Preliminary Study Of The Effect Of MiR-195on The Biological Behavior Of Human Glioma And Its Possible Mechanism

Malignant glioma, as the most common malignant tumor of the central nervoussystem, features a high rate of recurrence, mortality and disability, which is severelyharmful to human health. Despite intense efforts have gone into finding effectivetreatments, high-grade malignant gliomas, especially glioblastoma (GBM), arepresently still incurable. Temozolomide (TMZ) as a first-line chemotherapeuticagent, in addition to radiotherapy and surgical resection, improved both the overallsurvival and progression-free survival in patients with newly diagnosed GBM. Butmedian survival of GBM patients has not been significantly prolonged, which isclosely related to the biological behavior characteristics of glioma cells such asunlimited proliferation, apoptosis obstacle, enhanced invasion and resistance tochemotherapy drugs. Therefore, trying to find a molecular target that can effectivelyinhibit malignant biological behavior of glioma cells, will have great scientificsignificance and clinical application value in the treatment of glioma.MicroRNAs (miRNA) are small, non-coding21-23nucleotide RNAs thatregulate gene expression by binding to the untranslated regions of their targetmRNA molecules, then repressing transcription or inducing mRNA degradation. AmiRNA can regulate hundreds of target genes with which it forms a complexnetwork. Through the network, a miRNA is widely involved in embryonicdevelopment, cell proliferation, differentiation, apoptosis, cell cycle, angiogenesisand other biological processes. Moreover, target genes of miRNAs often focus onone or several signal pathways, which makes them regulate cell function and phenotype more rapidly and forcefully. MiRNAs have the characteristics of timesequence, conservatism and tissue specificity. Besides, they can exist stably invarious body fluids, even in formalin-fixed and paraffin-embedded (FFPE) tissues.Therefore, it is expected to be an ideal molecular marker for tumor diagnosis,treatment and prognosis. Recently, a large number of studies show that theexpression of specific miRNA is closely linked to the regulation of malignantbiological behaviors of glioma cells, such as unrestrained proliferation, abnormalapoptosis, migration, invasion and drug resistance.MicroRNA-195(miR-195) is an essential member of themiR-15a/15b/16/195/497family, located on chromosome17p13.1. The distancebetween miR-195and tumor suppressor gene P53is only650kb, where loss ofheterozygosity frequently occurs. Researches show that down-regulation of miR-195exists widely in various malignant tumors, including liver cancer, lung cancer, coloncancer, gastric cancer, breast cancer, primary peritoneal cancer, bladder cancer,tongue squamous cell carcinoma and so on. It plays a critical role in suppressingtumor growth through regulating a variety of biological behaviors of malignanttumor cells. Nevertheless, the biological role and the related mechanism of miR-195in glioma were not yet clear. According to the prediction based on the databases ofmiRNA target genes： miRDB, TargetScan and PicTar and domestically andinternationally confirmed miR-195target genes, we speculated that miR-195playeda crucial role in the regulation of G1/S transition of tumor cell cycle and the DNAdamage response, thereby affecting the malignant biological behavior of tumor cells.Therefore, our present study chose miR-195as the research object. The expressionlevels of miR-195in FFPE glioma tissues, glioma cells and TMZ-resistant strainwere examined. Then by changing the expression of miR-195, we observed its effect on the biological behaviors of glioma cells, including proliferation, migration,invasion, clone formation, tumor formation in vivo and chemotherapy resistance. Inlight of the findings above and the information collected from the database used forpredicting miRNA target gene, the possible molecular mechanism of the miR-195regulating the malignant biological behaviors of glioma cells was explored. Theresearch will provide an in-depth understanding of the biological functions ofmiR-195and the regulatory mechanisms of malignant biological behaviors ofglioma cells. It is of theoretical value and practical significance.The current study was performed from three aspects as follows.1Expression and clinical significance of miR-195in FFPE human gliomatissues