Scutellarin inhibits the glioma cell proliferation by downregulating BIRC5 to promote cell apoptosis

Abstract The expression changes of baculovirus inhibitor of apoptosis repeat‐containing protein5 in brain glioma after administration of Scutellarin was detected. To explore the effort of scutellarin on anti‐glioma by downregulating BIRC5.The effect of scutellarin on tumour growth and animal survival was detected by administering scutellarin to nude mice subcutaneous tumour formation and SD rats in situ tumour formation models. A significantly different gene BIRC5 was found by using the combination of TCGA databases and network pharmacology. And then qPCR was performed to detect the expression of BIRC5 in glioma tissues, cells and normal brain tissues and glial cells. CCK‐8 was used to detect the IC50 of scutellarin on glioma cells. The wound healing assay, flow cytometry and MTT test were used to detect the effect of scutellarin on the apoptosis and proliferation of glioma cells. The expression of BIRC5 in glioma tissues was significantly higher than that in normal brain tissues. Scutellarin can significantly reduce tumour growth and improve animal's survival. After scutellarin was administered, the expression of BIRC5 in U251 cells was significantly reduced. And after same time, apoptosis increased and cell proliferation was inhibited. This original research showed that scutellarin can promote the apoptosis of glioma cells and inhibit the proliferation by downregulating the expression of BIRC5.

anti-inflammatory, 9 anti-cancer 10 and neuroprotection effects. 11 It has been reported that scutellarin plays a significant role in the treatment of ischemic brain injury diseases, such as cerebral infarction, and the increased production of reactive oxygen species is an important mechanism for the occurrence of ischemic brain diseases. Therefore, the pharmacology action of scutellarin is related to the nature of scavenging reactive oxygen species. 11 Scutellarin aglycone can improve the neurological dysfunction of middle cerebral artery embolism rats with focal cerebral ischemia in a dose-dependent manner and reduce the area of cerebral infarction. 12 In addition, scutellarin has a neuroprotective effect on cerebral ischemia-reperfusion injury, 13 but there are few reports of this effect in glioma.
Baculovirus inhibitor of apoptosis protein 5 (BIRC5) is a newly discoveredmemberoftheinhibitorofapoptosisprotein(IAP)family, and its function is the strongest among the inhibitors of apoptosis that have been identified thus far. 14 BIRC5islocatedat17q25andis 14.7 kbinlength.Ithas3intronsand4exons,andcontainsarepeti-tivesequenceofbaculovirusIAPS.BIRC5isabundantlyexpressedin fetal tissues and various human malignant tumour tissues, but not in normal tissues or well-differentiated adult tissues. 15 Previous stud-ieshaveshownthatBIRC5ishighlyexpressedingliomaandassociated with poor prognosis in patients with glioma. 16,17 In the present study, scutellarin was proven to have significant anti-gliomaeffects,andBIRC5wasreportedasanoncogeneofglioma. Scutellarin exerts an anti-tumour effect by downregulating the BIRC5gene.BIRC5downregulationsignificantlyinhibitstheproliferation of glioma cells, in which glioma cell apoptosis is activated.

| Microarray data
Thegeneexpressionprofileandclinical-relateddataofGBMwere downloaded by entering the GDC data in TCGA database; these data which consisted of 330 high-grade glioma samples and five normal brain tissue samples. The series matrix and platform files were downloaded and saved as .txt files. The downloaded files were processed by the R software package, and the data were also evaluated by the R software package.

| Screening of differentially expressed genes
R language software and an annotation package were used to download series matrix and platform files. The ID of the matching probe name was converted into the international standard gene name, and thensavedasatxtfile.ALIMMApackagewasusedforgenedifferential expression analysis. The relevant command codes were input intoR,andthentheLIMMAsoftwarepackagewasusedtoanalyse the differentially expressed genes of high-grade glioma and normal brain tissues in the microarray data set. Samples with fold change (FC)correctionvaluesof>6 and p < 0.05wereconsideredtobedif-ferentiallyexpressedmRNAgenes.TheXLSresultsweresavedfor subsequentanalysis.

| Integration of microarray data
The list of differentially expressed genes of the microarray dataset accessedthroughtheLIMMApackagewassavedasanXLSfile.The gplots package was downloaded and the R language software was usedtoruntheinstructioncode.Subsequentanalysisyieldedalist of upregulated or downregulated genes in the transcriptomics data.

