The Therapeutic Effect Of Cell-penetrating PR Peptide To High-grade Meningiomas

PartⅠ、Purpose：RIZ1gene is located on human chromosome1p36region (1). RIZ gene encodes twoproteins, RIZ1and RIZ2. RIZ1protein contains the PR domain in the amino-terminalcompared to RIZ2.RIZ1possesses histone methyltransferase activity, which can promote lysinemethylation on histone H3(2-4). When RIZ1is upregulated in breast cancer, colorectalcancer and liver cancer cell lines, tumor growth significantly slowed down (4-8). In theprevious experiments, we used tissue microarray to detect the expression RIZ1inmeningiomas, and found that RIZ1expression was rarely found in malignant meningiomas.Furthermore, when malignant meningioma IOMM-LEE cell lines overexpress RIZ1, cellsgrowth was significantly inhibited in vitro, and the cell cycle arrest emerged in G2/Mphase. In malignant meningioma cells, RIZ1protein expression was negatively associatedwith c-Myc. Overexpression of RIZ1in meningioma cell lines can decrease the expressionof c-Myc. The results of the study have been published in "Oncogene"(9).Based on the findings above, RIZ1expresses of two proteins, RIZ1and RIZ2. Theonly difference between RIZ1and RIZ2is that RIZ1contains200more amino acids in theamino-terminal (ie, PR-domain), and the rest of the sequence is the same. RIZ1hasinhibition to malignant meningioma. Is PR domain alone able to produce this tumorinhibition? If it does work, is the effect the same to the different grades of meningiomacells? What are the possible mechanisms? In this experiment, we conducted a study onthese questions. PartⅡ、methodsFirst, we used different grades of meningioma resection specimens for culturingprimary cells, and performed primary cells Identification and detection of RIZ1expression.Then we use the HIV-TAT protein transduction system which can transduce protein into thetarget cells and maintain the biological function of the connection protein, and TAT proteinitself has no toxicity to cells. After the TAT was transduced into after meningioma primarycells, we examined influence to meningiomas primary cells proliferation and apoptosis indifferent dosing concentration and time. In the study of tumor suppressor mechanism, wefirst noted that RIZ1is a member of methyl transferase enzyme family8(KMT8), whichcan regulate the expression of related downstream molecules through methylation of theninth lysine on histone H3. In other tumors,1st/2nd/3rd lysine methylation can beobserved. We explore the molecule mechanisms of tumor suppressor activity of TAT-PRprotein through microarray and methylation target detection. Finally, we examined whetherexpression of RIZ1is really different in meningioma resection specimens byimmunohistochemistry.PartⅢ、resultIn primary meningioma cells, it was found that RIZ1expression decreased while thegrade of meningioma increases. Cancer gene c-Myc expression elevated with the grade ofmeningiomas increased. TAT-PR domain inhibited the cell proliferation of high-gradeprimary meningiomas and induced apoptosis of primary high-grade meningioma cells. Innude mice experiments, tumor growth was inhibited in the protein treatment group,compared with the control group. In this experiment, we observed in the treated group ofWHO III grade primary meningioma cells that:1,1st and2nd methylation of H3K9,2,c-Myc expression level decreased. By the Gene expression microarray, we plotted thewhole gene expression patterns changing with time by protein dispose and found that theexpression of cell cycle-related and tumor proliferation associated molecules were changed.In meningioma resection specimens, although specimens are few, but the trend can still beobserved that RIZ1expression declined with the grade of meningioma elevated.PartⅣ、conclusionTAT-PR domain protein induced proliferation and apoptosis of high-grade primary meningioma cells, and the tumor inhibitory effect have been validated in vivo experiments.This tumor inhibition may be established on the basis of low RIZ1expression at high gradeof meningiomas. This tumor suppressing activity may be achieved by methylation ofPR-domain towards downstream apoptosis-related molecules, such as c-Myc.The experiment can be divided into eight parts: Part I: meningioma primary cellculture. Part II: Expression of RIZ1at different grades of meningioma primary cells. PartIII: Construction of PR domain that can be transduced into cells. Part IV: PR domain’simpact on proliferation and apoptosis of primary meningioma cells. Part V: Nude mousetumorigenicity assay. Part VI: PR domain affect proliferation and apoptosis of primarymeningioma cell by H3K9methylation pathways. Part VII: whole genome expressionprofile chip investigate the downstream molecules of PR domain. Part VIII: meningiomaresection specimens of different grades for verification RIZ1expression.PartⅠ、Primary meningioma cells culture1、Purpose：we aim to culture different grades of primary meningioma cells,providing support for the following experiments.2、Methods：After surgery, specimens were taken from the part with