| Glioma is the most common primary tumor of the central nervous system. Owing to the invasive growth and rapid cell proliferation, the malignant glioma is hardly to be cured by the currently available combined therapeutic approaches, such as surgical resection, postoperative radiotherapy, chemotherapy and immunotherapy etc. Its high recurrence rate seriously affects the life quality and overall survival of the patients, and the prognosis is poor. This situation fully reflects our poor understanding of the etiology, pathogenesis and treatment strategy of gliomas. Therefore, a more comprehensive understanding of the molecular pathology of gliomas must be sought for optimizing treatment strategies and development of novel therapeutic approaches.Our previous study on the gene expression profiles of 63 glioma samples with different types and grades by Atlas cDNA microarray analysis demonstrated that the expression of SEPT7 gene, a member of septin family, was downregulated in a variety of gliomas and it was validated further in an additional group of glioma samples. On the basis of this finding, we constructed SEPT7 eukaryotic expression vector and transfected it into glioma cell lines in which SEPT7 was downregulated or deleted. It was shown that SEPT7 could inhibit the proliferation and invasion of gliomas, and induce apoptosis of tumor cells as well in vitro and in vivo. These evidences indicated that SEPT7 could be a new tumor suppressor gene. Moreover, we have previously profiled miRNA expression in glioma cell lines, and found that some human miRNAs were aberrantly expressed in gliomas, including overexpression of miR-21,miR-221/222,miR-19a/b and miR-30a-5p etc. In order to find the microRNAs in gliomas that could regulate SEPT7, we searched several microRNA target prediction database, such as TARGETSCAN and MIRANDA and 50 different miRNAs were predicted to have SEPT7 as putative target, including miR-19a/b and miR-30a-5p. The result was coincident with that found from HuMiTar algorithm for prediction of microRNA targets developed by Department of Mathematics, Nan Kai University and our lab. So the present study is to focus on exploring the modulation of SEPT7 and the regulation of malignant phenotype in gliomas by miR-19a/b and miR-30a-5p. The present study was divided into three partsIn the first part of this study, miR-19a/b and miR-30a-5p expression was examined in 8 malignant glioma cell lines and 43 freshly resected glioma samples by Real time PCR. It was found that these microRNAs were upregulated in gliomas, and their expression was positively correlated with the tumor grade. We also carried out In Situ Hybridization in tissue array to detect the microRNA expression in 75 glioma samples and 5 normal brain tissues and the result was coincident with that of Real time PCR examined.The second part of this study was focused on the validation of SEPT7 being the target of miR-19a/b and miR-30a-5p. Real time PCR was conducted to demonstrate the overexpression of miR-19a/b and miR-30a-5p in transfected glioma cells. The Luciferase reporter assay was used to identify the direct regulation of miR-19a/19b and miR-30a-5p on SEPT7. Besides SEPT7, PTEN and P53, the two tumor suppressor genes known to play important roles in gliomagenesis, also have been predicted as potential targets of miR-19a and miR-30a, respectively. So we detected the direct regulation of PTEN or P53 by miR-19a/b or miR-30a-5p simultaneously with Luciferase reporter assay. Expression of SEPT7 in glioma cells transfected with AS-miR-19a/b and AS-miR-30a-5p (Antisense oligonucleotides against miR-19a/b and miR-30a-5p) was determined by RT-PCR and Western blot analysis, It was shown that in cells transfected with AS-miR-19a/b or AS-miR-30a-5p, the expression of miR-19a/b or miR-30a-5p was significantly reduced, whereas the expression of SEPT7 was upregulated and no significanct alteration of mRNA expression of SEPT7 was observed. This finding indicates that the inhibition of SEPT7 expression is at posttranscriptional level. We also observed in the cells transfected with AS-miR-19a/b and PGL3-SEPT7 or PGL3-PTEN, the luciferase activity became stronger, and the luciferase activity was much stronger in the cells transfected with AS-miR-19a+ AS-miR-19b and PGL3-SEPT7 or PGL3-PTEN. We also found that in the cells transfected with AS-miR-30a-5p and PGL3-SEPT7 or