Abstract
Glioblastoma multiforme(GBM)was the most common and lethal primary brain tumor in adults.The current gold standard of therapy for GBM consisted of surgical resection when feasible followed by chemo and radiotherapy.Despite combined with them，GBM remained a uniformly lethal disease with a dismal prognosis.Median patient survival continued to be 14 months post-diagnosis.Thus the field was in great need for new and novel tumor-specific therapies.
Uncontrolled proliferation was one of the most important characters of glioblast-oma，inhibition of glioblastoma proliferation can improve the outcome of patients.However，the mechanism of uncontrolled proliferation was still unknown.We detected the expression level of genes in human glioblastoma and human normal brain by using Gene Chip Technology and found that RND3 gene was down regulated significantly in glioblastoma compared to normal brain tissues.The proliferation of glioblastoma was inhibited when RND3 was over expressed in U251 cells which was one of the most popular glioblastoma cells in glioblasotma research.Glioblastoma cells which stable over expressed RND3 and its control were injected into nude mice brain and data showed that the tumors were smaller when RND3 was over expressed.These data indicated that RND3 inhibited glioblastoma cells proliferation and glioblastoma growth both in vitro and in vivo.
RND3 is a member of the Rnd subfamily with unusual properties in that it lacks any detectable GTP ase activity and it does not bind GDP.Therefore，RND3 does not behave like a conventional molecularswitch.Article has reported that RND3 controlled cells proliferation but the mechanism was unclear.In the RND3 gene knock out mouse brain，we found that Notch1 signaling was activated，and most important，we found that RND3 can form complex with Notch1 active form NICD and decreased NICD expression level in the nucleus，indicated that RND3 regulated Notch1 signaling by decreasing NICD expression level in the nucleus.
Notch1 signaling was a critical signaling which controlled glioblastoma proliferatio，however the mechanism that Notch1 signaling controlled proliferation was still unclear.So we hypothesized that RND3 regulated glioblastoma cell proliferation by regulating Notch1 signaling.
In this study，we setted four parts to test our hypothesis:1)the expression of RND3 in human normal brain and glioblastoma samples；2)the effect of RND3 in human glioblastoma proliferation；3)the effect of RND3 in Notch1 signaling in human glioblastoma cells；4)the mechanism of RND3 regulated Notch1 signaling.
2.1 RND3 expression level in human glioblastoma and the relationship with the survival time of glioblastoma patients
Objective
To investigate the expression level of RND3 in human glioblastoma，and detect the prognostic value of RND3 in glioblastoma patients.
Methods
Laz staing was used to detect the expression level of RND3 in the mouse and Realtime PCR was used to detect the expression level of RND3 in mouse brain.Immunofluorescence was detected the location of RND3 in human glial cells，nerve cells and glioblastoma cells.Western blot was used to detect RND3 protein expression level in 15 human glioblastomas and 15 normal brain tissues；Realtime PCR was used todetect mRNA level of RND3 in 13 GBM and 13 normal brain tissues.
RND3 expression level in 49 human gliobalstoma was detected and RND3 expression level on the basis of immunohistochemistry results was quantified and sort from low to high，separated into low group(n＝25)and high group(n＝24).The survival time was the months that patients can survive after surgery and subjected to Kaplan-Meier curve assessment.Data were expressed as means±standard errors of the means.Statistical analysis was performed with SigmaPlot(version 11.0 and spss 13.0).Differences between means were assessed with the student t test or mann-Whitney's test；In multiple comparisons，oneway analysis of variance(ANOVA)was used；Pearson's test was used to detect the correction of two groups and compare quantitative values of expression；Survival curves were plotted by the Kaplan-Meier method and compared by log-rank test.
Results
(1)RND3 was highly expressed in the brain:Laz staing showed that RND3 was highly expressed in mouse brain and Realtime PCR showed that mRNA level of RND3 was high expression in mouse brain.
(2)RND3 was high expression in human glial cells and nerve cells:Immuflurance data showed that RND3 was highly expressed in human glial cells and nerve cells，and RND3 was mainly expressed in the nucleus.
(3)RND3 was mainly expressed in the nucleus:Immustaing result showed that RND3 was mainly expressed in the nucleus in human glioblastoma.
(4)RND3 was down regulated in human glioblastoma tissues compared to normal brain tissues:Immustaing result showed that RND3 expression was lower in the human glioblastoma tissues compared to normal brain tissues，and the expression of RND3.Western blot data showed that RND3 protein expression level was lower in the 4 human glioblastoma tissues compared to its paired normal brain tissues；Realtime PCR data showed that mRNA expression level of RND3 was lower in the 4 human glioblastoma tissues compared to their paired normal brain tissues，there was significant difference between glioblstoma tissues and paired normal brain tissues(P＜0.01)；Western blot data showed that protein expression level of RND3 was lower in 15 human glioblastoma tissues compared to 15 normal brain tissues，there was significant difference between glioblstoma tissues and paired normal brain tissues(P＜0.001).
