| Background and objectiveGlioma is the most common primary tumor in the central nervous system.Glioma accounts for 30%to 40%of all brain tumors,accounting for about 80%of intracranial malignancies.In all gliomas,there are more than 50%of patients are the glioblastoma.Glioma has a high incidence,high recurrence rate,high mortality and low cure rate.The World Health Organization(WHO)classification of tumors of the central nervous system classifies gliomas into WHO Ⅰ to Ⅳ grades,of which grade Ⅲand Ⅳ are called high-grade gliomas.Patients with high-grade glioma have poor prognosis with the median survival time after diagnosis rang:ing from 12 to 16 months.In 2017,the European Association for Neuro-Oncology published the updated recommendations for the diagnosis and treatment of adult diffuse gliomas,including glioblastoma.According to this guideline,the goal of glioma surgery is to remove all tumor tissue as much as possible.However,whether macroscopically resectable tumours share a less aggressive biology than those for which gross total resection cannot be achieved,and hence have an intrinsically better prognosis,continues to be debated.In addition to surgical treatment,for newly diagnosed adult patients aged<70 years who have a favourable general and neurological performance status,the addition of concomitant and maintenance temozolomide chemotherapy to conventional radiotherapy became the standard therapy.But all patients with glioblastoma eventually relapsed.Median survival was only 6 to 9 months since the first progression or recurrence of glioblastoma.In fact,there is no evidence that there is any therapeutic intervention has a significant impact on survival of recurrent glioblastoma patients now.Therefore,new treatment methods for glioma are urgently needed.MicroRNA(miRNA)is a kind of non-coding microRNA,which can inhibit mRNA translation or degrade mRNA by binding to complementary target mRNA,thus playing a vital role in cell differentiation,proliferation and survival.As research moves along,miRNA plays a key role in the development and progression of tumors.They can not only directly regulate various signaling pathways of tumors,but also regulate growth of tumors through influencing the tumor micro-environment.The purpose of this study is to search for new miRNA that play a key role in the development and progression of glioma,to elucidate their mechanism of action in glioma,and at the same time to look for drugs that can affect the expression of these miRNA,so as to make preliminary preparations for clinical translation.Part Ⅰ:The role of mir-584-5p in gliomaWe first accessed and downloaded the miRNA expression spectrum data of glioma in the TCGA database,and selected the miRNA whose expression decreases and whose function have not been validated in glioma.We used qRT-PCR to detect the expression of these miRNAs in the glioma tissues and normal brain tissues we collected from the first affiliated hospital of Zhengzhou university,and finally we choosed miR-584-5p with the most significant difference between glioma and nomal brain tissues as the research object.We use qRT-PCR to detect the expression of mir-584-5p in 100 glioma tissues,34 adjacent tumor tissues and 8 normal brain tissues.The results showed that the expression of mir-584-5p was related to the WHO grade of glioma,prognosis and age of glioma patients.The expression of miR-584-5p in glioblastoma(WHO grade Ⅳ),patients with poor prognosis and older patientswas lower.CCk-8 experiment and plate colony formation experiment showed that mir-584-5p could inhibit the proliferation of glioma cells U251 and U87MG.The results of scratch test and Transwell test showed that mir-584-5p could inhibit the migration of glioma cell lines U251 and U87MG.Flow cytometry showed that mir-584-5p could promote apoptosis of U87MG cells.The results of low adhesion plate pellet test showed that mir-584-5p could inhibit the sternness of U251 and U87MG cell lines.The results of tumor formation in nude mice showed that mir-584-5p could inhibit the ability to form tumors of U87MG cells.Part Ⅱ:search and verify the target protein of mir-584-5pWe intersected the target mRNA of mir-584-5p predicted by TargetScan and miRwalk,and analyzed the expression of these genes in glioma using cBioPortal website to select the genes whose expression decreased in glioma.Then we searched literature to find if any of these genes had been reported to have an antitumor effect in other kide of cancer.Finally,eleven genes were selected for further testing.After overexpression of miR-584-5p in U251 and U87MG cell lines,the expression of these 11 genes was detected by qRT-PCR,and only the expression of Jagged 1 gene decreased in both cell lines.The dual-fluorescence results showed that miR-584-5p can identify and combine with the 3’UTR of Jagged 1,and then degrade the mRNA of Jagged 1.The results of Western Blot showed that after overexpression of miR-584-5p,the expression Jaggedl at protein level in U251 cell lines decreased.Moreover,the expression of Jaggedl in tumor tissues of U87MG nude mice after overexpression of miR-584-5p was also significantly lower than that of the control group.Part Ⅲ:the role of mir-584-5p’s target gene Jaggedl in glioma The expression of Jaggedl mRNA in 100 glioma tissues,34 tissues adjacent glioma and 8 normal brain tissues was detected by qRT-PCR.The results showed that the expression of Jaggedl was related to the WHO grade of glioma and the prognosis of glioma patients.Glioblastoma(WHO grade IV)and patients with poor prognosis have higher expression of Jaggedl.Immunohistochemical results also showed that Jaggedl protein was highly expressed in high-grade glioma and correlated with the poor prognosis of patients.CCk-8 experiment and plate colony formation experiment showed that Jaggedl can promote the proliferation of glioma cells U251 and U87MG.The results of scratch test and Transwell test showed that Jaggedl can promote the migration of glioma cell lines U251 and U87MG.Flow cytometry showed that Jaggedl could inhibit the apoptosis of U251 and U87MG cells.The results of low adhesion plate pellet test showed that Jaggedl could promote the sternness of U251 and U87MG cell lines.The results of tumor formation experiments in nude mice showed that Jaggedl can promote the tumorigenic ability of U87MG cells.Part Ⅳ:Compound C could be able to elevate miR-584-5p to play a role in the inhibition of gliomaWe used Compound C with terminal concentrations of 5 μM,10μM and 20μM to stimulate glioma cell lines U251 and U87MG for 12h,24h and 48h,respectively.The expression of miR-584-5p was detected by qRT-PCR,and the expression of miR-584-5p in the two cell lines increased with the increase of Compound C dosage and the extension of medication time.The best concentration of Compound C is 10μM and the best duration of treatment is 48h.Moreover,the Jaggedl protein of U251 cell line was significantly down-regulated under this condition.After tumorigenesis 11 days with U87MG cell lines in nude mice,the growth of tumor was significantly inhibited when injected with Compound C 10mg/Kg intraperitoneally.The tumorigenesis tissues of Compound C group and PBS control group were made into wax blocks and cut into slices,then the expression of Jagged1 in the tissues was detected by immunohistochemistry.The results showed that the expression of Jagged1 in PBS group was higher than that in Compound C group.Conclusion:1.The expression of miR-584-5p was down-regulated in glioma tissues and these down-regulation was related to the poor prognosis and age of glioma patients.MiR-584-5p could inhibit the proliferation,migration and stemness of glioma cells and inhibit the tumorigenic ability of glioma cell line U87MG.2.MiR-584-5p could identify and combine with the 3’UTR of Jaggedl,resulting in the down-regulated expression of Jaggedl.In glioma tissues,miR-584-5p was negatively correlated with the expression of Jagged 1.3 Jaggedl was highly expressed in glioma tissues and was associated with the prognosis of glioma patients.Jaggedl could promote the proliferation,migration and stemness of glioma cells and promoted the tumorigenic ability of glioma cells U87MG.4.Compound C could upregulate the expression of mir-584-5p and decrease the expression of Jaggedl.Furthermore,Compound C could also inhibit the tumorigenic ability of U87MG cells. |
