MiR-223-3p Regulates Cell Growth And Chemosensitivity To Temozolomide By Targeting STMN1 In Glioma

Human gliomas, accounting for 35～60% of all brain tumors, is considered as one of the most malignant and aggressive tumors of the central nervous systim. Despite of the treatment with a combination of neurosurgery, radiotherapy, and chemotherapy, gliomas remains lethal with a median patient survival of 14.6 months, being considered as one of the worst 5-years survival rates of all human cancers. The poor prognosis of gliomas patients largely attributed to the rapid tumor growth, invasion and high rate of recurrence. Temozolomide (TMZ), a DNA-methylating antineoplastic agent that easily crosses the blood-brain barrier, is considered as a promising chemotherapy schema for gliomas treatment. TMZ can inhibit cell proliferation and induce cell apoptosis. Patients receiving a treatment combined radiotherapy with chemotherapy using TMZ tend to have significantly longer overall and progression-free survival, however, a major limitation to this therapy was deemed to be acquiring chemoresistance to TMZ in cancer cells.STMN1 (Stathmin) is a 19kDa cytosolic protein that regulates microtubule dynamics through either sequestering free tubulin heterodimers or promoting microtubule depolymerization and catastrophe. It has been reported that overexpression of STMN1 was observed in a wide variety of human malignancies, such as prostate cancer, breast cancer, non-small cell lung cancer, hepatoma, gastric cancer, and colorectal cancer. The expression of STMN1 gene has been associated with the proliferation and metastasis of cancer cells and correlated with invasion. In our previous study, we found that the expression of STMN1, as compared with that in the parental cell line U87, was up-regulated in U87TR cultured in medium with TMZ (110 ug/mL) to maintain the multidrug resistant (MDR) phenotype. Thus, STMN1 may be a potential target of chemotherapy in gliomas.MicroRNAs (miRNAs) are small noncoding RNAs of 18-22 nucleotides in length that regulate expression of specific target proteins by either transcriptional inhibition or degradation of corresponding mRNA. miRNAs have been revealed to play important roles in a great variety of tumorigenic function such as proliferation, invasion, angiogenesis and metastasis. Aberrant expression of miRNA has been shown in most human malignancies, including gliomas. In a bioinformatics analysis, we found that the STMN1 might be a potential target for miR-223-3p. miR-223-3p was described as a modulator of hematopoietic lineage differentiation and was found deregulated in several types of cancers.Thus, our study was aimed at illuminating the potential involvement of STMN1 in gliomas. We found that STMN1 might play an important role in the progress of cell proliferation, invasion, migration and chemoresistance in gliomas cell, probably targeted by miR-223-3p.Chapter I The expressions of STMN1 in drug-resistance glioma cells and gliomas specimen.Objective:To investigate whether the expressions of STMN1 was involved in temozolomide resistance of glioma cells and analyze the relationship between STMN1 and gliomas.Methods:Temozolomide resistant cell line U87TR was established by culturing U87 cells to temozolomide with increasing concentrations persistently. CCK-8, qRT-PCR and Western blot to test U87TR cell line biological characteristics. STMN1 mRNA and protein expression were detected in U87TR cells, each grade glioma tissues were collected from Zhujiang Hospital Department and Nanfang Hospital Neurosurgery Department. Using qRT-PCR to detect the expression level of miR-223-3p in gliomas tissues and temozolomide-resistant cell lines U87TR. Spearman correlation to analyze the correlation between miR-223-3p and STMN1 in glioma tissues. We also detected STMN1 mRNA in normal brain tissue and different levels of glioma tissue by qRT-PCR, immunohistochemistry to analyze the expression of STMN1 in different grade glioma tissues.Results:A temozolomide-resistant cell line U87TR was constructed by using our established method. U87TR cells were significantly more resistant to TMZ treatment than U87 cells through measuring the cell survival rate with CCK-8 assay (Figure. 