CUL4B Promotes Cell Proliferation And Migration Of Lung Cancer Cells By Regulating MiRNAs

CUL4B acts as a scaffold that assembles DDB1, RBX1 and substrate receptors to form Cullin4B-Ring-based E3 ubiquitin ligases (CRL4B). Lack of CUL4B greatly compromises cell proliferation, extra embryonic development, hematopoiesis and neurogenesis. On the other hand, CUL4B is overexpressed in a variety of cancers and is crucial for the maintenance of the malignant behaviors of cancer cells. Mechanistically, CUL4B could repress a number of tumor suppressor genes including p16, PTEN, p21, and IGFBP3, by catalyzing H2AK119 mono-ubiquitination at their promoters. While CUL4B was demonstrated to possess oncogenic properties in those studies, the mechanisms by which CUL4B is upregulated remain to be elucidated.MicroRNA (miRNA) can negatively regulate their target genes by binding to the 3’ untranslated region (UTR) of mRNAs, thus inhibiting protein synthesis. Many tumor suppressors and oncogenes have been identified to be targets of miRNA, and aberrant miRNA expression is associated with pathogenic and prognostic implications in cancer. Recent studies showed that miRNAs themselves are subjected to epigenetic regulation. However, the mechanisms underlying the expressional regulations of a large number of miRNAs remain to be characterized.Given that miRNA and CUL4B play important roles in tumorigenesis, we investigated the role of CUL4B in lung cancer, and the regulation between miRNAs and CUL4B.PART ONECUL4B promotes lung cancer cell proliferation, migration and invasionCUL4B is highly expressed in a variety of tumors. However, the roles of CUL4B in lung cancer remain to be determined. In this section, we investigated the role of CUL4B in lung cancer, and obtained the following results:1) CUL4B is up-regulated in lung cancer tissues:We first determined the protein levels of CUL4B by Western blot analysis in 30 matched pairs of tumor and their adjacent non-tumor tissues from the patients with lung cancer. Consistent with our previous observation of other solid tumors, CUL4B levels were higher in tumor than in adjacent non-tumor in 66.6% (20/30) of tissue pairs.2) Upregulation of CUL4B in lung cancer occurred at post-transcriptional levels: To gain insight into the mechanism by which CUL4B is upregulated in lung cancer, we examined the mRNA by q-PCR in 30 paired specimens described above. No corresponding changes between CUL4B mRNA and protein levels were observed, suggesting that the upregulaton of CUL4B in lung cancer may have occurred at post-transcriptional levels.3) CUL4B promotes lung cancer cell proliferation and tumor growth:Upregulation of CUL4B in lung cancer suggests that CUL4B might function as an oncogene in lung cancer. We thus examined the effect of CUL4B on cell proliferation by knocking down its expression in lung cancer cell line A549 (shCUL4B). MTT assays showed significantly reduced growth in shCUL4B A549 cells. We further tested the roles of CUL4B in tumor growth in vivo in a xenograft model. Significantly reduced tumor volume and weight were observed in shCUL4B group than in control group.4) CUL4B promotes tumor cell migration and invasion:Wound healing assay showed that the healing of scatches in CUL4B knockdown H1299 cells was much slower than that in control cells. In addition, the number of cell that migrated across transwell membrane was considerably lower in CUL4B knockdown cells. Furthermore, matrigel invasion assay showed a great reduction in the number of invasive cells in the shCUL4B cells.PART TWOmiR-194 regulates CUL4B expression in lung cancer cellsThe fact that CUL4B is upregulated at post-transcriptional levels suggests that miRNAs might be involved in regulating CUL4B expression. In the section, we tested this hypothesis, and achieved the following results:1) miR-194 could target the 3’UTR of CUL4B:Three target prediction algorithms (TargetScan, miRanda, and miRDB) were utilized to predict the potential miRNAs that could target the 3’-UTR of CUL4B. miR-194 appeared to be the only miRNA predicted to target CUL4B by all three programs. Furthermore, the miR-194 binding sites within the 3’-UTR of CUL4B were highly conserved across different species. These results suggest that CUL4B might be a target of miR-194.2) miR-194 directly binds the 3’-UTR of