| Research background:Glioblastomas are the most common primary malignant neoplasms in the human central nervous system.The survival period is mostly less than one year.Despite of advances recently in surgery,chemoradiotherapy and molecular therapy,prognosis is still poor for many patients.Strong invasiveness and high recurrence rate are the main causes of death.Therefore,understanding the biological characteristics and molecular mechanisms of glioblastoma can make a foundation for prevention and early diagnosis,and provide new ideas and approaches for clinical treatment and improvement of prognosis.As a research hotspot in recent years,microRNA(miRNA)is still in its infancy in the research of cancers.But as oncogene or tumor suppressor gene,it is more and more concerned on neoplasms development,treatment and prognosis by scientists.And it is expected to be able to provide the necessary targets in the diagnosis and treatment of glioblastomas.miRNA have been confirmed to be involved in almost all important biological processes of cancer,including cell proliferation,apoptosis,cell migration and invasion,cell differentiation and drug resistance.Their production and expression are influenced by genetic variation,epigenetic regulation,and caner microenvironment.The roles of various miRNA in gliomas have been reported.Among them,miR-29,miR-124,miR-181 and miR-7 inhibit neoplasms invasion and angiogenesis;while,miR-21,miR-10b,miR-221/222 and miR-93 promote tumor invasion and angiogenesis.miR-16,as a member of the microRNA family,was considered to be a tumor suppressor when it was first discovered in chronic lymphocytic leukemia(CLL).In recent years,the studies found:the expression of miR-16 was decreased in the bile duct cancer,bladder cancer,non-small cell lung cancer and other solid neoplasms.It suggested that miR-16 could participate in the occurrence and development of tumor by being reduced.However,some researchers had confirmed that the expression of miR-16 was elevated in some cancers,such as gastric cancer and ovarian cancer.In these neoplasms,miR-16 was a proto-oncogene.These studies suggest that miR-16 may have organ specificity and play a role of tumor suppressor or proto-oncogenes in different tumors.So far,the role of miR-16 in glioblastomas has been less reported.The current findings support miR-16 was tumor suppressor in glioblastomas.The way of action is to combine with target gene specifically.It inhibits cancer from different aspects such as cell proliferation,apoptosis,cell cycle,cell invasion and metastasis.In order to investigate the role of miR-16 in glioblastoma and its possible molecular mechanisms,the target genes of miR-16 were predicted and screened by TargetScan and miRanda databases in this article.The results showed that the 3’－terminal of Wip-1 could specifically bind to the 5’-terminal of miR-16.It suggested that there was a certain targeting relationship between miR-16 and Wip1.Wipl is a protein phosphatase of the PP2C family,acting on the serine/threonine site.Wip1 as a proto oncogene plays an important role in the development of many kinds of cancers.ATM and p53,as recognized cancer suppressor genes,have been proved to be target genes of Wipl.Wipl dephosphorylates ATM and P53,making them inactive,then promotes the development and progression of cancers.Based on the above research background,this paper proposes a scientific hypothesis:miR-16 inhibits glioblastoma growth by targetly conbining with Wip1 and regulating Wip]-ATM-p53 signaling pathway.In signal transduction,proteins can be activated by obtaining phosphoryl groups,and be inactivated by removing phosphoryl groups.As mentioned earlier,Wipl is a protein phosphatase acting on serine/threonine site.It performs oncogene function by dephosphorylating the target proteins.ATM and P53 are both target proteins of Wipl and can be inhibited by Wip1 dephosphorylation.Whether the expression levels of phosphorylated ATM(p-ATM)and phosphorylated P53(p-P53)have changed in glioblastomas will be a question to be discussed in this study.Therefore,in vitro cell function experiments were carried out to verify the effects of miR-16 and Wipl-ATM-p53 signaling pathways on the proliferation,apoptosis and invasion of glioblastoma cells.In vivo nude mice orthotopic transplantation tumor experiments were carried out to further verify the inhibition of miR-16 on glioblastomas.To preliminarily explore the molecular mechanism of miR-16 inhibiting glioblastomas.Objective This study was designed to investigate the role of miR-16 targetly regulating Wip1-ATM-p53 signaling pathway and its possible molecular mechanism in inhibiting the growth and invasion of glioblastomas.Methods:Three cell lines(SHG44.U87 and U251 cells)were used to establish the high expression groups of miR-16 and the negative control groups by cell transfection.Clone formation test.EDU cell proliferation test,cell invasion test,cell apoptosis test and cell cycle test were carried out respectively.Using bioinformatics database to analyze the miR-16 gene regulatory network,and predict Wipl is a target gene of miR-16.The target relationship between miR-16 and Wipl was verified by dual luciferase reporter gene test.In vitro cell experiments were divided into two groups(miR-16 mimic group and control group).The mRNAs expression of Wip 