EGR1 promoted anticancer effects of Scutellarin via regulating LINC00857/miR‐150‐5p/c‐Myc in osteosarcoma

Abstract Scutellarin, an active flavone extracted from Erigeron breviscapus, is known to exhibit antitumour activity in many cancers. However, the effects of Scutellarin on osteosarcoma remain unclear. In this study, we found that Scutellarin suppressed osteosarcoma cell growth, induced cell apoptosis and inhibited tumorigenesis. Mechanistically, our data revealed that EGR1 was significantly increased under Scutellarin treatment. Increased EGR1 enhanced tumour‐suppressive effects of Scutellarin on osteosarcoma cells via transcriptionally downregulating LINC00857 expression. Additionally, we found that LINC00857 acted as a competitive endogenous RNA of miR‐150‐5p and inhibited the activity of miR‐150‐5p, which resulted in c‐Myc increase. Scutellarin could suppress c‐Myc protein levels through decreasing LINC00857 expression in osteosarcoma. Thus, these findings demonstrate that EGR1/ LINC00857/miR‐150‐5p/c‐Myc axis plays a key role in promoting anticancer effects of Scutellarin and Scutellarin might have potential clinical implication in osteosarcoma clinical treatment.

for many years because of its vasodilation, anti-inflammation, antioxidation, anticoagulation and antithrombotic activities. 4 Recently, Scutellarin was reported to be an antitumour factor to promote cell apoptosis and inhibit cell growth, invasion and migration in many human tumours including hepatocellular carcinoma, tongue squamous carcinoma, renal cancer and lung cancer, [5][6][7][8] but the role of Scutellarin in OS cells has been poorly understood.
Early growth response gene-1 (EGR1) is an immediate early gene, which owns a DNA-binding domain composed of three zinc-finger motifs and appears to activate transcription by binding to DNA as a monomer. 9 EGR1 can be expressed rapidly under the induction of various stimuli such as growth factors, hormones and neurotransmitters, and then regulate downstream target genes. 10 Emerging data suggest that EGR1is dysregulation in several human cancers and plays an essential role in tumorigenesis. 11 Notably, the antitumour mechanism of many natural compounds is closely related to the expression of EGR1. For example, Coptis chinensis induced apoptosis of HCC cells by promoting EGR1-induced NAG-1 promoter activity. 12 However, the role of EGR1 in the treatment of osteosarcoma by Scutellarin is still unknown.
Long non-coding RNAs (lncRNAs) are longer than 200 nucleotides RNAs, which have no protein-coding capacity. 13 Many reports have shown that lncRNAs play vital regulatory roles in many human diseases, especially in malignant tumours. 14 LINC00857 (long intergenic non-protein-coding RNA 857) has been reported to play a carcinogenic factor in several human cancers and regulated cancer cell growth, death and metastasis. For example, LINC00857 was increased in hepatoma cell lines and depletion of LINC00857 inhibited the progression of hepatocellular carcinoma. 15 Although several studies have suggest that LINC00857 play a key role in cancer, its functions in response to Scutellarin remain unclear.
Here, we found the potential of Scutellarin to inhibit growth of osteosarcoma cells at the cellular level and in animal models.
Mechanistically, we uncovered that Scutellarin induced EGR1 increase in osteosarcoma cells, which led to cell apoptosis increase and cell growth decrease. Subsequently, our data indicated that LINC00857 was decreased under Scutellarin treatment and contributed to Scutellarin-induced c-Myc downregulation via acting as a miR-150-5p sponge. Except that, we also found that EGR1 bound to the region of LINC00857 promoter, suppressing LINC00857 expression under Scutellarin treatment. Taken together, these results suggest that EGR1/Linc00857/miR-105-5p/c-Myc signal axis plays an essential role in antitumour effects of Scutellarin on osteosarcoma.

| Cell culture and transfection
The human osteosarcoma cancer cell lines 143B and U2OS were provided by the American Type Culture Collection. The cells were cultured in Dulbecco's modified Eagle's medium containing 10% foetal bovine serum and streptomycin (100 μg/ml) and were cultured at 37°C in a humidified atmosphere with 5% CO 2 . The siRNA for LINC00857, EGR1, c-Myc and miR-105-5p mimics, and inhibitors were ordered from GenePharma. For plasmid transfection, Lipofectamine 3000 (Invitrogen) was used in accordance with manufacturer's instructions.

| MTT and Hoechst 33342 staining assay
Briefly, we seeded osteosarcoma cells into 96-well plates at the density of 7 × 10 4 cells per well. After 48 h of treatment, 20 μl of MTT reagent (5 mg/ml) was added immediately to each well and incubated for another 4 h. Then, we removed the culture medium and added 200 μl dimethyl sulfoxide to each well. At the end, absorbance value at 570 and 630 nm was assessed by using microplate reader (PerkinElmer).
For Hoechst 33342 staining assay, osteosarcoma cells were cultured in six-well plates and treated with or without Scutellarin for 48 h. Then, these cells were stained with Hoechst 333342 solution (Sigma) and incubated for 30 min. After washing with PBS for three times, the morphologic changes of cells were recorded by fluorescence microscope (Olympus).

