The Study Of MiR-21 Mediates Human Glioma Growth

Human glioma was the most frequent human brain tumor and one year survival rate of most high-grade glioma was 46%. The oncogenesis of glioma and effective therapy method was the most core theme of neurosurgery and euro-oncology. Recently, some tumor oncogenesis correlated molecular events, including EGFR over expression, PTEN and p53 gene mutant, were discovered with the primary development in molecular-biology and molecular genetics study in glioma. It had been proved that, glioma oncogenesis was a complex process which including cell cycle progression disequilibrium, abnormal expression of signaling pathway key components, cell apoptosis, death or cell metabolism disorder. With thorough study of brain tumor, the molecular-pathology difference that contributes to the occurrence of primary and secondary glioma was still obscure. Thus, to study the signaling pathway key components' abnormal expression could better assess the essence of glioma than studying one single gene expression disorder.MiRNA was a class of～22bp small non-coding RNA which can regulate gene expression at the post-transcription level by perfect or imperfect complementarity to human the seed sequence in 3'UTR of its target gene. Each miRNA may have several or more targets and one protein expression could be regulated by several miRNA. The miRNAs that had been verified contributed to 1/3 of gene expression in nature. Therefore, because of miRNA's fine and extensive gene expression regulation status, it had been on focus in tumor molecular biology study field.The present study emphasis on miRNA expression disorder, tumor malignant phenotype changes by targeting disorder expressed miRNA by antisense oligonucleotide, EGFR/PI3K-AKT signaling pathway interactions with miRNA and possible mechanism. The present study was clarified into 4 parts:MethodIn chapter one, miRNA microarray chip was used to analyze the miRNA expression profile in one astrocytoma and five glioblastoma cell line to scan a class of up-regulated or down-regulated miRNAs. Among these, miR-21 was the only miRNA which was over expressed across 6 glioma cell line. Real-time PCR and in-situ hybridization method was used to identify over expression status of miR-21 in 60 glioma samples, 10 medullablastoma samples and 6 glioblastoma cell lines. miR-21 genomic coding region was amplified by PCR and the PCR products were sequenced to detect the mutant status. In chapter two, miRNA target prediction model was established and combined with the classic database, we scan several candidate target mRNA. The 3'UTR of the predicted target mRNAs were cloned into a luciferase reporter and luciferase assay was conducted to identify the real target of miR-21.In chapter three, Oligofectamine reagent was used to transfect antisense miR-21 oligo nucleotide suppress U251 glioma cell line growth in vitro and the malignant phenotype was observed. Real-time PCR and in-situ hybridization method was used to prove AS-miR-21 's effect; immuno-fluoresence and western blot were conducted to detect PI3K-AKT signaling pathway components, malignant phenotype related proteins, and the notable identified tumor suppressor Septin-7 expression; MTT assay, Annexin V, flow-cytometry assay, 2D,3D martrigel growth assay were used to detect the U251 cell proliferation, apoptosis, cell cycle distribution and invasion changes after knocking down miR-21 expression in vitro.In chapter four, U87 and U251 nude mice xenograft tumor model was established and the mixture of AS-miR-21 and oligofectamine was injected into the xenograft tumors every three days. The tumor volume was calculated to evaluate the anti-tumor effect of AS-miR-21. Immuno-histochemistry stain and TUNEL assay were laid out to further prove the results of the present study results in vitro.ResultsIn chapter one, the miRNA expression profile indicated, 8 miRNAs, including miR-21, were 2 folds higher over expressed across the cell lines; 18 miRNAs, including miR-127, were 50% less expressed than in the normal brain control. Real-time PCR results proved that, miR-21 was over-expressed in glioma samples. In addition, in-situ hybridization method miR-21 condition in 60 glioma samples was positively correlated to the pathological classification criteria and the PI3K-AKT, EGFR signaling pathway components; and its expression was negatively correlated to PTEN, Septin-7 expression across the 60 WHO classified gliomas.In chapter two, the previous established math model shown that, PTEN and Septin-7 were the possible targets of miR-21. The score of PTEN gene mRNA was 93.837, freedom was -7, the predicted seed sequence started form the 23rd base of PTEN mRNA 3'UTR; The score of Septin-7 gene mRNA was 94.0019, freedom was -8, the predicted seed sequence started form the 515th base of Septin-7 mRNA 3'UTR. Luciferase assay replied that, luciferase activity of the pGL-3-luc and AS-miR-21 co-transfection groups were significantly higher than it in the pGL-3-luc control group.In chapter three, Real-time PCR and in-situ hybridization method shown, AS-miR-21 can suppress the miR-21 expression in U251 cell effectively. MTT curve indicated, the U251 cell survival rate is lower than that in the AS-miR-21 treated group; IF and WB results proved that EGFR, PI3K-AKT signaling pathway components proteins and Bcl-2 protein were down regulated; tumor suppressors including Septin-7, TIMP-1 were up regulated; flow-cytometry assay verified, in the AS-miR-21 treated tumor cell group, cell cycle was blocked at G0/G1 phase and the early phase was induced accordingly.In chapter four, every 3 days the mixture of oligofectamine and AS-miR-21 was injected into the U87 and U251 xenograft tumor model. By the end of observation interval, it had been proved that, the AS-miR-21 treated xenograft tumor volume was significantly inhibited than that in the control and scramble treated groups. IHC shown, EGFR, PI3K-AKT signaling pathway components proteins, tumor malignancy related protein including Ki67, CyclinD-1, MMP-9 and Bcl-2 protein were down regulated; tumor suppressors including Septin-7, TIMP-1 were up regulated. TUNEL assay indicated that the apoptosis nuclei index was higher than in the control and scramble groups. The in vivo study results were equal to the results in vitro.ConclusionA class of miRNA is abnormally expressed in human gliomas and miR-21 can be treated as a molecular signature of human glioma and glioblastoma.There are direct binding sites reside in the 3'UTR of PTEN and Septin-7 mRNA.Oligofectamine mediated AS-miR-21 transfection can effectively suppress the miR-21 expression in U251 glioblastoma cell line. ASmiR-21 can inhibit EGFR, PI3K-AKT signaling pathway activity; suppress glioma cell proliferation, invasion, and migration; block cell cycle at G0/G1 phase; induce tumor cell apoptosis.Oligofectamine mediated AS-miR-21 treatment in human gliobalstoma xenograft model can get the similar results as the in vitro study.MiR-21 can be a gene therapeutic target candidate in human glioma. Antisense oligonucleotide transfetion is an effective approach to inhibit over expressed miRNA and it has optimistic clinic trail prospects.