completestructural and less necrosis, then use serum-free DMEM in the ice box for rapidlytransporting them within30minutes. We use serum-free wash buffer PBS to clean thesurface of the tumor tissue and blood impurities. Under the microscope, with the assistanceof microsurgical instruments, we removed necrotic tissue of the tumor, then placed theminto a small amount of PBS within3.5cm dish on ice and cut them into pieces about1*1mm, and the tumor tissues were added considerable trypsin, DNases, then the tissues wereplaced in37°C incubator being digested for30minutes. After digestion,2volumes of the10%FBS serum media were used to neutralize trypsin, then40microns with70micronscell strainers were used to filtered, and the obtained cells were centrifuged for5minutes at900rpm/min, resuspended, counted using a cell counter,5*106/ml in a cellconcentration of species were cultured in10cm dishes at37°C,5%CO2concentration.The culture medium was changed every2days. When the cells reached80%confluency,0.125%trypsin were used to digest, then the cells were cultured in new dishes.3、Result：After specimen collection, primary cell culture, we get four cases ofmeningioma primary cells, according to2007WHO grading standards, one grade I case, two grade II cases, one grade III case. All of the cases of primary cells were grown well,and the cells are mostly bipolar or multi-morphology of endothelial cell morphology.Multi-benign meningiomas were passaged to the10th generation, and the cell volumegradually increased, with proliferation significantly slowing down. But the malignantmeningioma cells show opposite reaction. We took3-5th generation of primary cell forsubsequent experiments.4、Conclution：After primary culture, we got four cases of meningioma primary cells,one grade I case, two grade II cases, one grade III case. PartⅡ、the expression levels of RIZ1in different primarymeningioma cells1、Purpose：to detect whether the expressions of RIZ1in the four cases ofmeningioma primary cells are different.2、Methods：First, in order to determine the localization of RIZ1in cells, and to verifythe specificity of the RIZ1antibody, we conducted RIZ1antibody identification. We usedRIZ1full-length cDNA plasmid Tools to transfect293T cells, so RIZ1transient expressionincreased, then we conducted RIZ1immunofluorescence experiment within their specificcellular localization. The4cases of meningioma primary cells were culture to the thirdgeneration. By using two methods, namely, immunofluorescence and immunoblotting, wedetected expression of RIZ1and c-Myc in four cases of primary cells.3、Result：During RIZ1plasmid transfection experiments, we found that RIZ1localizes in the nucleus. Results detected by immunofluorescence and Western blot showedthat with levels of meningioma elevated, expression of RIZ1decreased, but c-Mycexpression level rised, the grade III meningiomas lost RIZ1expression.4、 Conclution： In the4cases of meningioma primary cells, with levels ofmeningioma elevated, expression of RIZ1decreased, but c-Myc expression level rised. PartⅢ、construction of PR-domain protein capable oftransduction1、Purpose：In order to verify whether the PR domain specially contains anti-tumoreffect for meningioma cells, we need to build the PR domain protein that can cross the cellmembrane.2、Methods：We chosed TAT protein transduction system to transport protein intocells. First, by PCR amplification and cloning technology, we amplified the PR domainalone. Then by Nco I and EcoR I double digestion,the PR domain was connected to thepTAT-HA expression vector. The plasmid was transformed into DE3bacteria with theamplification of large number of bacteria, which was induced expression of the protein.After purification of the desired protein, protein gel electrophoresis was used to examinedthe molecular weight and purity of the protein. Concentration of protein quantified by BCAassay, then the protein was stored at-80℃.3、Result：After plasmid construction and protein purification steps, we obtained thedesired protein. By protein electrophoresis, we observed that the protein molecular weightis about32KD.4、Conclution：PR domain was successfully connected to TAT protein transductionsystem PartⅣ、The impact of PR on primary meningioma cellproliferation and apoptosis1、Purpose：to verified whether the PR domain have anti-tumor effects on primarymeningioma cells2、Methods：First we deal meningioma primary cells with protein concentration of0.2μM for2hours. HA-tag immunofluorescence staining was used to verify transductionprotein’s localization in cells. For drawing the proliferation experiment, we selected fivedegrees of protein concentration (0,0.05,0.1,0.2,0.4μM) for the four cases of primary cellsfor six-day growth curve. Each group of cells were detected by immunofluorescence forproliferation target ki67and PCNA, In the apoptosis assay, we deal meningioma cells withthe concentration of0.4μM.After24hours we detected apoptotic cells by flow cytometry.3、Result：We found that transduction proteins mainly located in the nucleus ofmeningioma primary