circCAMSAP1 promotes osteosarcoma progression and metastasis by sponging miR-145-5p and regulating FLI1 expression

doi:10.1016/j.omtn.2020.12.013

. 2020 Dec 23:23:1120-1135.

doi: 10.1016/j.omtn.2020.12.013. eCollection 2021 Mar 5.

circCAMSAP1 promotes osteosarcoma progression and metastasis by sponging miR-145-5p and regulating FLI1 expression

Zizheng Chen^{1

2

3}, Wenbin Xu^{1

2}, Deguang Zhang⁴, Junjie Chu⁴, Shuying Shen^{1

2}, Yan Ma^{1

2}, Qingxin Wang³, Gang Liu^{1

2}, Teng Yao^{1

2}, Yizhen Huang^{1

2

3}, Huali Ye^{1

2}, Jiying Wang^{1

2}, Jianjun Ma^{1

2}, Shunwu Fan^{1

2}

Affiliations

¹ Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, 3 East Qingchun Road, Hangzhou 310016, Zhejiang Province, China.
² Key Laboratory of Musculoskeletal System Degeneration and Regeneration Translational Research of Zhejiang Province, Hangzhou 310016, Zhejiang Province, China.
³ Zhejiang University School of Medicine, Hangzhou 310016, China.
⁴ Department of Head and Neck Surgery, Institution of Micro-Invasion Surgery of Zhejiang University, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, 3 East Qingchun Road, Hangzhou 310016, Zhejiang Province, China.

PMID: 33664993
PMCID: PMC7901030
DOI: 10.1016/j.omtn.2020.12.013

circCAMSAP1 promotes osteosarcoma progression and metastasis by sponging miR-145-5p and regulating FLI1 expression

Zizheng Chen et al. Mol Ther Nucleic Acids. 2020.

. 2020 Dec 23:23:1120-1135.

doi: 10.1016/j.omtn.2020.12.013. eCollection 2021 Mar 5.

Authors

Affiliations

¹ Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, 3 East Qingchun Road, Hangzhou 310016, Zhejiang Province, China.
² Key Laboratory of Musculoskeletal System Degeneration and Regeneration Translational Research of Zhejiang Province, Hangzhou 310016, Zhejiang Province, China.
³ Zhejiang University School of Medicine, Hangzhou 310016, China.
⁴ Department of Head and Neck Surgery, Institution of Micro-Invasion Surgery of Zhejiang University, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, 3 East Qingchun Road, Hangzhou 310016, Zhejiang Province, China.

PMID: 33664993
PMCID: PMC7901030
DOI: 10.1016/j.omtn.2020.12.013

Abstract

Osteosarcoma is the most common primary malignant bone tumor in adolescents. While chemotherapy combined with surgery can improve the prognosis of some patients, chemo-resistance is still a huge obstacle in osteosarcoma treatment. Accumulating evidence demonstrates that circular RNAs (circRNAs) are involved in cancer progression and metastasis, but their specific role in osteosarcoma remains mostly undescribed. In this study, we performed circRNA deep sequencing and identified 88 distinct circRNAs from a human osteosarcoma cell lines group (143B, HOS, SJSA, and U2OS) and the human osteoblast hFOB 1.19 (control). We found that circCAMSAP1, also named hsa_circ_0004338, is significantly upregulated in human osteosarcoma tissues and cell lines, and it is positively correlated with osteosarcoma development. Silencing of circCAMSAP1 effectively suppresses osteosarcoma cell growth, apoptosis, migration, and invasion. Furthermore, we validated that circCAMSAP1 functions in osteosarcoma tumorigenesis through a circCAMSAP1/miR-145-5p/friend leukemia virus integration 1 (FLI1) pathway. FLI1 promotes osteosarcoma tumorigenesis and miR-145-5p suppresses FLI translation. circCAMSAP1 directly sequesters miR-145-5p in the cytoplasm and inhibits its activity to suppress osteosarcoma tumorigenesis. Moreover, the regulatory role of circCAMSAP1 upregulation was examined and validated in rats. In summary, our findings provide evidence that circCAMSAP1 act as a "microRNA sponge" and suggest a new therapeutic target of human osteosarcoma.

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Conflict of interest statement

The authors declare no competing interests.

