Aberrantly expressed genes in HaCaT keratinocytes chronically exposed to arsenic trioxide

The purpose of this study is to search for aberrant genes in HaCaT keratinocytes after chronic exposure to arsenic trioxide. The objective of the investigation was to discover the mechanism of arsenic carcinogenicity in human epidermal keratinocytes. We hypothesize that a combined strategy of DNA microarray, qRT-PCR and gene function annotation will identify aberrantly expressed genes in HaCaT keratinocyte cell line after chronic treatment with arsenic trioxide. HaCaT cells were chronically exposed to 0.5µg/mL arsenic trioxide (As2O3) up to 22 passages and RNA was extracted. Microarray data analysis identified 14 up-regulated genes and 21 down-regulated genes in response to arsenic trioxide Two experimental groups: 1. The treatment group was sub-cultured up to passage 22 to establish a chronic exposure state. 2. The passage control group was also sub-cultured up to 22 passages but with no exposure to arsenic trioxide. 4 technical replicates with 3 replicates making a total of 8X3 =24 samples HaCat Cell untreated (passage control): 1. H1_H001, H1_H002, H1_H003 2. H2_ H004, H2_H005, H2_H006 3. H3_ H007, H3_H008, H3_H009 4. H4_ H010, H4_H011, H4_H012 HaCat Cell treated with 0.5µg/ml of arsenic trioxide: 5. A1_H013, A1_H014, A1_H015 6. A2_H016, A2_H017, A2_H018 7. A3_H019, A3_H020, A3_H021 8. A4_H022, A4_H023, A4_H024 Cell Type: Human Skin Keratinocyte: 1.5 ×105 HaCaT cells were cultured in 7.5 ml of complete DMEM containing 10% Fetal Bovine Serum (FBS) and 1% penicillin, streptomycin in T-25 culture plate. Cells were incubated in a humidified atmosphere with 5% CO2 at 37 ºC. The treatment groups were exposed to 0.5µg/mL As2O3 (equivalent to LC 0.5), and passaged at 90% confluent. Total RNA was extracted from 4 technical replicates of unexposed HaCaT cells and HaCaT cells chronically exposed to arsenic trioxide up to passage 22 using RNA STAT-60 (TEL-TEST, INC, Friendswood, TX, USA).

本研究旨在探究慢性暴露于三氧化二砷（arsenic trioxide）后人HaCaT角质形成细胞（HaCaT keratinocytes）中的异常表达基因，核心目标为阐明三氧化二砷致人表皮角质形成细胞癌变的分子机制。本研究提出如下假说：联合应用DNA微阵列（DNA microarray）、实时定量聚合酶链反应（qRT-PCR）与基因功能注释（gene function annotation）策略，可筛选出慢性暴露于三氧化二砷后人HaCaT角质形成细胞系中异常表达的基因。

将HaCaT细胞以0.5μg/mL三氧化二砷（As₂O₃）进行慢性暴露，直至传至第22代，随后提取总RNA。微阵列数据分析显示，三氧化二砷处理组共筛选得到14个上调基因与21个下调基因。

本实验设置两组：
1. 处理组：持续传代至第22代，构建慢性三氧化二砷暴露模型；
2. 传代对照组：同样传代至第22代，但未接触三氧化二砷。

本实验共设置8个实验组别，每组包含3个生物学重复，总计8×3=24个样本。

未处理的HaCaT细胞（传代对照组）样本如下：
1. H1_H001、H1_H002、H1_H003
2. H2_H004、H2_H005、H2_H006
3. H3_H007、H3_H008、H3_H009
4. H4_H010、H4_H011、H4_H012

经0.5μg/mL三氧化二砷处理的HaCaT细胞样本如下：
5. A1_H013、A1_H014、A1_H015
6. A2_H016、A2_H017、A2_H018
7. A3_H019、A3_H020、A3_H021
8. A4_H022、A4_H023、A4_H024

细胞培养相关信息如下：
细胞类型：人皮肤角质形成细胞。将1.5×10^5个HaCaT细胞接种于7.5mL含10%胎牛血清（FBS, Fetal Bovine Serum）与1%青霉素-链霉素的完全达尔伯克改良伊格尔培养基（DMEM, Dulbecco's Modified Eagle Medium）中，于T-25培养板内培养。细胞置于37℃、5% CO₂的饱和湿度培养箱中孵育。处理组细胞以0.5μg/mL As₂O₃（相当于半致死浓度LC₀.₅）进行暴露，待细胞汇合度达90%时进行传代。

采用RNA STAT-60试剂（TEL-TEST公司，美国德克萨斯州弗里斯特伍德市），分别从未暴露的HaCaT细胞与慢性暴露于三氧化二砷至第22代的HaCaT细胞的4个技术重复样本中提取总RNA。