Array detection and expression verification of DNA damage repair related genes in oral leukoplakia cancerization

doi:10.3969/j.issn.1674-8115.2021.03.003

Abstract

Abstract: Objective

·To study the gene expression profile of DNA damage repair in the occurrence and development of oral leukoplakia (OL) and to verify the expression of mRNA and protein of key genes, ataxia-telangiectasia mutated (ATM) and histone variant H2A family member X (H2AX).

Methods

·Affymetrix HTA 2.0 array was used to detect the high-throughput transcriptome of normal mucosa (n=3), low-risk leukoplakia (n=4), high-risk leukoplakia (n=4) and early squamous cell carcinoma (n=6). Gene Ontology function analysis was used to screen out the genes related to DNA damage repair [|log₂(chang fold, FC)|≥2.0 and P<0.05]. Real-time quantitative PCR (qPCR) and Western blotting were used to verify the mRNA and protein expression of key genes (ATM and H2AX) in human normal oral mucosal keratinocytes (HOK), OL (Leuk1) and squamous cell carcinoma cell lines (HN13), respectively.

Results

·DNA damage repair genes abnormally expressed in the occurrence and development of leukoplakia. There were 7 genes with |log₂FC|≥2.0 from normal mucosa to low-risk leukoplakia, 7 genes with |log₂FC|≥2.0 from low-risk leukoplakia to high-risk leukoplakia, and 52 genes with |log₂FC|≥2.0 from high-risk leukoplakia to squamous cell carcinoma. qPCR revealed that the expression of ATM and H2AX in HOK cells, Leuk1 cells and HN13 cells increased stepwise (P<0.05). Western blotting revealed that the expression of γ-H2AX protein was also up-regulated in the HOK cells, Leuk1 cells and HN13 cells (P<0.05). ATM protein was up-regulated in Leuk1 cells compared with HOK cells, but down-regulated in HN13 cells (P<0.05).

Conclusion

·DNA damage repair genes, ATM and H2AX, are involved in the occurrence and development of OL.

Key words: oral leukoplakia (OL), squamous cell carcinoma, DNA damage repair, ataxia-telangiectasia mutated (ATM), histone variant H2A family member X (H2AX)

CLC Number:

R781.5⁺9

Wei LIU, Min-wen ZHU, Lan WU. Array detection and expression verification of DNA damage repair related genes in oral leukoplakia cancerization[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(3): 302-307.

Figures/Tables 6

Tab 1 Primer sequence for ATM and H2AX genes by qPCR

Gene	Sequence
ATM	F: 5'-CGCCGCGGTTGATACTACT-3'
	R: 5'-ACGGCAGCAGATAAGCAGAT-3'
H2AX	F: 5'-CAACAAGAAGACGCGAATCA-3'
	R: 5'-CTGGATGTTGGGCAGGAC-3'
β-actin	F: 5'-CCAACCGCGAGAAGATGAC-3'
	R: 5'-GAGGCGTACAGGGATAGCACA-3'

Tab 2 Comparison of demographic data between the three groups

Item	Normal mucosa group (N=3)	Leukoplakia group (N=8)	Early squamous cell carcinoma group (N=6)	F/χ² value	P value
Age/year	52.7±8.7	54.5±10.1	55.1±9.8	0.691	0.911
Gender/n				0.032	0.984
Male	2	5	4
Female	1	3	2
Smoking/n	1	3	3	0.314	0.855
Alcohol intake/n	1	3	3	0.314	0.855

Fig 1 DNA damage repair genes were screened from all genes in the development and progression of leukoplakia

Fig 2 Clustering heat map of 52 different genes between oral early squamous cell carcinoma and high-risk leukoplakia

Fig 3 qPCR confirmed the differential expression of ATM and H2AX in normal mucosa, leukoplakia and squamous cell carcinoma cell lines

Fig 4 Western blotting examined the protein expression of ATM and H2AX in HOK cells, Leuk1 cells and NH13 cells

TrendMD

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