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  • br Materials and methods br Results

    2022-05-17


    Materials and methods
    Results
    Discussion In general, we have demonstrated that the CRISPR/dCas9-based system, fused with 2 × PP7 and PCP-EZH2, could stably and precisely methylate the H3K27 at specific locus of C/ebpα to silence gene expression and inhibit adipogenic differentiation in 3T3 cells. Therefore, it confirmed that the CRISPR/dCas9-based EZH2 system can be a novel and powerful tool used to gain insights to the events of gene express change and regulate cell phenotypes. Recent studies have revealed that dCas9 system may have widespread off-target events in combination with multiple sgRNAs [14,15], which potentially result in off-target changes in gene expression. The novel system could achieve significant genetic inhibition with only one sgRNA, reducing the risk of off-target effect caused by multiple sgRNAs. The interference of any mRNA transcript, especially for essential genes, would distort the homeostatic mechanism of the endogenous transcripts even the cell death. However, downregulating expression of endogenous genes by CRISPR/dCas9-based system owns a significant advantage to avoid it. CRISPR/dCas9-based system we constructed work through the specific combination between PP7 and PCP to carry EZH2 to the targets. Through the specific gene programming, our dCas9-based system may manipulate multiple epigenetic modification in genome-wide. With the discovery of more epigenetic D-NMAPPD or transcriptional module such as HAT [16]、LSD1 [17]、TET1 [18]、DNMTs [19]、KRAB [20], the system could be further modified to achieve specific effects in epigenetic editing by replacing the EZH2 with these epigenetic writers. In spite of the advantages of this system, there are still some problems within the system. The system includes three vectors, which leads to relatively complex cell-line construction procedure. Simultaneously, the oversize coding sequence of dCas9 and PCP-EZH2 in plasmid vector remains a big hurdle for transfection and virus packaging. In future, we will try to figure out the core region of dCas9 and EZH2 to minimize the vector burden and improve efficiency.
    Introduction Laryngeal carcinoma is the second most prevalent malignancy of the head and neck [1] and the 11th-most common cancer. The majority of laryngeal carcinoma are laryngeal squamous cell carcinomas (LSCC). The larynx plays important physiological functions, such as phonation, respiration, deglutition, and sensation of smell and taste, etc. Thus, advanced laryngeal lesions are accountable for high morbidity and mortality of the patient [2,3]. There is a dire need to clarify the mechanism of LSCC tumorigenesis and develop efficacious treatment of this disease. The long non-coding RNAs (lncRNAs) are an integral part of the human genome with length of over 200 nucleotides [4]. Emerging studies have shown that aberrant lncRNA expression may be one of the major contributors to tumorigenesis [5]. In studying LSCC, Li et al. found that the overexpression of certain lncRNAs are linked to PTEN methylation [6]. Wu et al. found that lncRNA H19 was prominently upregulated in laryngeal cancer and that H19 expression was inversely correlated with the survival of laryngeal patients. The upregulation of lncRNA H19 promoted laryngeal carcinoma via miR-148a-3p, DNMT1, and DNA methylation [7]. XIST (X inactive specific transcript) is a lncRNA associated with the polycomb group protein enhancer of zeste homolog 2 (EZH2), a methyltransferase in the core catalytic element of polycomb repressive complex 2. XIST was found to play a critical role in the regulation of tumorigenesis, cancer cell proliferation, migration, invasion, and metastasis [8,9]. The upregulation of XIST has been implicated in female cancers [10]. Dysregulation of XIST has been demonstrated in different cancers. For example, it has been shown that dysregulation of XIST leads to instability of heterochromatin and alteration of gene expression [10]. Another study demonstrated that XIST knockdown exerted tumor-suppressive effects in human glioblastoma stem cells through upregulating miR-152 [11]. However, the expression and biological function of XIST in laryngeal cancer is unclear.