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  • In present study we measured the

    2020-02-13

    In present study, we measured the methylation and calcium channel blocker levels of CDKL2 in HCC tissues and cell lines. Meanwhile, correlations between the methylation and mRNA expression, clinicopathological features were evaluated.
    Materials and methods
    Results
    Discussion DNA methylation is a heritable epigenetic modification that can contribute to HCC development (Mah and Lee, 2014). Aberrant DNA methylation of CDKL2 was first reported to be associated with HCC in a small sample of Taiwanese HCC tissues by methylation microarrays (Shen et al., 2012). However, the authors did not perform any clinical associations with their data due to the limited samples. In the present study, methylation levels of CDKL2 were measured in the three HCC cell lines and a large cohort of 178 HCC tissues, 169 adjacent non-tumor tissues and 24 normal liver tissues. We observed that the methylation levels of CDKL2 were higher in HCC cell lines and tissues than those in normal liver tissues. However, the mRNA expression was decreased both in HCC cell lines and HCC tissues, and showed an inversely correlation with DNA methylation. Moreover, the mRNA expression of CDKL2 could be upregulated by 5-aza-2′-deoxycytidine treatment in HCC cell lines. Several studies showed that CDKL2 probably played an important role in tumor progression. The genome-wide association studies (GWASs) in 2010 first found a meaningful SNP of CDKL2 in breast cancer (Bonifaci et al., 2010). Linna Li et al. also observed that CDKL2 could promote epithelial-mesenchymal transition (EMT) and tumor formation in a breast cancer xenograft model (Li et al., 2014). Recently, a few significantly differentially methylated CpG sites of CDKL2 were found in a genome-wide DNA methylation analysis of prostate cancer (Rubicz et al., 2016), as well as in HCC by methylation microarrays (Shen et al., 2012; Shen et al., 2015). Our results showed that the methylation levels of CDKL2 were significantly increased gradually from normal liver tissues to adjacent non-tumor liver tissues and further to HCC tumor tissues, suggesting that CDKL2 methylation participated in the progression of HCC. Early appeared and easily captured in carcinogenesis make DNA methylation an attractive source of potential biomarkers (Mikeska et al., 2012). Many studies reported that DNA methylation could be served as potential biomarkers for tumor diagnosis, such as p16, SOCS1, GSTP1, CDH1 (Mikeska et al., 2012; Mah and Lee, 2014). We found a high frequency of CDKL2 hypermethylation in HCC tissues, and the ROC analysis showed that the hypermethylation of CDKL2 could well distinguish HCC tissues from adjacent non-tumor tissues with a high specificity and sensitivity. Additionally, CDKL2 methylation could be detected in HCC plasma samples using pyrosequencing (Shen et al., 2012). These results suggested that CDKL2 methylation could be a potential noninvasive diagnostic biomarker for HCC, although further studies were needed. It was acknowledged that gene specific hypermethylation played an important role in the tumor progression through mediating transcriptional silencing (He et al., 2014; Schelleckes et al., 2017). Consistent with previous studies that CDKL2 not only participated in cognitive function but also tumor progression (Gomi et al., 2010; Fang et al., 2018), our results showed CDKL2 methylation could modulate gene expression based on two points bellowed. Firstly, there was a significantly negative correlation between the CDKL2 methylation and its mRNA expression in tissue samples. Secondly, the mRNA expression of CDKL2 could be upregulated by demethylation agent treatment in HCC cell lines. This expression-regulation-by-methylation pattern of CDKL2 might be a vital mechanism in hepatocarcinogenesis. Several limitations should be considered in the present study. First, compared with prospective study, our cross-sectional research was not powerful enough to establish the causal relationship between the CDKL2 hypermethylation and HCC progression. Second, plasma samples and early HCC patients were needed to value the performance of CDKL2 methylation as a potential biomarker of liquid biopsy of HCC. The last, our study was mainly based on FFPE samples which led the lack of specimens for mRNA expression analysis. In a word, further investigations were needed to explore the precise mechanism and the clinical significance of CDKL2 methylation in HCC.