Molecular Mechanism Of Ars2-regulated Apoptosis In Glioma Cells Via Microrna-6798-3p

Malignant glioma is one of the most common and deadly brain tumor,which is often associated with high morbidity and high mortality.Glioblastomas are the most common and primary of malignant gliomas.In the last decade,few effective therapeutics for glioma have been generated.With the treatment of aggressive surgical,chemotherapies,and radiation,patients' life expectancy is on average 14 months after diagnosis.Genetic alterations of gliomas affect genes that regulate cell proliferation,apoptosis,angiogenesis,migration,and invasion.However,the molecular mechanism of genetic alteration in regulating cell proliferation and apoptosis are poorly understood.Therefore,Comprehensive elucidation of genetic alterations in glioma may provide novel targets for diagnostic,prognostic,even therapeutic purposes of glioma.Ars2(Arsenic-resistance protein 2)is a nuclear protein which is encoded by human SRRT gene.c DNA of Ars2 were firstly cloned from a hamster cell line,studies suggested that Ars2 was a protein that could confer sodium arsenite resistance to cancer cells.Recently Ars2 has been reported as a factor in the RNA silencing mechanism,which is related to the antiviral defense flies,and is related to cell proliferation in mammals.In mammals,a truncated version of Ars2 was associated with arsenic resistance.However,its full-length protein regulates cell proliferation possibly via the effect on RNA metabolism.Ars2 is also considered as a new component of the nuclear CBC(nuclear cap-binding complex),which is important for the biogenesis of mi R-155,mi R-21 and let-7 and critical for cellular proliferation.In other words,the knockdown of Ars2 could effectively down-regulate the transcription of some micro RNAs.Recently,Ars2 was found to be highly expressed in human hepatocellular carcinoma(HCC)andcholangiocarcinoma,and studies have suggested that Ars2 can be used as a diagnostic and prognostic indicator,or even a target for therapy.However,little is known about the function of Ars2 on these processes in glioma.In this study,we found that Ars2 played an important role in human glioma cells.Knockdown of Ars2 inhibited tumorigenicity and induced apoptosis in glioma cells.Functional studies revealed that the knockdown of Ars2 induced the downregulation of mico RNA-6798-3p(mi RNA-6798-3p),which in turn leaded to the up-regulation of p53 and p21,and ultimately leaded to cell apoptosis.These findings suggest that Ars2 could be a new therapeutic target for glioma.The main results obtained in this study are listed as follows:1.Overexpression of Ars2 Is Associated with Poor Prognosis in Gliaoma PatientsTo determine whether Ars2 can be a prognostic indicator for the poor survival glioma patients,Kaplan-Meier analysis was used to analyze the progression-free survival for the Frence database.The results showed that the 3-year survival rate of patients with high expression of Ars2(78 cases)was 10%,while the 3-year survival rate of patients with low expression of Ars2(195 cases)was 39%.The 5-year survival rate of patients with high expression of Ars2 was 6%,and the 5-year survival rate of patients with low expression of Ars2 was 28%.We also found that Ars2 is highly expressed in glioma cell lines and low expressed in the normal human astrocytes(HA).That is to say Ars2 was widly expressed in glioma cells.These results indicated that overexpression of Ars2 was associated with poor prognosis in glioma patients.And we speculate that Ars2 may be a new targer for therapeutic purpose of glioma patients.2.Ars2 Regulates Apoptosis Through p53/p21 Dependent Pathway in Glioma CellsTo further investigate how Ars2 functions in regulating glioma cell apoptosis in detail,flow cytometry assay and western blot assay was applied to determine the effects of Ars2 knockdown on apoptosis in glioma cell lines U87 and LN229.Flow cytometry assay showed that Ars2 konckdown induced apoptosis in both U87 and LN229 cells.Western blot assay revealed that Ars2 knockdown leaded to marked increase in protein levels of p53 and its downstream p21.In addition,caspase inhibitor(z-VAD-fmk)was used to further investigate the apoptosis induced by Ars2 knockdown.Results showed that the apoptosis induced by Ars2 depletion was abrogated by z-VAD-fmk in U87 and LN229 cells.To further confirm that the effect of Ars2 konckdown on cell apoptosis is not an off-target,we overexpressed Ars2 in Ars2 knockdown cells.Flow cytometryassay and western blot assay showed that overexpression Ars2 in Ars2 depletion cells essentially abrogated cell apoptosis in glioma