Realtime PCR result showed that mRNA level of RND3 was lower in 13 human glioblastoma tissues compared to 13 normal brain tissues，there was significant difference between glioblstoma tissues and paired normal brain tissues(P＝0.001＜0.05).
(5)RND3 has a negative relationship with survival time of glioblasotma patients:The survival time of patients，whose tumor has a low RND3 expression level，was shorter than the survival time of patients whose tumor had a high RND3 expression level，there was significant difference between these two groups(P＜0.001).
Conclusion
RND3 was a critical protein in human glioblasotma.
Key words RND3 glioblastoma survival time
2.2 RND3 regulated glioblastoma cells proliferation and tumor growth
Objective
To detect the effect of RND3 in human glioblastoma cell proliferation.
Methods
RND3 gene kouck out mouse was generated，and whether glioblastoma can be found in the mouse brain，the proliferation of thebrain was also detected；The expression level of RND3 on the basis of immunohistochemistry results were quantified and sorted from low to high，separated into low group(n＝10)and high group(n＝10)，the tumor size was quantitatied in the surgery and mann-Whitney's test was used to detect the difference between RND3 low group and RND3 high group；Western blot was used to detect the expression level of RND3 and p-Histone H3 in 30 human samples and Pearson t test was used to detect the correlation of RND3 and p-Histone H3；Western blot was used to detect the expression level of RND3，NICD and p-histone H3 in U87 and U251 cells；U251 glioblastoma cells stably expressing either GFP-RND3(U251-GFP-RND3)or containing vector alone as a control(U251-GFP)，either siRND3(U251-siRND3)or containing vector alone as a control(U251-siCtrl)were generated；a variety of assays(BrdU，groth culve，p-Histone H3 expression level)were used to detect the proliferation of U251-GFP，U251-GFP-RND3，U251-siCtrl and U251-siRND3 cells，differences between means were assessed with the student t test.We also tested the effects of RND3 over expression on the ability of U251 cells to generate solid tumors in vivo.An intracranial glioblastoma model(n＝9 for each group)，which involved the injection of RND3-expressing or control U251 cells into the cerebral cortex of Athymic-nu/nu male mice，was used for this purpose；At 15 weeks after injection，the tumors were harvested and differences between means were assessed with the student t test.
Results
(1)RND3 can regulate brain cell proliferation:No glioblastoma was found in RND3 gene knock out mouse brain，however，we found that the mouse brain was bigger when RND3 gene was knocked out and the proliferation of brain cells was increased.
(2)RND3 has a negative relationship with tumor size of glioblastoma patients and p-Histone H3 expression level:The tumor was smaller in the RND3 high expression group compared to RND3 lowexpression group，there was significant difference between these two groups(P＝0.015＜0.05)；RND3 has a negative relationship with p-Histone H3 expresssion level(R＝-0.4598)and there was significant difference(P＝0.0053＜0.05)
(3)U251 cells were chosen as the mainly cells which was used in this project:Data showed that RND3 can be detected in U251 and U87 cells，and RND3 expression was lower in U251 cells compared to U87 cells，because we mainly focus on the inhibition effect of RND3 in glioblastoma，so we choose U251 cells as the main cells used in the project.
(4)All the RND3-positive clones expressed RND3 at a significantly higher or lower level than vector control or parental cells:All the cells have even green fluorescen when spot by the laser at 488；RND3 was over expressed in GFP-RND3 group compared to GFP group and there was significant difference between GFP group and GFP-RND3 group(P＜0.001)；RND3 was down regulated in siRND3 group compard to siCtrl group and there was significant difference between siCtrl group and si-RND3 group(P＜0.001).
(5)RND3 can negatively regulate glioblastoma proliferation in vitro:Cellgrowth culve showed that the cell number was smaller in GFPRND3 group，and there was siginificant difference compared to GFP group(P＜0.001)；The cell number was bigger in siRND3 group and there was siginificant difference compared to siCtrl group(P＜0.001).BrdU positive cells decreased when RND3 was up-regulated(GFPRND3)and there was siginificant difference compared its control group(GFP)(P＜0.001).BrdU positive cells inceased when RND3 was downregulated(siRND3)and there was siginificant difference compared to its control goup(siCtrl)(P＜0.001).The expression of p-Histone H3 decreased when RND3 was up-regulated(GFP-RND3)；p-Hisotne H3 expression level inceased when RND3 was down-regulated(siRND3).
