The Biological Clock Gene CLOCK Promotes The Proliferation And Migration Of Glioma Cells

In the living entities, circadian rhythm serves as an important regulatory system apart from the nervous, humoral and immune systems, controlling the biochemical, physiological or behavioral processes. Disruption of the circadian rhythm may negatively affect cellular function, potentially leading to increased susceptibility to diseases, including cancers. The circadian rhythm is regulated by transcription-translation feedback loops among a small set of core circadian genes. It has been shown that up to 10% of all genes in the mammalian genome are under the regulation of circadian genes, and the circadian genes may function as oncogenes or tumor suppressors. miRNAs are small non-coding RNAs that play essential roles in post-transcriptional regulation of gene expression. They can bind to partially complementary recognition sequences of mRNA, causing either degradation of mRNA or inhibition of translation, thus effectively silencing their mRNA target. Alterations of miRNAs have also been reported in the initiation and progression of tumors including gliomas. MiR-124 was frequently reported to be downregulated in glioma and play a glioma suppressive role by targeting specific genes, such as SNAI2. Although the roles in tumorigenesis of the clock genes and microRNAs are known, mechanisms of cross-talking between circadian clock and microRNAs in regulation of gliomagenesis are poorly understood.To study the molecular function of a core circadian gene CLOCK in glioma, we first measured the CLOCK protein level in 16 glioma tissues and two normal brain tissues and found that CLOCK protein expressed in glioma tissues of grade III and grade IV is higher comparing with normal brain tissues and grade Ⅱ glioma tissues. An increase of CLOCK protein was also found in glioblastoma cell lines U87MG, T98G, A172, and U251 compared to the human normal glial cell lines, HASP and HEB. Then we performed a loss-of-function analysis using CLOCK-targeting siRNA oligos in U87MG and T98G cells for functional assays. The results indicated that depletion of CLOCK suppressed the proliferation, survival, and migration of T98G and U87MG cells.Next, we use two algorithmic methods (TargetScan and PicTar) predicted that miR124 and miRl81b, were to putatively bind to the 3’UTR of Clock. We adopted luciferase reporter assays and mutation asssys validated that miR-124 but not miR-181b could target to CLOCK 3’UTR.To further exploring the mechanisms of cross-talking between CLOCK and miR-124 in regulation of gliomagenesis, we transfected CLOCK siRNA or miR-124 mimics into T98G, and found that both CLOCK knockdown and miR-124 over-expression could down-regulate the NF-κB activity, which has been reported playing important role in tumorigenesis. It suggested that the aberrant expression of miR-124 and CLOCK may be associated with NF-κB pathway in glioma.In summary, here we show that the expression level of CLOCK was significantly increased in high-grade human glioma and glioblastoma cell lines. In contrast it has been found that miR-124 expression level was attenuated in the glioma tissue samples and glioma cell lines by Silber, Lim et al., and Tan et al. in our lab verified the attenuation of miR-124 in glioma cell lines. We further identified that miR-124 directly targets to CLOCK 3’UTR and both over-expression miR-124 or knockdown of CLOCK induced the activation of NF-κB in glioma. Our data suggest that as a target of glioma suppressor miR-124, CLOCK positively regulates glioma proliferation and migration by reinforces NF-κB activity.