RESUMO
Colorectal cancer (CRC) and gastric cancer (GC) rank the top five common and lethal cancers worldwide. Early detection can significantly reduce the mortality of CRC and GC. However, current clinical screening methods including invasive endoscopic techniques and noninvasive fecal occult blood test screening tests/fecal immunochemical test have shown low sensitivity or unsatisfactory patient's compliance. Aberrant DNA methylation occurs frequently in tumorigenesis and cell-free DNA (cfDNA) methylation has shown the potential in multi-cancer detection. Herein, we aimed to explore the value of cfDNA methylation in the gastrointestinal cancer detection and develop a noninvasive method for CRC and GC detection. We applied targeted methylation sequencing on a total of 407 plasma samples from patients diagnosed with CRC, GC, and noncancerous gastrointestinal benign diseases (Non-Ca). By analyzing the methylation profiles of 34 CRC, 62 GC and 107 Non-Ca plasma samples in the training set (n=203), we identified 40,110 gastrointestinal cancer-specific markers and 63 tissue of origin (TOO) prediction markers. A new integrated model composed of gastrointestinal cancer detection and TOO prediction for three types of classification of CRC, GC and Non-Ca patients was further developed through logistic regression algorithm and validated in an independent validation set (n=103). The model achieved overall sensitivities of 83% and 81.3% at specificities of 81.5% and 80% for identifying gastrointestinal cancers in the test set and validation set, respectively. The detection sensitivities for GC and CRC were respectively 81.4% and 83.3% in the cohort of the test and validation sets. Among these true positive cancer samples, further TOO prediction showed accuracies of 95.8% and 95.8% for GC patients and accuracies of 86.7% and 93.3% for CRC patients, in test set and validation set, respectively. Collectively, we have identified novel cfDNA methylation biomarkers for CRC and GC detection and shown the promising potential of cfDNA as a noninvasive gastrointestinal cancer detection tool.
RESUMO
Evidence demonstrates that DNA methylation is associated with the occurrence and development of various neurological diseases. However, the potential target genes undergoing DNA methylation, as well as their involvement in the chemotherapy drug oxaliplatin-induced neuropathic pain, are still unclear. Here, Lrfn4, which showed hypermethylation in the promoter regions, was screened from the SRA methylation database (PRJNA587622) following oxaliplatin treatment. MeDIP and qPCR assays identified that oxaliplatin treatment increased the methylation in Lrfn4 promoter region and decreased the expression of LRFN4 in the spinal dorsal horn. The assays with gain and loss of LRFN4 function demonstrated that LRFN4 downregulation in spinal dorsal horn contributed to the oxaliplatin-induced mechanical allodynia and cold hyperalgesia. Moreover, oxaliplatin treatment increased the DNA methyltransferases DNMT3a expression and the interaction between DNMT3a and Lrfn4 promoter, while inhibition of DNMT3a prevented the downregulation of LRFN4a induced by oxaliplatin. We also observed that the transcriptional factor POU2F1 can bind to the predicted sites in DNMT3a promoter region, oxaliplatin treatment upregulated the expression of transcriptional factor POU2F1 in dorsal horn neurons. Intrathecal injection of POU2F1 siRNA prevented the DNMT3a upregulation and the LRFN4 downregulation induced by oxaliplatin. Additionally, intrathecal injection of DNMT3a siRNA or POU2F1 siRNA alleviated the mechanical allodynia induced by oxaliplatin. These findings suggested that transcription factor POU2F1 upregulated the expression of DNMT3a, which subsequently decreased LRFN4 expression through hypermethylation modification in spinal dorsal horn, thereby mediating neuropathic pain following oxaliplatin treatment.