The Role Of ZMYND11 In Glioblastoma Multiform

Glioblastoma multiform(GBM)is the most common and aggressive type of glioma(WHO grade Ⅳ)accounting for 25%-50% of intracranial Glioma.GBM is regarded as the most deadly brain cancer of human worldwide.The morbidity of GBM is about 1/100,000.GBM is commonly developed in patients older than 20-year-old,especially frequently in people on the age of 40 to 70 years old.Male patients are a little more than female patients.Over 60 percent of GBM patients are primary type,nearly 40 percent of GBM are developed malignantly from low grade glioma.It was reported that patients with a diagnosis of GBM obtained an overall survival time of 8-15 month and those with recurrent GBM survived only 3-9 month.Despite postoperative chemotherapy and radiotherapy,less than 10 percent of GBM patients could survive over 2 years,the survival rate of 5 years was less than 5 percent.Recently,an increas ing number of researches on genetic and target therapy of GBM have been performed worldwide.However,the mechanism and specific protein on occurrence of GBM remain unknown.We selected a kind of anti-cancer nucleus protein called ZMYND11 after looking up a lot of reference.It is short for Zinc finger MYND domaincontaining protein 11 with a molecular weight of 69 kDa.Besides,ZMYND11 contains several other functional domains.ZMYND11 is encoded by ZMYND11 gene,which is located at 10p15.3 and contains 15 exons.ZMYND11 is expressed in different organs of animals and human.In 1995 ZMYND11 was found to specifically bind the human adenovirus E1 A protein and inhibit transactivation and oncological role of E1 A.Moreover,it was revealed that the particular domains of ZMYND11 protein could recognize and bind H3K36me3 on H3.3K36me3.By this mechanism,ZMYND11 could suppress the activity of RNA polymerase and the elongation process of H3.3K36me3-related oncological proteins as a co-regulator.Furthermore,ZMYND11 was described to suppress the development and progression of breast cancer.So far no research on ZMYND11 and GBM has been reported yet.To explore the role of ZMYND11 in GBM,we collected the samples of GBM tissue from 20 GBM patients and non-neoplasm brain tissue from 20 cases with severe traumatic brain injury during surgery and found that the expression level of ZMYND11 in GBM samples was remarkable lower than that in non-neoplasm.In addition,our study detected that ZMYND11 inhibited the proliferation,invas ion and promoted the apoptosis of GBM using cellular functional trials.Nevertheless,the reason why ZMYND11 was downregulated in GBM remains unclear.We selected miR-196a-5p by looking up websites and references and speculated that miR-196a-5p were highly likely to target ZMYND11.MiR-196a-5p was explored to be upregulated in GBM and closely associated with poor outcome of GBM patients.Then we utilized luciferase reporter assay to confirm that miR-196a-5p could bind directly the 3’-UTRs of ZMYND11 and discovered the reverse relationship between miR-196a-5p and ZMYND11 expression both in GBM samples and cell lines.Rescue experiment showed that ZMYND11 downregulation could reverse the suppressive effect on U87 cells induced by decreased miR-196a-5p.Part one Expression compare of ZMYND11 in Glioblastoma multiform(GBM)tissues and non-neoplasm brain tissues and analysis ofsignificance of ZMYND11 expression in patients’ prognosisObjective :To compare ZMYND11 expression in GBM tissues and non-neoplasm brain tissues,and explore the relationships between ZMYND11 expression and survival time of GBM patients.Methods:The tumor samples from 20 GBM patients and non-neoplasm brain samples from 20 severe traumatic brain injury cases were collected intraoperatively and divided into tumor group and control group.The follow-ups were performed after discharge.The relative protein expression and the relative mRNA expression of ZMYND11 in two groups mentioned-above were detected using Western blot and q RT-PCR tests.In addition,the 20 GBM cases were divided into high level group and low level group.Finally,the relationship between expression level of ZMYND11 and survival of GBM patients was analyzed.Results:1.Clinical data of GBM patients and sample providers.Clinical information and data: 20 GBM patients composed the tumor group,including 12 males and 8 females,whose ages ranged from 44 to 68,54.9 years old on average.Control group consisted of norma l brain tissue from 20 cases with severe traumatic brain injury,14 males and 6 females were included,whose ages ranged from 21 to 65,35.7 years old on average.The period of following up ranged from 3.6 to 16.1 month,9.74 month on average.None of GBM patients underwent any chemotherapy or radiotherapy before surgery.2 of 20 GBM cases experienced no chemotherapy or radiotherapy,postoperatively.16 cases received chemotherapy,18 patients underwent radiotherapy.2.The expression level of ZMYND11 was marked decreased in GBM tissue compared with non-neoplasm brain tissue.Diagnosis of GBM was confirmed by at least two experienced o pathologists in our hospital.The results of both of the Western blot and q RT-PCR tests showed that the expression level of ZMYND11 was lower in GBM than that in non-neoplasm tissue.3.The expression level of ZMYND11 in GBM was associated with outcome of GBM patients.To investigate the clinical significance of the expression level of ZMYND11 to GBM patients,we analyzed relationship between the patients’ survival and ZMYND11 expression level.It was found that patients with higher level of ZMYND11 obtained a longer medium survival than cases with lower level of ZMYND11.Conclusion:1.The expression level of ZMYND11 was remarkably lower in GBM tissuses than that in non-neoplasm brain tissue.2.The expression level of ZMYND11 was positively associated with the medium survival time.Part two The effect of ZMYND11 on proliferation,invasion