LAD1在食管鳞癌发生发展中的作用及其分子机制

Role of LAD1 in the Development of Esophageal Squamous Carcinoma and Its Molecular Mechanism

本研究旨在揭示锚定细丝蛋白基因ladinin-1(LAD1)对食管鳞癌(esophageal squamous carcinoma,ESCC)细胞增殖、迁移、侵袭和细胞凋亡的影响及其可能的作用机制。对ESCC组织及癌旁组织进行转录组测序并结合生物信息学数据库分析,发现LAD1在ESCC组织中高表达,通过RT-qPCR验证LAD1在ESCC细胞系中的表达。收集31例ESCC患者的癌组织和对应癌旁组织标本,用免疫组织化学染色检测LAD1在ESCC组织中的表达并分析LAD1与ESCC组织病理特征的相关性。利用siRNA敲低KYSE-30和KYSE-150细胞中LAD1的表达,通过CCK-8实验、平板克隆实验、划痕愈合实验、Transwell实验、流式细胞术检测ESCC细胞功能的变化。通过UCSC和JASPAR数据库预测可能调控LAD1的上游转录因子(transcription factors,TFs),并通过RT-qPCR和免疫组织化学染色验证其在ESCC细胞和组织中的表达。最后采用RT-qPCR实验和双萤光素酶实验验证转录因子与LAD1的调控关系。结果显示,相比于HET-1A细胞,LAD1在KYSE-30和KYSE-150细胞中高表达(P<0.01或P<0.001),与转录组测序结果一致。免疫组织化学结果显示,LAD1在ESCC中高表达,并与病理分化程度呈负相关(P<0.01或P<0.001)。敲低LAD1能抑制ESCC细胞增殖、迁移、侵袭并促进细胞凋亡(P<0.01或P<0.001)。UCSC和JASPAR数据库预测结合RT-qPCR实验和免疫组织化学染色推测KLF5、EHF、ELF3可能是LAD1的上游转录因子,利用RT-qPCR实验和双萤光素酶实验进一步验证,发现LAD1可能受到KLF5、EHF、ELF3的调控。本研究提示,LAD1在ESCC细胞和组织中高表达,并与病理分化程度相关联;敲低LAD1能抑制ESCC细胞增殖、迁移、侵袭能力,促进细胞凋亡;KLF5、EHF、ELF3是调节LAD1的上游转录因子。

The aim of this study was to reveal the effects of anchoring filament protein gene ladinin-1(LAD1)on the proliferation,mi-gration,invasion and apoptosis of esophageal squamous carcinoma(ESCC)cells and its possible mechanisms of action.Transcriptome sequencing of ESCC tissues and paracancerous tissues combined with bioinformatics database analysis revealed that LAD1 was highly ex-pressed in ESCC tissues,and the expression of LAD1 in ESCC cell lines was verified by RT-qPCR.Cancerous tissues and correspon-ding paracancerous tissue specimens from 31 ESCC patients were collected for immunohistochemical staining to detect the expression of LAD1 in ESCC tissues and analyze the correlation between LAD1 and histopathological features of ESCC.siRNA was used to knock down LAD1 expression in KYSE-30 and KYSE-150 cells,and functional changes in ESCC cells were detected by CCK-8 assay,plate cloning assay,scratch healing assay,Transwell assay,and flow cytometry.Upstream transcription factors(TFs)that may regulate LAD1 were predicted on the UCSC and JASPAR databases,and their expression in ESCC cells and tissues was verified by RT-qPCR and immunohistochemical staining.Finally,the regulatory relationship between TFs and LAD1 was verified by using RT-qPCR assay and dual-luciferase assay.The results showed that LAD1 was highly expressed in KYSE-30 and KYSE-150 cells compared to HET-1A cells(P<0.01 or P<0.001),consistent with the transcriptome sequencing results.Immunohistochemical results showed that LAD1 was highly expressed in ESCC and negatively correlated with the degree of histopathological differentiation of ESCC(P<0.01 or P<0.001).Knockdown of LAD1 suppressed ESCC proliferation,migration,invasion and promoted apoptosis(P<0.01 or P<0.001).UCSC and JASPAR database predictions combined with RT-qPCR experiments and immunohistochemical staining speculated that KLF5,EHF,and ELF3 might be upstream transcription factors of LAD1,and RT-qPCR experiments and dual-luciferase experiments were utilized to further verify that LAD1 might be regulated by KLF5,EHF,and ELF3.This study suggests that LAD1 is highly expressed in ESCC cells and tissues and correlates with the degree of pathological differentiation of ESCC tissues;knockdown of LAD1 inhibits the proli-feration,migration,and invasive ability of ESCC cells and promotes apoptosis;KLF5,EHF,and ELF3 are the upstream transcription factors regulating LAD1.