Insights into Molecular Mechanisms and Therapeutic Potential of Arborvitae Essential Oil (Thuja plicata) on Cervical Cancer Cells

This analysis aimed to assess the effect of arborvitae (Thuja plicata) essential oil (AEO) in an in vitro model of cell lines derived from cervical cancer, namely HeLa and SiHa, by examining its influence on gene expression modulation. The working cell lines were HeLa, and SiHa. Total RNA sequencing was performed on the Illumina NovaSeq 6000 platform by Novogene Bioinformatics Technology Co., Ltd, Beijing, China, with two independent replicate sequences for each cell model. Methodology: The execution of the bioinformatics pipeline analysis was carried out as follow: R (ver. 4.3.1), RStudio (ver. 2023.09.1 +494) and Galaxy (https://www.usegalaxy.orgaccessed on 02 February 2024) open-source platforms were used to analyze the Illumina raw data. The Quality Check was conducted through the FastQC tool (v0.74+galaxy0). Afterward, all the reads were processed by the Rsubread package (v2.14.2); at that point, the reads were trimmed and aligned to the Human Genome Reference (GRCh38.p14 v44). The obtained output was the BAM files, of which the number of reads was counted by the FeatureCounts tool (v2.0.3); at the final step, the Differential Expression Analysis was settled by the DESeq2 package for R (ver. 1.42. 0). The differential gene expression measurements were normalized by DESeq2's median of ratios (median of ratio of gene counts relative to geometric mean per gene) method. All genes with an adjusted p-value (p-adj) minus or equal to 0.05 and fold change (Log2FC) up to 2 or less than minus 2 were selected as differentially expressed genes (DEGs). Conclusions: Our study found that AEO regulates genes related with cell cycle regulation, cell growth, differentiation, and cell death in both cell lines, mainly through downregulation; however, its effect appears to be mediated by different pathways in each cell line. Overall design: Cervical cancer-derived cell lines underwent total RNA extraction (SiHa and HeLa) after 4 hrs AEO or DMSO (vehicle control) treatment; this process was addressed in duplicate. After that, the replicates were compared as a single end of each cell line vs. the control for the development of the differential expression analysis.

本研究旨在评估侧柏（Thuja plicata）精油（arborvitae essential oil, AEO）在源自宫颈癌的细胞系（HeLa与SiHa）的体外模型中对基因表达调控的影响。所用细胞系为HeLa与SiHa。总RNA测序由中国北京诺禾致源生物信息科技有限公司（Novogene Bioinformatics Technology Co., Ltd）在Illumina NovaSeq 6000平台完成，每个细胞模型设置两次独立重复测序。

生物信息学分析流程执行如下：采用R语言（版本4.3.1）、RStudio（版本2023.09.1+494）以及Galaxy平台（https://www.usegalaxy.org，2024年2月2日访问）对Illumina原始测序数据进行分析。首先通过FastQC工具（v0.74+galaxy0）完成质量控制；随后使用Rsubread软件包（v2.14.2）处理所有测序读段，对读段进行修剪并比对至人类基因组参考序列（GRCh38.p14 v44），最终得到BAM格式文件；再通过FeatureCounts工具（v2.0.3）统计读段数量；最后通过R语言的DESeq2软件包（v1.42.0）完成差异表达分析。差异基因表达量通过DESeq2的中位数比率法（即每个基因计数相对于其几何均值的中位数比率）进行标准化。筛选校正后p值（p-adj）≤0.05且倍数变化（Log2FC）≥2或≤-2的基因为差异表达基因（DEGs）。

本研究发现，AEO在两种细胞系中均能调控与细胞周期调控、细胞增殖、分化及细胞死亡相关的基因，且主要通过下调基因表达发挥作用；不过其在两种细胞系中的调控通路似乎存在差异。

实验整体设计：分别用AEO或二甲基亚砜（DMSO，溶剂对照）处理SiHa与HeLa宫颈癌细胞系4小时后，提取总RNA，设置两次生物学重复。随后将每个细胞系的重复样本以单端测序数据形式分别与对照组进行比较，以开展差异表达分析。