| Colony formation assay
143B and U2OS cells were collected and prepared into single-cell suspension in complete culture medium. After cell counting, 143B and U2OS cells were cultured in six-well plates with 200 or 400 cells per well. After 24 h, Scutellarin was added to each well. These cells were cultured for 2 weeks and then were stained by 0.04% crystal violet, and proliferation ability was assessed by taking pictures of the above cells.

| Annexin V-FITC staining and flow cytometry
The protocol was provided by the manufacturer, and its instructions were followed (Yeasen). In brief, the cells were pretreated with Scutellarin, harvested by centrifugation at 300 g in 4°C and suspended in 100 μl binding buffer. Then, 5 μl Annexin V-FITC and 10 μl propidium iodide (PI) staining solution were added for a 10-min incubation at room temperature in darkness. Then, the cells were suspended with 400 μl binding buffer and mixed gently. The flow cytometry (BD Biosciences) analysis was conducted to test cell apoptotic events.

| Animal experiments
Animal experiments were conducted according with the National Institute of Health Guide for the Care and Use of Laboratory Animals with the approval of the Animal Research Committee of Dalian Medical University. 143B cells were subcutaneously injected into the nude mice. After 7 days, the mice were randomly divided into two groups. The control group was treated with 100 μl PBS by intragastric administration. The test group was treated with 100 μl SCU (60 mg/kg) diluted by PBS through the same administration every day. All mice need to be observed every 2 days, and the tumour volume was measured. The 143B tumour volume was calculated using the formula V = 1/2 (length × width 2 ). After 12 days of administration of Scutellarin or normal PBS, the mice were killed via cervical dislocation, and then, the tumours were taken pictures, measured weights and fixed for further experiments after carefully removed.

| Dual-luciferase reporter assay and ChIP assay
The dual-luciferase reporter assay and ChIP were performed as previously described. 16

| Statistics and data analyses
The data were expressed as the means ± SD, and the statistical evaluation was performed using one-way analysis of variance (ANOVA).
Values of p < .5 were considered statistically significant.

| Scutellarin decreases osteosarcoma cell growth and increases cell apoptosis
The major chemical structure of Scutellarin is shown in Figure 1A.

| Scutellarin upregulated EGR1 expression in osteosarcoma cells
To uncover the antitumour mechanism of Scutellarin, osteosarcoma cells were treated with or without 500 μM Scutellarin for 48 h. The mRNA profiles were investigated by RNA sequencing analysis. As shown in Figure  with RNA sequencing analysis, Scutellarin significantly increased the EGR1 expression ( Figure 2F-N). Taken together, these results suggest that EGR1 is increased under Scutellarin treatment in osteosarcoma.

| EGR1 contributes to the antitumour effects of Scutellarin in osteosarcoma cells
To investigate the role of EGR1 in Scutellarin-induced tumour sup- U2OS cells. Then, we treated these cells with or without 500 μM Scutellarin. The efficiency of knockdown was measured by Western blotting. We found that EGR1 was decreased when the cells were

