Effects And Mechanism Of Long Non-coding RNA GAS5 On Biological Behaviour Of Huamn Glioma Cells

Background:Glioma is the most common primary brain tumor in humans. The highly malignant glioma represents over 50% of all types of primary glioma. The vast majority of glioma develops quite rapidly and invades normal brain tissue quickly. Traditional treatments such as surgical procedures, adjuvant radiotherapy and chemotherapy have inherent limitations. Advancements in gene therapy and immunotherapies in the past decades have not significantly increased the overall survival of patients with glioma. Discoving a key gene in the initiation and progression of glioma will further assist in identifying therapeutic targets for glioma treatment.More than 90% of transcribed RNA is classified as non-coding RNA. long non-coding RNA(lnc RNA) are transcripts with lengths greater than 200 bases. Recent studies show lnc RNAs possess multiple biological functions which play a critical role in biological behaviour of cancer cells. Emerging evidence reveals that lnc RNAs which function as oncogenes and tumor suppressors are closely associated with tumor proliferation, apoptosis, migration and invasion et al. The main underlying mechanisms include transcriptional or posttranscriptional regulation. In recent years, an accumulating number of reports have revealed that lnc RNAs may regulate the level of genes through competitively binding with mi RNAs.Our preliminary studies showed lnc RNA GAS5(Growth Arrest-Specific 5) could function importantly in glioma. This study was aimed to explore the role of GAS5 in cell growth, cell cycle, migration and invasion of human glioma cells and underlying mechanism. Objective:To investigate the effects of GAS5 on proliferation, cell cycle, migration and invasion of human glioma cells, and further studies were conducted to explore the mechanism of GAS5 playing the role on human glioma cells. Methods:1. MTS assay, flow cytometry, wound healing assay, the invasion assay and tumorigenicity in nude mice experiments were applied to study the function of GAS5 after overexpression or knock down of GAS5.2. Bioinformatics techniques were used to predict the possible micro RNAs which might interact with GAS5;3. Real-time quantitative PCR(QRTPCR) was performed to validate which target micro RNA could be downregulated by GAS5.4. To determine whether target micro RNA is able to negatively regulate GAS5, the expression of GAS5 was detected by QRTPCR after introducing target micro RNA mimincs or inhibitor into U251 cells.5. The expression of GAS5 and mi R-18a-5p in tissue microarrays(including glioma tissue and normal brain tissue) were detected by ISH(In situ Hybridization) for studying the relationship between GAS5 and target micro RNA.6. Bioinformatics techniques were applied to predict the biding site between GAS5 and target micro RNA and further the biding site would be validated.7. QRTPCR was conducted to detect the expression of pre-mi RNA or pri-mi RNA after overexpression of GAS5.8. RNA precipitation was performed to confirm whether this reciprocal repression of GAS5 and target micro RNA involves RISC.9. Western blot was applied to detect the target gene of target micro RNA for revealing the pathway. Results:1. Overexpression of GAS5 attenuated U87 and U251 cells proliferation, migration and invasion in vitro. Knock down of GAS5 increased the ability of proliferation, migration and invasion in both U87 and U251 cells. Compared with vector control, GAS5 significantly suppressed tumor growth in nude mice.2. Bioinformatics techniques predicted 46 micro RNAs could interact with GAS5.3. After expression profiling of 46 micro RNAs, mi R-18a-5p repressed by GAS5 was validated. Meanwhile, Knock down of GAS5 increased mi R-18a-5p expression.4. Overexpression of mi R-18a-5p reduced the GAS5 level while knock down of mi R-18a-5p increased the GAS5 expression.5. ISH showed upregulation of GAS5 and downregulation of mi R-18a-5p in normal brain tissue while upregulation of mi R-18a-5p and downregulation of GAS5 in glioma tissue.6. Bioinformatics techniques predicted one biding site between GAS5 and mi R-18a-5p. The GAS5 construct with deletion of binding sites(d-GAS5) had no effect on the expression of mi R-18a-5p. MTS experiments concluded d-GAS5 inhibits U251 cell growth less than GAS5.7. GAS5 overexpression had no effect on the expression of pri-mi R-18a-5p or pre-mi R-18a-5p, suggesting a possible post-transcriptional regulation involved.8. RNA precipitation found that both GAS5 and mi R-18a-5p are associated with the RISC complex through which GAS5 is able to reduce mi R-18a-5p level and vice versa.9. Western blot results showed the target gene of mi R-18a-5p, Neogenin, was upregulated after overexpression of GAS5. Conclusion:1. GAS5 inhibits glioma cells proliferation, migration and invasion.2. The reciprocal repression of GAS5 and mi R-18a-5p involves RISC.3. Gas5 may serve as a tumor suppressor by downregulating mi R-18a-5p level and further increasing the expression of the target gene of mi R-18a-5p, Neogenin.