Hsa_circ_0001982 promotes the proliferation, invasion, and multidrug resistance of osteosarcoma cells

Abstract Background Osteosarcoma (OS) is the most common bone cancer mostly seen in people aged 10–25 years. This research aims to clarify the function of hsa_circ_0001982 in osteosarcoma (OS) and its effect on drug resistance, preliminarily exploring its mechanism. Methods The expression of hsa_circ_0001982 and miR‐143 in OS clinical tissues and cells was detected by real‐time fluorescence quantitative polymerase chain reaction (qRT‐PCR), MTT, colony formation assay, and transwell assay assessed cell proliferation, colony formation, migration, and invasion, respectively. The targeted relationship of hsa_circ_0001982 and miR‐143 was verified by a dual‐luciferase reporter assay. Result The expression of hsa_circ_0001982 was significantly increased in OS tissues and cells (MG63), as in well as chemoresistant OS tissues and cells (MG63/Dox). Overexpression of hsa_circ_0001982 promoted proliferation, colony formation, migration, invasion, and multidrug resistance in MG63 cells. By contrast, knockdown of hsa_circ_0001982 markedly reduced the resistance of MG63/Dox cells to doxorubicin (IC50 evidently reduced). Bioinformatic prediction showed that miR‐143 was a target miRNA of hsa_circ_0001982, and a dual‐luciferase reporter assay proved this. Further experiments revealed that miR‐143 expression was notably downregulated in OS tissues, chemoresistant OS tissues, and MG63/Dox cells. Moreover, miR‐143 was negatively correlated with hsa_circ_0001982 in OS cells and tissues. Conclusion The regulation of malignant behaviors such as proliferation, invasion, migration, and multidrug resistance of OS cells by hsa_circ_0001982 may be achieved by targeting miR‐143. Moreover, hsa_circ_0001982 is a potential target for early diagnosis and targeted therapy of OS.


| INTRODUC TI ON
Osteosarcoma (OS) is the most common bone cancer mostly seen in people aged 10-25 years with some 51 million annual new each year.
It usually metastasizes to the lung via the blood transport route at an early stage and results in high mortality. [1][2][3][4] At present, surgical resection of the lesion, combined with preoperative and postoperative adjuvant therapy, is the first-line treatment for OS, which can prolong the survival of OS patients. However, the treatment effect for patients with metastatic and recurrent OS is not optimistic leaving a low 5-year survival rate. 5 Moreover, the advent of multidrug resistance (MDR) has also become a major challenge to tackling OS, making cancer cells resistant not only to single cytotoxic drugs, but also to a range of drugs with different structures and cellular targets. 6,7 Therefore, on the one hand, a more definite molecular regulatory mechanism of malignant biological behavior of OS cells and multidrug resistance is required. On the other hand, it is urgent to explore new and effective biomarkers for improving the diagnosis, treatment, and prognosis of OS.
Circular RNAs (circRNAs) were originally found to be novel closed circular non-coding RNA molecules stably present in mammals, which are composed of both exon splicing and intron splicing, without a 5′ end cap and a 3′ end tail. 8,9 However, recent studies have shown that in some cancers, circRNA has translation function and can encode proteins and peptides. 10 CircRNAs contain binding sites for multiple microRNAs (miRNAs) and can enrich miRNAs. They can also regulate the expression levels of target genes by interfering with the binding of miRNAs to target genes, so as to participate in the occurrence and development of a variety of diseases, and thus are considered a potential disease marker. 11 Numerous studies have demonstrated that circRNAs act as oncogenes or suppressors in the development and progression of a variety of tumors. [12][13][14] Of note, some circRNA molecules such as circ-NT5C2 and circ100876 are involved in malignant behaviors such as proliferation and invasion of OS. 15,16 Hsa_circ_0001982 (circRNA RNF111), as a member of circRNAs, plays a role in a variety of malignancies. For example, Qiu et al. found that hsa_circ_0001982 was upregulated in breast cancer (BC) tissues and cells under hypoxia, and its high expression promoted glycolysis, cell viability, migration, and invasion of BC cells. 17,18 Besides, its noticeably high expression was also discovered in colorectal cancer 19 and gastric cancer 20 tissues and cells where it promoted the malignant behavior of tumors and served as a potential tumor marker. At the same time, hsa_circ_0001982 was also found to be associated with chemoresistance of malignant tumors. For example, hsa_circ_0001982 was distinctly highly expressed in adriamycinresistant 21 and paclitaxel-resistant BC tissues and cells, and its high expression promoted paclitaxel resistance in BC. 22 However, there is no study on the role and mechanism of hsa_circ_0001982 in OS malignant progression and its multidrug resistance. Therefore, in this study, function experiments were performed to explore the expression of this circRNA in OS, what effects it has on OS cell biological functions such as proliferation, colony formation, migration, invasion, and multidrug resistance, and how it affects. This provides some rationale for subsequent studies and clinical treatment of OS.

| Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR)
Total RNA was extracted from cells or tissues using Trizol reagent (Invitrogen, USA) and reversely transcribed into cDNA according to the instructions of the reverse transcription-PCR kit (Takara, Japan).
A SYBR Green PCR kit (Takara, Japan) was used to detect the relative expression levels of hsa_circ_0001982, RNF111, and miR-143, with GAPDH and U6 as internal references, respectively. The relevant primer sequences are listed in Table 1, and the data were processed using the 2 − ΔΔCt method. 23

| Cell proliferation assay
Cells in the logarithmic phase were collected and adjusted to a concentration of 5 × 10 4 cells/ml. Next, the cells were mixed well to inoculate 100 μl to a 96-well plate. After the cells had completely adhered and cultured for 24 h, a proliferation assay was carried out according to the instructions of the MTT kit. Briefly, 10μl MTT solution (5 mg/mL) was added to each well for a 4-h culture. Then, the supernatant was aspirated and 110μl formazan solution was added to each well and shaken at low speed for 10 min for full dissolution.
Then, the absorbance value at 490 nm was detected by a microplate reader and the cell proliferation rate was calculated. And, six replicate wells were set for each group.

| Determination of drug Sensitivity and cytotoxicity
Cells at the logarithmic phase were collected and adjusted to a concentration of 5 × 104 cells/ml and inoculated 100 μl to a 96-well plate for a 12-h culture. The cells were mixed with 10 μl of paclitaxel, cisplatin, methotrexate, and doxorubicin/adriamycin at different concentrations for a 48-h culture. Then, 10 μl of MTT solution (5 mg/ml) was added to each well and cultured for 4 h. The supernatant was removed, and 110 μl of formazan solution was added to each well and shaken at low speed for 10 min for full dissolution. Five duplicate wells were set for each group and microplate reader determined the absorbance at 490 nm. Finally, the half inhibitory concentration (IC50) was calculated.

| Cell colony formation assay
Cells in logarithmic growth were collected and the density of the cell suspension was adjusted to 1 × 10 3 cells/ml. Then, the cells were seeded into a culture dish containing DMEM medium and cultured statically at 37°C in 5% CO 2 for about 14 days. For a further step, crystal violet solution (Beyotime, China) was added to each well for staining the cell clones, and finally, photographs were taken for recording.

| Transwell assay
First 100 μl of diluted Matrigel (BD Biosciences, USA) was added to Transwell insert (Corning, USA) and allowed to solidify for 3-5 h in a 37°C incubator. Then, cells were suspended in a serum-free medium to achieve a concentration of 5 × 10 5 cells/mL. Next, 100 μl of

| Statistical analysis
Experimental data were expressed as mean ± standard deviation (SD), and statistical analysis was performed using SPSS 23.0 software. Comparison between two groups was conducted by using t test, and comparison of multiple groups by one-way analysis of variance. p < 0.05 indicates a significant difference.

| Hsa_circ_0001982 is upregulated in OS tissues and cells
To

| Hsa_circ_0001982 promotes MG63 cell proliferation, invasion, and migration
To further explore the function of hsa_circ_0001982 in OS, hsa_ circ_0001982 in MG63 cells was knocked down and overexpressed, respectively. As shown in Figure 2A

| Hsa_circ_0001982 improves multidrug resistance of MG63 Cells
Paclitaxel, cisplatin, doxorubicin, and methotrexate are chemotherapy drugs commonly used in OS clinic trials. 24 To investigate the relationship between hsa_circ_0001982 and chemoresistance in OS, we administered these drugs to OS cells in different groups

| Hsa_circ_0001982 targets miR-143 expression in OS tissues and cells
To further explore the regulatory mechanism of hsa_circ_0001982 in OS, we identified miR143 with complementary base matches to hsa_circ_0001982 by performing CircInteractome (Figure 4A).
The results of the luciferase reporter assay demonstrated that co-transfection of miR-143 mimics greatly inhibited the luciferase activity of the hsa_circ_0001982-WT vector but had no effect on Bcl-2. 33 Additionally, miR-143 is also associated with chemoresistance in OS. 34 In the study by Tang et al., hsa_circ_0001982 functions in BC by regulating miR-143 expression. 18 Herein, we speculated

| CON CLUS IONS
Hsa_circ_0001982 is significantly highly expressed in OS tissues and cells as well as chemoresistant OS tissues and cells. Functionally, hsa_circ_0001982 can promote OS cell proliferation, colony formation, invasion, migration, and the development of multidrug resistance, which may be achieved by targeting inhibition of miR-143 expression. As a result, hsa_circ_0001982 may be used as a potential target for early diagnosis and targeted therapy of OS.

CO N FLI C T O F I NTE R E S T
The authors declared that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.