1,ATM and p53 was detected by qRT-PCR.The proteins of Wipl,ATM,P53,p-ATM and p-P53 in each group were detected by Western blot test.Nude mice were divided into 5 groups:miR-16 high expression group(agmir group),miR-16 low expression group(antagomir group),corresponding control group(NC group)and blank control group(control group).The proteins of Wipl,ATM,P53,p-ATM and p-P53 in orthotopic transplantation of nude mice were detected by Western blot.The mRNAs and protains expression of Wipl,ATM and p53 in orthotopic transplantation of nude mice was detected by qRT-PCR and immunohistochemistry.Findings:Compared with the control group,when the expression of microRNA-16 was over-expressed,the clone number of SHG44,U87 and U251 cells was significantly lower(t=11.300,p<0.01;t=8.812,p<0.01;t=11.857,p<0.01).The proliferation of cells was significantly decreased(t=26.67,p<0.01;t=10.776,p<0.01;t=10.096,p<0.01).The invasive ability of cells was weakened(t=6.683,p<0.01;t=8.1815 p<0.01;t=7.578,p<0.01).The early apoptosis rate was significantly increased(t=4.918,p<0.05;t=4.006,p<0.05;t=3.573,p<0.05).The number of cells in G1 phase was significantly increased(t=6.392,p<0.05;t=9.802,p<0.05;t=8.975,p<0.01),and the number of cells in S phase was significantly decreased(t=4.643,p<0.05;t=6.284,p<0.05;t=5.009,p<0.01).The differences were statistically significant(p<0.05).The results of dual luciferase reporter gene showed that luciferase activity of wild-type Wipl was decreased significantly in miR-16 overexpression group(t=4.481,p<0.05),compared with the control group.Luciferase activity of mutant Wipl was not significantly different(t=0.693,p>0,05).Wipl is a specific target of miR-16.The results of qRT-PCR in vitro three cells tests showed:compared with the control group,when the expression of microRNA-16 was over-expressed,the expression of Wip1 mRNA was decreased(t=8.589,p<0.01;t=8.034,p<0.01;t=7.727,p<0.01).The ATM mRNA expression was inreased(t=4.304,p<0.05;t=7.407,p<0.05;t=6.530,p<0.05).The expression of p53 mRNA was also increased(t=4.879,p<0.01;t=5.946,p<0.01;t=4.8]8,p<0.01).The results of western blot test in vitro three cells tests showed:compared with the control group,when the expression of microRNA-16 was over-expressed,the expression of Wipl protain was decreased(t=9.918,p<0.01;t=5.646,p<0.01;t=5.609,p<0.01).The expression of p-ATM protain was inreased(t=4.785,p<0.01;t=9.484,p<0.01;t=4.464,p<0.05).The expression of p-P53 protain was also increased(t=3.848,p<0.05;t=3.024,p<0.05;t=4.222,p<0.05).While,the expression of ATM protein was not significantly different(t=1.938,p>0.05;t=2.152,p>0.05;t=2.670,p>0.05).The expression of P53 protein was also not significantly different(t=2.589,p>0.05;t=2.189,p>0.05;t=1.859,p>0.05).Through nude mice orthotopic neoplasm transplantation experiment,compared with normal brain tissue,the expression of miR-16 in intracranial glioblastomas of nude mice was decreased significantly in·the blank control group(t=2.836,p<0.05).The tumor sizes of miR-16 higher expression group were significantly smaller than that of the control group(t=9.643,p<0.01),and the tumor sizes of miR-16 lower expression group were significantly larger than that of the control group(t=13.29,p<0.01).The qRT-PCR method showed that the Wipl mRNA expression in miR-16 higher expression group was lower than that in the control group(t=8.515,p<0.05),while the ATM and p53 mRNAs expression was higher(t=5.430,p<0.05;t=5.213,p<0.05).On the contrary,Wipl mRNA expression was higher compared lower expression of miR-16 group with the control group(t=3.690,p<0.05),however,the ATM and p53 mRNAs expression was lower(t=3.106,p<0.05;t=2.808,p<0.05).Western blot test illustrated that Wipl protein expression was lower compared the miR-16 higher expression group with the control group(t=5.383,p<0.01),while the expression of p-ATM and p-P53 proteins were higher(t=4.35,p<0.01;t=2.648,p<0.05).Contrarily,the Wipl protein expression was higher compared miR-16 lower expression group with the control group(t=6.200,p<0.01),while the expression of p-ATM and p-P53 protein were less(t=5.828,p<0.01;t=4.537,p<0.01).But the expression of ATM protein in every group had no significant difference(t=1.133,p>0.05;t=0.149,p>0.05),the expression of P53 protein in every group had no significant difference else(t=0.019,p>0.05;t=0.157,p>0.05).The result of immunohistochemistry method instructed that Wipl protein expression was lower compared the miR-16 higher expression group with the control group(t=2.428,p<0.05),while the expression of p-ATM and p-P53 proteins were higher(t=2.291,p<0.05;t=2.494,p<0.05).Conversely,the Wipl protein expression was higher compared miR-16 lower expression group with that control group(t=2.352,p<0.05),while the expression of p-ATM and p-P53 proteins were less(t=2.397,p<0.05;t=2.013,p<0.05).But the expression of ATM protein in every group had no significant difference(t=1.423,p>0.05;t=0.935,p>0.05),the expression of P53 protein in every group had no significant difference else(t=0.158,p>0.05;t=0.458,p>0.05).Conclusions:(1)miR-16 inhibits SHG44,U87 and U251 cells proliferation and invasion.(2)Wipl is a target gene of miR-16.miR-16 inhibits the proliferation and invasion of glioblastomas by targeting the Wipl-ATM-p53 signal pathway. |
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