Circ_001422 aggravates osteosarcoma progression through targeting miR‐497‐5p/E2F3 axis

Circular RNAs exert vital functions in the pathogenesis of osteosarcoma (OS). Circ_001422 has been confirmed to be involved in regulating OS progression, but its specific mechanism has not been clearly studied. This work aimed to analyze circ_001422's role in OS cell biological behaviors and the possible molecular mechanisms. This work carried out reverse transcription‐quantitative polymerase chain reaction for detecting circ_001422, E2F3 and miR‐497‐5p levels, whereas Cell counting kit‐8 together with Transwell assays for measuring cell growth, migration as well as invasion abilities. Relation of miR‐497‐5p with E2F3, as well as circ_001422 with miR‐497‐5p was analyzed through dual‐luciferase reporter gene assay. Protein level was identified by western blot. According to our results, circ_001422 expression within OS tissue significantly increased compared with corresponding healthy samples. Inhibition of circ_001422 significantly decreased OS cell growth, invasion and migration. From mechanism research, miR‐497‐5p was proved as circ_001422's target, and E2F3 was miR‐497‐5p's target. Besides, miR‐497‐5p downregulation or E2F3 overexpression abolished circ_001422 inhibition‐mediated inhibition on OS cell proliferation, invasion and migration. Collectively, this study has first suggested circ_001422's role in enhancing OS proliferation, migration as well as invasion via miR‐497‐5p/E2F3 axis. Our results will offer new ideas and new anti‐OS targets.

circ_0008259 can suppress OS occurrence and development via the miR-21-5p/PDCD4 axis. [12] Circ-LRP6 expression increases in OS cells as well as tissues, whereas its downregulation can inhibit the OS cell growth, migration and invasion by miR-141-3p/HDAC4/HMGB1 axis. [13] Circ_001422, the newly reported circRNA, has been reported to promote OS tumorigenesis. [14] However, the mechanism of the involvement of circ_001422 in OS is not completely clear at present. MicroRNAs (miRNAs) are able to modulate target levels through incomplete complementation with target gene 3'UTR, thereby regulating malignant biological behavior of cancer cells. [15] MiR-497-5p, belonging to miR-15 family, shows low expression within diverse tumor tissues like gastric cancer, OS, hepatocellular carcinoma (HCC) as well as non-small-cell lung cancer (NSCLC), which is considered as the vital tumor suppressor. [16][17][18][19] However, the current functional research on miR-497-5p within OS remains lacking. In recent years, studies found that circRNAs may be miRNAs' "molecular sponges" and inhibit their function. [12,13] For instance, as revealed by Ma and colleagues, circ_UBAP2 sponged miR-637 as the ceRNA to regulate HMGB2 expression to facilitate OS progression. [20] Zhang et al. showed that circ_0002137 modulated OS development via miR-433-3p/IGF1R axis. [21] As reported by Liu and coworkers, circ_0081001 upregulated BACH1 expression through miR-494-3p, thus promoting OS cell invasion and growth. [22] Here, miR-497-5p was reported as circ_001422's potential target. However, it remains unclear about circ_001422/miR-497-5p axis' effect on OS development. E2F transcription factor 3 (E2F3), which belongs to the E2F transcription factor (TF) family, regulates the progression of the cell cycle and has been confirmed with high levels within diverse cancers and can accelerate cancer cell growth. [23][24][25] E2F3, which belongs to the E2F TF family, participates in cancer genesis and progression, and its expression is increased within diverse cancers like bladder cancer (BLCA), laryngeal cancer, OS and breast cancer. [26][27][28][29] In addition, various miRNAs like miR-22, miR-194-5p or miR-195-5p, can regulate cancer genesis and progression via E2F3, suggesting that E2F3 may be the possible anticancer therapeutic target. [26,27,30] TargetScan online prediction identified E2F3 as miR-497-5p's potential target. However, it remains unclear about miR-497-5p/ E2F3's effect on OS.
The present work examined circ_001422's role in OS cell biological behavior and investigated how circ_001422/miR-497-5p axis affected OS for the first time. Our results will offer the possible targets to diagnose and treat OS clinically.

| Nuclear and cytoplasmic fractions
This work utilized ReadiPrep Nuclear/Cytoplasm Isolation Kit (AAT Bioquest) for determining circ_001422 localization. Briefly, cytosol extraction buffer was used to process cells, followed by centrifugation for 20 s to collect the supernatant as a cytoplasmic extract.
Thereafter, resuspend the pellet in X1 high salt buffer (150 µL). The samples were then centrifuged to collect the supernatant as a nuclear extract. Subsequently, we conducted RT-qPCR for analyzing cytoplasmic/nuclear fraction of circ_001422.

| Pull-down assay
Briefly, circ_001422 was labeled with biotin, followed by overnight incubation using streptavidin beads under 4°C. Thereafter, the mixture was then centrifuged washed with wash buffer I. The bead-biotin complex was assed to the lysate and incubate under ambient temperature for a 1-h period. The sample was then rinsed by Wash Buffer II, beadbound RNA was captured and RT-qPCR assay was then determined.

| Bioinformatics analysis
To study the potential miRNAs associated with circ_001422, online bioinformatics database StarBase 2.0 (http://starbase.sysu.edu.cn/) was used. The most likely complementary combination of miRNAs were introduced. Among these predicted target miRNAs, we found that miR-497-5p has binding sites for circ_001422. TargetScan (www.targetscan. org/vert_71/) was used to predict the potential targets of miR-497-5p.
Among these predicted target, we found that E2F3 has binding sites for miR-497-5p.