cells. Cell proliferation assay, proliferation target testing andapoptosis assay showed similar results that transduction proteins have better effects on high-level of meningioma than the low-level. During the experiment, we found a fewphenomena worthy of further study:1, transduction protein has the most obviousanti-proliferative and pro-apoptotic for grade3meningioma primary cells, but nosignificant anti-tumor effect for grade1meningiomas, grade2meningiomas cells showedintermediate effect.2, For the2cases of grade2meningioma primary cells, transductionprotein had better effect on M12than M11, and previous experiments revealed that theexpression of the endogenous RIZ1of M12is significantly lower than M11.3, theproliferation target of ki67and PCNA expression level supported the proliferation curvefor the four cases of meningioma primary cells.4、Conclution：PR domain can solely produce anti-tumor effects to the high-levelprimary meningioma cells PartⅤ、PR domain might affect proliferation and apoptosis ofprimary meningioma cell by H3K9methylation pathways1、Purpose：In the previous study, we found that transduced PR domain protein mightinduced inhibition for the high-level meningioma tumor cells, and its mechanism deservedfurther study. RIZ1is a family member of histone methyltransferase (COMT)8, which canconduct methylation of the ninth lysine in histone H3, related in molecules regulation onthe expression of downstream molecular pathways. PR domain of RIZ1as a tumorsuppressing component needs to be confirmed whether it has methyltransferase activity.2、Methods：Using a protein concentration of0.4μM as dosing to handle grade3primary meningioma cells, the processing time is0hours,4hours and12hours.Respectively, the cells have been extracted protein for immunoblot assay to detect theexpression of histone methylation: H3K9me1(histone H3methylation occurred on theninth lysine), H3K9me2(dimethyl of occurrence), H3K9me3(trimethyl of occurrence).We found that as time changed, the expression of c-Myc and of endogenous RIZ1change.3、Result：We found that with the change of the time, the expression of H3K9me1andH3K9me2significantly increased, whereas the expression of c-Myc is gradually reduced,and the expression of RIZ1and H3K9me3showed no obvious trend.4、Conclution：In the experiments, we observed that with histone H3methylation on the ninth lysine ranking1st and2nd increased, the expression of c-Myc is graduallydecreased, which reminders the existence of signal paths. The PR domain itself asfunctional configuration of RIZ1or coordinating with other molecules might promoteH3K91st Bit2nd Bit methylation which might regulate the expression of c-Myc in turn toregulate the expression and the downstream molecules. Eventually cause inhibition to highlevels of meningiomas. PartⅥ、By full spectrum of gene expression microarray, wesupposed to explore the PR domain downstream moleculars.1、Purpose：From the previous step, we can find that PR domain’s methyltransferaseactivity might promote changes in the expression of downstream molecule c-Myc throughhistone. As to explore addition pathways, we decided to carry through the wholespectrum of gene expression microarray research.2、Methods：Using a protein concentration of0.4μM as dosing to handle grade3primary meningioma cells, the processing time is0,4,12,24,48hours, while taking samevolume of the PBS-treated as control group. After microscopic graphs taken, total RNAwas extracted for total gene expression profiling test.3、Result：Compared with the control group, We found that the cell morphology ofgrade3primary meningioma changed from regular polygon to typical diamond, and also tothe extension of cell antennae. As the whole spectrum of gene expression microarrayshows, in progress with time, the number of genetic changes also progressive increase,which includes changes in tumor suppressor genes and cell cycle protein expression.4、Conclution：Transduction PR domain proteins may induce a series of tumorsuppressor gene expression levels increased, and changes in cell cycle proteins, which maybe caused by inter-process of histone methyltransferase activity. Whether there are otherregulation mechanisms needs further research. PartⅦ、Resection specimens from different grades of eningiomawere used to verify the amount of change in expression of RIZ1.1、Purpose：From the previous experiments, we found that the transduced PRdomain’s tumor inhibition for the high grade of meningiomas is based on the low RIZ1expression of high-grade meningiomas. In the clinical specimens, whether the RIZ1expression in high-grade meningioma cells decrease still need to be confirmed. weexplored this problem in this part of the experiment.2、Methods：After collection of different grades of meningioma resection specimens,we studied the changes in the expression of RIZ1and c-Myc by immunohistochemistry.3、Result：We found that as grades of meningiomas elevated, expression of RIZ1shows a downward trend and expression of c-Myc shows rising trend.4、Conclution：In high-grade meningiomas, RIZ1relatively decreased.