MiR-145 Regulates UHRF1 And Reverses DNA Methylation Pattern Alteration In Lung Cancer Cells

Lung cancer, with poor prognosis and low survival rate, was one of the most severe cancers in China. As an important epigenetic modification, DNA methylation was found to be in abnormal state in majority of cancers including lung cancer, such as hypomethylation of repeated sequence and hypermethylation of promoter of tumor suppressor genes. In previous studies, altered expression of DNMT1, DNMT3A, DNMT3B and UHRF1 were believed to contribute to the cancer specific DNA methylation pattern. Moreover, the altered expression of UHRF1/DNMTs in cancers was confirmed in this study through the analysis of data and the detection in the cancer cell lines and lung cancer clinical samples. However, the underlying mechanism of overexpression of UHRF1/DNMTs in cancers was largely unclear. As an important regulator, microRNA might participate in the regulation of UHRF1/DNMTs. So we wanted to find miRNAs that could be responsible for elevated expression of UHRF1/DNMTs in majority of cancers. Firstly, we screened out the miRNAs that were predicted to target UHRF1/DNMTs through miRNA target prediction databases. Then we screened out the miRNAs that were down regulated in majority of cancers through the analysis of the expression data of these miRNAs in cancers downloaded from databases. Moreover, combined the primeval miRNA mimics transfection experiment with the detection of the expression of these miRNAs in cell lines and clinical samples, we chose miR-145, a famous tumor suppressor, for further experiments. After the transfection experiment, we found that miR-145 could inhibit the expression of UHRF1 and DNMT1 in both protein level and mRNA level and could repress UHRF1 by directly targeting its 3’-UTR. Besides, we found that miR-145 could inhibit cell proliferation through the restriction of cells on the G1 phase of cell cycle. Furthermore, overexpression of miR-145 could reduce 2.8% of global DNA methylation level in A549 cell and could also reduce the DNA methylation level of majority of genome elements except enhancer. On the other hand, the DNA methylation level of the promoter of some tumor suppressor genes, like CDH13, DLC1, RASSF1 and CDH1, would decline in some extent by the influence of miR-145. Interestingly, we also found that the DNA methylation level of repeated sequence LINE1 and HERV-K increased by 7.9% and 7.1% in A549 cell treated with miR-145 respectively. These results indicated that the cancer specific DNA methylation pattern could be reversed in some extent by miR-145. Meanwhile, through the detection in cell lines and lung cancer clinical samples, we found that miR-145-promoter, the upstream promoter of miR-145 itself, and miR-143/145-promoter, the co-promoter of miR-143 and miR-145, had a higher methylation level in tumor cell than in normal cell. Moreover, the DNA methylation level of both promoters, especially miR-143/145-promoter, had a significant reverse correlation with the expression level of miR-145. By knockdown of UHRF1 in A549, we demonstrated that UHRF1 could regulate miR-145 expression through the influence on the DNA methylation level of these two miR-145 promoter regions. Furthermore, we found that stable overexpression of miR-145 in A549 cell could reduce 16.3% of the DNA methylation level of endogenous miR-143/145-promoter, but not miR-145-promoter. Together, this study suggested that miR-145 formed a regulatory loop with UHRF1 and DNA methylation and the abnormity of their regulation balance might lead to the overexpression of UHRF1 and tumorigenesis.