| Statistical analysis
Results were represented by mean ± SD (X ± SD) and data measure-  Figure 1A).
Relationship of circ_001422 level with clinicopathological parameters of OS was analyzed. circ_001422 level was not related to patient age, gender, or tumor sizes. Compared with lowexpression group, patients in circ_001422 high expression group had higher clinical stage and higher metastasis rate (Table 2).
Compared with hFOB1.19 OB, circ_001422 expression was significantly increased within OS cells. Particularly, HOS and SaOS-2 cells exhibited higher circ_001422 level, and they were selected for the following functional experiments ( Figure 1B).
Next, we analyzed subcellular localization of circ_001422's

| Silencing circ_001422 suppressed OS cell growth and invasion
For exploring circ_001422's effect on OS cells, this work transfected si-circ_001422 in HOS and SaOS-2 cells, followed by qRT-PCR for detecting transfection efficiency. As a result, following transfection with circ_001422 siRNA, circ_001422 expression was significantly reduced, indicating that the transfection was effective and might be applied in later analyses (Figure 2A). Since si-circ_001422 1# has the higher knockout efficiency, it was selected for the next experiment.
Then, CCK-8 along with Transwell assay was carried out for analyzing how silencing circ_001422 affected OS cells. According to 3.3 | MiR-497-5p serves as circ_001422's target circRNAs often play a role of miRNA sponges for regulating the expression of miRNAs, thereby participating in cellular functions. [13,14] The StarBase database estimated miR-497-5p containing circ_001422 binding sites ( Figure 3A). For verifying relation of circ_001422 with miR-497-5p, this work performed luciferase reporter gene assay. As a result, miR-497-5p mimic significantly reduced normal group (WT) luciferase activity by luciferase assay, but not the mutant group (MUT) ( Figure 3B). RNA pull-down analysis also confirmed that circ_001422 interacted with miR-497-5p ( Figure 3C). miR-497-5p expression in OS cells and tissues was analyzed. miR-497-5p expressed decreased within OS tissues whereas negative relation to circ_001422 level ( Figure 3D−E).

| E2F3 serves as miR-497-5p's target
For exploring miR-497-5p's mechanism in OS occurrence and development, the TargetScan bioinformatics database was utilized for predicting miR-497-5p's possible target. According to predicted results, there were binding sites in E2F3 3'UTR for miR −497-5p ( Figure 5A). To test this prediction, this work conducted the luciferase reporter assay. As a result, cells cotransfected with E2F3-WT miR-497-5p had evidently reduced luciferase activity relative to miR-NC group ( Figure 5B). To verify miR-497-5p's effect on the possible target molecule E2F3, as well as on the expression of E2F3, this work carried out WB and RT-qPCR assays.

| DISCUSSION
CircRNAs are increasingly found to be related to tumor occurrence and development, becoming potential new targets for tumor therapy. [6,7] Many articles indicate that circRNAs also act as important players in tumorigenesis and development in OS. [8][9][10] For example, circ-LRP6 showed high expression within OS, which enhanced OS proliferation, migration and invasion. [13] Additionally, circ_0000658 level decreased within OS, while circ_0000658 downregulation suppressed OS malignant biological behavior as a tumor suppressor gene. [31] As found by a previous study, circ_001422 was confirmed to promote OS metastasis and development by miR-195-5p/FGF2/PI3K/Akt axis. [14] This experiment also confirmed that circ_001422 was abnormally increased within OS, while circ_001422 upregulation within tissues was tightly associated with high patient clinical stage as well as metastatic events. These results suggest that circ_001422 is likely to be involved in the metastasis of OS, which is consistent with presious findings, suggesting that circ_001422 has a tumorpromoting effect in tumors.
We conducted dual-luciferase reporter assay and targetScan bioinformatics software, which verified E2F3 as miR-497-5p's target. E2F3 has been widely suggested to participate in tumor cell occurrence and development. [23,25] For example, E2F3 showed high expression within BLCA, and it showed positive relation to cancer malignancy. [30] E2F3 level within prostate cancer was related to patient survival. [35] E2F3 also exhibited abnormally high expression within endometrial cancer tissues, which was related to pathological grade and tumor stage. [36] Currently in this work, E2F3 expression markedly increased within OS tissues, which exhibited negative relation to miR-497-5p level whereas positive relation to circ_001422 level within OS tissues.
As an important participant in the ceRNA network, circRNAs can adsorb miRNAs through sponges, thereby affecting the latter downstream target gene levels, resulting in changes in cell phenotypes. [12][13][14] Here, we have demonstrated miR-497-5p as circ_001422's target, while E2F3 as miR-497-5p's target. We were curious that whether circ_001422/miR-497-5p/E2F3 axis was related to OS development. As revealed by diverse experiments, circ_001422 silencing alone remarkably inhibited growth, invasion together with migration in OS cells, while the inhibitory effect was partly abolished through inhibiting miR-497-5p or upregulating E2F3.
Collectively, this work confirmed the promoting activity of circ_001422 for OS cell migration and invasion in vitro, and preliminarily determined its mechanism of promoting metastasis.
More importantly, we demonstrated for the first time that

CONFLICT OF INTEREST STATEMENT
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
The data used to support the findings of this study are available from the corresponding author upon request.