Study On Effects And Mechanism Of MiR-125b In Glioma

This study, involved tissue specimens, glioma cell lines and the nude mice model,was designed to investigate miR-125b expression in glioma, as well as its influenceon the biological functions of glioma and the potential molecular mechanisms.Thisexperiment was divided into three parts:1. miR-125b expression in serum and tumors in glioma patients and its clinicalsignificance.miRNAs is a kind of18to25nt, small non-coding single-stranded RNAmolecule, which can regulate gene expression via binding to mRNA at thepost-transcriptional level. Studies have confirmed that miRNAs were closelyassociated with tumors. In this study we collected tumor tissue from patients withglioma undertook surgery in our hospital. The result of deep sequencing test showedthat compared with normal brain tissue,34miRNAs were downregulated in glioma bymore than4times, whereas46miRNAs were upregulated more than four times.Among them, miR-125b was significantly upregulated. To further validate the resultof deep sequencing, we applied qRT-PCR detection of miR-125b expression in serumand glioma in patients. We found that miR-125b expression were significantly higherin serum and tumor tissue than that of normal adults (p <0.01). In order to furtherinvestigate the clinical significance of miR-125b, we analyzed Ki67score of gliomaspecimens and its correlation with miR-125b expression. The results showed thatmiR-125b was positively correlated with Ki67scores, and the time of tumorrecurrence in patients with high expression of miR-125b was significantly earlier thanthat of patients with low expression of miR-125b. These results suggested thatmiR-125b expression was upregulated in glioma patients, and associated withmalignant degree and prognosis of disease.2. miR-125b promoted proliferation, invasion, and apoptosis resistance of gliomacells.In the first part of the study, we found that miR-125b was associated withmalignant degree and prognosis of glioma. However, its impacts on the biologicalfunctions of glioma are still undefined. Therefore, in this part, we investigatedsystematically the effects of miR-125b on proliferation, invasion, and apoptosis resistance of glioma cells both in vitro and in vivo.The agonist for miR-125b agomir-125b and the negative control agomir-NCwere used to treat U87and U251cells respectively, and the MTT examination wasperformed at every24hour time point. We found that the agomir-NC transfectionlead to no change of cell proliferation while agomir-125b leads to increased cells at48hours (P<0.01),72and96hours (P<0.05). In addition, the agomir-125b transfectionleads to increased clone formation in compared to the control group and agomir-NCgroup (P<0.05). miR-125b decreases the glioma cell apoptosis induced by TMZ. Wefound that TMZ could induce cell apoptosis in TMZ treated group, which was notaffected by agomir-NC transfection. However the agomir-125b treatment coulddecrease the cell apoptosis. In western blot study, the apoptosis related proteins PARPand Caspase-3also decreased following agomir-125b treatment, in compared to theTMZ group. These data suggested that miR-125b can increase the anti-apoptoticability of glioma cells. Furthermore, the U87cells transfected with agomir-NC,agomir-125b or control vectors were injected into the nude mice. We found that therewere no differences between the tumor growth rate between control group and theagomir-NC group; in contrast, the agomir-125b group showed much faster tumorgrowth in compared to the two groups (P<0.05). This suggested for the involvementof miR-125b in tumor growth in vivo.Though the above results showed that miR-125b could accelerate growth ofglioma cells, invasion and antiapoptotic abilities, and thus had important researchsignificance, the specific mechanism need to be further clarified.3. miR-125b influenced biological behavior of glioma via targeting Cx43.To investigate the mechanism underlying miR-125b influences on biologicalbehavior of glioma, potential miR-125b targets were identified using the databases,Targetscan, microRNA.org and miRBase. To determine if the identified sequencedownregulates Cx43mRNA translation following miR-125b binding, Cx433’UTRscontaining the target site (Cx43-3’UTR-wt) or a mutated site (Cx43-3’UTR-mut),were constructed in a luciferase reporter system. Subsequently, miR-125b and eitherluciferase empty vector or Cx43-3’UTR-wt or Cx43-3’UTR-mut plasmids wereco-transfected into HEK-293cells. HEK-293cells were incubated in RPMI1640medium containing10%fetal bovine serum at37°C in an incubator containing5%CO2. Full-length Cx433’UTR containing the miR-125b binding site, was PCR amplified and cloned into HindIII and SacI sites of the pMIR-REPORT miRNAsexpression reporter vector. The resulting vector was named Cx43-3’UTR-wt. A pointmutation of Cx43-3’UTR-wt was identified using the Easy Mutagenesis System andsubcloned also, with the resulting vector named Cx43-3’UTR-mut. HEK293T cellswere seeded into24-well plates and divided into three groups. Empty vector (400ng)was added to the control group, Cx43-3’UTR-wt plasmid (400ng) to the wild-typegroup, and Cx43-3’UTR-mut plasmid (400ng) to the mutation group. Plasmids andmiR-125b were transfected using Lipofectamine2000, and20ng pRL-TK was addedas an internal reference. Within each group, parallel wells were used for the additionof mimic-NC, a negative control for miR-125b mimic (miR-con). After36hours oftransfection, the fluorescence intensity of cells was measured using theDual-Luciferase Reporter Assay System. Luciferase assays found significantly lowerfluorescence intensities in HEK-293cells transfected with Cx43-3’UTR-wt,compared with negative controls (miR-con)(P <0.05). However, no significantdifference was detected between HEK-293cells transfected with Cx43-3’UTR-mutor empty vector (P>0.05; Figure3B). Moreover, western blot assays showed that inU87cells, miR-125b mimic (mimics of synthesized miR-125b, showing similareffects to natural miR-125b) significantly reduced (P <0.05), but miR-125b inhibitorsignificantly increased (P <0.05), Cx43levels.To determine if Cx43participates in miR-125b influences on glioma, wetransfected a plasmid containing the Cx43gene into U87cells. We found that,co-transfected with miR-125b, the clonogenic ability of Cx43overexpression U87cells was significantly lower than in the negative control (P <0.05). Moreover, theanti-apoptotic ability of Cx43overexpression U87cells was significantly lower thanthe negative control (P <0.05).These results suggest that miR-125b could bind with Cx43mRNA3’UTR anddownregulated its protein expression at the post-transcriptional level. Overexpressionof Cx43could block the effects of miR-125b on glioma.