Construction of warning system for lung cancer by whole genome sequencing screening for sensitive methylation sites of non-small-cell lung carcinoma

Abstract

Objective To explore the construction of early warning system for non-small cell lung cancer (NSCLC) by whole genome sequencing. Methods The experiment was divided into two groups: normal tissue and NSCLC tissue. The expression of excision repair cross-complementation group 1 (ERCC1), tubb3 and RRM1 mRNA in NSCLC cells was detected by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR). The distribution of methylation in NSCLC cells was detected by whole genome sequencing. CpG methylation analysis was performed using sequencing data. Histone modification data were used to analyze the relationship between histone modification and DNA methylation. The expression of histone methylated eggs and methylated related enzymes in normal and NSCLC tissues were detected by protein immunoassay. Results Compared with the control cells, ERCC1 mRNA [(0.78±0.14) vs. (0.12±0.04), χ2=6.370, P<0.05] and RRM1 mRNA [(0.52±0.07) vs. (0.05±0.01), χ2=5.360, P<0.05] expression in NSCLC cells was down-regulated compared with control cells. Methylation level in NSCLC group increased at transcription start site and decreased in intergene region (χ2=3.140, P<0.05). Nine CpG methylation sensitive sites were found in the NSCLC group (RUNX3, MIR196A1, HOXA11, OTP, GATA4, PTPRU, SLC15A3, ZIC1 and TFAP2B). NSCLC group compared with control group, the lower histone acetylation [(H3K9ac [(43.57±8.84) vs. (10.64±4.35), χ2=8.730, P<0.05] and H3K27ac [(40.52±8.64) vs. (9.67±3.58), χ2=5.470, P<0.05) ] and histone methylation [(H2az [(42.56±9.74) vs. (12.47±6.05), χ2=7.420, P<0.05]. H3K4me1 [(37.47±6.42) vs. (15.46±7.34), χ2=5.380, P<0.05], H3K4me2 [(50.37±10.24) vs. (9.47±6.54), χ2=9.270, P<0.05]. H3K4me3 [(52.37±6.49) vs. (10.58±5.88), χ2=1.690, P<0.05] and H3K79me2 [(34.55±6.42) vs. (11.23±6.94), χ2=3.450, P<0.05]. Compared to the control group, NSCLC group DNMT1 express cut [(1.88±0.24) vs. (0.12±0.01), χ2=5.430, P<0.05], DNMT3a expression cut [(1.75±0.36) vs. (0.49±0.11), χ2=7.890, P<0.05], expression of DNMT3b cut [(0.88±0.14) vs. (0.13±0.05), χ2=1.360, P<0.05], expression of H3K4me3 cut [(2.53±0.35) vs. (0.35±0.08), χ2=5.440, P<0.05), H3K9me2 expression cut (0.55±0.07) vs. (0.05±0.01), χ2=3.270, P<0.05]. Conclusion Histone modification is closely related to DNA methylation, and the expression of histone methylation and methylation-related enzymes is also affected. Methylation sensitive sites can be used as biomarkers for early detection of NSCLC. Key words: Whole genome sequencing; Non-small-cell lung carcinoma; Methylation; Histone modification; Lung cancer cells