| EGR1 transcriptionally suppresses LINC00857 expression in osteosarcoma cells
Increasing studies have indicated that LncRNAs play important roles in cancer. 17,18 Therefore, we want to know whether LncRNAs are involved in Scutellarin-induced tumour suppression. To this end, we first detected the expression of eight LncRNAs after Scutellarin treatment and found that Scutellarin significantly downregulated LINC00857 expression in 143B cells ( Figure 4A). To further confirm this, we treated 143B and U2OS cells with or without 500 μM Scutellarin for 48 h and the expression of LINC00857 was measured by qRT-PCR. As shown in Figure 4B,C, we found that LINC00857 was gradually downregulated following Scutellarin increase.
Combined with our previous results, we want to know whether EGR1 regulates LINC00857 expression in response to Scutellarin treatment. To prove it, we first knocked down EGR1 in 143B and U2OS cells and treated these cells with or without 500 μM Scutellarin for 48 h. Then, we detected the expression levels of LINC00857 by qRT-PCR. We found that knockdown of EGR1 upregulated LINC00857 expression and reversed Scutellarin-induced LINC00857 downregulation ( Figure 4D,E).
To further prove whether EGR1 suppressed LINC00857 expression relying its transcriptional activity, we first inspected the upstream sequence of LINC00857 using the JASPAR software and identified three potential binding sites of EGR1 on LINC00857 promoter, which were named BS1, BS2 and BS3. To verify it, we cloned the promoter of LINC00857 and different truncations by PCR. We then inserted them into the pGL3-based luciferase reporter plasmids, which were named P1-P3 and transfected them into 143B cells with or without Scutellarin treatment ( Figure 4F). As illustrated in Figure 4G, the luciferase activities of P1 and P2 were decreased in response to Scutellarin treatment. However, the decrease was abolished when P3 was transfected, suggesting that the region of P2 and BS1 was essential for Scutellarin-induced LINC00857 downregulation. To further confirm that BS1 was indeed responsive to EGR1, we first transfected P2 into143B cells with or without EGR1 knockdown and luciferase activities were measured. As shown in Figure 4H, loss of EGR1 elevated the luciferase activities, which were suppressed by Scutellarin.
Then, the luciferase reporter plasmids containing BS1 WT and BS1Mut were constructed ( Figure 4I). The BS1 WT and BS1 Mut were individually transfected into 293T cells as indicated and luciferase activities were measured. We found that the luciferase activity of BS1 WT but BS1 Mut was significantly decreased in response to EGR1 ( Figure 4J). Similarly, we also obtained that Scutellarin dramatically inhibited the luciferase activity of BS1 WT but BS1 Mut in 143B cells ( Figure 4K).
Subsequent ChIP assay showed that the chromatin fragment corresponding to the assumed EGR1 binding site (BS1) was specific in anti-EGR1 immunoprecipitate of osteosarcoma cells, and the binding was enhanced after treatment with Scutellarin ( Figure 4L).
Taken together, these results demonstrate that EGR1 can bind to the promoter of LINC00857 and inhibit its expression in response to Scutellarin treatment.

| LINC00857 contributes to Scutellarininduced c-Myc downregulation via acting as a miR-150-5p sponge
Previous studies have indicated that the depletion of LINC00857 suppresses cell proliferation and induces cell apoptosis via decreasing some oncogenic proteins including c-Myc in oesophageal adenocarcinoma. 19 Thus, we ask whether LINC00857 contributes to Scutellarin-induced c-Myc downregulation in osteosarcoma cells.
To this end, we first detected protein levels of c-Myc in response to Scutellarin treatment. As shown in Figure 5A, we found that  Figure 5E).
To further verify the relationship between LINC00857 and miR-150-5p, we first inserted the wild-type LINC00857 or the mutant of the binding site in the reporter plasmid ( Figure 5E).
We then transfected them into 143B cells with or without miR-150-5p mimics treatment and found that miR-150-5p significantly decreased luciferase activity of wild-type LINC00857 ( Figure 5F). The expression of c-Myc was detected by Western blotting. We found that the loss of EGR1 abolished Scutellarin-induced c-Myc downregulation and the phenotype was reversed by LINC00857 inhibition ( Figure 5J). Consistently, we found that knockdown of EGR1 prevented Scutellarin-induced cell viability decrease, and the phenotype was reversed by LINC00857 inhibition or miR-150-5p overexpression ( Figure 5K). These data indicate that LINC00857 contributes to Scutellarin-induced c-Myc downregulation via acting as a miR-150-5p sponge and EGR1 promotes anticancer effect of Scutellarin on osteosarcoma cells through regulating LINC00857/ miR-150-5p/c-Myc axis.

| DISCUSS ION
Scutellarin is an active flavone extracted from Erigeron breviscapus.
Many reports have shown that Scutellarin exerts anticancer actions  increase induced by Scutellarin is still unknown and we will uncover it in the future.
Accumulating studies have revealed that c-Myc has been characterized as a vital oncogene, which is correlated with cell proliferation, apoptosis and metastasis in various human cancers. In human hepatocellular carcinoma, the downregulation of c-Myc inhibited tumour proliferation. 33 In osteosarcoma, c-Myc has been shown to promote the growth of tumour cellsss. 34 Overall, we found that protein levels of c-Myc were decreased in response to Scutellarin treatment in osteosarcoma cells, which relied on the LINC00857 decrease by Scutellarin. LINC00857 was reported to act as an oncogene in many cancers including hepatocellular carcinoma, lung cancer, pancreatic cancer and gastric cancer. 15,20,35,36 Many studies have suggested that LINC00857 can function as an miRNAs sponge to regulate signalling pathways and biological functions. 21 Consistently, our study revealed that LINC00857 contributes to Scutellarin-induced c-Myc downregulation via acting as a miR-150-5p sponge. Moreover, we found that miR-150-5p or c-Myc knockdown eliminated increases in LINC00857-induced cell viability under Scutellarin treatment. Thus, our data suggest that EGR1/LINC00857/miR-105-5p/c-Myc axis plays an important role in Scutellarin-inhibited osteosarcoma cell growth.

ACK N OWLED G EM ENTS
This work was supported by grants from the National Natural

CO N FLI C T O F I NTE R E S T
The authors declare no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data will be made available on request to the corresponding authors.