cells.To further confirm whether p53/p21 pathway is involved in the apoptosis induced by Ars2 knockdwon,si RNAs with p53 and p21 were employed.The results suggest that knockdown of Ars2 induces cell apoptosis through p53/p21 pathway in glioma cells.3.Ars2 Regulates Apoptpsis in Glioma Cell Through Regulating mi RNA-6798-3pSince Ars2 plays an important role in the biogenesis of micro RNA(mi RNA),the high-throughput screening of all genomic mi RNAs using Ribo Arraymi DETECT Micro RNA Assay was employed to analysis the expression of mi RNAs in U87 cells transfected with vector control sh RNA(sh Con)or sh Ars2-1#.Mciro RNAs whose expression levels was significantly down-regulated at least 2-fold were selected.These25 mi RNAs were considered as potential candidates in sh Ars2 cell compared to sh Con cells.We determined that mi RNA-6798-3p was the significantly down-regulated mi RNA in U87 Ars2 knockdown cells by q RT-PCR analysis.And this was also confirmed in another glioma cell line LN229 cells.In order to further verify whether mi RNA-6798-3p was involved in the cell apoptosis induced by Ars2 depletion,mi RNA-6798 mimic was employed.Flow cytometry assay showed that cotransfection with mi RNA-6798-3p mimic markedly abrogated Ars2 knockdown-mediated apoptosis in U87 and LN229.And werstern blot assay showed that cotransfection with mi RNA-6798-3p mimic attenuated C-Caspase 3,p53,and p21 expression caused by Ars2 knockdown.These results suggested that Ars2 regulates glioma cell apoptosis through regulating mi RNA-6798-3p.4.Ars2 is Required for Tumorigenesis of Glioblasoma Cells in vivoTo further evaluate whether depletion Ars2 inhibit tumor growth in vivo,orthotopic glioma xenograft model was employed in this study.Kaplan-Meier survival analysis showed that knockdown of Ars2 with sh Ars2-1# significantly prolonged the survival time of NOD/SCID mice compared with that of vector control sh RNA(82.2 � 16.5 days versus 42.2 � 3.9 days).We also found that tumor volumes in mice bearing syngeneic U87 cells infected with vector control sh RNA displayed large tumors extending throughout the whole right hemisphere,whereas NOD/SCID mice bearing sh Ars2-1#cells displayed almost invisible tumors.To evaluate the morphological changes in tumor section of NOD/SCID mice bearing syngeneic U87 cells after Ars2 depletion,H&E staining was used.Histological examination revealed that brain sections from vectorcontrol sh RNA cells displayed a large number of tumor cells.Brain sections from NOD/SCID mice bearing sh Ars2-1# cells displayed fewer tumor cells.Immunohistochemical(IHC)staining was used to stain the brain of mice bearing sh Con and sh Ars2-1#.We found that the expression of Ki-67 was increased in the brain of mice bearing sh Con cells,while the expression of Ki-67 was not observed in the brain of mice bearing sh Ars2-1# cells.In order to further determine whether the knockdown of Ars2 was successful in the orthotopic glioma xenograft model,IHC staining was also employed.Ars2 positive cells were markedly decreased in brain section after depletion of Ars2.These results indicated that Ars2 play an important role in tumorigenicity.In addition,we also found that Ophiopogonin D(OP-D)can be a novel autophagy inhibitor in breast cancer cells.The main results obtained in this study are listed as follows:5.OP-D Induces Inhibition of Autophagic Degradation in Breast Cancer CellsOP-D,a steroidal glycoside,extract from Ophiopogon japonicas.It was reported that,OP-D can modulate multiple oncogenic signaling pathways to inhibite cells proliferation in lung cancer cells.And we found that OP-D can induce the inhibition of autophagic degradation in breast cancer cells.Treating breast cancer cells with OP-D leads to an increase in EGFP-LC3 puncta fornation in MCF-7 and MDA-MB-231 cells.OP-D treatment also results in an increase of SQSTM1,LC3B-? and LAMP1.However,the protein level of BECN1 remianed unchanged.Co-treated with or withour Bafilomycin A1(Baf)and Rapamycin(Rapa)shows that the function of OP-D is similar to Baf.That is to say,OP-D induces the inhibition of autophagic degradation in breast cancer cells.6.OP-D Inhibits Autophagic Degradation through Inhibiting the Acetylation of?-TubulinAccording to the results of EGFP-LC3 transfection,we can easily found that after OP-D treated,the EGFP-LC3 puncta almost distribute the whole cell.It is reported that the puncta movment depened on the Tubulin.And we also found that the acetylation of?-Tubulin was disturbed by using western blot.That is to say,OP-D inhibites the autophagic degradation in breast cancer cells by inhibiting the acetylation of ?-Tubulin.