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@#[摘 要] 脑胶质瘤（胶质瘤）具有高发病率、高病死率、高复发率及低治愈率的特点，胶质瘤细胞的无限增殖能力和高侵袭迁移 能力是胶质瘤治疗的难点，近年来微小核糖核酸（microRNA，miRNA）的出现为研究胶质瘤的发生及侵袭迁移机制提供了新的思 路。miRNA是一类内生的、长约20~24个核苷酸的非编码小RNA，可通过与其靶基因mRNA的3’UTR区域互补结合，从而在转 录后水平调控基因的表达。多项研究证实，miR-10b在胶质瘤组织和胶质瘤患者血清中高表达，并影响患者预后情况。miR-10b 可能通过影响其靶基因如PTEN、CDKN、 P53、HOXD10等的表达，参与胶质瘤细胞的增殖、侵袭和迁移等过程。深入研究miR10b对胶质瘤的调控作用及其机制，对该病的诊断、治疗和预后评估等均具有重要意义。
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Objective To analyze miR-10b expression level and the gene upstream methylation level in schwannomas so as to explore and identify the potential target genes for miR-10b in schwannomas .Methods The miR-10b with its potential target genes including HOXB 3 ,HOXD10 ,PTEN ,PIK3CA ,MAPRE1 and HADC4 were quantitatively analyzed by PCR in 13 cases of schwannomas and 6 cases of human vestibulocochlear nerves . We studied the correlation between the differentially expressed genes and the clinical characteristics of schwannomas . Finally ,the differences in miR-10b gene upstream methylation levels were measured and analyzed by pyrosequencing between schwannomas and normal vestibulocochlear nerves .Results Compared with that of normal nerves ,the expression level of miR-10b was significantly higher (P=0 .0003) while the level of PTEN was lower (P=0 .0047) in schwannomas .Negative correlation existed between the levels of miR-10b and PTEN (P=0 .001 , r= -0 .689) . Moreover ,the methylation level of the miR-10b gene promoter was downregulated in schwannomas ;it had negative correlation with the expression level of miR-10b (P= 0 .011 , r= -0 .571) .There was a significant difference in tumor mass diameter between miR-10b higher expression group and lower group (P=0 .016);however ,there was no difference in age or recurrence rate (P>0 .05) .Conclusion The downregulation of methylation level of the promoter leads to higher expression of miR-10b gene ,and it may targetedly inhibit the expression of PTEN .
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Objective To explore the of miR-10b cordribution in patients with non-small cell lung cancer (NSCLC) and adjacent tissues,and to investigate the effect of miR-10b on the malignant change of lung cancer cell A549 by regulating the expression of Kruppel-like factor 4 (KLF4).Methods Fourty patients with NSCLC were selected with lung cancer and in miR-10b expression adjacent tissues of lung cancer cell A549 transfected miR-10b mimics,changes of CCK-8 assay were used to detect the proliferation of lung cancer cells;real time PCR and Western blot were used to examine the cell KLF4 mRNA and protein levels;soft agar colony formation assay was used to detect the expression of miR-10b on the proliferation of lung cancer cell A549 tumor malignant.Results miR-10b expression in lung cancer A549 cells and lung cancer tissue were higher than that of normal lung epithelial 16HBE cells and cancer adjacent tissues;overexpression of miR-10banalogue in A549 cells,KLF4 protein levels significantly decreased,KLF4 mRNA was not changed significantly;miR-10b expression significantly increased the growth of A549 cells.Conclusions The distribution of miR-10b in different cell types and tissues may be different,which may promote the proliferation and malignancy of lung cancer cells by inhibitingthe expression of KLF4.