| Identification of scutellarin component targets
The biologically active components of scutellarin were obtained from the traditional Chinese medicine systems pharmacology database and analysis platform (http://lsp.nwu.edu.cn/tcmsp.php).
After searching the database and eliminating duplicates, a total of 12 main compounds were obtained. Next, UniProt (http://www. unipr ot.org/)wasusedtoconfirmgeneinformation,includingname and gene ID. at a density of 4 × 10 5 cells/mL, and placed in an incubator. After 24 h, the supernatant containing non-adherent cells was removed and fresh medium was added. Once cells had reached close to confluence(80%-90%),themediumwaschangedevery3-5 days,with a density of ~4 × 10 5 cells/cm 2 , the cells were passaged three times, and the suspended cells were discarded. Subsequently, purely ad-herentU251cellswereculturedforfurtheranalysis.Thegrowthstatus of the cultured cells was observed under an inverted microscope (ECLIPSE Ti, Nikon).

| Cloneformation
TheU251cellsweredigestedwith0.25%trypsinwhencellviability was good, and the cells were counted on a cell counting board. A total of 500 cells were added to each well in the 6-well plate and placed in the cell incubator at 37°C overnight. The state of the cells was observed under an inverted microscope the next day. After all the cells had adhered to the wall, the medium containing dead cells was abandoned, the fresh 10% FBS complete medium and scutellarin were added to the corresponding concentration, and the 6well plate was put back into the incubator for culture. The state of the cells was observed every 2 days and the culture medium was changedevery4 days.Whenthecellcommunityexceeded50cells, a picture was captured under the microscope and it was recorded; it wasthenwashedwithPBSthathadbeenwarmedupinadvance,and theremainingPBSwasdriedintheholewithapipette.Cellswere thenfixedfor20 minwith4%paraformaldehydepreheatedby1 mL ineachhole,andanappropriateamountofPBSwasaddedtoclean thecellsthreetimes.Atotalof200 μL 0.5% crystal violet dye solu-tionwasaddedtoeachholetodyefor20 min;theholeswerethen washed with pure water three times and covered using an orifice plate cover and left to dry overnight. In addition, the cell communities of >30 pixels in each orifice plate were calculated using ImageJ software (National Institutes of Health) and were statistically analysed compared with the Control group.

| Woundhealingassay
U251 cells were digested and counted. Next, 1 × 10 6 cells were seeded to each well of a 6-well plate and incubated overnight in an incubator at a constant temperature of 37°C. Using the 10-μL pipette, the monolayer cells were scratched with the word '#' on the orificeplate,thecompletemediumcontaining10%FBSwasadded, the 6-well plate was gently shaken and the medium discarded. The steps above were repeated three times to remove the cells caused by scratches. Fresh and complete medium containing scutellarin was added, and the culture flask was placed in an incubator with 5% CO 2 at a constant temperature of 37°C. The images of eight visual fields ineachgroupat0,24and48 hweretakenatthesamepositionusing an inverted microscope. In addition, the area of each scratch at 0, 24 and48 hweremeasuredusingImageJsoftware,andcomparedwith the Normal and Control groups.

| Cellapoptosisdetectionbyflowcytometry
U251cellswereseededina6-wellplatewith10 6 cells per well, and incubated in a cell incubator with 5% CO 2 37°C, overnight. After   Finally, all nude mice were euthanized by excessive isoflurane inhalation. The standard of death was cardiac arrest.

| Scutellarin administration
A total of 60 rats and 60 nude mice were randomly divided into 4 groups, namely the DMSO negative control group, temozolomide positive control group, scutellarin low-dose treatment group (20 mg/kg) and scutellarin high-dose treatment group (50 mg/ kg), 18 15 in each group. The intraperitoneal injection was administered once a day, and the body weight, water and diet and the tumour volume-related indicators of rats and nude mice were monitored.

| PET/CT scan
Before the PET/CT test, the rats and nude mice were fasted for 8 h and were water-free for 4 h. Following isoflurane inhalation anaesthesia, each rat and nude mouse was injected intraperitoneally with 18F-

| Haematoxylin-eosin (H&E) staining
The glioma tissue was dehydrated by automatic dehydrator, made transparent with xylene, soaked in wax and embedded in paraffin. PBS was then used for rinsing every 5 min for a total of six times.
Subsequently,~150 μL fluorescent secondary antibody was added to each tissue slide, followed by rinsing with PBS every 5 min for a total of five times after incubation at room temperature for 2 h.
Finally, the tablets were sealed using a water-based sealing agent, and the images were observed and collected under the fluorescence microscope.