Figures

**Figure 1**
circCAMSAP1 is relatively highly expressed in osteosarcoma tissues and cells and predominantly localized in cytoplasm (A) Heatmap and hierarchical clustering analysis based on osteosarcoma cell lines and hFOB 1.19. (B) The expression of circCAMSAP1 in human osteosarcoma and chondroma tissue was detected by real-time PCR. Data are represented as mean ± SEM (n = 10). ∗∗p < 0.01. (C) The expression levels of circCAMSAP1 in hFOB 1.19 and osteosarcoma cell lines (143B, HOS, U2OS, and SJSA) were detected by real-time PCR. Data are represented as mean ± SEM (n = 3). ∗∗p < 0.01, ∗∗∗p < 0.001. (D) The expression of circCAMSAP1 in human osteosarcoma and chondroma tissue was detected by RNA fluorescence *in situ* hybridization (FISH). Scale bars, 50 μm. (E) Schematic illustration shows the CAMSAP1 exon 2–7 circularization forming circCAMSAP1 (left arrow). The presence of circCAMSAP1 was validated by real-time PCR, followed by Sanger sequencing. Right arrow represents “head-to-tail” circCAMSAP1 splicing sites. (F) The expression of circCAMSAP1 and CAMSAP1 mRNA in 143B and HOS cells treated with or without RNase R was detected by real-time PCR. The relative levels of circCAMSAP1 and CAMSAP1 mRNA were normalized to the value measured in the mock treatment. Data are represented as mean ± SD (n = 3). ∗∗∗p < 0.001. (G) The presence of circCAMSAP1 was validated in 143B and HOS osteosarcoma cell lines by real-time PCR. Divergent primers were amplified circCAMSAP1 in cDNA, but not in genomic DNA. GAPDH was used as a negative control. (H) RNA FISH with junction-specific probes demonstrated the cellular localization of circCAMSAP1. circCAMSAP1 probes were labeled with Alexa Fluor 488, and nuclei were stained with DAPI. Scale bars, 50 μm. p values were determined by a Student’s t test.

**Figure 2**
circCAMSAP1 regulates migration, invasion, and proliferation of osteosarcoma cells *in vitro* (A) The expression levels of circCAMSAP1 and CAMSAP1 mRNA in 143B and HOS cell lines after transfection of small interfering RNA (siRNA) or negative control (NC)-siRNA, detected by real-time PCR. Data are represented as mean ± SEM (n = 3). ∗∗∗p < 0.001. (B) Downregulation of circCAMSAP1 resulted in more apoptotic 143B and HOS cells than those transfected with the NC-siRNA. Apoptosis rates were determined with annexin V-FITC/PI staining. Data are represented as mean ± SEM (n = 3). ∗p < 0.05, ∗∗p < 0.01. (C) circCAMSAP1 knockdown suppressed cell migration ability, as demonstrated by the wound healing assay in 143B and HOS cell lines. Data are represented as mean ± SEM of three independent experiments. ∗∗p < 0.01, ∗∗∗p < 0.001. (D) Cell migration ability of 143B and HOS cell lines after transfection of siRNA or NC-siRNA was analyzed with the Transwell migration assay. Data are represented as mean ± SEM of three independent experiments. ∗∗p < 0.01, ∗∗∗p < 0.001. (E) Cell invasion ability of 143B and HOS cell lines after transfection of siRNA or NC-siRNA was analyzed with the Transwell Matrigel invasion assay. Data are represented as mean ± SEM of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (F and G) circCAMSAP1 knockdown suppressed cell proliferation, as demonstrated by the CCK-8 assay in 143B and HOS cell lines. Data are represented mean ± SEM (n = 3). ∗p < 0.05. (H) circCAMSAP1 knockdown suppressed cell growth, as demonstrated by the colony formation assay in 143B and HOS cell lines. Data are represented as mean ± SEM of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. p values were determined by a Student’s t test.

