Molecular mechanisms of ligament flavum hypertrophy: analysis based on methylation sequencing and transcriptome integration

doi:10.12307/2025.291

Abstract

Abstract: BACKGROUND: Ligament flavum hypertrophy is the main cause of lumbar spinal stenosis, which is the result of multiple pathological factors working together. Currently, the molecular mechanism and pathway of action of ligament flavum hypertrophy are unclear, and there is a lack of effective non-surgical treatment options.
OBJECTIVE: To investigate the molecular mechanisms of ligament flavum hypertrophy using methylation sequencing and transcriptome integration analysis methods.
METHODS: Five normal ligament flavum tissue samples and five hypertrophic ligament flavum tissue samples were collected. Abnormal methylation sites and methylation status were recorded by methylation sequencing and abnormally expressed genes were recorded by transcriptome integration analysis.
The genes that showed a negative correlation between methylation level and expression level at the intersection of the two were selected. GO and KEGG enrichment analyses were used to study the major functional pathways and molecular functions of differentially expressed genes. Key genes regulating ligamentum flavum hypertrophy were screened using protein-protein interaction analysis.
RESULTS AND CONCLUSION: Methylation sequencing of the two groups of the ligament flavum showed 37 173 hypermethylation sites and 10 583 low methylation sites. Transcriptome integration analysis found 720 abnormally expressed genes, of which 463 were upregulated and 257 were down-regulated. There were 383 overlapping genes, of which 192 genes showed a negative correlation between the methylation level and the expression level. GO functional pathway analysis results showed that molecular function was enriched to 10 terms, cellular component was enriched to 15 terms, and biological process (BP) was enriched to 30 terms. The results of KEGG pathway enrichment analysis showed that 192 genes were mainly enriched to 9 pathways, such as PI3K-Akt signaling pathway, Rap1 signaling pathway, and focal adhesion signaling pathway. The protein-protein interaction analysis identified five genes, PPARG, EGFR, CNR1, TNF and COL11A2, which may be the key genes that regulate ligamentum flavum hypertrophy, and they can influence the occurrence and development of ligamentum flavum hypertrophy mainly through the regulation of tissue fibrosis, cell proliferation and differentiation, inflammatory factor levels, and collagen fiber expression.

Key words: Iigamentum flavum hypertrophy, methylation sequencing, transcriptome integration analysis, lumbar spinal stenosis, DNA methylation

CLC Number:

He Yang, Tang Buyuan, Lu Changhuai. Molecular mechanisms of ligament flavum hypertrophy: analysis based on methylation sequencing and transcriptome integration [J]. Chinese Journal of Tissue Engineering Research, 2025, 29(5): 1013-1020.

Figures/Tables 13

2.1 甲基化位点原始数据质控及处理 2.1.1 过滤以及标化甲基化数据使用BMIQ(Beta Mlxture Quantile dilation)方法对检测后得到的甲基化位点进行beta值的标准化处理，用beta值的密度图来看图上是否有异常样本(图1)，用箱式图(图2)来验证数据标化的情况。结果显示，样本都正常，标化后样本数据整齐。 2.1.2 甲基化数据主成分分析与奇异值分解通过降维度以及奇异值分解来看数据集中变异情况以及它的重要组成部分的来源(图3，4)，可以看出如果用全部样本，黄韧带肥厚组与黄韧带正常组之间距离不大，而且它们的主要差异并非来源于组别。为了得到差异来源于组别，选取了比较集中在一起的样本B1，B3，B5以及A1，A3，A5，再做主成分分析和奇异值分解(图5，6)，可以看出当只选取B1，B3，B5与A1，A3，A5样本时，组别距离大，差异来源主要是组别差异。 2.2 甲基化测序结果使用P < 0.01筛选出47 756个差异的探针位点，其中高甲基化位点37 173个，低甲基化位点10 583个。使用火山图展示差异结果，见图7。 2.3 转录组整合分析结果筛选阈值为P < 0.01和|logFC |> 1。一共找到720个差异表达基因，其中463个上调基因，257个下调基因。使用火山图展示差异结果，见图8。 2.4 差异甲基化基因与差异表达基因四象分析及富集通路分析两者之间重叠的基因有383个，其中192个基因的mRNA表达水平与其甲基化水平负相关，见图9。针对重叠基因中呈负相关的192个基因进行GO、KEGG功能通路富集分析，筛选条件为P < 0.05。GO功能分析结果表明，GO的分子功能(molecular function，MF)富集到10个条目，细胞组分(cellular component，CC)富集到15个条目，生物学途径(biological process，BP)富集到30个条目，见图10。KEGG通路富集分析结果表明，192个基因主要富集到PI3K-Akt信号通路、Rap1信号通路、Focal adhesion信号通路、FoxO 信号通路等9条通路，见图11。 2.5 PPI互作分析筛选调控黄韧带肥厚的关键基因。通过Strings数据库进行PPI互作网络分析，将degree≥ 2的基因导入 Cystocape进行可视化，见图12。进一步采用degree法、Stress法和MCC法计算出Top=10的基因并可视化分析，其中有5个重叠基因(PPARG、EGFR、CNR1、TNF和COL11A2 )，这5个基因可能是调控黄韧带肥厚的关键基因，见图13。"

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