| Statistical analysis
All experimental data of this original research were statistically ana-lysedusingSPSSv.21software(IBMCorp.).Comparisonsbetween two groups were made using an independent sample t-test, and comparisons between three or more groups were made using oneway anova.Dataareexpressedasthemean ± SD.p < 0.05wasconsidered to indicate a statistically significant difference.

| Effects of scutellarin on body weight and survival time of tumour-bearing rats in situ
First, SD rats were used to establish an orthotopic tumour model. In order to evaluate whether the glioma model was successful, rats were randomlyselectedforbraindissectionat7 daystoseeifthetumour had formed, and the remaining rats then underwent a PET-CT scan to confirm the formation of tumours in the brain. The results showed that allratshadtumoursinthebrainat7 days( Figure 1A).Next,alltherats were treated with scutellarin. The weight of the rats was found to have significantlydecreased7 dayspost-operatively( Figure 1B),whichwas consistent with the clinical primary glioma patients. 19 Butinterestingly, during the treatment phase after tumour formation, the weight of the ratsintheDMSOgroupcontinuedtodecrease( Figure 1C),andthe ratsintheDMSOgroupdiedonthefirstdayoftreatmentwithscutellarin ( Figure 1D).Thisshowedthatintheearlystageofgliomagrowth, the tumour consumes a lot of nutrition and leads to the weight loss of rats. After treatment with scutellarin, the growth of glioma is inhibited and the body weight of rats gradually recovers. No significant difference was observed between the scutellarin and other groups, which showed that scutellarin has no other obvious side effects on rats. Next, the survival of all rats was evaluated, and it was shown that scutellarin effectively increased the survival rate of rats compared with theDMSOnegativecontrol( Figure 1D);then,ascomparedwiththe temozolomide group, a certain difference in the survival improvement of rats was observed following treatment with scutellarin ( Figure 1E).
In summary, scutellarin can improve the survival of rats with glioma, and has a certain anti-glioma effect.

| Effect of scutellarin on body weight, tumour volume and survival time of subcutaneous tumourbearing nude mice
In order to further study about the anti-cancer effect of scutellarin in vivo, nude mice were used to establish a subcutaneous tumour model. And in order to evaluate the success of the nude mouse model, one nude mice were randomly selected from each group for dissection. The results showed that the subcutaneous mass was glioma ( Figure 2B).Thenudemicewithsuccessfultumourmodel

| Effects of scutellarin on U251 cell proliferation
The CCK-8 assay was used to detect the IC50 of scutellarin on U251cells.After48 hoftreatment,theIC50ofscutellarininU251 cellswasfoundtobe338.4 μM( Figure S3A,B)andthatinLN229 cells 297.2 μM ( Figure 1A). Simultaneously, it could be seen that scutellarinhadadose-dependenteffectonU251andLN229cells ( Figure 3C and Figure S1B). In order to study whether the dose of IC50 can cause damage to normal glial cells, CCK-8 was per-formedtodetecttheviabilityofHEBcellsatadoseofIC50.The resultsshowedthatneither297.2nor338.4 μMcouldsignificantly affect the viability of HEB cells ( Figure S1C,D), nor did they af-fecttheproliferationrateofHEBcells( Figure S1E).Aplatecloning assay was performed to study the effect of scutellarin on U251 cell proliferation. As compared with the Control group, the number of U251 cell clones in the scutellarin group was significantly decreased ( Figure 3D,E),whichindicatedthatscutellarincouldin-hibittheU251cellproliferation.