andapoptosis of GBMObjective:To explore the influence of ZMYND11 expression to GBM cellular proliferation,invasion and apoptosis after successfully upregulating ZMYND11 expression in U87 cell line.Methods:After using transfection with lentivirus to increasing the expression of ZMYND11 in U87 cell line and confirming the effectiveness of transfection,we performed CCK-8 test,tranwell test and flow cytometry analys is to detect the role of increas ing-ZMYND11 in cellular proliferation,invasion,apoptosis and cycle of U87 cells.Results:1.The expression level of ZMYND11 was increasing successfully in U87 cell line after transfection.To detect the role of ZMYND11 in U87 cells,we transfected U87 cells with lentivirus of ZMYND11-upregulation and corresponding control lentivirus.The Western Blot assay showed that expression level of ZMYND11 was upregulated effectively and obviously in U87 cells after transfected with lentivirus of ZMYND11 overexpression.2.ZMYND11-upregulation inhibited the cellular proliferation of GBMCCK-8 assay showed that proliferative capability of U87 cells was markedly inhibited in ZMYND11-upragulation group,compared with control group.We thought ZMYND11 inhibited cellular proliferation of GBM.3.ZMYND11 repressed mitosis in GBM cells.Cell cycle analysis by flow cytometry revealed that ZMYND11 overexpression induced increased cells in G0/G1 phase for U87 cells with a corresponding decrease in G2/M phase for U87 cell,suggesting that ZMYND11 promote mitosis in GBM cells.Cell apoptosis analys is by flow cytometry showed that the percentage of cells in early to mid-stage of apoptosis was significantly higher in ZMYND11 overexpression group than that in control group.This result indicated that ZMYND11 promoted the apoptosis of GBM.4.ZMYND11 suppressed cellular invasion of GBM.Transwell assay indicated that ZMYND11 upregulation effectively reduced the invas ion viability of U87 cells relative to control group.It implied that ZMYND11 suppressed cellular invasion of GBM.5.ZMYND11 inhibited effectively the growth of GBM.Besides intro trial,the cell xenograft trial was performed.The result showed that the mean weight of the tumors extracted from the ZMYND11 upregulation group was significantly lower than that of the control group and the mean volumes of the xenograft tumors in the ZMYND11 increasing group were all obviously smaller than those in control group during the whole of trial(Fig.2F).These results manifested that ZMYND11 upregulation suppressed effectively GBM growth in vivo.Conclusion:1.Overexpression of ZMYND11 inhibited effectively the proliferation and mitosis of GBM.2.Increased ZMYND11 repressed significantly the invas ive ability of GBM.3.Upregulation of ZMYND11 promoted markedly the cellular apoptosis of GBM.4.ZMYND11 overexpression suppressed effectively the growth of GBM.Part three miR-196a-5p promoted the cellular proliferation and inva-sion,inhibited apoptosis by targeting ZMYND11Objective: To explore the mechanism of miR-196a-5p promoting GBM cellular proliferation,invasion and anti-apoptosis by recognizing and interacting with ZMYND11 in GBM cell.Methods: We tested the expression level of ZMYND11 and miR-196a-5p in 20 patients mentioned above using Western blot and q RT-PCR tests to identify the correlation between miR-196a-5p and ZMYND11 in GBM tissue.Furthermore,we downregulated and upregulated miR-196a-5p expression in U87 with lentivirus and plasmid vector transfection and measured expression level of ZMYND 11 in U87 cells transfected using Western Blot.In addition,we performed luciferase reporter assays with GV272 vector transfection to confirm that miR-196a-5p directly interacted with 3’UTR of ZMYND11.To investigate whether inhibition of ZMYND11 was a mechanism of miR-196a-5p promoted progression of GBM,we performed rescue experiment by downregulating miR-196a-5p and ZMYND11 with lentivirus and siRNA,respectively.Results:1.The expression level of miR-196a-5p correlated negatively with expression level of ZMYND11 in GBM.Compared to the expression level of ZMYND11 of 20 patients mentioned above,we found the reverse correlation between miR-196a-5p and ZMYND11 in the 20 samples.Furthermore,we downregulated and upregulated miR-196a-5pexpression in U87 with lentivirus and plasmid vector transfection and measured ZMYND 11 expression in U87 cells transfected using Western Blot.We found that the miR-196a-5p expression correlated negatively with ZMYND 11 expression in U87 cells.2.miR-196a-5p exerted its oncological function by inhibiting ZMYND11 in GBM.By luciferase reporter assays,we discovered that the activity of luciferase remarkably decrease in MT-ZMYND11 group,compared to control group and MUT-ZMYND11 group.This result indicated that miR-196a-5p could directly interact with a certain site in 3’UTR of ZMYND11 gene.The rescue experiment result revealed that proliferative and invas ive ability of U87 cells increased significantly in miR-196a-5p knockdown plus ZMYND 11 s iRNA group,while cells apoptosis decreased remarkably.It was indicated that knockdown of ZMYND11 could abrogate the suppressive effect of miR-196a-5p down-regulation on GBM cells.Conclusion:1.miR-196a-5p inhibited directly the expression of ZMYND11 in GBM,so that miR-196a-5p expression correlated with ZMYND11 in GBM.2.ZMYND11-downregulation induced by miR-196a-5p promoted the proliferation,invasion and suppressed cellular apoptosis in GBM.Summary:1.ZMYND11 expression level was lower s ignificantly in GBM tissue than that in non-neoplasm brain tissue.The GBM patients with relatively higher level of ZMYND11 obtained a longer medium survival time than those with lower level of ZMYND11.2.Increasing ZMYND11 expression suppressed the proliferation and invasion of GBM and promoted the cellular apoptosis of GBM.3.In GBM miR-196a-5p promoted proliferation,invas ion and anti-apoptosis by targeting ZMYND11.