(6)RND3 can inhibit glioblastoma growth in vivo:Glioblastomacan be seen in 9 nude mice in GFP group(n＝9)，however，there was only 1 glioblastoma can be seen in a nude mouse in GFP-RND3 group(n＝9).There was significant difference between GFP group and GFPRND3 group(P＜0.001).
Conculsion
RND3 negatively regulated glioblastoma cell proliferation.(https://www.daowen.com)
Key Words RND3 Prolifertion BrdU p-Histone H3
2.3 RND3 regulated GBM proliferation by negatively regulating Notch1 signaling
Objective
To detect the mechanism of RND3 regulating GBM proliferation.
Methods
Microarray was used to detect all the genes expression level in RND3 gene knockout mouse and its control.Western blot and Realtime PCR were used to detect Hes1 mRNA and protein level in the brain of RND3 gene knock out mouse and its control，and student t test was used to test the difference between these two groups.Realtime PCR was used to detect HES1，HEY1 and HES5 mRNA expression level when RND3 was up or down regulated in human glioblasotma cells.Western blot was used to detect HES1 protein expression level when RND3 was up or down regulated in human glioblasotma cells，student t test was used to detect the difference between up or down regulated RND3 group and their controls.Immustaing was used to detect the expression of Notch1 in human samples.Realtime PCR was used to detect the mRNA level of Notch1 target gene，HES1 and HES5，in human samples.Western blot was used to detect the protein level of Notch1，HES1 and HES5 in human samples.Flag-NICD or siNotch1 was transfected into U251 cells to active or inhibit Notch1 signaling and Compound E was a drug used to inhibited Notch1 signaling，p-Hisotne H3 expression level，cell numberand BrdU positive cells were detected when Flag-NICD，siNotch1 or Compound E was given to the cells.In rescue experiments，Flag-NICD was used to active Notch1 signaling after RND3 was transfected to U251 cells，cells number and BrdU positive cells were detected，Stastics analysis was performed with one way anova compared to the Ctrl group.In other hands，cells were given DMSO or Compound E，and named DMSO and Compound E group，then each group was given siCtrl and siRND3 sepratedly，cell number and BrdU were used to detect the proliferation of cells，P value was for comparison to control group one way anova；cells were given siCtrl or siNotch1，and named siCtrl and siNotch1 group，then each group was given siCtrl and siRND3 sepratedly，cell number was detected，P value was for comparison to control group(siCtrl)and from one way anova.
Results
(1)RND3 can regulate Notch1 signaling in the brain:Microarray data showed that Notch1 signaling target gene expression level increased in the mouse brain when RND3 was knocked out.Western blot and Realtime PCR data showed that HES1，the target gene of Notch1 signaling，expresson level increased both in protein level and mRNA level when RND3 was knocked out and there was significant difference compared to control group(P＜0.05).
(2)RND3 can negatively regulate Notch1 signaling in glioblastoma:HES1 mRNA level increased when RND3 was knocked down and there was significant difference compared to control group(P＝0.0077＜0.05)；HES1 mRNA level decreased when RND3 was over expressed and there was significant difference compared to control group(P＝0.0077＜0.05).HES1 protein level increased when RND3 was knocked down and HES1 protein level decreased when RND3 was over expressed.Endogenous-induced transcription of the reporter was inhibited when RND3 was over expressed in U251 cells and there was significant difference compared to control group(P＝0.0001＜0.05)；Endogenousinduced transcription of the reporter was actived when RND3 was down expressed in U251 cells and there was significant difference compared to control group(P＝0.0002＜0.05).We got the similar result in U87 and 293T cells.
(3)Regulating Notch1 signaling can regulate glioblastoma proliferation:Immustaing result showed that RND3 higher expression in human glioblastoma compared to normal brain tissues；Western blot showed that the expression of Notch1 and HES5 was higher in human glioblastoma，and there was significant difference compared to normal brain tissues.The cell number increased when Notch1 signaling was actived by Flag-NICD，and there was significant difference compared to its control(P＜0.05)；Cell number decreased when Notch1 signaling was inhibited by compounE or siNotch1，and there was significant difference compared to their control groups(P＜0.05).BrdU positive cells increased when Notch1 signaling was actived by Flag-NICD and there was significant difference compared to their control groups(P＜0.05)；BrdU positive cells decreased when Notch1 signaling was blocked by either Compound E or siNotch1 and there was significant difference compared to their control groups(P＜0.05).