and cell lines:Objective: To investigate the expression of miR-195in human brain gliomaFFPE tissues and cell lines and analyze the correlation between miR-195expressionand pathological grades, laying a foundation for further exploring the biologicalfunction of miR-195in glioma in vitro and in vivo.Methods: The expression of miR-195was detected in human glioma cell linesSHG-44, U87, U251, human astrocyte cell line,28cases human differentpathological grades glioma FFPE samples and2cases normal brain tissues byreal-time fluorescent quantitative reverse transcriptase polymerase chain reaction(qRT-PCR).Results:(1) qRT-PCR results showed that compared to normal humanastrocytes (HA), miR-195levels of three cell lines SHG-44, U87, U251weresignificantly reduced, with the highest degree of reduction in SHG-44and the lowestin U251(P<0.05).(2) The expression level of miR-195in28cases of glioma FFPEtissues was lower than that in normal brain tissues (P<0.05). There was no significant difference between grade I and grade II (P>0.05). There were differencesbetween grade I and grade III or grade IV (P<0.05). The difference also existedbetween grade II and grade III or grade IV (P<0.05). The expression of miR-195ingrade III or IV was significantly more than that in grade I or grade II (P<0.05). Theanalysis of correlation showed that the negative correlation existed betweenmiR-195expression level and pathological grades of human gliomas (r=0.798,P<0.001).2Effects of miR-195on proliferation, apoptosis, migration and invasion ofhuman glioma cells in vitro and in vivo:Objective: To observe the effects of upregulation of miR-195on proliferation,apoptosis, invasion and migration of human glioma cells in vitro and in vivo.Methods: Control group (Blank group), nonsense sequence group (NC group)and miR-195over expression group (miR-195group) were designed in theexperiment. First, Cy5labeled miR-195mimics and nonsense sequences weretransfected into human glioma cell lines U251and SHG-44respectively by cationicliposome LipofectamineTM2000. Transfected cells were observed with confocallaser microscopy. The transfection efficiency was screened by flow cytometry andthe optimal concentration for transfection was identified. The expression of miR-195was determined by qRT-PCR. Second, cell proliferation was detected by CCK-8assay and colony formation assay. Apoptosis rate was assessed byAnnexinV-FITC/PI assay and Hoechst33342/PI staining. Cell cycle was analyzed byflow cytometry. Cell migratory ability was evaluated by wound healing assay. Cellinvasiveness was gauged by Transwell Matrigel invasion assay. The expressionlevel of Bcl-2protein was detected by immunocytochemical assay. Finally, we further investigated whether these effects could also be detected in subcutaneousglioma models of nude mice.Results: After miR-195mimics were transfected into U251and SHG-44,strong red fluorescence can be observed by using the laser confocal. The besttransfection concentration of miR-195mimics was50nm, whose transfectionefficiency can be reached above90%. qRT-PCR results showed that compared withBlank group, miR-195contents in miR-195group of U251and SHG-44were up9.98,65.97times respectively (P<0.05). CCK-8assay and clone formationexperiment results showed that compared with the NC group and the Blank group,by overexpressing miR-195, the ability of cell proliferation was significantlydecreased with colony forming ability attenuated (P<0.05). Flow cytometry andHoechst33342/PI double staining were used to detect apoptosis. The results showedthat compared with the Blank group and the NC group, apoptosis and necrosis cellsin miR-195group rose sharply.(P<0.05). Wound healing assay results showed that,in U251, overexpression of miR-195can inhibit cell migration (P<0.05), but inSHG-44, there were no statistical differences on migration rate among three groups(P>0.05). Transwell invasion assay results showed that, in U251, overexpression ofmiR-195group can reduce the number of cells through the Matrigel membrane(P<0.05), but in SHG-44, the number of cells through the Matrigel membrane inmiR-195group was more than that in the other two groups (P<0.05). Cell cycleanalysis by flow cytometry showed that, in comparison with Blank group and NCgroup, the G0/G1phase cells markedly increased, while the S phase cells decreasedsignificantly in miR-195group (P<0.05). By microRNA target prediction websitesand