**Figure 3**
circCAMSAP1 abundantly sponges miR-145-5p in osteosarcoma cells (A) Schematic illustration showing the overlapping results of the target miRNAs of circCAMSAP1 as predicted by miRanda, TargetScan, RNAhybrid, and CircInteractome. (B) The relative level of six miRNA candidates in the hFOB 1.19, 143B, and HOS lysates was detected by real-time PCR. Data are represented as mean ± SEM (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (C and D) Lysates prepared from 143B and HOS cells were subjected to an RNA pull-down assay and tested with quantitative real-time PCR. Relative levels of circCAMSAP1 were normalized to the levels of input. n = 3. ∗p < 0.05 versus control (lacZ) probe. (E) Luciferase reporter assay for the luciferase activity of LUC-circCAMSAP1 or LUC-circCAMSAP1 mutant in HEK293T cells co-transfected with miRNA mimics. Data are represented as mean ± SD for three experiments. ∗∗p < 0.01. (F) Upper panel: schematic illustration demonstrates complementary to the miR-145-5p seed sequence with circCAMSAP1. Lowercase letters indicate mutated nucleotides. Lower panel: HEK293T cells were co-transfected with miR-145-5p mimics and a luciferase reporter construct containing wild-type (WT) or mutated circCAMSAP1. Data are represented as mean ± SD (n = 3). ∗∗p < 0.01. (G) RNA FISH images demonstrate the co-localization of circCAMSAP1 and miR-145-5p in 143B and HOS cell lines and osteosarcoma tissue samples. circCAMSAP1 probes were labeled with Alexa Fluor 488; miR-145-5p probes were labeled with Cy3. Nuclei were stained with DAPI. Scale bars, 200 and 50 μm. p values were determined by a Student’s t test.

**Figure 4**
miR-145-5p is downregulated in osteosarcoma cells and inhibits cell migration, invasion, and proliferation *in vitro* (A) miR-145-5p was downregulated in human osteosarcoma tissue compared with chondroma tissue, as detected by real-time PCR. Data are represented as mean ± SEM (n = 10). ∗p < 0.05. (B) RNA FISH images demonstrated that miR-145 expression was lower in human osteosarcoma tissue than in chondroma tissue. miR-145-5p probes were labeled with Cy3. Nuclei were stained with DAPI. Scale bars, 100 and 50 μm. (C) miR-145-5p was downregulated in osteosarcoma cell lines (143B, HOS, and U2OS) compared with hFOB 1.19, detected by real-time PCR. Data are represented as mean ± SEM (n = 10). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (D) The expression of miR-145-5p in 143B and HOS cell lines was detected by real-time PCR. Data are represented as mean ± SEM (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (E and F) 143B and HOS cells were transfected with miR-145-5p mimic or inhibitor, and apoptosis rates were determined with annexin V-FITC/PI staining. The percentage of apoptosis cells is shown as mean ± SEM from three independent experiments. ∗p < 0.05, ∗∗p < 0.01. (G and H) miR-145-5p mimic/inhibitor can suppress/promote 143B and HOS cell line proliferation, as determined by the CCK-8 assay. Data are represented as mean ± SEM (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (I and J) Cell migration and invasion of HOS and 143B cells, transfected with miR-145-5p mimic or inhibitor, were evaluated by Transwell migration and Matrigel invasion assays. Data are represented as mean ± SEM (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (K and L) miR-145-5p mimic or inhibitor regulated cell migration ability, as demonstrated by the wound healing assay in 143B and HOS cell lines. Data are represented as mean ± SEM of three independent experiments. ∗p < 0.05, ∗∗p < 0.01. (M and N) miR-145-5p overexpression or knockdown regulated cell growth, as determined by the colony formation assay. Data are represented mean ± SEM of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. p values were determined by a Student’s t test.

**Figure 5**
miR-145-5p targets FLI1 in osteosarcoma cells and tissues (A) Relative expression mRNA levels of four gene candidates in osteosarcoma cell lines (143B, HOS, U2OS, and SJSA) compared with hFOB 1.19, as detected by real-time PCR. Data are represented as mean ± SEM (n = 10). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (B) FLI1 expression levels were upregulated in human osteosarcoma tissue compared with chondroma tissues, as detected by real-time PCR. Data are represented as mean ± SEM (n = 10). ∗∗p < 0.01. (C) FLI1 expression was higher in human osteosarcoma than in chondroma tissue. Representative images are shown. Scale bars, 100 μm. (D) Upper panel: schematic illustration demonstrates complementary to the miR-145-5p-5p seed sequence with FLI1. Lowercase letters indicate mutated nucleotides. Lower panel: HEK293T cells were co-transfected with miR-145-5p mimics and a luciferase reporter construct containing WT or mutated FLI1. Data are represented as mean ± SEM (n = 3). ∗p < 0.05, ∗∗p < 0.01. (E) Quantitative real-time PCR analysis of FLI1 mRNA levels in RNA sample by miR-145-5p miRNA pull-down. Data are represented as mean ± SEM (n = 3). ∗∗∗p < 0.001. (F and G) miR-145-5p inhibition increased FLI1 and c-Myc and decreased IGFBP3 protein (E) and mRNA levels (F), and miR-145-5p overexpression reduced FLI1 and c-Myc and increased IGFBP3 protein (E) and mRNA levels (F). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (H) Knockdown of FLI1 promoted apoptosis in 143B and HOS cells. Apoptosis rates were determined by annexin V-FITC/PI staining. Data are represented as mean ± SEM (n = 3). ∗∗∗p < 0.001. p values were determined by a Student’s t test.