(4)RND3 regulated proliferation by negatively regulating Notch1 signaling:The cell number decreased when RND3 was over expressed and there was significant difference between GFP group and GFP-RND3 group(P＜0.001)，however，when we transfected Flg-NICD to active Noth1 signaling，the cell number of GFP-RND＋Flag-NICD group was increased and there was no significant difference compared to GFP group(P＝0.292＞0.05).The proliferation index was smaller when Notch1 signaling was blocked by siNotch1 or Compound E，and there was significant difference compared to their control group(P＜0.001).
Conclusion
RND3 negatively regulated GBM proliferation by negatively regulating Notch1 signaling.
Key Words RND3 Notch1 proliferation glioblastoma
2.4 RND3 regulated Notch1 signaling by decreasing NICD expression in glioblastoma
Objective
To detecte the mechanism of RND3 regulated Notch1 signaling.
Methods
Immunofluorescence was used to detect the location of RND3，Notch1，CSL and MAML 1 in glioblastoma cells.Realtime PCP was used to detect the Notch1，CSL，MAML 1 expression level when RND3 was over expressed.Western blot was used to detect the expression level of NICD，CSL，MAML 1 in the U251 cells when RND3 was over expressed and co-ip was used to detect whether RND3 form complex with NICD，CSL and MAML 1.Chip assay was used to check whether RND3 over expression can decrease NICD bind to HES1 promoter.In rescue experiments，Flag-NICD was transfectd to U251 cells which over expressed RND3，Realtime PCR was used to detect HES1 mRNA level，Stastics analysis was performed with one way anova compared to the Ctrl group.In other hands，cells were given DMSO or Compound E，and named DMSO and Compound E group，then each group was given siCtrl and siRND3 sepratedly，Realtime PCR was used to detect HES1 mRNA level，Stastics analysis was performed with one way anova compared to the Ctrl group.immustaing and western blot was used to detect the expression level of RND3 and Notch1 in human sample and Stastics analysis was performed with Pearson test.MG-132was used to block ubquitin and check whether RND3 decreased NICD expression level by ubquitin.
Results
(1)RND3 and NICD protein was mainly expressed in the nucleus:Immunofluorescence result showed that RND3 and Notch1 was mainlyexpressed in the nucleus both in glioblastoma cells and glioblasotma tissues.
(2)RND3 can form complex with NICD:Data showed that we can detect Myc-RND3 when we pull the protein by using Flag-NCD and we also can detecte Flag-NICD when we pull the protein by using Myc-RND3.
(3)RND3 over expression decreased NICD protein expression level:Western blot data showed that NICD expression level decreased in the nucleus when RND3 was over expressed in U251 cells.Realtime PCR data showed that there was no significant difference of Notch1 mRNA level in GFP-RND3 gourp and its control.
(4)RND3 and CSL protein was mainly expressed in the nucleus:Immunofluorescence result showed that RND3 and CSL were mainly expressed in the nucleus in glioblastoma cells.
(5)RND3 can form complex with CSL:Data showed that we can detect Myc-CSL when we pull the protein by using GFP-RND3 and we also can detecte GFP-RND3 when we pull the protein by using Myc-CSL.
(6)RND3 over expression decreased CSL protein expression level:Western blot data showed that CSL expression level decreased in the nucleus when RND3 was over expressed in U251 cells.Realtime PCR data showed that there was no significant difference of CSL mRNA level in GFP-RND3 gourp and its control.
(7)RND3 and MAML 1 protein was mainly expressed in the nucleus:Immunofluorescence result showed that RND3 and MAML 1 were mainly expressed in the nucleus in glioblastoma cells.
(8)RND3 can form complex with MAML 1:Data showed that we can detect Flag-MAML 1 when we pull the protein by using GFP-RND3 and we also can detecte GFP-RND3 when we pull the protein by using Flag-MAML 1.
(9)RND3 over expression decreased MAML 1 expression level:Western blot data showed that MAML 1 expression level decreased in the nucleus when RND3 was over expressed in U251 cells.Realtime PCR data showed that there was no significant difference of MAML 1 mRNA level in GFP-RND3 gourp and its control.
(10)RND3 regulated NICD was critical for RND3 regulating Notch1 signaling:Chip result showed that over expressed RND3 can decreased NICD which bind to the promoter of HES1，and Flag-NICD abolished the effect of RND3 in the Notch 1 signaling；when blocked Notch1 signaling，the effect of RND3 lost.
(11)MG-132 abolish the effect of RND3 in NICD:The expression of NICD decreased when the expression of RND3 increased，the expression of NICD didn't decrease when MG-132 and RND3 were both given to the cells.
Conclusions
RND3 regulated Notch1 signaling by decreasing NICD-CSL-MAML 1 complex expression level，RND3 formed complex with NICD and decreased NICD expression level was critical for RND3 regulting NICDCSL-MAML 1 complex.
Key words RND3 NICD CSL Maml1 HES1