MiR-101is Down-regulated In Glioblastoma Resulting In STMN1-induced Proliferation, Migration, And Invasion

Adult glioblastoma multiforme (GBM) is the most common intracranial malignanttumor with its high morbidity and mortality. Despite of having surgery and standardadjuvant chemoradiotherapy, the median survival of patients is still less than15months.Now it is generally accepted that the high relapse rate of GBM is the main reason leadingto an unfavorable prognosis. GBM is more likely to have local recurrence rather thandistant metastasis, so this kind of a high relapse rate mostly has close connections with theinherent invasiveness of GBM cells on the tissues adjacent to tumors and its own strongcapabilities of proliferation and migration. At present the main methods in treating GBM isthat surgical resecting substantial part of glioblastoma and the edematous tissuesurrounding it, which can effectively alleviate the symptoms of intracranial hypertensionand reduce the tumor burden. Although assisted by the standard postoperativechemotherapy, this kind of treatment does not completely get rid of the tumor cellsinvading into the adjacent tissue or the surrounding normal brain tissue. It is still possiblefor the recurrence of the tumor. Postoperative recurrence time is different in patientswith different pathological grade of glioma. There are also differences of recurrence timeamong the same grade of glioma with the same kind of treatment after surgery, whichmight has close relationship with the diversity of invasive ability of tumor cells. Therefore,it is of great possible to assist the existing treatment by inhibiting the capability ofproliferation, migration and invasion of glioma cell.MicroRNAs is an endogenous non-coding single-stranded nucleotide, the length of it isabout19-25nucleotides. It has been confirmed that the seed sequence of match with the3’UTR by complete or incomplete sequence-specific complementary base pairing, whichcan affecting the stability of the target mRNA and inhibiting the translation process ofmRNA. That is to say microRNA may regulate gene expression in post-transcriptionallevel. At present, there are more and more evidences that changes in levels of microRNAexpression connect with human neoplastic diseases. About50%microRNA located atfragile sites on chromosomes, it is usual that these loci in tumor cells are missing oramplification, and the abnormal expression of microRNA have something to do with theprognosis of clinical patients. MicroRNA extensively participate in a variety of biologicalprocesses such as cell proliferation, migration, invasion, apoptosis, autophagy, stress andso on. MiR-101is a member of the numerous microRNAs. In recent years, it has been widely reported that the the expression of mir-101in tumor cells is down-regulation and itcan inhibit the tumor growth both in vivo and in vitro. Comprehensive analyses ofmiRbase, TargetScanHuman and other databases and literature confirm that target genes ofmiR-101include EZH2, COX-2, SOX-9, MCL-2, APP, etc. Mir-101can regulate a varietyof signaling pathways by changing the expression of these genes in the process oftumourgenesis.2011another three new targets of miR-101in breast cancer have beenfound which include STMN-1.STMN-1(stathmin-1), also known as OP (oncoprotein-18), is a phosphoprotein ofmicrotubule depolymerization, Sobel et al reported it the first time in1983. Our previousanalysis using gene chip technology and bioinformatics analysis techniques of72gliomatissues have filtered out29chemoresistance-related genes, STMN-1is one of them,whose expression in glioma tissue is significantly higher than in normal brain tissue. In theearly stage of mitosis, by the phosphorylation its S25and S38serine sites, STMN-1takespart in correct formation of spindle and proper separation of sister chromosomes, which isrelated to the proliferation of tumor cells.STMN-1is also involved in the process ofprogrammed cell death, apoptosis and autophagy, it has been showed in our preliminaryresults that the interference of STMN-1’s expression in U251cells lead to the increase ofits sensitivity to TMZ and this phenomenon may be related to apoptosis. Furthermore, inthe process of cell migration, phosphorylation of the no.16serine site of STMN-1helpSTMN-1regulate the function of microtubule through Rac1-Pak1pathway, which affectscell movement. Therefore, both the expression levels and phosphorylation levels ofSTMN-1have a great deal to do with the process of oncogenesis, besides, the diagnosis,prognosis evaluation of malignant tumors.In this experiment we want to choose the most appropriate experimental cell line byanalyzing the different expression of mir-101in four glioblastoma cell lines.Respectively building overexpression and interference lentiviral vectors of miR-101andSTMN-1is another important part of our experiment. Cells transfected with the lentivirusmentioned are detected the ability of migration and invasion. Next, establish dualluciferase reporter lentivirus containing STMN1-3’UTR wild-type and mutant-type. Withmir-101up-regulated U251cells transfected with the dual luciferase reporter virus, thedetector can verify the fluorescence intensity of glioma cells with different approaches, wecan clear whether the seed sequence of miR-101bond STMN1-3’UTR directly. It canconclude that the processes of proliferation, migration and invasion in glioma cells are regulated by a potential