**Figure 6**
Overexpression of FLI1 reverses miR-145-5p-mimic-induced attenuation of cell migration, invasion, and proliferation in osteosarcoma cells (A) 143B and HOS cells were co-transfected with miR-145-5p and FLI1 plasmids or control vector, and apoptosis rates were determined with annexin V-FITC/PI staining. The percentage of apoptosis cells is shown as mean ± SEM from three independent experiments. ∗∗∗p < 0.001. (B) Cell migration and invasion of HOS and 143B cells, co-transfected with miR-145-5p and FLI1 plasmids or control vector, were evaluated by Transwell migration and Matrigel invasion assays. Data are represented as mean ± SEM (n = 3). ∗p < 0.05, ∗∗∗p < 0.001. (C) Proliferation of osteosarcoma cells, co-transfected with miR-145-5p and FLI1 plasmids or control vector, were determined by the CCK-8 assay. Data are represented as mean ± SEM (n = 3). ∗p < 0.05, ∗∗p < 0.01. (D) FLI1 overexpression promoted the migration ability of miR-145-5p overexpression cells, as demonstrated by the wound healing assay in 143B and HOS cell lines. Data are represented as mean ± SEM of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (E) FLI1 overexpression promoted the growth of miR-145-5p overexpression cells, as determined by the colony formation assay. Data are represented as mean ± SEM of three independent experiments. ∗∗∗p < 0.001. p values were determined by a Student’s t test.

**Figure 7**
Knockdown of miR-145-5p reverses shCircCAMSAP1-induced attenuation of cell migration, invasion, and proliferation in osteosarcoma cells (A) 143B and HOS cells were transfected with control vector or shCircCAMSAP1, respectively. The relative level of miR-145-5p was detected by quantitative real-time PCR analysis. Data are represented as mean ± SEM (n = 3). (B) The protein expression of FLI1, c-Myc, and IGFBP3 in 143B and HOS cells was detected by western blot analysis. Cells were co-transfected with shCircCAMSAP1 and miR-145-5p sponge or control vector. (C) The mRNA expression of FLI1, c-Myc, and IGFBP3 in 143B and HOS cells was detected by quantitative real-time PCR analysis. Cells were transfected with control vector and shCircCAMSAP1 with or without miR-145-5p sponge. Data are represented as mean ± SEM (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (D) Proliferation of osteosarcoma cells, co-transfected with control vector and shCircCAMSAP1 with or without miR-145-5p sponge, were determined by a CCK-8 assay. Data are represented as mean ± SEM (n = 3). ∗∗p < 0.01. (E) 143B and HOS cells were co-transfected with control vector and shCircCAMSAP1 with or without miR-145-5p sponge, and apoptosis rates were determined with annexin V-FITC/PI staining. The percentage of apoptosis cells is shown as the mean ± SEM from three independent experiments. ∗∗p < 0.01, ∗∗∗p < 0.001. (F) Cell migration and invasion of 143B and HOS cells, co-transfected with control vector and shCircCAMSAP1 with or without miR-145-5p sponge, were evaluated by Transwell migration and Matrigel invasion assays. Data represent the mean ± SEM (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (G) miR-145-5p knockdown promoted the growth of circCAMSAP1 knockdown cells, as determined by the colony formation assay. Data represent the mean ± SEM of three independent experiments. ∗∗∗p < 0.001. (H) miR-145-5p knockdown promoted the migration ability of circCAMSAP1 knockdown cells, as demonstrated by the wound healing assay in 143B and HOS cell lines. Data represent the mean ± SEM of three independent experiments. ∗∗p < 0.01, ∗∗∗p < 0.001. p values were determined by a Student’s t test.

