| Objective:Glioblastoma is a common primary malignancy in clinic,and high incidence,high mortality rate,high recurrence rate and low cure rate are the remarkable characteristics of glioblastoma.A standardized treatment programme for international unification is surgical removal of the lesion as much as possible,and supplemental chemotherapy(Temozolomide)and(or)radiotherapy(division and/or standard brain radiotherapy).However,even adopting the standardized treatment programme,the median survival time was still less than 15 months and the prognosis was not significantly improved for high grade glioblastoma.With the development of biotechnology and the study of molecular biology,molecular pathology and genetics deeply,more and more tumor treatment target genes and inference for prognosis of molecular markers have been found.MiR-19a was first found in lymphocytic leukemia and it could promote the proliferation of cancer cells and improve the defense capability of cancer cells.In recent years,more and more research has been found that miR-19a was abnormal up-regulated in many tumors of digestive system,respiratory system,urinary system,immune system,blood system and endocrine system and so on.Therefore,the purpose of this study was to explore the abnormal expression of miR-19a in human glioblastoma,the relationship between miR-19a and the proliferation,invasion and migration of glioblastoma,whether it affected the prognosis and how to regulate the downstream target genes.We hope that this study could provide a new diagnosis and treatment for glioblastoma patients and neurosurgeons.Methods:1.The expression of miR-19a in human glioblastoma and the correlation with clinicopathological types(1)30 cases of glioblastoma(the pathological diagnosis was glioblastoma after operation)and 15 cases of normal brain tissue were obtained from neurosurgery department of First Affiliated Hospital of Dalian Medical University between July 2014 and July 2017.They were transported in liquid nitrogen and frozen in-80?refrigerator.According to the classification of glioblastoma by WHO,the samples were collected and analyzed statistically,and followed-up simultaneously.(2)qRT-PCR was used to detect the differentially expression of miR-19a in normal brain tissue and glioblastoma.2.Biological function of miR-19a in glioblastoma cell lines in vitro(1)qRT-PCR was used to detect the expression of miR-19a in cell lines,U87 and U251 cell lines were selected for follow-up experiment.Transfected lentivirus into U87 and U251 making miR-19a down-regulated and getting a stable cell line sh-miR-19a.qRT-PCR was used to detect transfection efficiency.(2)The differences in proliferation and invasion were compared between sh-miR-19a and control by CCK8,plate cloning and scratch test.3.Gene chip Analyzed the gene chip between sh-miR-19a and control,compared the expression of mRNAs in cell line,screened and detected the significantly mRNA TET2,and subsequent studies were followed by TET2.4.Nude mouse tumorigenesis test in vitro(1)Injected enough dosage sh-miR-19a cell line and control cell line into the right side of axillary of 6-8 weeks nude mouse,observed,measured and recorded the growth and size of tumors every day in order to research the effect of miR-19a on tumor formation in vivo.(2)Immunohistochemistry(IHC)and qRT-PCR were used to detect the expression of TET2 and miR-19a in tumor tissues in vivo.5.The study on the mechanism of miR-19a mediated TET2 Western blot was used to detect the cell cycle-related proteins of control,nc and sh-miR-19a.Results:1.miR-19a is up-regulated in the tissue and cell line of glioblastoma qRT-PCR was used to detect the expression of miR-19a in 30 cases of glioblastoma,15 cases of normal brain tissue and glioblastoma cell lines.Compared with normal brain tissue,miR-19a was significantly up-regulated in glioblastoma and glioblastoma cell lines.However,the expression levels were not exactly the same in all the glioblastoma cell lines.The expression of miR-19a was most obvious up-regulated in U87 and U251.2.The expression level of miR-19a was positively correlated with the pathological classification of tumorWe calculated the correlation between various factors and the expression level of miR-19a by analysing the clinical data of samples,and found that 14 cases(46.7%)were female patients and 16 