Hypomethylation Of Wnt5a Promoterincreased Invasion Ability Of Hypotaurine Synthesis Deficient Glioma Cells

Objectives: Glioma is one of the most common central nerve system malignancies.Over ? of the intracranial tumors are gliomas.The higher the glioma grades,the worse the patient's prognosis.Take the glioblastoma as an example,the median survival time was only about 15 months even with the intervention of post-operation chemotherapy.Scarcely can we find the metastasis of glioma.The lethal nature of glioma is its invasive growth.The higher the glioma grade,the stronger the invasion ability.Many studies indicated that Wnt signal pathway had close relationship with glioma invasion especially Wnt5 a.The exact functions of Wnt5 a in tumor invasion are still elusive.It was well acknowledged that the promoter region epigenetic modification affected the expression of Wnt5 a.Recently,hypotaurine was found to contribute to glioma invasion.Hypotaurine could also inhibit histone demethylase and play roles in regulating epigenetic modification.This study attempted to elucidate the relationship between intracellular hypotaurine and Wnt5 a expression in human glioma cells.The aim of this study was to test the hypothesis that hypotaurine could increase the promoter region methylation status of Wnt5 a and inhibite the expression of Wnt5 a,resulting in increasing glioma's invasion ability indirectly.Methods: 1.Cell line: U251 cells were adopted in this study.The cells were subjected to 2-aminoethanethiol dioxygenase gene knock down through RNA interference.Simultaneously,the mock control cells were also constructed using the same strategy.The two cells carried puromycin resistance and were kept in nitrogen.The ADO gene knockdown efficiency was confirmed through real-time polymerase chain reaction(RT-PCR)technique.2.Media: The cells were cultured in DMEM high glucose media.Culture temperature and carbon dioxide concentration were 37°C and 5% individually.Except otherwise stated,extra added components were based on study aims.0.5?g/ml puromycin was necessary for keeping resistance.3.Hypotaurine quantification: The cells were culture in 10 cm dishes.When the cells reached to 70-80%confluence,cool PBS was used to rinse the cells in triplicates.The rinsing volume was 10 ml for each time.To get rid of the residual water,centrifugation with 3000 rpm was applied for 1min.1ml 21:79 methanol/water(containg 1.5?M L-cystine(13C6,99%;15N2)internal standard)was added to each plate.The cells were collected by scraping and transferred to 2ml cool chloroform in a15 ml centrifugation tube.The tube was sonicated for three times with power of 50 w and each time lasted for 15 s.After soaked in ice bath 30 min,the tube was centrifuged at 13000×g for 20 min.A 700?l supernatant was allocated to 1.5ml tube and subjected to lyophilized at-50? under vacuum condition or stored at-80? for instant use.The protein layer was collected to dry naturally.The protein dry weight was used to calibrate cell numbers.For intracellular hypotaurine quantitation,the dried sample was dissolved in 80?l 21:79 methanol/water.Subsequently,20?l Acc QTag Ultra Derivatization reagent was added and incubated at 55? for10 min.Liquid chromatography separation was finished by Angilent 1290 Infinity system using Agilent C18 column(2.1mm×100mm,1.8?m).The mobile phase A was 0.5% formic acid solution with 5m M ammonia formate.Phase B was pure acetonitrile.The elution grades were 0-1.0min,5% B;1.0-20 min,5%-25% B;20.0-25.0,25%-57%B;25.1-27.0 min,99% B;27.1-30 min,5%B.The flow rate was0.25 m L/min.The injection volume was 1?L.The column temperature was 50?.Quantitation of hypotaurine was achieved by Agilent 6460 Triple Quad LC/MS system.A multiple reaction monitor mode was employed and the collision energy was 120 V with monitor ion pairs of 283?171.4.Real time PCR: The cells were cultured in 10 cm dishes.When the cells reached to 70-80% confluence,the culture media were discarded.The plate was rinsed with cool PBS in triplicates.Commercial acquired total Ribonucleic Acid(RNA)extract kit was used for RNA extraction.The operation was in accordance with the instructions provided by the manufacturer.The integrity of the isolated RNA was evaluated by specific equipment and that OD260/280 was in the range of 1.8-2.0 was thought to be qualified.Reverse transcript kit was used for reverse transcription to synthesizecomplementary deoxyribonucleic acid.Realtime PCR was carried out using MX 3000 p real time PCR system.The reaction was based on SYBR insersion method.Every cells performed at least using 3 biological replicates.Housekeeping gene a-actin was used as calibrator.The difference was calculated by 2^(-??Ct)algorithm.5.Western blot(WB):Cells were harvested and treated by lysis solution to release proteins.After the proteins were quantified,sodium dodecyl sulfate polyacrylamide gel electrophoresiswas performed to separate the proteins.The gel containing the proteins was subjected to transmembrane using semi-dry method.After transmembrane,the PVDFcontaing target proteins was cut.The membrane was subjected to rinsing,blocking and incubating with 1st and 2nd antibodies.Electro-Chemi-Luminescence imaging technique was employed to take the photos.6.RNA sequencing analysis: The cells were cultured in 10 cm dishes.When the cells reached to 70-80% confluence,the culture media were discarded.The plate was rinsed with cool PBS in triplicates.Commercial acquired total Ribonucleic Acid(RNA)extract kit was used for RNA extraction.The operation was in accordance with the instructions provided by the manufacturer.The integrity of the isolated RNA was evaluated by specific equipment and that OD260/280 was in the range of 1.8-2.0 was thought to be qualified.RNA sequencing analysis was conducted by Novogene.7.Invasion ability analysis: QCM? 24-Well Cell Invasion Assay kit was used for invasion analysis.After the cells reached to Logarithmic phase,they were subjected to trypsinization and counting.The cell density was modified to 104-5/m L.the cellswere incubated in the up-wells with each well containing 2ml cell solutions.The stimulator regents were added in the corresponding lower-wells e.g.the hypotaurine.Culture temperature and carbon dioxide concentration were 37°C and 5% individually.The more the cells' invasion ability,the more the fluorescent intensities.Results: 1.After cell thawing,the shape and growth of all the cells were acceptable.The ?ADO cells expressed lower level of ADO gene than that of the control Vct cells.LC/MS analysis indicated that ADO gene silence resulted in the less synthesis of intracellular hypotaurine.Thus,the genetical manipulation realized the aim of impairing cell hypotaurine synthesis.2.RNA-seq analysis indicated that 96 gene were upregulated and 218 gene were downregulated in the ?ADO cells.Additional bioinformatics analysis exhibited that the most perturbated metabolic pathway due to ADO gene silence was ECM-receptor pathway.This pathway had close relationship with cell invasion in different types of tumor.3.In the ECM-receptor pathway,Wnt5 a gene expression was significantly elevated.4.In vitro cell analysis indicated that hypotaurine could inhibit the expression of Wnt5 a.Through methylation specific PCR analysis,the lower expression of Wnt5 a was due to the promoter region hyper-methylation status.5.Higher Wnt5 a expression contributed to compromised cell invasion ability in vitro.Conclusions: Hypotaurine could inhibit the expression of Wnt5 a,resulting in impaired invasion ability.This was caused by hypotaurine.Because,hypotaurine could inhibit histone demethylases.The inhibition resulted in hyper methylation of Wnt5 a promotor region.This study indicated that Wnt5 a and hypotaurine metabolism could be potential targets for glioma treatment.