CUL4B mRNA and regulates CUL4B protein expression:To determine the role of miR-194 in regulating CUL4B, we first transiently transfected miR-194 mimics or inhibitors into H1299 and A549 cells, and found that overexpression of miR-194 significantly down-regulated CUL4B at protein level, while inhibition of endogenous miR-194 resulted in an increased amount of CUL4B proteins. To determine whether miR-194 acts directly on the 3’-UTR of CUL4B, luciferase reporter vectors containing the 3’-UTR of CUL4B with and without point mutations in the seed sequence of miR-194 were cotransfected with mimics or inhibitors of miR-194 into H1299 cells. The results showed that transfection with miR-194 mimics dramatically reduced the luciferase activity of CUL4B 3’UTR, whereas transfection with miR-194 inhibitor significantly increased the luciferase activity. In contrast, miR-194 mimics and inhibitors had no effect on the luciferase activity of the reporters in which miR-194 binding sites were mutated. Furthermore, RNA pull down assay confirmed that miR-194 directly binds to 3’-UTR of CUL4B mRNA.3) The inhibitory effect of miR-194 on mobility of lung cancer cells was mediated by its repression of CUL4B:To determine whether CUL4B serves as a functional effector of miR-194 in lung cancer cells, we examined the rescue effect of exogenous CUL4B on miR-194-mediated cell mobility. Transwell assay showed that ectopic expression of CUL4B could significantly rescue the suppressive effect of miR-194 mimics.4) p53-miR-194 axis was involved in regulating CUL4B expression:Because miR-194 is transcriptionally upregulated by p53, we next tested whether CUL4B expression was affected by the function of p53. As expected, the protein level of CUL4B was higher in p53 null H1299 cells than in A549 cells with wild type p53. Furthermore, Nutlin-3 treatment, which stabilizes p53, resulted in a reduction in CUL4B protein. In contrast, the protein levels of CUL4B showed no response to Nutlin-3 treatment in p53 null H1 299 cells. Knockdown of p53 further confirmed its negative regulation of CUL4B. We next determined whether the negative regulation of CUL4B by p53 is mediated by miR-194 by examining the levels of p53, CUL4B and miR-194 at different time points after Nutlin-3 treatment. The results showed that the spike of miR-194 expression coincided with the drop in CUL4B protein levels. Furthermore, luciferase activity of CUL4B 3’-UTR is much higher in H1299 cells than in A549 cells, whereas the expression of miR-194 was lower in H1299 cells than in A549 cells. In contrast, no obvious difference in luciferase activity of mutant CUL4B 3’-UTR was observed between these two cell lines. More importantly, inhibition of miR-194 could block the decreased CUL4B protein levels and the decrease in luciferase activity of CUL4B 3’-UTR caused by Nutlin-3 treatment. These results indicate that CUL4B is negatively regulated by the p53-miR-194 axis.5) miR-194 was downregulated in human lung cancer tissues and negatively correlated with CUL4B:We determined the levels of miR-194 in 30 paired specimens using q-PCR. Downregulation of miR-194 was observed in 63.3%(19/30) of tumor tissues. Importantly, miR-194 levels were negatively correlated with CUL4B protein levels in these tumor tissues.PART THREECUL4B and miR-194 form a double negative feedback loop in lung cancer cellsTo further investigate the molecular mechanisms by which CUL4B promote oncogenesis, we performed high-throughput q-PCR analyses of cancer-related miRNAs in CUL4B knockdown A549 and control cells, and obtained the following results:1) CUL4B represses transcription of miR-194 in lung cancer cells:High-throughput q-PCR analyses of cancer-related miRNAs showed that all members of two miR-194 clusters (miR-192-194-2 and miR-215-194-1) were upregulated in CUL4B knockdown cells, and these results were further verified by q-PCR in both CUL4B knockdown H1299 and A549 cells. Moreover, the expression of pri-miRNA of these two clusters (pri-miR-194-1/2) was also significantly increased in CUL4B knockdown cells, suggesting that CUL4B may repress the initial step of miR-194 biogenesis.2) CUL4B directly binds to and represses the transcription of miR-194 gene clusters:To characterize the molecular mechanisms responsible for the repression of miR-194 by CUL4B, we performed