| Human glioma, originating from glia or their precursors within the central nervous system, is the most common form of primary brain tumor in adults, and for highly malignant gliomas(WHO III and IV) there is no successful treatment; patients have an average survival time of approximately 12–15 months after iagnosis.Glioma are highly invasive and infiltrate normal brain tissue, and as a result, surgical resection is always incomplete. Treatment for glioma consist of surgical resection combined with radiotherapy plus chemotherapy or target therapy, which has been found to significantly increase survival. As yet, the mechanisms that underlie the correlation between high grade and poor prognosis are still unknown, because all grades of glioma are characterized by inappropriate proliferation, invasion of normal brain tissue, and disruption of normal brain functions. So only the molecular mechanism is well-defined can we find out novel therapeutic approaches.mi RNAs are 21 to 23 nucleotides, endogenous small noncoding RNA sequences that negatively regulate gene expressions at a post-translational level by binding to regions of sequence complementarity to 3′UTR of target m RNAs,resulting in either the degradation or translational inhibition of the m RNA. Many mi RNAs have been proved to be protooncogenes or tumor suppressors in various human cancers, including in glioma.3-5Recent studies have revealed that mi RNAs, such as mi R-7, 6,7 mi R-124,8-10 mi R-128,11 mi R-107,12 and mi R-181 b,13-15 are globally dysregulated in glioma.However, the particular molecular mechanisms through which mi RNAs mediate glioma carcinogenesis and metastasis are still largely unknown. So the identification and characteri-zation of the role of novel mi RNAs in glioma may provide new insight into understanding the molecular mechanisms of glioma development. Micro RNA-302c-3p(mi R-302c-3p) is an important member of mi R-302/367 cluster which has five highly homologous mi RNA members: mi R-302 a, mi R-302 b, mi R-302 c, mi R-302 d, and mi R-367.Human mi R-302/367 gene is located in chromosome 4, q25 region.Early studies have focused on the role of mi R-302/367 as an important regulator in embryonic stem cells, including stem cell renewal and the maintenance of pluripotent stem cell differentiation. Recent researchers show that mi R-302/367 also has an important regulatory function in tumors, immunological diseases and cardiovascular disease, by regulating different target genes in different disease and playing different biological effects.The tumorigenicity is a complex and involve different cellular processes. The Extensive studies in recent years have focused on the mi RNAs which is almost involved in the whole process of differentiation of tumor. Among them,the mi R-302/367 plays an important role in a variety of tumors. Based on Leivonen’s study, mi R-302 c was reduced expressed in their experimental models and others found that it may be involved in hepatocellular carcinoma metastasis, which suggested that mi R-302 c might play an important role in glioma progression In this study, we first detected the expressions of mi R-302c-3p in glioma patients’ tissues, and a series of in vitro and in vivo studies were then conducted to investigate the mechanisms and impact of mi R-302c-3p in glioma. By bioinformatics analysis(RNA22) and experimentally validation, we found metadherin(MTDH) was a direct target of mi R-302c-3p. Upregulation of MTDH was also detected in glioma tumors compared with normal brain tissues and is inversely correlated with mi R-302c-3p expression. Finally, we find that overexpression mi R-302c-3p could dramatically inhibit glioma cells proliferation and invasion both in vitro and in vivo.This topic will be studied from three aspects:1. The expression level of mi R-302c-3p in human glioma tissues and normal brain tissueObjective:To examine the expression level of mi R-302c-3p and MTDH in 27 human brain glioma tissues and 6 normal brain tissues, and to analyze the different expression of mi-302c- 3p in glioma was associated with glioma WHO grade. By using dual-luciferase reporter assay we can demonstrate if the MTDH is a direct target of mi R-302c-3p and for further exploring the biological function of mi R-302c-3p in glioma in vitro and in vivo.Methods:Real-time fluorescent quantitative reverse transcriptase polymerase chain reaction(q RT-PCR) method was performed to compare the expressing levels of mi R-302c-3p and MTDH in human brain glioma tissues and normal brain tissues. By using dual-luciferase reporter assay we demonstrated the relationship between MTDH and mi-302c- 3p.Results:(1) mi R-302c-3p was down-regulated in human glioma tissues The expression of mi R-302c-3p were examined in both glioma tissues and normal brain tissues by q RT-PCR.The expression level of mi R-302c-3p was significantly decreased in tumor tissues compared with normal brain tissues(0.39±0.04 vs. 1.06±0.03,P<0.001). Furthermore, the expression levels of mi R-302c-3p in tumors with high grades(WHO III and WHO IV)were significantly lower than those in those with low grades(WHO I and WHO II)(0.27±0.03 vs.0.66±0.05,P<0.001);(2) the correlation analysis: MTDH is up-regulated in human glioma tissues and its expression correlates with mi R-302c-3p expression in glioma To corroborate the potential importance of MTDH in gliomas, we compared the level of MTDH expression in 27 tumors versus 6 normal brain tissues. The expression of the MTDH accessed by q RT-PCR was significantly increased in gliomas by compared with the normal brain tissues(p=0.0027). MTDH is overexpressed in 85.2%(23/27) of tumors compared with normal tissues. To further evaluate the correlation between MTDH and mi R-302c-3p expression in gliomas, we compared the expression of MTDH and mi R-302c-3p in 27 gliomas.Expression of MTDH and mi R-302c-3p exhibited a significant inverse correlation(P =0.003,R2 =0.4065), which significantly suggested that the mi R-302c-3p target status of MTDH.