The alteration of epigenetic machineries such as histone methylations has been identified as essential mechanisms driving initiation and progression of human cancers including the hepatocellular carcinoma[18, 20]. As one major types of histone modification associated with epigenetic events, the COMPASS complex is responsible for catalyzing the trimethylation of H3K4 and target gene transcription associated with the prognosis of hepatocellular carcinoma patients[21, 22]. However, the functional regulation of the COMPASS complex and downstream signaling pathways during HCC pathogenesis remains poorly elucidated. In the present study, we first demonstrated the highly elevated expression of MKL1 gene in the clinical tissues from HCC patients by bioinformatic analysis, combined with validation in three HCC cell lines. Moreover, the silencing of MKL1 expression resulted into significantly repressed proliferation, migration, invasion and colony formation but promoted apoptosis in HCC cells. In addition, we confirmed by cancer cell line-based xenograft model that MKL1 gene silencing impaired the tumorigenesis of HCC cells. Finally, we showed that MKL1 silencing caused greatly reduced expression of three COMPASS complex components and repressed downstream NF-kF signaling in HCC cells. These results provided novels insights into the molecular mechanism underlying HCC development, especially the regulation of epigenetic events mediated by histone lysine methylation.
MKL1 (Megakaryoblastic leukemia 1) was previously identified as one part of translocation mutation associated with acute megakaryoblastic leukemia, which is one member of the myocardin transcriptional coactivator family featured with several conserved domains such as C-terminal transactivation domain, a leucine zipper-like domain and a glutamine-rich domain[26]. Reports in recent years showed that MKL1 has been widely implicated in various biological and pathogenic processes such as cell migration, chromatin reorganization, histone post-translational modification, dendritic spine morphology, angiogenesis, immune responses and also cancer development[27–30]. For instance, MKL1 could be recruited onto the promoter of the matrix metalloproteinase 2 (MMP2) gene in ovarian cancer cells as a response to hypoxia, and promote MMP2 gene transcription and ovarian cancer cell migration and invasion by recruiting the SET1 and BRG1, which serve as histone methyltransferase and chromatin remodeling protein respectively[31]. Also, MKL1 was involved in endothelial-to-mesenchymal transition that mediated the liver fibrosis and cirrhosis[23]. Moreover, the depletion of MKL1 gene effectively repressed the growth of hepatocellular carcinoma xenograft through enhancing the oncogene-induced senescence[24]. In the present study, we demonstrated the significantly increased MKL1 expression in HCC clinical tissues and cell lines, followed by direct evidences showing the regulation of HCC proliferation, migration, invasion, apoptosis and in vivo tumorigenesis by MKL1 silencing. The regulation of HCC proliferation by MKL1 was also validated by the reduced Ki67 expression in tumors caused by MKL1-silenced HCC cells in nude mice. Our investigation further validated the expressional alterations and roles of MKL1 in HCC pathogenesis, which also provided further insights into the cellular functions of MKL1 high expression in HCC initiation and progression.
As introduced above, the modulation of histone methylation and chromatin remodeling has been shown as one key mechanism of MKL1-regulated target gene transcription activation[21, 31]. Previous investigations showed that the transcription-enhancing functions of MKL1 protein was mainly mediated by the recruitment of the COMPASS complex and resultant histone lysine methylation[21, 31]. The COMPASS complex is composed of three major protein components, including SET1 protein responsible for catalyzing the lysine methylation in histones wrapped by target gene sequences, and WDR5 and ASH2 serving as histone methyltransferase adaptors[21, 32, 33]. In this study, we showed that the expression of SET1, WDR5 and ASH2 proteins in HepG2 and Huh-7 cells were all significantly down-regulated by silencing of MKL1 gene expression, which convincingly verified the effects of MKL1 protein in regulating the COMPASS complex functions in context of hepatocellular carcinoma. Interesting, the regulation of SET1, WDR5 and ASH2 proteins by MKL1 was also recently observed in hepatic stellate cells during TGF-(tumor growth factor b)-induced fibrogenic response and fibrosis in the liver[34]. Combined together, it is reasonably to conclude that the MKL1-regulated COMPASS complex serves as an essential diver of HCC pathogenesis, through promoting liver fibrosis and modulating hepatocellular carcinoma cell proliferation, migration, invasion and apoptosis.
The regulation of target gene transcription by MKL1 and the COMPASS complex has also been regulated by their reciprocal interactions with the NF-kB signaling pathway. On one hand, the NF-κB protein was required for the association of MKL1 protein with the promoter regions of target genes, thus initiating the following COMPASS complex-induced histone modification and transcription activation[21, 31]. On the other hand, the introduction of MKL1 protein could also enhance the nuclear enrichment of the NF-kB protein and strength the binding of NF-kB protein with target gene promoter, therefore promoting further elevated expression of NF-kB target genes[21]. Here in this study, we also showed that the expression of p65 protein, one major subunit of the NF-kB protein, was remarkably decreased in both the HepG2 and Huh-7 cells, indicating the involvement of NF-kB signaling and its interaction with the COMPASS complex during MKL1-regulated HCC pathogenesis. Of note, the dynamic alteration and molecular mechanisms underlying the interaction between MKL1 protein, the COMPASS complex and NF-kB in HCC cells deserve further investigation.