The Study On The Effect Of MiRNA-210and The Target Gene EFNA3on The Growth And Invasion Of Malignant Peripheral Nerve Sheath Tumor (MPNST)

BackgroundMaligant peripheral nerve sheath tumors (MPNST) are rarely sarcomas which believed to be derived from Schwann cells or pluripotent cells of the neural crest. Half of all MPNSTs are closely association with benign peripheral nerve tumors-neurofibromatosis type1(NF1), which occur in the general population at a frequency of only0.02%. Although the incidence of MPNST in the general polulation is very low, MPNST occur at a prevalence ranges from4%to10%of all soft tussue sarcomas in childhood, and are often fatal. The mainstay of therapy for patients with MPNST relies largely upon surgery, but the treatment approach often fails to completely remove the tumor. The other treatment approaches such as chemotherapy and irradiation for MPNSTs had also been demonstrated to be discouraging. Therefore, novel therapeutic methods for patients with MPNSTs are urgently needed. With the development of molecular biology, molecular targeted therapy was emerged and considered to be a promising and effective therapeutiv option for MPNSTs.microRNAs (miRNAs) are short and small non-coding RNAs with18-25nucleotides in length that involve in the inhibition of the expression of a variety of human genes by either mRNA degradation or translational repression. miRNAs had been demonstrated to be regulated many biological processes, such as cell proliferation, differentiation, cell death and metabolism, etc. Abnormal expression of miRNAs were closely associated with the carcinogenesis and progession of malignant tumors, and has potential diagnostic and prognostic value in various maligancies. In addition, emerging studies suggested that miRNA targeting offers a very useful and powerful tool for the treatment of many malignant tumors.miRNA-210is among the most important miRNA that involves in many biological processes includes cell proliferation, DNA repair, metabolism, cell migration, etc. Accumulated data had demonstrated that miRNA-210is the most consistently induced miRNA under hypoxia, which mediated by hypoxia-induced factor (HIF). As the expression of miRNA-210is correlated with accumulation of HIF-1α under hypoxia condition, miRNA-210have been demonstrated to be frequently up-regulated in cancer, such as glioblastoma, clear cell renal cell carcinoma, lung cancer, breast cancer, etc. Moveover, miRNA-210may play an oncomir role in cancer initiation and progression involving regulation of cellular growth, apoptosis, migration and invasion in many malignant tumors. However, until now, the actions and its underlying mechanisms of miRNA-210remain unknown in MPNST.Therefore, to study the roles of miRNA-210in the cell proliferation and invasion of MPNST and underlying the molecular mechanisms will definitely help clarify MPNST pathogenesis and develop potential therapeutic targets for MPNST.Part Ⅰ. The effect of miRNA-210on proliferation and invasion in MPNSTAims:(1). To examine the expression of miRNA-210in MPNST.(2). To investigate the roles of miRNA-210in proliferation and invasion of MPNST.Methods and results: (1). Real time-PCR was used to detect the expression of miRNA-210in SK-N-SH cells and MPNST cell lines include ST88-14, sNF96.2, T265p21and YST-1, the results suggested that miRNA-210was largely increased in ST88-14and sNF96.2cells.(2). The pre-miRNA-210, pre-con, anti-miRNA-210or anti-con were transfected into ST88-14and sNF96.2cells, respectively. MTT and colony formation assays indicated that the cell viability and coloning efficiency increased in ST88-14and sNF96.2cells after transfected with pre-miRNA-210than transfected with pre-con. In contrast, introduction of anti-miRNA-210decreased the cell viability and coloning efficiency in ST88-14and sNF96.2cells.(3).Flow cytometry results suggested that introduction of pre-miRNA-210into ST88-14and sNF96.2cells significantly increased the percentage of S phase cells. Correspondingly, introduction of anti-miRNA-210into ST88-14and sNF96.2cells significantly reduced the percentage of S phase cells.