| Background:Glioblastoma multiforme (GBM) is the most common, aggressive and lethal intracranial tumor in adults andhas the characteristics of rapidinfiltrative growth, the inherent complexity of the tumor and easy to relapse. Current therapeutic options, including surgery, radiation and adjuvant chemotherapy, are not particularly effective in the treatment of GBM. Over the past decade, the median survival of GBM has remained 12 months or less. Gliomas generally originated in neural stromal cells or neural parenchymal cells, according to a variety of methods can be gliomas are classified according to their origin and divided among glioma clinical astrocytoma most common. World Health Organization divided glioma into Ⅰ-Ⅳ grades, generally considered the grade I to be classified as the benign glioma, can be cured by surgery, while its less frequent malignant progression, prognosis better; and the higher class II, III degree malignant glioma, but also has some aggressive; Ⅳ grade was the highest degree of malignancy, compared to class Ⅱ, Ⅲ glial more aggressive, patients prognosis is also not good. Primary glioblastomas occur in the brain parenchyma, and secondary glioblastomas from low-level of malignant glioma, such as Class Ⅱ, Ⅲ gliomas. Although there are surgical resection and put comprehensive treatment of chemotherapy combined treatment to glioma patients, however, the survival of patients after surgery is still only 12 months. The reason may be due to glioma stem cells had resistance to radiotherapy and chemotherapy. In recent years, With the continuous development of histopathologic and molecular biology, more and more scholars to explore the mechanism of glioma, they also studied glioblastoma clinical and pathological heterogeneity, abnormal expression of genes and signal transduction pathway disorders, these studies will help us to understand the occurrence and development mechanism of glioma, which is very important to the clinical diagnosis and treatment of glioma patients. Meanwhile, the hot and difficult academic research is to find new treatment methods and drugs glioma. Numerous studies confirm that a variety of microRNA play a regulatory role in the development and progression of glioma, which is currently the focus of academic research.Numerous studies have confirmed the invasion and metastasis ability of glioma cell itself has a root cause of the occurrence and recurrence of glioma.MicroRNAs are a class of small endogenous noncoding RNAs consist of 20-25 nt, which modulate target gene expression by binding with target mRNA sequences in the 3’UTR with full or partial complementarity way that cleavage of target mRNAs or inhibit mRNA translation. Therefore, miRNA and its corresponding target genes form a complex regulatory network that has a very important significance for the body’s cells and a series of life events in the process. Recent studies have found that more than 50% of miRNAs targeted at vulnerable sites of the genome, or tumor-associated genomic regions, suggesting that miRNAs may be related to the occurrence and development of tumors. More and more studies confirm that miRNAs could affect cancer development and progression, these effects mainly for the following three aspects:first, some miRNAs are cancer-promoting genes in the development of cancer; second, part of the miRNAs may inhibit tumor development and progression, it was tumor suppressor gene; third, some oncogenic virus encoded miRNAs also can participate in the relevant the occurrence and development of malignant tumors, but the survival of the virus in the body of its host, also may regulate the expression of miRNA in the body cells, and thus participate in the development and progression of tumors. MiR-10b belongs to the family of miRNAs, a large number of studies have confirmed its related to cancer invasion and metastasis. Among these miRNAs, miR-10b was associated with tumor invasive potential in hepatic cancer, pancreatic cancer, glioma, esophageal cancer and neurofibromatosis. But the exact function of miR-10b about invasion and metastasis in brain are still less study.MiR-10b belongs to the family of miRNAs, a large number of studies have confirmed its related to cancer invasion and metastasis. RhoC was overexpression in a variety of malignant tumors, such as stomach cancer, pancreatic cancer, non-small cell lung cancer, breast cancer, ovarian cancer, colon cancer, bladder cancer, bladder epithelial cell cancer, liver cancer. With RhoC have been discovered in different types of cancer, which in the process of tumor development role has gradually clear. Numerous studies have demonstrated that abnormally high levels of RhoC expression was related to the invasion and migration of tumor cells in a variety of malignancies. Further study found that, RhoC could activate a series of kinases to promote metastasis of tumor cell. Present research has confirmed the high expression of RhoC in a variety of human malignant tumors, especially in some of the higher invasion and metastatic malignant tumors, but its research in human gliomas were less.Objective:To explore the role of miR-10b in GBM invasionand metastasis the possible molecular mechanism, and provide new effective target for clinical treatment of glioma.Methods:(1) We detected the expression of miR-10b and RhoC mRNA by qPCR in 63 cases of glioma patients serum and cancer tissues, and 15 cases of corresponding normal tissue. We detected the expression of RhoC protein by western blot in cancer tissues and corresponding normal tissue.