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. 2020 Apr 15;10(2):391–418. doi: 10.1016/j.jcmgh.2020.04.002

Figure 12.

Figure 12

Elevated CSF1R expression is associated with miR-34a promoter methylation and the invasion front of metastatic primary CRCs. (A) Genomic region 2.0 kbp upstream of the transcriptional start site (TSS) (position indicated by arrow) within the human miR-34a gene. Vertical bars represent CpG dinucleotides. The position of the p53 binding site (BDS) is indicated. The horizontal bars indicate PCR amplicons used for MSP and bisulfite-sequencing PCR (BSP), respectively. (B) Representative results of MSP analysis. (C) Bisulfite sequencing analysis of the miR-34a promoter in DLD1_par and DLD1_5FU cells. 9 subcloned amplification products were sequenced for each cell lines. Each horizontal line represents 1 individual clone, and each circle 1 single CpG dinucleotide. Open circles represent nonmethylated and black circles methylated CpGs. (D) qPCR analysis of pri-miR-34a in DLD1_5FU cells after treatment with 5-aza for 72 hours or alternatively with 5-aza for 72 hours combined with TSA for the last 24 hours. (E) Western blot analysis of cell lysates isolated from DLD1_5FU cells after treatment with 5-aza for 72 hours or alternatively with 5-aza for 72 hours combined with TSA for the last 24 hours. (F) Left: quantification of CSF1R protein expression in human CRC samples of 78 patients. The methylation status of miR-34a in these samples had been determined previously.65 Right: representative immunohistochemical detections of CSF1R protein in miR-34amCPG low and high tumors, respectively. Scale bar = 50 μm. (G) Evaluation of CSF1R protein expression at the invasion front in M0 and M1 CRCs (left chart) and examples of representative immunohistochemical detections (right panel). The presence of an invasion front was confirmed by DH, a certified pathologist. Results were analyzed using the chi-square test. Scale bar = 50 μm. In panels D, F, and G, mean values ± SD are provided. ∗P < .05. bisulfite dH2O, no DNA input in bisulfite reaction; HT29, negative control; M, methylation-specific PCR product; MiaPaCa2, positive control; PCR dH2O, no DNA in PCR; U, unmethylated allele spec. PCR-product; untreated, no bisulfite added;