**Figure 8**
Knockdown of circCAMSAP1 suppresses tumorigenesis *in vivo* (A and B) Nude mice were injected with 5 × 10⁶ 143B stable cells. Tumors were dissected and photographed after 5 weeks. (C) miR-145-5p knockdown promoted the tumor growth rate of circCAMSAP1 knockdown cells, and tumor volumes were evaluated by the equation v = ab²/2. Data represent the mean ± SEM (n = 6). ∗p < 0.05. (D) Average tumor weight in each group at the end of the experiment (day 35). Data represent the mean ± SEM (n = 6). ∗p < 0.05, ∗∗p < 0.01. (E) Histological analysis of tumor tissues by hematoxylin and eosin staining. FLI1, cleaved caspase-3, c-Myc, IGF1, and IGFBP3 expression levels were examined by immunohistochemistry. Scale bars, 100 μm. (F) Western blot analysis of FLI1, c-Myc, and IGFBP3 in tumors from xenograft mice. (G) Quantitative real-time PCR analysis of FLI1, c-Myc and IGFBP3 expression in tumors from xenograft mice. ∗∗p < 0.01, ∗∗∗p < 0.001. (H) Schematic illustration of the circCAMSAP1/miR-145-5p/FLI1 axis. p values were determined by a Student’s t test.

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References

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[1] Chou A.J., Geller D.S., Gorlick R. Therapy for osteosarcoma: where do we go from here? Paediatr. Drugs. 2008;10:315–327. - PubMed

[2] Chou A.J., Geller D.S., Gorlick R. Therapy for osteosarcoma: where do we go from here? Paediatr. Drugs. 2008;10:315–327. - PubMed

[3] Kaste S.C., Pratt C.B., Cain A.M., Jones-Wallace D.J., Rao B.N. Metastases detected at the time of diagnosis of primary pediatric extremity osteosarcoma at diagnosis: imaging features. Cancer. 1999;86:1602–1608. - PubMed

[4] Kaste S.C., Pratt C.B., Cain A.M., Jones-Wallace D.J., Rao B.N. Metastases detected at the time of diagnosis of primary pediatric extremity osteosarcoma at diagnosis: imaging features. Cancer. 1999;86:1602–1608. - PubMed

[5] Tang N., Song W.X., Luo J., Haydon R.C., He T.C. Osteosarcoma development and stem cell differentiation. Clin. Orthop. Relat. Res. 2008;466:2114–2130. - PMC - PubMed

[6] Tang N., Song W.X., Luo J., Haydon R.C., He T.C. Osteosarcoma development and stem cell differentiation. Clin. Orthop. Relat. Res. 2008;466:2114–2130. - PMC - PubMed

[7] Provisor A.J., Ettinger L.J., Nachman J.B., Krailo M.D., Makley J.T., Yunis E.J., Huvos A.G., Betcher D.L., Baum E.S., Kisker C.T., Miser J.S. Treatment of nonmetastatic osteosarcoma of the extremity with preoperative and postoperative chemotherapy: a report from the Children’s Cancer Group. J. Clin. Oncol. 1997;15:76–84. - PubMed

[8] Provisor A.J., Ettinger L.J., Nachman J.B., Krailo M.D., Makley J.T., Yunis E.J., Huvos A.G., Betcher D.L., Baum E.S., Kisker C.T., Miser J.S. Treatment of nonmetastatic osteosarcoma of the extremity with preoperative and postoperative chemotherapy: a report from the Children’s Cancer Group. J. Clin. Oncol. 1997;15:76–84. - PubMed

[9] Bielack S.S., Hecker-Nolting S., Blattmann C., Kager L. Advances in the management of osteosarcoma. F1000Res. 2016;5:2767. - PMC - PubMed

[10] Bielack S.S., Hecker-Nolting S., Blattmann C., Kager L. Advances in the management of osteosarcoma. F1000Res. 2016;5:2767. - PMC - PubMed

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circCAMSAP1 promotes osteosarcoma progression and metastasis by sponging miR-145-5p and regulating FLI1 expression

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circCAMSAP1 promotes osteosarcoma progression and metastasis by sponging miR-145-5p and regulating FLI1 expression

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