| Aims:Glioma is the most common primary brain tumors,and its incidence ranks first in all brain tumors.Type IV glioma shows the most malignant among all types of gliomas.Although generally carried out in a variety of clinical treatments including surgery, radiotherapy and chemotherapy,the prognosis of patients with gliomas remains poor, and the5-year survival rate was only10%.Since IV glioma has strong invasiveness as well as resistance to existing chemotherapy,more research is urgently needed to optimize treatment. Exploration of novel therapeutic targets for the development and progression of gliomas may show promises for improving the clinical treatment of gliomas.Recently, several studies have shown that RAS/RAF signaling pathway plays an important role in the development and progression of glioma. Raf-1kinase inhibitor protein (RKIP)is a member of phosphatidylethanolamine family,which was involved in regulation of cancer. Recent studies showed that RKIP could inhibit Raf-1-MEK1/2-ERK1/2and NF-κB signaling pathway, offset their pro-survival and anti-apoptosis activities,resistance to apoptosis,and inhibit the growth of tumor cells.Overexpression of RKIP can enhance the sensitivity of tumor cells to chemotherapeutic drugs. However, the detailed role of RKIP in gliomas as well as the involved mechanism is unclear.MicroRNA is a class of19-25length small RNA. Since first discovered in C.elegans in1998,the expression and function of microRNAs have been widely concerned.MicroRNAs do not encode proteins,but inhibit gene expression at a post-transcriptional level, through directly binding to the3’-untranslated region of its target mRNA, inhibiting mRNA translation or inducing their degradation. In recent years, the potential application of microRNAs in diagnosis and treatment for malignant glioma has attracted more and more attention. Many studies showed that microRNAs were involved in the regulation of glioma cell biological processes,such as survival,apoptosis,proliferation, cell cycle progression, migration, invasion and angiogenesis.In addition, there is a significant difference in microRNAs expression between glioma tissues and adjacent tissues,suggesting that microRNAs may be used as an important tool for the diagnosis of gliomas.Recent studies have found that microRNA-98plays an important regulatory role in the development, progression as well as drug resistance in various cancers,such as prostate cancer, squamous cell carcinoma, lung cancer, and so forth.However, the role of microRNA-98in glioma has not been reported. The present study found that HMGA2was a direct target of microRNA-98.HMGA2is a member of high mobility protein family A group,which is involved in the regulation of epithelial-mesenchymal transition in tumor cells through affecting transcription factor activity, and thus participates in tumor metastasis.However, the role of HMGA2in glioma cells remains largely unknown.This study aims to investigate the role of RKIP in gliomas may be closely related to the regulatory effect of microRNA-98on HMGA2. First, we collected26cases of glioma tissues, and found that the expression of RKIP was significantly reduced compared to adjacent tissues,suggesting that RKIP may play an important role in gliomas. After that, we further investigated the relationship between RKIP and microRNA-98in glioma, as well as the targeting relationship between microRNA-98and HMGA2in glioma cells. Methods:(1)26cases of glioma tissues and glioma U251,U87and SHG44cell lines were collected, and determined the expression levels of RKIP, microRNA-98and HMGA2using Real-time PCR and Western blotting in glioma tissues and glioma cells,respectively, Adjacent tissues and normal glial HEB cells were used as controls.(2)Dual luciferase reporter assay was performed to determine whether microRNA-98could direct target HMGA2.(3)Glioma U87and U251cells were transfected with lentivirus to overexpress RKIP.Western blotting was performed to determine the transfection efficiency. Real-time PCR was used to detect the expression level of microRNA-98,and western blotting was performed to examine the protein expression of HMGA2.(4)In glioma U87and U251cells, BrdU assay was performed to determine the cellular proliferation after overexpression of RKIP.(5)In glioma U87and U251cells, Transwell assay was performed to determine the cellular invasion after overexpression of RKIP.(6)Glioma U87and U251cells were transfected with microRNA-98mimic, and real-time PCR was performed to determine the transfection efficiency. Besides, co-transfection of microRNA-98and RKIP was performed. Then the expression level of microRNA-98was determined in each group.(7)Groups were set as follows:Control, RKIP overexpression, microRNA-98+RKIP overexpression. Western blotting was performed to examine the protein level of HMGA2in each group.(8)Groups were also set as follows:Control, RKIP overexpression, microRNA-98overexpression, microRNA-98+RKIP overexpression. BrdU assay was performed to determine the cellular proliferation and Transwell assay was used to determine the cellular invasion in each group.Results:(1)The expression levels of RKIP and microRNA-98were notably reduced in glioma tissues, while the expression of HMGA2was significantly increased in glioma tissues, when compared to adjacent tissues. Besides, it is positive correlated between microRNA-98and RKIP,while negatively correlated between microRNA-98and HMGA2in glioma tissues.(2)The expression levels of RKIP and microRNA-98were notably reduced in glioma SHG44,U251,U87cells, while the expression level of HMGA2was significantly increased in glioma SHG44,U251,U87cells. Besides, these differences were significant in U251and U87cells.(3)Dual luciferase reporter assay showed that microRNA-98could directly bind to the3’-UTR in HMGA2mRNA. (4)In glioma U87and U251cells, overexpression of RKIP promoted the expression of microRNA-98while inhibited the expression of HMGA2as well as the invasion ability, without any effect on cellular proliferation.(5)Co-transfection of microRNA-98and RKIP further promoted the expression of microRNA-98compared with single transfection of microRNA-98in glioma U87and U251cells.(6)Co-transfection of microRNA-98and RKIP further inhibited the HGMA2expression compared with single transfection of RKIP in glioma U87and U251cells.(7)Co-transfection of microRNA-98and RKIP showed no effect on cell proliferation compared with single transfection of microRNA-98or RKIP,respectively, in glioma U87and U251cells.(8)Co-transfection of microRNA-98and RKIP further inhibited cellular invasion ability compared with single transfection of RKIP or microRNA-98,respectively, in glioma U87and U251cells.Conclusions:(1)The expression levels of RKIP and microRNA-98were significantly downregulated in glioma tissues as well as glioma cell lines.(2)It was positive correlated between RKIP and microRNA-98expression in glioma tissues.(3)Overexpression of RKIP promoted the expression level of microRNA-98,inhibited cell invasion,while showed no effect on cellular proliferation in glioma cells.(4)The expression level of microRNA-98was significantly upregulated in glioma tissues and glioma cell lines.(5)It was negatively correlated between HMGA2and microRNA-98expression in glioma tissues.(6)HMGA2was a direct target of microRNA-98.(7)It was suggested that RKIP might inhibit glioma cell invasion through inhibition of HMGA2by upregulating microRNA-98expression.(8)RKIP/microRNA-98/HMGA2signaling may become an important therapeutic target for glioma. |
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