Objective: The morphology and functions of the human trabecular meshwork (HTM) are dysregulated in glaucoma, and the molecular mechanisms of this dysregulation remain unknown. According to an established in vitro model, whose function was to study the regulatory networks sustaining the response of HTM cells to the increased substrate stiffness, we systematically analyzed the expression pattern of long noncoding RNAs (lncRNAs), the important regulatory RNAs in cells. Methods: Bioinformatics analysis was performed to identify the dysregulated lncRNAs in response to increased substrate stiffness using transcriptome sequencing data (RNA-seq). Then we interfered with the expression of several dysregulated lncRNAs in HTM cells to explore their molecular targets. The cross-linking immunoprecipitation and sequencing method (CLIP-seq) was used to identify enhancer of zeste homolog 2 (EZH2)-targeted RNAs in HTM cells. The chromatin IP and sequencing method (ChIP-seq) was used to identify the targets of EZH2 and histone H3 at lysine 27 (H3K27me3). Results: The response of thousands of dysregulated lncRNAs to increased substrate stiffness was identified through RNA-seq. Functional prediction of these lncRNAs revealed that they potentially regulated key biological processes, including extracellular matrix (ECM) organization. By interfering with the expression of lncRNA SHNG8, ZFHX4-AS1, and RP11-552M11.4, the results demonstrated that those lncRNAs extensively regulated the expression levels of ECM-associated genes. Moreover, we found that EZH2 expression was significantly decreased at high substrate stiffness. Using CLIP-seq to identify EZH2-targeted RNAs in HTM cells, we found that SNHG8 was bound by EZH2. According to the CLIP-seq data of EZH2, we found that EZH2 binding sites were observed in the transcripts of SNHG8-regulated genes, but not in the ChIP-seq results of EZH2 and H3K27me3. Conclusion: Our results suggest that SNHG8 and EZH2 may cooperate to regulate the expression of a subset of genes by influencing their RNA abundance, explaining how they support HTM cell morphology and high density. This study contributes to the understanding of the alteration of HTM during the progression of glaucoma by identifying functional lncRNAs, especially SNHG8, and suggests novel therapeutic targets to treat glaucoma.