Natural Antisense Transcript-Mediated Regulation of HOXA10-AS in Oral Squamous Cell Carcinoma

IF 2.7 3区医学 Q1 DENTISTRY, ORAL SURGERY & MEDICINE

Journal of Oral Pathology & Medicine Pub Date : 2025-03-04 DOI:10.1111/jop.13613

Kanaka Sai Ram Padam, Satyajit Dey Pereira, Naveena A. N. Kumar, Raghu Radhakrishnan

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引用次数: 0

Abstract

Background

The oncogenic role of HOXA10-AS and HOXA10 in cancer has been well documented. However, the epigenetic role of HOXA10 and the natural antisense-mediated regulation of HOXA10-AS in oral squamous cell carcinoma progression is not understood.

Methods

A total of 35 oral squamous cell carcinoma specimens and 35 adjacent normal clinical specimens were collected and categorized on the basis of their lymph node status. HOXA10-AS and HOXA10 expression were analyzed using RT-qPCR. Methyl-capture sequencing was performed using lymph node-negative (n = 6) and lymph node-positive (n = 5) matched cases. The promoter activity of HOXA10 was determined using a luciferase assay. ChIP-qPCR was performed to determine histone mark localization in the distal promoter region of HOXA10. A protein–protein interaction network of genome-wide antisense targets was constructed using StringDB, and functional enrichment was performed using the R package ClusterProfiler. Transient siRNA-mediated transfection was performed to target specific exons of the HOXA10-AS gene, followed by subsequent cell proliferation, cell cycle, and cell migration assays and validation of cancer signaling pathways through western blotting.

Results

HOXA10-AS and its antisense target HOXA10 were significantly overexpressed in the lymph node-positive samples. The transcriptionally active distal promoter of HOXA10 consists of a constitutively unmethylated CpG island region (CUR). H3K4me3, H3K27ac, and H3K27me3 histone mark deposition at the adjacent methylated loci of the distal promoter suggest the nature of euchromatin-driven regulation. Genome-wide mapping revealed 11 potential targets of HOXA10-AS. Targeted specific knockdown of HOXA10-AS exons significantly reduced the expression of HOXA10 and deregulated its downstream targets, contributing to decreased cell cycle progression and epithelial-to-mesenchymal transition.

Conclusion

HOXA10-AS regulates the expression of HOXA10 through a natural antisense-mediated mechanism and is epigenetically regulated by constitutively unmethylated marks in the distally enhancing promoter of HOXA10.

Abstract Image

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天然反义转录介导的HOXA10-AS在口腔鳞状细胞癌中的调控。

背景：HOXA10- as和HOXA10在癌症中的致癌作用已被充分证实。然而，HOXA10的表观遗传作用以及HOXA10- as在口腔鳞状细胞癌进展中的自然反义调控尚不清楚。方法：收集35例口腔鳞状细胞癌标本及35例相邻正常临床标本，根据其淋巴结状态进行分类。RT-qPCR分析HOXA10- as和HOXA10的表达。对淋巴结阴性（n = 6）和淋巴结阳性（n = 5）匹配的病例进行甲基捕获测序。用荧光素酶测定HOXA10的启动子活性。采用ChIP-qPCR方法确定HOXA10远端启动子区组蛋白标记的定位。使用StringDB构建全基因组反义靶点的蛋白-蛋白相互作用网络，并使用R包ClusterProfiler进行功能富集。短暂sirna介导的转染针对HOXA10-AS基因的特定外显子，随后进行细胞增殖、细胞周期和细胞迁移试验，并通过western blotting验证癌症信号通路。结果：HOXA10- as及其反义靶点HOXA10在淋巴结阳性标本中显著过表达。HOXA10转录活性远端启动子由一个组成性未甲基化的CpG岛区（CUR）组成。H3K4me3、H3K27ac和H3K27me3组蛋白标记沉积在远端启动子邻近的甲基化位点，表明了常染色质驱动调控的性质。全基因组图谱揭示了HOXA10-AS的11个潜在靶点。靶向特异性敲除HOXA10- as外显子显著降低了HOXA10的表达，并解除了其下游靶标的调控，导致细胞周期进程和上皮细胞向间质细胞的转变减慢。结论：HOXA10- as通过天然的反义介导机制调控HOXA10的表达，并受HOXA10远端增强启动子中组成性未甲基化标记的表观遗传调控。

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来源期刊

Journal of Oral Pathology & Medicine 医学-病理学

CiteScore

5.90

自引率

6.10%

发文量

121

审稿时长

4-8 weeks

期刊介绍： The aim of the Journal of Oral Pathology & Medicine is to publish manuscripts of high scientific quality representing original clinical, diagnostic or experimental work in oral pathology and oral medicine. Papers advancing the science or practice of these disciplines will be welcomed, especially those which bring new knowledge and observations from the application of techniques within the spheres of light and electron microscopy, tissue and organ culture, immunology, histochemistry and immunocytochemistry, microbiology, genetics and biochemistry.