(2) The miR-10b shRNA lentivirus was transfected to human glioma U87 cells, we detected the expression of miR-10b after transfection; detected the multiplication efficiency of U87 cells by MTT assay; flow cytometry was used to detect the cell cycleand apoptosis after transfected; detected the invasion by Transwe Ⅱ assay; detected the proliferation of U87 cells by colony formation assay; detected the transfer ability of U87 cells by healing assay; detected the formation mechanism of apoptosis by Caspase-3-activity assay kit.(3) To predict potential targets of miR-10b by bioinformatics software, and then WT-RhoC and Mut-RhoC were transfected into the U87cells which has been stably transfected miR-10b inhibitor. We detected the expression of miR-10b after transfection; to detect the luciferase expression after transfection; to detect the expression of RhoC protein by western blotResult:(1) miR-10b and RhoC mRNA in Glioma serum and cancer tissueswere significantly higher than the it in corresponding normal tissues; the expression of RhoC protein in gliomas tissues was significantly higher than that in normal tissues; the expression of RhoC protein in level III and IV grade gliomas were significantly higher than that in I and II grade gliomas; glioma cancer tissue from patients with clinical and pathological grading RhoC protein was positively correlated.(2) We had suppressedthe expression of miR-10b in U87 glioma cells, and then we detected the growth of U87cells by MTT after transfecting 48 h and 72 h.The results showed that the survival rate of U87 cells in miR-10b shRNA group at all time points had no significant effect compared to negative control group (NC); We used flow cytometry to detect cell cycle after transfection 48 h, the results showed that the cell cycle of U87 cells did not significantly change after transfection of miR-10b inhibitor, there was no significant difference in the proportion of cells in G1/G1 phase, G2/M phase and S phase. We detected apoptosis in each group after transfection 48 h by Annexin V-PI double staining, the results show no significant apoptosisof U87cells after transfection inhibitors of miR-10b, miR-10b can not induce U87 cells apoptosis. We have used Transwe II assay to detected the apoptosisafter transfection48 h, the results showed that the average number of invasive cells in miR-10b shRNA group was significantly less than the NC group. We have detected cell proliferative activity in each group by the colony formation assay after transfection48 h, the results showed that inhibition of miR-10b expression, ability to form cell clones compared with no significant change compared to NC group. We have detected cell migration in each group by cell scratch test, the results show cell migration in the U87cells of non-transfected miR-10b inhibitorgroup stronger than the transfection miR-10b inhibitor group. We’ve detected that caspase-3 activity in each group after transfected by caspase-3 activity assay kit, results showed caspase-3 activity in miR-lOb shRNA-transfected group had no significantly affected compared with the NC group.(3) We detected the expression of luciferase in each group after transfected, the results showed that when the expression of miR-10breduced could significantly inhibit the expression of wild-type RhoC, but had no significant effected on the mutant RhoC. We examined the expression of RhoC protein by western blot assay, the results show that the expression of miR-10b reduction could significantly inhibit the expression of WT-RhoC RohC protein in wild-type cells, but had no significant effected on the mutant Mut-RhoC.Conclusion:This study examined the expression of miR-10b and RHOC in peripheral blood and tumor in glioma patients, and analyzed the impact of the miR-10b expression changes in glioma cell,and analyzed its relationship with invasion and metastasis. We also exploredthe effec of miR-10b signal activation on regulation of the RHOC expression of stromal tumor cells, and explored theinfluence on the biological behavior of glioma cells.This study was to explore the mechanism of human glioma cell invasion and metastasis, which was targetd RhoC expression by of miR-10b. This study explore the occurrence and development mechanism of glioma and lay the foundation to clinical diagnosis and targeted therapy of glioma. |
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