pathway of miR-101---STMN-1. Experiment was divided into twoparts: the first part, measure the expression of miR-101in the collected glioma tissuespecimens and then analyze the relationship between the expression and the pathologicalgrade of the tumors, furthermore, combining the construction of lentiviral vector withQ-PCR, WB and dual fluorescence target validation technique to verify that the negativerelationship between miR-101and STMN-1is surely existed; the second part, glioma celllines transfected with miR-101overexpression and STMN-1siRNAlentivirus respectively,followed by detection of cell proliferation, migration and invasion.PARTⅠ: Detect the expression of miR-101in human glioma tissues and glioma celllines and confirm the immediately negative regulatory relationship between miR-101and STMN-1in glioma cell lines.Objective: To detect the relationship between miR-101expression levels of gliomatissue samples with the pathological grade of tumor, verify the direct binding mechanismbetween miR-101and STMN-13’UTR in glioma cells, which lead to their negativeregulation.Methods: Real-time quantitative PCR is used to analyze of the expression ofmiR-101in37cases of glioma tissue specimens; both real-time PCR and western-blottechnique are applied to detect the expression of STMN-1’s mRNA levels and proteinlevels in U251cells transfected with miR-101overexpression or inhibition lentivirusrespectively; build pL/TO/IRES/Luc-STMN1-3’UTR and pL/TO/IRES/Luc-STMN1-3’UTR-mut lentiviral vectors then use dual luciferase reporter system technology to verifythe seed sequence of miR-101s can be directly combined with STMN-13’UTR in U251cells,which is to say STMN-1is the regulatory target of miR-101.Results: The expression of miR-101in four alternative GBM cell lines are in theorder of:. U251> A172> U87> T98. The results of37glioma tissues analyzed byreal-timePCR show that the changes in the expression of miR-101among normal brain tissues,low-grade glioma tissues(WHOⅠ-Ⅱgrade) and high-grade gliomas(WHOⅢ-Ⅳgrade)tissues reach statistically significant differences (P <0.01), that is to say,the higher the tumor grade is, the lower intracellular miR-101expression it has. But thereis no significant statistical difference about the expression of miR-101betweenWHOⅠandⅡgrade level or between WHOⅢ and Ⅳ grade level (P>0.05). Aftertransfected with miR-101-inhibit and miR-101-overexpression lentivirus, the changes about mRNA and protein expression levels of STMN-1in U251cells were detected byreal-time PCR technology and western-blot technique. The results show a clear negativeregulatory relationship between miR-101and STMN-1. With built STMN1-3’UTR-WTand STMN1-3’UTR-mut double fluorescent lentivirus, miR-101overexpression andnegative control lentivirus according to experimental groups co-transfected into U251cells, the transfected cells were measured of the firefly luciferase’s RLU and the Renillaluciferase’s RLU. According to data analysis, STMN1-3’UTR is capable of interacting withmiR-101.Conclusion: The expression of miR-101is negatively correlated with the tumors’pathological grade, there are significant statistical differences between low-grade gliomasand high-grade gliomas; the seed sequence of miR-101can directly combine withSTMN1-3’UTR, resulting in a poor stability of STMN-1mRNA and the degradation ofit. Thereby, further reducing in the expression of STMN-1protein is inevitable. PARTⅡWhether miR-101can regulate proliferation, migration and invasion ofU251cell by targeting the STMN-1.OBJECTIVE: To confirm that miR-101actually regulates the proliferation, migration and invasion of U251cell through STMN-1.Methods: Use prepared miR-101-overexpression, miR-101-interference, STMN-1-siRNA and the corresponding negative control (NC) virus to transfect U251cell line; detect cell proliferation ability by CCK-8assay and draw the proliferation curve;evaluate the capacity of cell migration with the scratch assay experiment; capability of invasion is measured by Transwell assay.Results: After adding CCK-8reagent into the96-well plates we use microplatereader to detect OD450nm, the result showed that ability of cell proliferation in U251+miR-101-overexpression group is lower than U251+miR-101-interference group, U251+miR-101-NC group and U251group, the changes in OD450nm valuereach significant statistical difference (P <0.05), but no statistically significant difference is among the rest three groups. U251cells were transfected with miR-101-interference, miR-101-overexpression and NC lentivirus, cell scratch test was done after72h,24h later the results showed that overexpression of miR-101in U251cellsmakes changes in the value of the scratch area significantly less than the other thr ee groups and there is a statistically significant difference (P <0.05). Use transwellassay to detect the invasion capability of U251cells transfected with miR-101-overexpression, NC and siRNA-STMN-1lentiviral, after16h the results show that the number of invasive cells in U251+miR-101-overexpression group and U251+siRNA-STMN-1were fewer than, respectively, both NC group and normal U251group,which reach statistically significant difference (P <0.05). There is also apparent statistically significant difference (P <0.05) between the first two groups.Conclusions: miR-101can inhibit the expression of STMN-1in post-translation level,consequently inhibiting the proliferation, migration and invasion of U251cell.