PGL3-P53, the luciferase activity was significantly enhanced. Therefore, it can be concluded that SEPT7 and PTEN are targets of miR-19a/b and miR-30a-5p can directly target SEPT7 and P53.In the third part, we transfected AS-miR-19a/b to the human glioma cell lines SNB19, LN229, U251 and AS-miR-30a-5p to U251, LN308, U87 glioma cells. In order to observe the tumor-promoting effect of miR-19a/b (oncomiR)and miR-30a-5p (oncomiR), and tumor-suppressing effect of SEPT7, we set up cell groups in which AS-miR-19a/b combined with SEPT7 siRNA (SEPT7 si) or PTEN siRNA (PTEN si), and AS-miR-30a-5p combined with SEPT7 si or P53 siRNA (P53 si)were transfected. Thus. the glioma cells were grouped as follows:control cells, cells transfected with scramble oligonucleotides, with AS-miR-19a, with AS-miR-19b, with AS-miR-19a+ AS-miR-19b, with SEPT7 si, with AS-miR-19a+AS-miR-19b+SEPT7 si and with AS-miR-19a+AS-miR-19b+PTEN si (detected only in LN229 cells with the wild type of PTEN), and control cells, cells transfected with scramble oligonucleotides, with AS-miR-30a-5p, with SEPT7 si, with AS-miR-30a-5p+SEPT7 si, with AS-miR-30a-5p+P53 si (detected only in U87 cells with the wild type of P53). Real time PCR was conducted to detect the expression of miR-19a/b and miR-30a-5p in transfected cells. The cell proliferation rate was determined by 3-(4,5-Dime--thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and cell cycle kinetics was detected by flowcytometry. The cell apoptosis was examined by Annexin V assay and invasive ability was evaluated by transwell assay. The results showed that the expression of miR-19a/b or miR-30a-5p in the cells transfected with antisense oligonucleotides was significantly downregulated. The cell proliferation activity and invasive ability were reduced, cells were arrested in G0/G1 phase, and apoptosis was induced in cell groups transfected with AS-miR-19a, AS-miR-19b or AS-miR-30a-5p as compared to those of the cells transfected with scramble oligonucleotide and control cells, and the phenotypic changes were more obvious in the AS-miR-19a+AS-miR-19b group, whereas in the SEPT7 si group that the cell proliferative rate was promoted, cell cycle accelerated and cells became more invasive. It was also shown that the effects of AS-miR-19a+AS-miR-19b+SEPT7 si group and AS-miR-19a +AS-miR-19b+PTEN si had no significant difference, and there were also no statistical significant differences among these groups and AS-miR-19a or AS-miR-19b group. Similarly, the alteration of malignant phenotype of glioma cells including cell proliferation, invasion or apoptosis showed no significant difference among AS-miR-30a-5p+SEPT7 si group, AS-miR-30a-5p+P53 si group and control group. Therefore, it can be concluded that the tumor-suppressing effect of SEPT7 is almost the same as PTEN and P53; miR-19a/b and miR-30a-5p are oncomiRNAs. Both of them can promote the growth of glioma by negatively regulating SEPT7, PTEN and SEPT7, P53, respectively.Conclusion:The results demonstrate that miR-19a/b and miR-30a-5p expression are significantly increased in the majority of the gliomas and its expression is positively correlated with the tumor grade. Transfection of AS-miR-19a/b or AS-miR-30a-5p into the glioma cells is able to inhibit the glioma cell proliferation activity, invasive ability, arrest the cells in G0/G1 pahse, and induce cell apoptosis. We also showed that miR-19a/b affected the malignant phenotype of glioma cells through the regulation of SEPT7, PTEN and miR-30a-5p by targeting SEPT7 and P53, respectively. Thus, miR-19a/b and miR-30a-5p are identified as oncomiRs which implicate that they can be candidate targets for gene therapy of gliomas. To our knowledge, this is the first comprehensive study on the expression and the potential mechanism of miR-19a/b and miR-30a-5p in gliomagenesis. These evidences provide us the molecular pathologic machnism and a new class of targeted therapeutic strategy for human gliomas.However, since one microRNA may have hundreds of targeting genes involved in a variety of cellular processes, so exploring the genes associated with gliomagenesis regulated by miR-19a/b and miR-30a-5p should be expanded. On the other hand, SEPT7 gene can be modulated by multiple miRNAs besides miR-19a/b and miR-30a-5p, and that also needs to be studied further. |
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