MiR-373 Inhibits Glioma Cell U251 Migration And Invasion

Glioblastoma multiforme(GBM) is the most malignant glioma, characterized by remarkable aggressiveness. Despite multidisciplinary cooperation including surgical resection, radiotherapy, and chemotherapy, the prognosis of GBM is still gloomy as less than 10 % GBM patients survive 5 years. Unveiling the underlying mechanisms of its aggressiveness could promote the discovery of potential targets for effective treatment.miRNAs are small noncoding RNAs approximately 22 nucleotides in length that regulate gene expression post-transcriptionally. They are involved in almost all cellular processes, including cell proliferation, apoptosis, senescence, differentiation, migration, and invasion. Deregulations of miRNAs have been demonstrated in many diseases, especially cancer. A variety of mi RNAs have been suggested to function as oncogenes or tumor suppressors in almost all kind of tumors, including glioma. Identifying those deregulated mi RNAs and exploring their functions as well as potential targets may help us develop more effective biomarkers and therapies.miRNA-373(miR-373) is one of the miRNAs that are deregulated in a number of cancers. However, miR-373 seems to act in a cellular context specific way. While some investigations suggested that miR-373 functions as an oncogene, for example, in testicular germ cell tumors,breast cancer, esophageal cancer, and cervical cancer. Its tumor suppressive potential was also revealed, such as in lung cancer, pancreatic cancer, ovarian cancer and colon cancer. In glioma, however, the functions of miR-373 have not been fully investigated.In the present study, we aim to clarify the functions of miR-373 in GBM cell line U251 in vitro. We investigate whether miR-373 could inhibit glioblastoma cell line U251 proliferation, migration and invasion in vitro, and explore the underlying mechanisms.Objectives: We investigate whether miR-373 could inhibit glioblastoma cell line U251 proliferation, migration and invasion in vitro, and explore the underlying mechanisms.Methods: 1. The effect of miR-373 on cell proliferation was evaluated by MTT assay. Cell death and apoptosis were assessed using by flow cytometry.2. Wound-Healing Assay and Transwell Migration and Invasion Assay was performed to investigate the effect of miR-373 on U251 cells migration and invasion.3. Target prediction Online(miRanda and TargetScanHuman7.0) in combination with literature review were used to identify functional targets of miR-373. Dual luciferase reporter assays were performed to investigate that whether the 3’UTR of targeted mRNA is the direct target of miR-373.4. RNAi and functional rescue experiments were performed to investigate whether CD44 and TGFBR2 are the functional targets of miR-373 in U251.Results: 1. Although no significant growth inhibitory effect was observed after miR-373 overexpression or inhibition, we did find that U251 cells underwent considerable morphologic change after transfection of miR-373 mimics. Such phenomenon suggested that miR-373 may affect cell mobility.2. As shown by Wound-healing assay and Transwell assays, miR-373 inhibits U251 cell migration and invasion.3. Based on target prediction using online miRanda and TargetScanHuman7.0 and by reviewing published articles regarding miR-373, we focus on the following two verified targets that are involved in cell mobility, CD44 and TGFBR2. qRT-PCR confirmed that mi R-373 downregulated the mRNA expression of CD44 and TGFBR2. Western blot also confirmed that miR-373 downregulated the protein expression of CD44 and TGFBR2. Luciferase activity assays suggested that miR-373 regulates CD44 and TGFBR2 expression by targeting 3’UTR of them mRNA directly.4. Silenced CD44 and TGFBR2 using corresponding siRNA respectively exhibits the similar cellular phenotype as overexpression of miR-373 showing inhibited migration and invasion in U251 cells. Rescue experiments by overexpression CD44 or TGFBR2 using corresponding cDNA plasmids that lack the 3’UTR of CD44 or TGFBR2, respectively exhibited no significant inhibitory effects on cell migration or invasion.Conclusions: 1. miR-373 suppresses GBM cell line U251 migration and invasion.2. miR-373 down-regulates CD44 and TGFBR2, both CD44 and TGFBR2 are direct targets of miR-373 in U251.3. CD44 and TGFBR2 are functional targets of miR-373, down-regulation of which contributing to its migration and invasion suppressive function in U251.4. miR-373 inhibits TGFβ-induced migration and invasion in U251.