related biological information software, we found that Bcl-2was an importanttarget gene of miR-195. And immunocytochemistry results indicated that overexpression of miR-195can downregulate the expression of Bcl-2. The effects ofmiR-195on the formation of glioma subcutaneous model in nude mice suggestedthat tumor nodules in miR-195group appeared later and grew at a slower rate. Bothtumor weight and tumor volume decreased markedly in miR-195group as comparedwith those of the NC group and the Blank group (P<0.05). Subsequently, tumorspecimens immunohistochemical analysis showed that, compared with the NC groupand the Blank group, the expression of Ki67reduced remarkably in miR-195group(P<0.05).3Role and mechanism of miR-195in temozolomide-acquired resistance ofglioblastoma:Objective: To study the role and mechanism of microRNA-195intemozolomide-acquired resistance of glioblastoma.Methods: TMZ-resistant variants were developed by stepwise exposure toincreasing concentration of TMZ. The expression levels of miR-195in twoestablished resistant variants were detected by qRT-PCR. Then by changing theexpression level of miR-195in the resistant cells, we further examined the effects onproliferation and drug resistance of glioma cells by CCK-8assay. Cell apoptosis andcell cycle were analyzed by flow cytometry. The expressions of MGMT, P21, Bcl-2and Cyclin D1were detected by Western blot assay. Finally we establishedsubcutaneous glioma models in nude mice for observing the effects of up-regulatingmiR-195on the tumor growth and glioma susceptibility to TMZ.Results: For6months we finally obtained the stable TMZ-resistant variants bystepwise increasing concentration of TMZ, named as U251R, SHG-44R. qRT-PCRresults showed that the expression levels of miR-195in U251R and SHG-44R wereless than that in the parental cells. Since SHG-44R reduced more significantly (P<0.05), SHG-44was chosen for subsequent experiments. CCK-8results showedthat the upregulation of miR-195increased the sensitivity to TMZ in SHG-44R,knockdown of miR-195increased the resistance to TMZ in SHG44R (P<0.05). Cellapoptosis was detected by flow cytometry. The results showed that overexpressionof miR-195can notably increase the total apoptosis rate of glioma resistant strain(P<0.05). Downregulation of miR-195can reduce the total apoptosis rate, but therewas no significant difference on total apoptosis rate in all three groups (P>0.05).Cell cycle analysis suggested that when the level of miR-195in SHG-44R wasupregualted, glioma cells grew more slowly and had a longer passage time withG0/G1phase cells increased and S phase cells decreased (P<0.05). There was nosignificant difference between miR-195inhibitor group and control group (P>0.05).Western blot assay showed that overexpression of miR-195can downregulate theexpression of P21, Cyclin D1, Bcl-2. Conversely, knockdown of miR-195increasedthese protein expressions(P<0.05). Notably the expression level of MGMT was notaffected by the changes of miR-195level. Subcutaneous glioma model in nude miceand TUNEL staining showed that miR-195combined with TMZ can inhibit gliomagrowth and promote glioma cell apoptosis significantly (P<0.05).Conclusion:(1) miR-195was down-regulated in all the detected glioma FFPEspecimens and cell lines. And the expression level was negatively correlated withpathological grades of glioma. We may take it as a new effective glioma I-IV gradediagnostic marker and as a supplement to conventional histopathological gradingstandards.(2) Over expression of miR-195can attenuate the glioma cellsproliferation in vitro and in vivo, induce G0/G1arrest and downregulate Bcl-2expression. It also can increase apoptosis or necrosis of glioma cells. Nevertheless,its influence on migration and invasion of glioma cells may be associated with glioma cell types, and further studies were needed. In general, miR-195cannegatively regulate the malignant biological behaviors of glioma cells.(3) Theexpression of miR-195in glioblastoma resistant strains was much lower than that intheir parental cell lines. Upregulation of miR-195can inhibit proliferation, promotecell death and downregulate the expressions of P21, Cyclin D1and Bcl-2. MiR-195can restore in vitro and in vivo glioma cell sensitivity to TMZ treatment to a certainextent, which was not involved with MGMT. In summary, miR-195is expected tobecome a new potential gene therapeutic target of glioma.