Objective:To investigate the tissue methylation features of adenocarcinoma patients presenting as pulmonary nodules on CT scans.Methods:A retrospective analysis was conducted on 70 adenocarcinoma patients with pulmonary nodules diagnosed at the Shanghai General Hospital from June 1, 2022 to January 20, 2024. Participants were assigned to two groups using the random number table, with 40 in the discovery group and 30 in the validation group. In the discovery group, tissue samples were analyzed using reduced representation bisulfite sequencing (RRBS) technology to compare the average methylation levels between cancer tissues and paired adjacent non-cancerous tissues. Differentially methylated regions (DMRs) were screened for analysis of their distribution across various genomic functional elements, and hierarchical clustering was plotted. GO and KEGG pathway enrichment analyses were further conducted on the DMRs. Subsequently, candidate DMRs associated with lung adenocarcinoma were validated using TCGA lung adenocarcinoma cohort and targeted bisulfite sequencing technology in the validation group. The comparison of methylation levels between groups was conducted using t-tests or non-parametric tests, while rates and composition ratios were analyzed using chi-square tests or Fisher′s exact test.Results:In discovery cohort, the average methylation level in cancer tissues was lower compared to adjacent normal tissues [(42.369±4.627) vs (44.370±4.046), t=?2.059, P=0.043]. A total of 37 995 DMRs were identified, including 16 889 upregulated regions and 21 106 downregulated regions, predominantly locating in promoter regions (48.917%), introns (36.457%), and exons (10.812%). The DMR clustering heatmap revealed two distinct clusters corresponding to cancer tissues and adjacent non-cancerous tissues. GO analysis showed that DMRs associated genes were mainly located in the cell membrane and nuclear chromatin, and were primarily involved in RNA polymerase Ⅱ-related transcription and regulation. KEGG pathway enrichment analysis indicated that DMRs associated genes were mainly involved in neuroactive ligand-receptor interaction, cancer pathways, calcium signaling pathway, cAMP signaling pathway, and MAPK signaling pathway. Validation in the TCGA cohort confirmed 11 potential characteristic DMRs. In the validation group, TBS confirmed that the methylation levels of DMRs associated with MIR10B, DMRTA2, HOPX, TFAP2B and MARCH11 in cancer tissues were significantly higher than those in adjacent non-cancerous tissues [11.200(4.305, 27.088) vs 2.650(1.298, 4.645), Z=?4.539, P<0.05; 18.610(13.600, 33.025) vs 8.675(5.488, 13.085), Z=?4.554, P<0.05; 17.600(2.183, 76.015) vs 1.085(0.898, 1.835), Z=?5.131, P<0.05; 5.250(3.220, 7.693) vs 3.495(2.165, 4.383), Z=?2.861, P<0.05; 11.515(7.525, 21.033) vs 7.830(5.518, 11.488), Z=?2.440, P<0.05 ], and the differences were statistically significant. Conclusions:Lung adenocarcinoma tissue exhibits different methylation patterns compared with adjacent normal lung tissue. The identified DMRs are involved in the regulation of several key pathways. Results from the TCGA cohort and an independent validation group support the potential diagnostic value of DMRs such as MIR10B, DMRTA2, HOPX, TFAP2B, and MARCH11 in lung adenocarcinoma, though their clinical application requires further validation.

The blood-brain barrier (BBB) is a tight boundary formed between endothelial cells and astrocytes, which separates and protects brain from most pathogens as well as neural toxins in circulation. However, detailed molecular players involved in formation of BBB are not completely known. Dentin matrix protein 1 (DMP1)-proteoglycan (PG), which is known to be involved in mineralization of bones and dentin, is also expressed in soft tissues including brain with unknown functions. In the present study, we reported that DMP1-PG was expressed in brain astrocytes and enriched in BBB units. The only glycosylation site of DMP1 is serine89 (S89) in the N-terminal domain of the protein in mouse. Mutant mice with DMP1 point mutations changing S89 to glycine (S89G), which completely eradicated glycosylation of the protein, demonstrated severe BBB disruption. Another breed of DMP1 mutant mice, which lacked the C-terminal domain of DMP1, manifested normal BBB function. The polarity of S89G-DMP1 astrocytes was disrupted and cell-cell adhesion was loosened. Through a battery of analyses, we found that DMP1 glycosylation was critically required for astrocyte maturation both in vitro and in vivo. S89G-DMP1 mutant astrocytes failed to express aquaporin 4 and had reduced laminin and ZO1 expression, which resulted in disruption of BBB. Interestingly, overexpression of wild-type DMP1-PG in mouse brain driven by the nestin promoter elevated laminin and ZO1 expression beyond wild type levels and could effectively resisted intravenous mannitol-induced BBB reversible opening. Taken together, our study not only revealed a novel element, i.e., DMP1-PG, that regulated BBB formation, but also assigned a new function to DMP1-PG.
Animals ; Astrocytes ; cytology ; metabolism ; Blood-Brain Barrier ; cytology ; metabolism ; Cells, Cultured ; Extracellular Matrix Proteins ; genetics ; metabolism ; Female ; Glycosylation ; Male ; Mice ; Proteoglycans ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

In the original publication, the label of Fig. 2C should be read as "GFAP/lectin/DAPI" not "DMP1/GFAP/lectin/DAPI".

Fractures are frequently occurring diseases that endanger human health. Crucial to fracture healing is cartilage formation, which provides a bone-regeneration environment. Cartilage consists of both chondrocytes and extracellular matrix (ECM). The ECM of cartilage includes collagens and various types of proteoglycans (PGs), which play important roles in maintaining primary stability in fracture healing. The PG form of dentin matrix protein 1 (DMP1-PG) is involved in maintaining the health of articular cartilage and bone. Our previous data have shown that DMP1-PG is richly expressed in the cartilaginous calluses of fracture sites. However, the possible significant role of DMP1-PG in chondrogenesis and fracture healing is unknown. To further detect the potential role of DMP1-PG in fracture repair, we established a mouse fracture model by using a glycosylation site mutant DMP1 mouse (S89G-DMP1 mouse). Upon inspection, fewer cartilaginous calluses and down-regulated expression levels of chondrogenesis genes were observed in the fracture sites of S89G-DMP1 mice. Given the deficiency of DMP1-PG, the impaired IL-6/JAK/STAT signaling pathway was observed to affect the chondrogenesis of fracture healing. Overall, these results suggest that DMP1-PG is an indispensable proteoglycan in chondrogenesis during fracture healing.