| Malignant glioma has the highest morbidity among primary malignant tumors in the central nervous system.The characters of glioma can be listed as follows: fast cell proliferation,intensive invasion and vulnerability of recurrence.The survival of glioma patients has improved at variable degree with the novel approaches to surgery,radiotherapy and chemotherapy.However,despite several therapeutic approaches,gliomas prognosis remains poor.Therefore,it becomes urgent to find new therapy for glioma.In recent years,with the increasing study of glioma at molecular level,searching for glioma related genes and treating glioma at genetic level are becoming hot new focuses.Micro RNAs(mi RNAs)are non-coding RNAs which are about 22 nucletides in length.Mi RNAs can regulate gene expression by binding to 3’-untranslated regions(UTRs)of target gene m RNAs.Till now,many mi RNAs has been found playing important roles in the development of glioma.According to the role of mi RNAs in gliomas,mi RNAs are divided into oncogenetic mi RNAs and tumor-suppressor mi RNAs.Our previous study focused on the mi RNAs profiles of 8 glioma samples and their adjacent brain tissues.We found that 44 mi RNAs expressed lower in gliomas than non-tumor brain tissues.Among the 44 mi RNAs mi R-758-3p expressed obviously lower than other mi RNAs.So,we choose mi R-758-3p for the further study.By searching mi RNA target prediction database(TARGETSCAN,MICRORNA.ORG,MIRDB),we found that WHSC1 has a potential binding site with mi R-758-3p.In this study we focused on the expression level of mi R-758-3p in gliomas.By transfection of mi R-758-3p mimics we studied the effect of mi R-758-3p on the progression of glioma and its’ potential mechanism.Our study was divided into three parts.In the first part of our study,we detected the expression level of mi R-758-3p in6 glioma tissues and 6 non-tumor brain tissues by using real time PCR.The result show that mi R-758-3p expressed lower in glioma tissues than non-tumor brain tissues,which is consistent with mi RNA profiles.Then we detected the expression level of5 glioma cell lines in order to choose the right cell lines as researching subjects.Wefound that the expression of mi R-758-3p are lower in U87 and LN229 cell lines than in other three cell lines.So we choose U87 and LN229 as research subjects.In the second part of our study,we transfected U87 and LN229 with mi R-758-3p mimics,then we detected the transfection rates of mi-758-3p by using real time PCR and Flow cytometry.MTT method was purchased to evaluate the cell proliferation rate of glima cells after transfection;Flow cytometry was used to analysis the cell cycle and apoptosis rate after transfection;Transwell analysis was purchased to detect the migration and invasion ability of glioma cells;we build U87 nude mouse tumor xenograft model.The nude mice were divided into two groups with five mice in each treatment group.The xenograft models were injected in multisite injection manner.The tumor volume was conducted every 3 days.The results show that the mi R-758-3p group cells growth slower than the NC group(P<0.05).The apoptosis cells rate is higher than NC group(P<0.05).More cells were in habited at G0/G1 phase(P<0.05).The invasion ability is weaker than NC group(P<0.05).The tumors of mi R-758-3p group growth slower than NC group obviously.In the third part of our study,we searched for the potential target of mi R-758-3p by using TARGETSCAN,MICRORNA.ORG,MIRDB database.Real time PCR and Western blot were used for detecting the expression of the target gene at m RNA and protein level after transfection with mi R-758-3p mimics.Western blot was used to evaluate the expression of proteins,which are related to cell cycle,apoptosis and invasion.The results show that WHSC1 is one interesting target of mi R-758-3p.The m RNA expression of WHSC1 did not changed,while the protein expression of WHSC1 changed significantly.The results of luciferase assay show that mi R-758-3p inhabited the luciferase activity of the plasmid containing 3’UTR of WHSC1.At the same time the protein expression of Cyclin D1,MMP2,Bcl-2 are lower than NC group,while the protein expression of Bax is higher than NC group(P<0.05)Conclusion: mi R-758-3p expressed lower in glioma tissues than non-tumor brain tissues.Mi R-758-3p may inhabit cell growth,increase cell apoptosis,arrest cell cycle progression,reduce cell invasion ability by regulating the expression of WHSC1.Further more mi R-758-3p may influence the expression of many proteins which are related with cell cycle progression,apoptosis and invasion.The vivo experimentfurther demonstrated that mi R-758-3p may inhabit glioma growth. |
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