chromatin immunoprecipitation (ChIP) assay, and found that CUL4B could directly bind to the promoters of the miR-192-194-2 and miR-215-194-1 clusters, and this binding is accompanied by increased H2AK119ubl. In addition, knockdown of CUL4B markedly reduced the levels of CUL4B binding to the promoters of these two clusters as well as those of H2AK119ub1. Consistent with our previous observation, knockdown of CUL4B markedly reduced the enrichment of EZH2, as well as the associated H3K27me3 in these regions. These results suggest that CUL4B can repress the transcription of miR-192-194-2 and miR-215-194-1 clusters by promoting H2AK119 monoubiquitination and consequently the recruitment of EZH2 and H3K27 trimethylation.3) CUL4B and miR-194 formed a double negative feedback loop:The repression of miR-194 by CUL4B together with the finding that CUL4B is a direct target of miR-194 suggest the existence of a double negative feedback loop between miR-194 and CUL4B. To test this, miR-194 mimics were transfected into H1299 cells and the endogenous level of pri-miR-194 was analyzed. As expected, transfection of miR-194 mimics efficiently elevated pri-miR-194 as well as mature miR-192 and miR-215. Importantly, this upregulation was dramatically attenuated by co-transfection of CUL4B expression vector. Collectively, these data indicate that miR-194 and CUL4B can regulate each other in a double negative feedback loop.4) miR-194 repression mediates the oncogenic activity of CUL4B:Because CUL4B suppresses miR-194 expression, we predicted that miR-194 target genes may be downregulated in CUL4B knockdown cells. Indeed, knockdown of CUL4B significantly reduced the levels of RBX1 and N-cadherin as well as luciferase activity of their 3’-UTR reporters. Moreover, inhibition of miR-194 was able to rescue the decreased levels and luciferase activity of 3’-UTR of RBX1 and N-cadherin in shCUL4B cells. In addition, inhibition of miR-194 remarkably restored the mobility and invasiveness in CUL4B knockdown H1299 cells, indicating that CUL4B contributes to oncogenesis of lung cancer cells at least partly through repression of miR-194.PART FOURCUL4B up-regulates CDK2 by repressing the transcription of miR-371-373 clusterIn addition to miR-194, miR-371/372/373 were also upregulated in CUL4B knockdown A549 cells. Therefore, in this part, we analyzed the mechanisms responsible for the regulation of miR-371-373 cluster by CUL4B.1) CUL4B negatively regulates miR-371-373 cluster members:q-PCR analysis showed that knockdown of CUL4B in H1299, A549 and HeLa cells significantly increased the expression of all members of miR-371-373 clusters, whereas overexpression of CUL4B significantly decreased the expression of these miRNAs.2) CUL4B negatively regulates the transcription of miR-371-373 cluster. We next analyzed pri-miR-371-373 expression and found that the level of pri-miR-371-373 was significantly increased in CUL4B knockdown H1299, A549 and HeLa cells, and significantly decreased in CUL4B overexpressing cells. These results suggest that CUL4B represses the initial step of miR-372/373 biogenesis.3) CUL4B directly binds to and represses the transcription of miR-371-373 cluster: ChIP assay showed that CUL4B could directly bind to the promoter of the miR-371-373 cluster, and this binding is accompanied by increased H2AK119ub1. In addition, knockdown of CUL4B markedly reduced the enrichment of CUL4B, EZH2, as well as H2AK119ub1 and H3K27me3 in the promoter of miR-371-373 cluster, suggesting that CUL4B can repress the transcription of miR-371-373 cluster by promoting H2AK119 monoubiquitination and consequently the recruitment of EZH2 and H3K27 trimethylation.4) CUL4B modulates CDK2 protein expression by repressing miR-372/3 transcription:miR-372/373 have been reported to regulate the expression of CDK2 by targeting its 3’-UTR. Therefore, we analyzed CUL4B-miR-372/3-CDK2 axis in cells. Knockdown of CUL4B significantly decreased the levels of CDK2 protein. Conversely, CUL4B overexpression resulted in higher levels of CDK2. The down-regulation of CDK2 by CUL4B knockdown could be efficiently attenuated by miR-372/373 inhibitors. Moreover, knockdown CUL4B markedly reduced luciferase activity of wild type, but not mutant 3’-UTR of CDK2, suggesting that CUL4B may up-regulate CDK2 expression by repressing miR-372/373.