Mechanisms Of LRRC4As The Core Of It Regulated Multiphase Loops In Glioma

[Background]Leucine-rich repeat C4(LRRC4) gene was highly specific to brain tissue and it behaved as a tumor suppressor in the pathogenesis of gliomas. Methylation of the LRRC4promoter has been considered as one of the important mechanisms inactivating LRRC4in gliomas. Exogenous overexpression of LRRC4could inhibit glioma cells growth and arrest glioma cells in the G0/G1phase of the cell cycle. Induction of LRRC4expression inhibited glioma cell proliferation and invasion by downregulating the ERK/MAPK and PI-3K/AKT signaling pathways. MiRNAs are endogenous non-coding small RNAs that tend to negatively regulate genes by binding targets’3’UTR, which inhibit the process of proteins in the post-transcription level, even degradate mRNA.To explore the miRNA regulating network and the function of LRRC4as a tumor suppressor gene in glioma cells, miRNA microarray was applied to investigate the differentially expressed miRNAs in U251/LRRC4(U251stably expressing LRRC4) contrasting with U251/Control cells.[The expressions and prognosis of miRNAs regulated significantly by LRRC4in glioma]In the result of miRNA microarray, there were4downregulated miRNAs and3upregulated miRNAs by LRRC4more than2-fold which subsequently identified by qRT-PCR. The network of LRRC4-miRNA-target genes was constructed through analyzing remarkable function of LRRC4-regulating miRNAs’predicted target genes. It’s showed in the network that LRRC4could regulate the expression of multiple miRNAs, and miRNA could regulate many target genes, furthermore one gene could be regulated by many miRNAs. It’s interested that there was interaction between miRNAs (miR-182, miR-381and miR-590-3p) and LRRC4the target gene in the network. Data’s indicated that miR-182and miR-381was overexpressed in glioma tissues and cell lines, and the higher level of the expression, the worse the patients’prognosis was. In the contrary, miR-185was downexpressed in glioma tissues and cells, and the worse prognosis of patients was correlated to lower miR-185expression.[miR-182or381promoted BRD7expression and inhibited glioma cell growth though inhibiting LRRC4as a potential therapeutic marker, and the LRRC4-AP-2-miR-182-LRRC4loop played important role in the pathogenesis of glioma]It’s known that miRNAs took part in proliferation and growth in glioma cells. MiR-182or miR-381overexpression could promote glioma cell growth in vivo and in vitro. Therefore, they were considered to be potential therapeutic biomarker in glioma. MiR-182or miR-381silencing could arrest glioma cells in the GO/G1phase of the cell cycle and inhibit glioma cells growth identified by upregulating phosphorylated Rb and suppressing E2F3. LRRC4was the co-target gene of miR-182and miR-381. The expression of miR-182, miR-381and BRD7were inversely correlated with LRRC4expression in gliomas. miR-182and miR-381silencing was found to inhibit the expression of BRD7, upregulate phosphorylated Rb, suppress E2F3, arrest glioma cells in the G0/G1phase of the cell cycle, inhibit glioma cells growth and induce differentiation of glioma cells to astrocyte-like cell by upregulating LRRC4and suppressing LRRC4-mediated binding of AP-2/SP1/E2F6/c-Myc to BRD7in ERK/MAPK and PI-3K/AKT signal pathways.Transcription of miR-182was induced by transcription factor AP-2predicted by online softwares and confimed by ChIP. According to our previous results, miR-182was verified to inhibit the expression of LRRC4, and LRRC4might inhibit the expression and transcription of AP-2though negatively regulating the ERK/MAPK and PI-3K/AKT signaling pathways. It’s indicated that the LRRC4-AP-2-miR-182-LRRC4loop formed among LRRC4, miR-182and AP-2was involved in glioma development.[miR-185regulated genomic methylation level and recover expression of such hypermethylation genes as LRRC4in glioma cells by inhibiting DNMT1, and functioned as tumor suppressor through inhibiting CDC42and RhoA. The LRRC4-miR-185/SP1-DNMT1-LRRC4loop played an important role in glioma]MiRNA may be involved in the process of tumor development as a tumor suppressor gene. It’s certified that miR-185could inhibit glioma cell growth, motility and invasion identified by MTT, scratch test and transwell test. Therefore, miR-185could function as a tumor suppressor. In order to clarify the mechanism of miR-185suppressing glioma, bioinformatics and some relative assays were performed to demonstrate that miR-185could inhibit DNMT1. DNMTl is one of the most important DNA methyltransferase which maintains methylation. Then it’s showed that overexpression of miR-185could inhibit DNMT1and reduce global methylation by HPLC-DAD. Our previous study showed that nine genes (LRRC4, ANKDD1A, GAD1, HIST1H3E, PCDHA8, PCDHA13, PHOX2B, SIX3and SST) were hypermethylated in glioma. Here, miR-185was confirmed to re-express them by decreasing their methylation. Hence, miR-185was considered to inhibit glioma cells growth and migration by targeting DNMT1, reducing global methylation and recovering expression of such hypermethylation genes as LRRC4.MiR-185was predicted to participate in Rho GTPase activity based on GO analysis, while CDC42and RhoA were the main elements regulating Rho GTPase. Then CDC42and RhoA were identified to be the direct targets of miR-185. Further, CDC42and RhoA were inversely correlated with miR-185expression in gliomas. miR-185was clarified to mediate glioma cell growth and migration by inhibiting CDC42and RhoA and VEGFA indirectly.It’s verified that overexpressing LRRC4could increase the expression of miR-185, while miR-185could regulate global methylation by inhibiting DNA methyltranferase DNMT1and increasing the expression of such hypermethylation gene as LRRC4in the end. There may be form LRRC4-miR-185-DNMT1-LRRC4loop among LRRC4, miR185and DNMT1that participating in glioma development. In addition, DNMT1was positively regulated by SP1, and it could increase the expression of LRRC4, while LRRC4could also inhibit SP1by negatively regulate ERK/MAPK and PI-3K/AKT signal pathway. So that the LRRC4-SP1-DNMT1-LRRC4loop formed among LRRC4, SP1and DNMT1took part in the glioma formation.In conclusion, development of glioma is the pathological processing with multiple genes and multi-stages. Genes, miRNAs and DNA methylation play an important role in glioma formation. They may support or antagonize each other and construct complicated network in glioma. In sum of above study, at the time of LRRC4regulating miRNAs as a tumor suppressor, those miRNAs regulated by LRRC4were found to regulate the binding of transcription factors to DNA in their targets mediated signal pathways by directly targeting genes (such as LRRC4), or regulate methylation and expression of such hypermethylation genes as LRRC4by directly targeting DNA methyltransferase and controlling global methylation. And multipases loops which the core was LRRC4were formed. They were LRRC4-AP-2-miR-182-LRRC4, LRRC4-miR-185-DNMT1-LRRC4and LRRC4-SP1-DNMT1-LRRC4. These loops participated in glioma development with multiple positive feedback formation among them.