| Identification of differentially expressed genes and scutellarin targets in glioma
The transcriptomics and clinical data of glioma were downloaded from TCGA database. A total of 191 differentially expressed mRNAs were obtained using the edgeR package according to the screening of the dataset (corrected p < 0.05, logFC>6), (Table SI) results showed that all six cases were gliomas ( Figure S3). RT-qPCR wasperformedtodetecttheexpressionofBIRC5andCOL3A1ingliomatissuesandcells.ItwasfoundthatBIRC5hadanabnormallyhigh expression in glioma tissues and cells ( Figure 4D,E),whilenosignificant change was observed in the expression of COL3A1 ( Figure 4F,G).
In addition, the clinical follow-up data of glioma patients from TCGA database was analysed, and it was found that the 5-year survival rate ofpatientswithBIRC5upregulationwassignificantlylowerthanthat of patients with BIRC5 downregulation ( Figure 4H). Among them, BIRC5washighlyexpressedinrenalcellcarcinoma, 20 pancreatic cancer, 21 colorectal cancer 22 and breast cancer 23 and inhibited tumour cellapoptosis.ThemodeofactionbetweenBIRC5andquercetinor luteolin in scutellarin was studied by molecular docking. The results showedthatBIRC5canbindtothetwopharmacologicalcomponents of scutellarin, and the binding mode and binding site between them is shown in Figure 4I,J. In addition, their binding energies were found to all be <−5,suggestingthatthebindingactivitybetweenmoleculesis good. In order to further prove that scutellarin can target and regulate BIRC5, BIRC5 was overexpressed in scutellarin treated glioma cells.
TheresultsshowedthatSCU + OE-BIRC5couldgreatlyreducetheinhibition of cell proliferation induced by scutellarin ( Figure 4K).Atthe same time, it also reversed the apoptosis of glioma cells induced by scutellarin ( Figure 4L).Therefore,BIRC5isareliabletargetofscutellarin. In conclusion, scutellarin may play an anti-glioma role by regulating BIRC5andaffectingapoptosis.

| DISCUSS ION
Glioma accounts for 80% of all primary malignant tumours of the central nervous system. 24,25 At present, the standard treatment for glioma is maximal surgical resection, supplemented with radiotherapyandchemotherapy.However,post-treatmentgliomarecurrence occurs frequently, and the prognosis is unfavourable. 26 Therefore, identifying the mechanism of the occurrence and development of glioma is urgent, in order to explore new therapeutic drugs and methods.
Scutellarin is a flavonoid glycoside compound with a variety of pharmacological activities and has been clinically used to treat various cardiovascular and cerebrovascular diseases. 27 In recent studies, scutellarin has been reported to have marked anti-tumour effects.
Deng et al. 28  dose-dependent manner. The above findings showed that scutellarin can inhibit the proliferation and growth of glioma cells in vivo and in vitro. In order to find out the mechanism of scutellarin against glioma, the possible target of scutellarin was obtained by bioinformatics and network pharmacology methods. Our experimental results indicated that scutellarin may to exert its anti-glioma effect through BIRC5.
BIRC5isanewlydiscoveredmemberoftheIAPfamilyandhas the strongest function among the inhibitors of apoptosis found so far. 14 Apoptosis is a type of programmed cell death, and evading the regulation of the apoptotic cell death mechanism is an important tumour characteristic. 31 A large amount of evidence showed that changes in apoptosis are not only related to the tumour occurrence and development, but also affect tumour resistance to drug therapy. [32][33][34] Of note, it has been reported that glioma cells have blocked apoptosis and uncontrolled proliferation. 35 However,themechanism of tumour cell apoptosis escape is very complex, so understanding it and developing new drugs to improve the treatment of glioma is urgent.Inthepresentstudy,itwasfoundthatBIRC5expressionwas abnormallyincreasedinU251andLN229cells,andscutellarincould significantlyattenuatethesechanges.AscomparedwithHEBnormal glial cells, scutellarin also significantly decreased the expression levelofBIRC5ingliomacells.
In addition, we also studied whether scutellarin inhibits the proliferation of glioma cells through apoptosis, because the inhibition of apoptosis is an important factor in the uncontrolled proliferation of glioma cells. 36 These results showed that scutellarin significantly promoted cell apoptosis, while the proliferation of glioma cells was significantly weakened.
In conclusion, the present study confirmed that scutellarin can inhibit glioma growth, as well as the proliferation and migration of glioma cells. In addition, scutellarin can significantly reduce BIRC5 expression in order to reverse glioma cell apoptosis inhibition and to exert its anti-glioma effect. Therefore, scutellarin may become a new potential targeted drug for the treatment of glioma.

ACK N O WLE D G E M ENTS
Wewouldliketoacknowledgealltheauthorsfortheircontributions made to the manuscript.