent years, circRNAs has drawn the increasing attention owning to the development of bioinformatics and high-throughput sequencing technologies [23, 24, 25, 26]. With the following distinct characteristics, such as unique structure, conservation across species, cell type-specific and tissue-specific expression, and stable expression in saliva, blood and exosomes [27, 28, 29], more than ten thousand different circRNAs have been discovered and studied in various organisms, which also raised the hot topic on circRNAs in malignant tumor research [19, 30, 31]. However, although the studies on circRNAs are being developed on a high speed, there are still many questions unrevealed. In our project, according to the previous studies, we put forward a hypothesis that circRNAs may also have effect on OS progression with the cell type and developmental stage-specific characteristics and can be served as valuable clinical biomarkers for OS.
In the current study, we selected seven tumor-related and highly expressed circRNAs, including circHIPK3, circFOXO3, circRTN4, circITCH, circCdr1as, circFAT1 and circZFR, which have been reported previously to be involved in the progression of colorectal cancer, hepatocellular carcinoma, prostate cancer, gastric cancer and so on. In these seven circRNAs, circHIPK3 has been found to act as a competing endogenous RNA (ceRNA) of miRs with the activity to competitively sequester and quench of miRs, such as miR-7, miR-558 and miR-124, resulting in the dramatic association with the development of a variety of tumors, such as colorectal cancer, hepatocellular carcinoma, and bladder cancer [10, 32, 33]. Besides, circFAT1 and circRTN4 has been showed to be highly expressed in exosomes of colorectal cancer, indicating its potential function in tumorgenesis . CircZFR is another circRNA with significant higher expression in both bladder carcinoma tissues and papillary thyroid carcinoma tissues [35, 36]. What is more, it also has been found that circ-Cdr1as played an important role as a super sponge or competing endogenous RNA (ceRNA) of miR-7, which has the tremendous effects on the growth of many kinds of cancer, such as breast cancer, hepatocellular carcinoma, and cervical cancer [15, 16]. In addition, circFOXO3 and circITCH were reported to suppress tumor cell proliferation, migration and invasion. According to our result, circFAT1 induced the highest rate of apoptosis of osteosarcoma cells compared with other circRNAs (Additional file 6: Figure S1), we therefore choose circFAT1 for the further investigation.
CircFAT1 is derived from the FAT1 gene. Previous studies have demonstrated that the FAT1 gene plays potential roles in tumors and inflammation . The expression of this gene may be a crucial event in cancer invasion and metastasis . In our study, we showed that circFAT1 was highly expressed in osteosarcoma cell lines and tissues. Silencing of circFAT1 inhibits the proliferation, migration and induces apoptosis of osteosarcoma cell line as demonstrated by loss-of-function assays. Previous studies reported that sponge of miRNA is one kind of main mechanisms that circRNAs acts in cancer [15, 16]. Our further studies also revealed that circFAT1 exerts its regulatory functions through harboring miR-375 to rescue the expression of YAP1, a core factor in the Hippo pathway, which is also directly targeted by miR-375.
The Hippo pathway, a highly conserved cell signaling pathway in evolutionary history, plays a critical role in regulating cell proliferation, metastasis, tumorigenesis, and drug resistance in tumors [39, 40, 41, 42]. Multiple studies have investigated YAP’s oncogenic role, both in vitro and in vivo [43, 44]. Furthermore, upregulation of YAP gene expression has been detected in cancers such as melanoma, hepatocellular carcinoma, and neurofibromatosis [45, 46, 47]. YAP also plays a critical role in the initiation and progression of cancer mediated by other oncogenes such as Kras and beta-catenin [46, 47]. These findings strongly indicate YAP’s essential role in human cancers. For osteosarcoma, YAP/TAZ and its related target gene can be found frequently overexpressed in human OS cells [48, 49]. Reversely, the downregulation of YAP can inhibit the proliferation and migration of OS cell in vitro and impair tumor growth in vivo . On the level of mechanism, some studies showed that NF2 and Kibra, the activators of the Hippo pathway, could upregulate the level of YAP in osteo-tissues mediated by inhibition of Sox2 . The activation of Hedgehog signaling could also trigger the overexpression of YAP via Hippo pathway crosstalk . What is more, the mutation of RASSFs, NF2 and Mob1 tumor suppressor in OS frequently leads to the upregulation of YAP/TAZ . Many important studies have confirmed the critical role of YAP/TAZ in inducing chemo-resistance, radio-resistance even the molecular targeted therapy-resistance in OS [49, 51]. Our previous findings also demonstrated that TAZ, the homologous protein of YAP, is overexpressed in OS and highly related to the proliferation and metastasis of OS cells . Consistently, our current study further indicated that YAP plays a critical role in mediating OS metastasis and tumorigenicity. At the same time, our study revealed a new mechanism of the linkage with circular RNA and the Hippo pathway. Combined with the studies that noncoding RNA plays an important role in the Hippo pathway [52, 53], our study firstly clarified the mechanism that circFAT1 regulates YAP1 expression via sponging miR-375 and acts as an upstream factor in the Hippo/YAP pathway.
In summary, we characterized and functionally analyzed an abundant circRNA derived from exon 2 of the FAT1 gene. We further demonstrated that circFat1 is upregulated in human OS and can efficiently sponge miR-375 to promote YAP1 expression. We also demonstrate that circFAT1 knockdown effectively inhibited the aggressiveness and metastasis of OS cells by targeting the miR-375/YAP1 axis. Our findings reveal that the circularized protein-coding exons or “sponging miRNAs” have other regulatory effects, and thus provide a new therapeutic target for the treatment of OS.
The study was sponsored by National Natural Science Foundation of China (81802680, 81871797, 81472064 and 81601925), Key Project of Zhejiang Medical Science and Technology Plan (2015145597), Key Project of Zhejiang Provincial Natural Science Foundation (LZ15H06002), Medical Science and Technology Project of Zhejiang Province (2017179447), Key Project of Zhejiang Medical Science and Technology Plan (2016145597). No benefits in any form have been or will be received from a commercial party related directly or indirectly to the subject of this study.
Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Gang Liu contributed transwell, western blots and animal experiments. Kangmao Huang and Zhiwei Jie performed IF, clony formation experiments and collected and classified the human tissue samples. Yizheng Wu, Junxin Chen, and Zizheng Chen contributed RT– PCR and real-time PCR experiments. Shuying Shen performed primers design, FISH, circRNA pull-down, Northern experiments. Shuying Shen and Xiangqian Fang analyzed the data and wrote the paper. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The present study was approved by the Ethics Committee of Sir Run Run Shaw Hospital.
Consent for publication
The authors declare that they have no competing interests.
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