(3) MTDH was a direct target of mi R-302c-3p which inhibited its expression via binding to its 3?UTR On the basis of the prediction software, RNA22(https://cm.jefferson.edu/rna22v1.0-homo_sapiens/Get Inputs.jsp), we find that the3?UTR of MTDH contains a complementary site(position 2271~2293) for the seed region of mi R-302c-3p, and may be a potential target of mi R-302c-3p. To validate the mi RNA-target interaction and determine whether mi R-302c-3p inhibited the expression of MTDH through direct interaction with 3?UTR of MTDH m RNA in the intracellular environment in glioma, Luciferase assay was established. Results showed that mi R-302c-3p mimics suppressed the luciferase activities about 69.6% in the wild-type group(P = 0.005), while no obvious alteration was detected in the mutant group or the negative control group, which indicated that mi R-302c-3p could directly bind with MTDH3?UTR at the seed sequence.2.The research about effects of mi R-302c-3p inhitibted proliferation and invasion of human glioma cells in vitroObjective:To observe the overexpress of mi R-302c-3p affected the U87 MG glioma cells proliferation and invasion in vitroMethods:To assess the proliferation of U87 MG we maked U87 MG transduced by LV-302c-3p as transfected group,and transduced by LV-NC as negative control group;These cells were determined by using CCK-8. Briefly, 100μL 5×104 cell/ml cell suspensions were initially seeded onto each well in the 96-well plate. After 1, 3, 5, 7 days, 10 μL CCK-8 was added to each well and incubated at 37℃ for 4 h. Then, 100 μL of solution was aspirated from each well and poured in a 96-well plate. The absorbance in each well at 450 nm was measured with an ELISA reader.For cell invasion assay, a specialized Chamber with BD Bio Coat Matrigel(354480,BD Biosciences) was used according to the manufacturer’s instructions. Briefly, cells(5 ×10 4 cells/m L) were loaded into chamber inserts containing an 8-mm pore size membrane with a thin layer matrigel matrix. Cells migrating to the lower surface of the membrane during 24 or 48 h were fixed with 100% methanol and non-invading cells on the upper surface of the membrane were removed. The membranes, with migrated cells, were then stained with DAPI(D9564, Sigma), scanned, and digital images were obtained with a microscope. The number of invasive cells was then determined for 5 independent fields under a microscope. The mean of triplicate assays for each experimental condition was used for analysis.Results:(1) The CCK-8 assays were performed to assess cell proliferation of U87 MG cells transfected with LV-302c-3p or LV-NC. A significant reduction in the proliferation rate was observed on the U87 MG cells transfected with LV-302c-3p when compared to negative control.( 2) To explore the effect of overexpression of mi R-302c-3p on the invasive capacity of glioma cells, transwell assays were performed. U87 MG cells transfected with LV-302c-3p or LV-NC were seeded into the chambers with matrigel, and their invasive potential were determined after 24 h culture. The results showed that the invasive capacity of U87 MG cells overexpressing mi R-302c-3p was reduced by 82.4% compared with the control groups.2. The research about effects of mi R-302c-3p inhibits glioma cell tumorigenesis in vivoObjective:To further investigate the overexpression of mi R-302c-3p could dramatically inhibit glioma cells proliferation and invasion in vivo.Methods:To further investigate the role of mi R-302c-3p inhibition on tumorigenesis in vivo, we constructed a lentivirus vector to mediate the expression of mi R-302c-3p and established 2stable cell lines, which were named U87MG-302c-3p and U87MG-NC. Then, we implanted U87MG-302c-3p and U87MG-NC cells into nude mice. To further investigate the role of mi R-302c-3p inhibition on tumorigenesis in the brain, C6 were transfected with LV-302c-3p and then injected into the brain. Cells transfected with LV- NC were used as a control. After 21 th day post-injection, MR image analysis and histopathological analysis were performedResults:(1)We fing mice injected with U87 MGNC cells formed larger tumors compared with the U87MG- 302c-3p cells(P = 0.004,)(2) Results showed that the right brain injected with C6-NC cells formed much larger tumors compared with the C6–302c-3p cellsConclusion:(1) the expression level of mi R-302c-3p was significantly decreased in tumor tissues compared with normal brain tissues; Furthermore, the expression levels of mi R-302c-3p in tumors with high grades(WHO III and WHO IV) were significantly lower than those in those with low grades(WHO I and WHO II). mi R-302c-3p may be a candidate tumor suppressive mi RNA of glioma and might be a new prognostic biomarker for glioma patients. The expression of the MTDH accessed by q RT-PCR was significantly increased in gliomas by compared with the normal brain tissues; Expression of MTDH and mi R-302c-3p exhibited a significant inverse correlation, which significantly suggested that the mi R-302c-3p target status of MTDH.(2) The results showed that the invasive capacity of U87 MG cells overexpressing mi R-302c-3p was reduced by 82.4% compared with the control groups.(3) The mice injected with U87MG-NC cells formed larger tumors compared with the U87MG-302c-3p cells. The rats right brain injected with C6-NC cells formed much larger tumors compared with the C6–302c-3p cells. |
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