Stat3-mediated Antisense Mir-21 Regulation Of Htert Inhibition Of Glioma Cell Growth

Research background:MicroRNAs (miRNAs) are a class of small non-coding RNAs and consist of 20-25ribonucleotides. The mature miRNAs with full or partial complementarity to the targetmRNAs direct the cleavage of target mRNAs or act as repressors of translation, involvedin modulating the development, differentiation, proliferation and apoptosis of cells.Recent studies have shown a distinct connection between miRNAs and the developmentof cancer, some of which are specially expressed in glioblastoma. We have collecteddifferent pathological diagnosis of malignant glioma, normal brain tissue as well asglioma cell lines, and then screen the glioma-specific expression of miRNAs usingmiRNAs microarray methods to find specific expression in human gliomas of miRNAs,miR-21 is one of them. As an oncomiRs, miR-21, has been reported to be dyregulated ina variety of cancer. In this study, we showed that reduction of miR-21 inhibited cellgrowth in GBM cells, accompanying a decreased expression of human telomerase reversetranscriptase (hTERT) mediated by STAT3 transcription. Further, this effect was exertedin a STAT3–dependent manner. Taken together, these results suggest that modulation ofthe mechanism responsible for miR-21 in GBM could be used as a critical therapeuticstrategy for GBM intervention and warrants further investigation.Methods:1. Selected human glioma cell line U87 and LN229 cells, which were cultured in 10%newborn calf serum DMEM medium, and placed in 37℃, 5% CO2 incubator, takinglogarithmic phase for cell experiments. The experiments were divided into antisensemiR-21 group, scramble group and blank group. The effects of the proliferation, cellcycle, apoptosis and related target genes were evaluated by MTT assay, Flowcytometric method.2. Engineered of lentiviral vector based siRNA construct against hTERT and divided theexperiment into hTERT siRNA group, negative control siRNA gruop, and blankgroup. The effects of the proliferation, cell cycle and apoptosis were evaluated by MTT assay and Flow cytometric method after the lentiviral vectors infected into U87and LN229 cells. Western blot assay and RT-PCR assay were used to study theexpression of hTERT in glioma cells atfer transfecte with antisense miR-21.3. STAT3 inhibitor WP1066 was used to inhibit the tyrosine phosphorylation of STAT3STAT3 in U87 and LN229 cells. Western blot assay, RT-PCR assay, Luciferasereptors and Chromatin immunoprecipitation assay were used to study whether hTERTwas regulated by STAT3, and verified that STAT3 could be the critical turning pointin the effect of miR-21 suppressed hTERT.4. To correlate the growth inhibitory effects with anti-tumor effects of As-miR-21, weemployed a LN229 glioma cells xenograft model, and evaluated the targe geneswhich regulated by miR-21.ResultsResults:1. Reduction of miR-21 by antisense oligonucleotide inhibited cell growth, arrested thecell at G0/G1 phases, and induced cell apoptosis in U87 and LN229 GBM cell, with adecreased expression of STAT3, pSTAT3 as well as hTERT.2. Knockdown of hTERT expression using lentiviral vector mediated delivery ofhTERT-siRNA triggered cell apoptosis and cell cycle G1/G0 arrest in U87 andLN229 cells.3. ChIP showed the presence of STAT3-binding sites in the hTERT gene. Luciferasereporter assay revealed that the hTERT gene promoter was direct bound by STAT3.Furthermore, U87 and LN229 cells that treated with miR-21 antisenseoligonucleotide and IL-6 which could elevate the expression of activated STAT3showed a significant increase of hTERT expression, whereas cells that treated withmiR-21 oligonucleotide and STAT3 inhibitor WP1066 displayed a reduction ofhTERT expression.4. Knockdown of miR-21 considerably inhibited tumor growth and diminished theexpression of STAT3, pSTAT3 and hTERT in xenograft model, which furthercomfirmed that miR-21 could decresed the expression of STAT3 and hTERT. ConclusionsConclusions:1. Reduction of miR-21 by antisense oligonucleotide inhibited cell growth in U87 andLN229 GBM cells, with a decreased expression of hTERT. Further, we found thatSTAT3 was an important mediator between miR-21 and hTERT. Reduction ofmiR-21 repressed STAT3 expression and STAT3 phosphorylation.2. STAT3 inhibition led to a remarkable depletion of hTERT both at mRNA and proteinlevels by binding to the hTERT gene promoter. Moreover, activation of STAT3activity abrogated miR-21-induced hTERT expression whereas inactivation ofSTAT3 activity overrode As-miR-21-repressed hTERT expression. Finally,knockdown of miR-21 considerably inhibited tumor growth and diminished theexpression of STAT3 and hTERT in xenograft model.These results indicate for the first time that miR-21 inhibits cell growth throughregulating hTERT expression in a STAT3–dependent pathway in GBM cells.