1A). The mRNA expressions of MDR-associated protein genes MDR1, BCRP and MRP were detected by qRT-PCR in U87TR cells compared to parental cells U87 (Figure. IB), and Western blotting was performed to evaluate the protein expressions of MDR1 and BCRP. The cellular morphology of U87TR cells was changed significantly observed under microscopy, compared with that of the parent cells U87. The levels of MDR1, BCRP and MRP were significantly higher in the temozolomide-resistant cells than that in the U87 cells. In this study, we identified that STMN1 expression was up-regulated in U87TR cells compared to parental U87 cells. These results indicated that STMN1 might be a key therapeutic target for glioma.Conclusions:STMN1 expression involved in multi-drug resistance and was was up-regulated in glioma specimens.Chapter Ⅱ miR-223-3p regulates STMNl expression in glioma cells.Objective:To verify whether STMN1 was a direct target gene of miR-223-3p in glioma.Methods:Using bioinformatics to predict the binding sites of STMN1 3’UTR and miR-223-3p, miR-223-3p mimics, NC (miRNA mimics negative control), miR-223-3p NC inhibitor and miR-223-3p inhibitor were transfected into U251 and U87 glioma cells by lipofectin transfection. qRT-PCR and Western blot to test the STMN1 mRNA and protein levels of U251 and U87 cells transfected with nucleotides, and analyze the impact of miR-223-3p on STMN1 in glioma cell lines. Dual luciferase reporter gene system validation for regulatory impact of miR-223-3p on STMN1 expression.Results:miR-223-3p exert negative regulatory effects on STMN1 mRNA and protein expression in glioma cells.Conclusions:STMN1 gene mRNA 3’UTR is directly regulated target of miR-223-3p. STMN1 is a direct target of miR-223-3p in glioma.Chapter ⅢmiR-223-3p regulated glioma cell growth and drug resistance by targeting STMN1.Objective:To verify whether miR-223-3p regulates glioma cell proliferation, invasion, and metastasis by targeting STMN1, and whether the regulates temozolomide chemotherapy resistance of glioma cells.Methods:Chemical synthesis nucleotides miR-223-3p mimics, NC (miRNA mimics negative control) were transfected into human glioma cell lines U87 by liposome-mediated transfection. qRT-PCR and Western blot analysis the effect of miR-223-3p on STMN1 mRNA and protein expression. CCK-8 kit to detect the effects of miR-223-3p on glioma cell growth and drug sensitivity assay to detect the impact of miR-223-3p on glioma cells temozolomide chemotherapy resistance. Transwell assay to detect the effect of miR-223-3p on glioma cell invasion and metastasis. siRNA-STMN1 was transfected into human glioma cell line U251 by liposome-mediated transfection. qRT-PCR and Western blot to detect the mRNA and protein STMN1 genes interference efficiency by siRNA-STMN1. CCK-8 kit to detect the effects of siRNA-STMN1 on glioma cell proliferation and rug sensitivity assay to detect siRNA-STMN1 impact on temozolomide chemotherapy resistance. Transwell model to certify the siRNA-STMN1 influence on glioma cells glioma cell invasion and metastasis.Results:STMN1 mRNA and protein levels were significantly down-regulated in miR-223-3p mimics transfected U87 glioma cells than miR-NC transfected U87 cells. the expression of STMN1mRNA and protein in siRNA-STMN1 transfected U251 cells were significantly reduced than U251 transfected with siRNA-NC. miR-223-3p could inhibit the invasion, metastasis, proliferation of U87 cells and increase sensitivity to temozolomide chemotherapy. Also, siRNA-STMN1 could inhibit the proliferation, invasion and metastasis of U251 cells, and increase temozolomide chemotherapy sensitivity.Conclusions:miR-223-3p suppressed the proliferation, invasion and migration of glioma cells by targeting STMN1, and increased temozolomide chemotherapy sensitivity.