(4). Transwell assay indicated that overexpression of miRNA-210by introduction of pre-miRNA-210could increase the invasion of ST88-14and sNF96.2cells. However, inhibition of miRNA-210expression by introduction of anti-miRNA-210reduced the invasion of both cells.Conclusions:(1). miRNA-210was increased in MPNST cells.(2).miRNA-210promoted the proliferation and invasive ability of MPNST cells.Part Ⅱ. miRNA-210regulates expression of EFNA3in MPNSTAims: (1). To identify the potential target gene of miRNA-210in MPNST.(2). To investigate the molecular mechanism of miRNA-210in regulation of EFNA3.Methods and results:(1). Real time-PCR was used to examine the expression levels of potential target genes of miRNA-210include EFNA3, MNT, AIFM3, ZNF462, GIT2(PKL) in SK-N-SH and MPNST cells include ST88-14and SNF96.2, and results showed that EFNA3was significantly decreased in ST88-14and sNF96.2cells than in control.(2). Bioinformatics sofeware analyzed that the3’UTR of mRNA of EFNA3gene, a potential miRNA-210binding site was identified.(3).3’-UTR of EFNA3gene containing wild-type or mutated miRNA-210binding site were constructed into psi-CHECK2dual luciferase reporter vectors, named EFNA3-3’UTR-psi-CHECK2and Mut-EFNA3-3’UTR-psi-CHECK2respectively. The result showed that ectopic expression of miRNA-210significantly inhibited the luciferase activity in293T cells transfected with EFNA3-3’UTR-psi-CHECK2reporter vector. However, miRNA-210overexpression had no effect on the luciferase activity in293T cells tranfected with Mut-EFNA3-3’UTR-psi-CHECK2reporter vector.(4). Real time-PCR and Western blot were used to examine the effects of miRNA-210on EFNA3expression, ST88-14and sNF96.2cells were transfected with anti-miRNA-210or miRNA-210mimics or control miRNA, respectively. The results showed that introduction of miR-210mimics significantly increased miRNA-210expression and decreased EFNA3mRNA and protein expression in ST88-14and sNF96.2cells. However, anti-miRNA-210significantly decreased the miRNA-210expression and increased EFNA3expression in them, compared to the control.Conclusions:MiRNA-210is able to regulate EFNA3gene, implied that miRNA-210may inhibit proliferation and invasion of MPNST cells by targeting EFNA3gene.Part Ⅲ. The role of miRNA-210mediated inhibition of EFNA3on proliferation and invasion of MPNSTAims:To investigate the role of miRNA-210inhibited EFNA3expression on proliferation and invasion of MPNST.Methods and results:(1). EFNA3-Talen (L) and EFNA3-Talen (L) plasmids had been constructed and transfected into MPNST cells ST88-14and sNF96.2cells, and the stable EFNA3knockout cells include ST88-14-EFNA3-Talen and sNF96.2-EFNA3-Talen were established.(2). MTT assay was used to detected the effect of miRNA-210overexpression on ST88-14-EFNA3-Talen and sNF96.2-EFNA3-Talen stable cells, respectively. The results indicated that the growth rates of ST88-14-EFNA3-Talen and sNF96.2-EFNA3-Talen stable cells that were transfected with miRNA-210is not higher than miRNA-210transfected control cells.(3). Transwells experiment was used to examine the effect of miRNA-210overexpression on EFNA3-Talen stable ST88-14or sNF96.2cells and control cells. The results indicated that the invasive ability of miRNA-210transfected EFNA-Talen stable cells is stronger than miRNA-210transfected control cells.Conclusions:miRNA-210induced inhibition of EFNA3gene expression partly contribute to the proliferation of MPNST cells and EFNA3gene is a key gene involved in inhibition of invasion of MPNST cells.Main innovative points1. For the first time, we examined the expression of miRNA-210in MPNST cells and investigated the effect of miRNA-210in promoting proliferation and invasion of MPNST.2. For the first time, we identified that miRNA-210negatively regulate EFNA3gene in MPNST.3. For the first time, we investigated the function of miRNA-210/EFNA3cascade in the proliferation and invasion of MPNST, implied that miRNA-210/EFNA3cascade could be served as the potential therapeutical target.