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@#To investigate the effects of miRNA-10b(miR-10b)on breast cancer proliferation and apoptosis in MCF-7 and MDA-MB-231, the miR-10b mimics used to increase the endogenous expression of miR-10b, and miR-10b inhibitor used to decrease the endogenous expression of miR-10b, were stably transfected into MCF-7 and MDA-MB-231 breast cancer cells. The expression level of miR-10b was determined by real-time PCR. The effects of miR-10b on proliferation were evaluated by MTT assay, while cell cycle assay and the apoptosis rate were measured by flow cytometry. Bioinformatic software was used to predict the potential targets of miR-10b, and 3′UTR luciferase reporter and qRT-PCR assay were used to verify a direct target of miR-10b. The expression levels of caspase-3 and p21 protein were measured by Western blot. Results confirmed that over-expression of miR-10b could promote the proliferation of breast cancer cells and inhibit the apoptosis by up-regulating the endogenous miR-10b, while the miR-10b inhibitor could restrain the proliferation of breast cancer cells and increase the apoptosis by reducing the endogenous miR-10b. In conclusion, miR-10b could negatively regulate the expression of caspase-3 and p21 by targeting TP53INP1, hence highlighting its potential as an oncogene in breast cancer cells.
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Objective To analyze the expression of hsa-miR-10b in three cervical cancer cell lines , and to find the target genes of hsa-miR-10b.Methods PCR was applied to measure expression levels of hsa-miR-10b in C-33A, HeLa and CaSki .The relative studies on hsa-miR-10b were retrieved from PubMed .The sequence and genome char-acteristics of hsa-miR-10b were analyzed on line by miRbase and NCBI .The target genes of hsa-miR-10b were searched by TargetScan , PicTar and miRanda , and then demonstrated by Gene Ontology and Pathway Enrichment analysis.Results Compared with C-33A, the expression of hsa-miR-10b significantly reduced in HeLa and Caski (P<0.01).Current studies showed that hsa-miR-10b was related with multiple tumorigenesis .hsa-miR-10b was lo-cated in human chromosome 2q31.1 and highly conserved among different species .The target genes were enriched in the biological processes of transcription , gene expression regulation , cell proliferation and etc .(P<0.05).Pathway analysis showed that these target genes were responsible for biochemical erents mediated by to the signaling pathways of cancer, cell cycle, ARF and etc.(P<0.05).Conclusions hsa-miR-10b may have extensive functions , and closely related with the occurrence and development in cervical cancer .Prediction of target genes provides a theoreti-cal basis for the further study .
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Objective To investigate the effect of miR-10b on migration and invasion of human hepatocellular carcinoma (HCC) cell lines.Methods Transwell assay was used to evaluate the motility and invasiveness in different HCC cell lines,qRT-PCR was then used to detect the expression levels of miR-10b in different HCC cell lines.Artificial mimics of miR-10b were transiently transfected into HepG2 cells,which have low expression of miR-10b,and then the changes in migration and invasion were evaluated by Transwell assay.Antagomirs of miR-10b (miR-10b-AS) were transiently transfected into MHCC97H cells,which have high expression of miR-10b,and then the changes in migration and invasion were evaluated as well.Cell morphology changes were detected by immunofluorescence and electron microscopy,Results There was a significant correlation of miR-10b expression level with cell motility and invasiveness.Low-level expression of miR-10b was observed in HepG2 cells,which exhibited weak motility and invasiveness; whereas high-level expression of miR-10b was observed in MHCC97H cells,which exhibited strong motility and invasiveness.Up-regulation of miR-10b expression in HepG2 cells increased cell motility and invasiveness,whereas inhibition of miR-10b reduced cell motility and invasiveness in MHCC97H cells.Immunofluorescence and electron microscopy results showed that up-regulation of miR-10b in HepG2 cells significantly increased proliferation of filopodia and lamellipodia,whereas inhibition of miR-10b decreased filopodia and lamellipodia amount in MHCC97H cells.Conclusion miR-10b is involved in the invasion and metastasis of HCC cell through regulation of cytoskeleton in vitro and inhibition of miR-10b is likely to be a new molecular target to block metastasis.