Analysis of Septin 9 gene expression in laser microdissected colonic epithelial cells along the colorectal adenoma-carcinoma sequence

Background: Colorectal adenocarcinoma is a result of the malignant transformation of epithelial cells in the colonic mucosa caused by tumor-specific mutations and epigenetic aberrations. One of the recently identified epigenetic biomarkers is Septin 9 (SEPT9), whose aberrantly methylated circulating DNA sequence can be found in plasma samples and also in colonic biopsies is highly associated with the occurrence of colorectal cancer. Although the sensitivity of the Septin 9 methylation assay gives the possibility to be applied as an efficient screening method, its influence on the gene expression level in the colonic tissue still remains to be elucidated.

Aims: Our aim was to determine the local expression level alterations of Septin 9 gene in tissue samples by analyzing epithelial cells obtained from healthy tissues and also from different areas of adenoma and colorectal mucosa.

Methods: From 3 healthy colonic biopsies, 3 surgically removed low-grade adenomas, 3 left-sided, Dukes B stage colorectal cancers and from their histologically normal adjacent areas (1cm and 10cm distance from the tumor) approximately 1000 epithelial cells were collected by laser capture microdissection. After RNA isolation followed by reverse transcription, the gene expression levels of Septin 9 gene splice variants (Septin9_v1, v2, v3, v4, v4*, v5) were analyzed using real-time PCR. Furthermore, Septin9-specific immunohistochemistry analyses were also performed.

Results: The gene expression level of the splice variants altered differently in the analysed tissue regions. The septin9_v1 expression was higher in the healthy biopsies, than in the normal adjacent areas near the tumors, which further decreased in the adenoma and the tumor epithelial cells, while the expression of the other splice variants elevated sequentially during the progression. According to the immunohistochemistry analyses, Septin9 protein expression was higher in the healthy biopsies and in the tumor adjacent normal tissues, while in the adenoma and in the tumor samples weaker immunostaining could be observed.

Conclusion: According to our results, expression level alterations of Septin 9 could be found in the laser microdissected epithelial cells, which can contribute to the tumor development by the dysregulation of the cell cycle resulting abnormal proliferation.