cases(53.3%)were male patients(p=0.98);17 cases of patients(56.6%)were younger than 50 and 13 cases of patients(43.3%)were older than 50(p=0.868);22 cases of tumor(73.3%)located in the frontal temporal bobe,3 cases of tumor(10%)located in the paracele,4 cases of tumor(13.3%)located in the top occipital lobe and 1 cases of tumor(3.33%)located in the cerebellum(p=0.443);patients with the maximum diameter of tumor less than 5cm were 12 cases(40%)and more than 5cm were 18 cases(60%)(p=0.596);the expression level of miR-19a in ? and ? degree of glioblastoma was significantly lower than in? and ? degree of glioblastoma(p<0.05).Therefore,the expression level of miR-19a was positively correlated with the pathological classification of tumor.3.miR-19a promoted the proliferation,invasion and formation of glioblastomaIn order to study the influence of miR-19a on the migration ability of glioblastoma,we used scratch test and found that the migration ability of the glioblastoma cell line induced by down-regulated miR-19a was significantly decreased,especially in 8h and 16h(p<0.05).In order to study the influence of miR-19a on the proliferation of glioblastoma,we used plate cloning and CCK-8.The OD value of down-regulated group was significantly decreased compared with control from the second day by CCK-8 and the p value was less than 0.05 in the third day.The proliferation of down-regulated miR-19a glioblastoma cell line was significantly decreased compared with control by plate cloning(p<0.05).Two cell lines were divided into control group and down-regulated group.The two cell lines were injected into nude mouse respectively and after feeding 1 month,the volumn and weight of glioblastoma in down-regulated group were significantly smaller than control(p<0.05).It demonstrated that down-regulated miR-19a could obviously affect the formation and proliferation of glioblastoma.4.After down-regulated the miR-19a,the expression of TET2 was increased significantlyTo find the downstream target genes of miR-19a,gene chip was used to analysis the two group cell lines and obtained more than 1 thousand differentially expressed genes,TET2 with fold change equalling to 8.12 was the most obvious gene among them.When miR-19a was down-regulated,TET2 was up-regulated,we supposed that TET2 was the target gene of miR-19a,and this may provide a new research direction.5.The up-regulated TET2 was highly toxic to glioblastoma cellsTranfected lentivirus into glioblastoma cell line in order to increase the expression of TET2.We found that cell activity of up-regulated TET2 glioblastoma cell line was significantly decreased,and it proved that TET2 had some cytotoxicity to glioblastoma cells.6.The up-regulated TET2 affected the expression of apoptosis related protein in U87 and U251 cellsWestern blotting analysis showed that the up-regulated TET2 promoted the expression of activated caspase-3 and Bax protein in glioblastoma,and inhibited the expression of Bcl-2 gene.In addition,the data showed that the ratio of Bcl-2/Bax was significantly reduced by the quantitative data of Bcl-2 protein and the standardization data of Bax.7.The up-regulated TET2 significantly reduced the mesenchyme related molecules in U87 and U251 cellsThe results of scratch test showed that the migration ability of U87 and U251 cells was significantly suppressed by the up-regulated TET2.Further studies revealed that the up-regulated TET2 promoted the expression of mesenchyme related molecules(including Snail,Slug and N-cadherin).8.After up-regulated the expression of TET2,the ratio of p-AKT/AKT and ?-catenin/?-actin in two glioblastoma cell lines significantly reducedConclusions:1.miR-19a highly expressed in glioblastoma,and its expression level was significantly related to the WHO pathological grade and prognosis.2.Down-regulated miR-19a could significantly affect the proliferation and invasion of glioblastoma.3.Down-regulated miR-19a could significantly affect the tumorigenesis ability of glioblastoma in nude mouse.4.Down-regulated miR-19a led to a significant increase in TET2.5.TET2 had a high cytotoxicity to tumor cells by regulating apoptotic proteins and mesenchyme related molecules.6.TET2 could suppress glioblastoma by significantly decreasing the ratio of p-AKT/AKT and beta-Catenin/beta-actin. |
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