title: miR-221/222 is the regulator of Cx43 expression in human glioblastoma             cells.
abstract: The miR-221/222 cluster is significantly upregulated in malignant glioma             cells and regulates the expression of multiple genes associated with glioma cell             proliferation, invasion and apoptosis, which was shown in our previous studies.             Cx43 has been identified as a tumor suppressor and major component for the establishment             of gap junction intercellular communication (GJIC) in glial cells, which is frequently             reduced or deleted in high-grade gliomas. According to bioinformatic analysis,             connexin 43 (Cx43) may be one of the target genes of miR-221/222. The aim of the             present study was to validate Cx43 as a target gene of miR-221/222 and to determine             whether overexpression of miR-221/222 is one of the molecular mechanisms for the             reduced expression of Cx43 in malignant gliomas. We transfected miR-221/222 antisense             oligonucleotides (AS-miR-221/222) into U251 human glioblastoma cells using a lipofectamine             method. Northern blot analysis was conducted to detect the expression of the miR-221/222             cluster. Luciferase reporter assays were exploited to confirm Cx43 as a target             gene of miR-221/222. Cx43 expression was assessed by western blotting and immunofluorescence             staining. Scrape loading and dye transfer (SLDT) assays were used for examination             of GJIC. Proliferation and invasion of U251 cells were evaluated by MTT and transwell             assays, respectively. Cell cycle kinetics and apoptosis were determined with flow             cytometry. We found that expression of the miR-221/222 cluster was significantly             reduced while Cx43 expression was upregulated in U251 cells transfected with AS-miR-221/222,             and the GJIC deficiency in parental U251 cells was re-established. Moreover, the             luciferase activity determined by the luciferase reporter assay was enhanced in             AS-miR-221/222-treated cells, and cell proliferation and invasion were suppressed             while apoptosis was induced. We conclude that miR-221/222 function as oncogenic             microRNAs in human gliomas, at least in part, by targeting Cx43.
authors.affiliation: Department of Neurosurgery, Tianjin Medical University General Hospital, Tianjin, PR China.
authors.forename: Jianwei
authors.initials: J
authors.lastname: Hao
authors.forename: Chunzhi
authors.initials: C
authors.lastname: Zhang
authors.forename: Anlin
authors.initials: A
authors.lastname: Zhang
authors.forename: Kun
authors.initials: K
authors.lastname: Wang
authors.forename: Zhifan
authors.initials: Z
authors.lastname: Jia
authors.forename: Guangxiu
authors.initials: G
authors.lastname: Wang
authors.forename: Lei
authors.initials: L
authors.lastname: Han
authors.forename: Chunsheng
authors.initials: C
authors.lastname: Kang
authors.forename: Peiyu
authors.initials: P
authors.lastname: Pu
mesh_terms: D017209:Apoptosis / Q000235:genetics; D001483:Base Sequence; D002453:Cell Cycle / Q000235:genetics; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D018031:Connexin 43 / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D005909:Glioblastoma / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D035683:MicroRNAs / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020320:Oligoribonucleotides, Antisense / Q000737:chemistry / Q000378:metabolism
chemical_list: D018031:Connexin 43; D035683:MicroRNAs; D020320:Oligoribonucleotides, Antisense; C525709:MIR221, human; C525710:MIR222, human