The epigenetic writer LSD1 controls hepatic cell cycle progression by alteration of global expression profiles and protein signatures

Background and Aim:

Chronic liver disease is one of the major health problems worldwide. The common development process of chronic liver disease is characterized by fibrosis with a high risk to end up in hepatocellular carcinoma (HCC). The lysine-specific histone demethylase, modifying the histone 3 methylation state, is a major epigenetic writer that is known to affect tumour growth. However, its function in chronic liver disease and liver cancer has not yet been addressed. In the present study we therefore investigated its hepatic function by global transcriptomic and proteomic analysis in liver cancer cells and hepatic stellate cells (HSC).

Methods:

LSD1 function was inhibited in different hepatocellular carcinoma cell lines (Huh7, Hep3B, and HepG2) and in myofibroblastically activated human hepatic stellate cells (HSC, LX-2) by means of RNA interference or application of a specific LSD1 inhibitor. Cells were functionally characterized by cell growth, cell differentiation, and senescence assays using immunostaining and FACS analysis. The microRNA (miRNA) pattern in response to LSD1 inhibition was measured by qPCR arrays followed by signalling pathway analysis for target genes of significant miRNAs. Global transcriptomics was carried out by RNA ultra-deep sequencing and protein expression profiles were analysed by protein mass spectrometry.

Results:

LSD1 inhibition initiates cell growth arrest in both, hepatocellular carcinoma cells and myofibroblastically activated HSC. Notably, comprehensive bioinformatic analyses of global changes in transcriptomic and proteomic expression profiles demonstrated pronounced alterations of central mediators involved in cell cycle progression and DNA synthesis. Thus, expression levels of PLK1, TP53, various cyclin-dependent kinases (CDK4, CDK6) as well as a set of DNA polymerases are significantly changed in response to LSD1 inhibition. These results were even more pronounced on protein than on transcript level suggesting that posttranscriptional alterations are additionally involved. miRNA are non-coding RNAs, controlling gene expression posttranscriptionally. Indeed, miRNA profiling reveals a distinct change in miRNA pattern. Interestingly, in HSC LSD1 inhibition did not only alter miRNAs involved in cell cycle control such as miR-34 but additionally lead to an increase of antifibrotically acting miRNAs e.g. miR-29.

Conclusions:

Our research demonstrates that LSD1 functions as a central mediator of cell cycle control in both HSC and HCC cells by induction of tremendous changes in expression of the cell dividing apparatus.