Methyltransferase DNMT3B promotes colorectal cancer cell proliferation by inhibiting PLCG2

Abstract

Aberrant DNA methylation patterns in the promoter region of PLCG2 are associated with dysregulated signaling pathways and cellular functions. Its role in colorectal cancer cells is still unknown. In this study, qRT-PCR is used to measure DNMT3B expression in colorectal cancer. Western blot analysis and immunohistochemistry are used to analyze DNMT3B and PLCG2 protein levels in colorectal tissues and cell lines. Cell Counting Kit-8 (CCK-8) and colony formation assays are used to assess the proliferation of colorectal cancer cells. Methylation-specific PCR (MSP) and bisulfite-sequencing PCR (BSP) are used to measure DNA methylation level. Our results show that DNMT3B is overexpressed in colorectal cells in the TCGA datasets according to Kaplan-Meier plots. DNMT3B is significantly overexpressed in tumor tissues compared to that in adjacent nontumor tissues. Western blot analysis results demonstrate high expression of DNMT3B in tumor tissues. Compared to normal colonic epithelial cells, colorectal cancer cell lines exhibit elevated level of PLCG2 methylation. Overexpression of PLCG2 effectively prevents the growth of colorectal cancer xenograft tumors in vivo. PLCG2 is identified as a key downstream regulatory protein of DNMT3B in colorectal cancer. DNMT3B inhibits PLCG2 transcription through methylation of the PLCG2 promoter region. DNMT3B controls colorectal cancer cell proliferation through PLCG2, which is useful for developing therapeutic approaches that target PLCG2 expression for the treatment of colorectal cancer.

1. Introduction

Colorectal cancer (CRC) is the third most common and deadly tumor type worldwide, although great progress has been made in CRC treatment over the past few years [1]. Although there have been some advancements in early diagnosis, the current surgical and chemoradiotherapy treatments are not optimal [2]. This is attributed to the pathological stage, invasion, and metastasis of CRC cells, which compromise the effectiveness of the treatment [3].

DNA methylation plays a crucial role in the initiation and progression of CRC [4‒6]. It is an epigenetic modification involving the addition of methyl groups to DNA molecules. In CRC, DNA methylation typically involves the increased or decreased methylation of certain genes, potentially leading to changes in the expression levels of these genes. Generally, in CRC, tumor suppressor genes often undergo increased methylation, suppressing their function and diminishing their ability to resist cancer [7]. Conversely, some oncogenes may undergo decreased methylation, resulting in their overactivation and promoting the growth and spread of cancer cells [8]. Furthermore, the patterns and extent of DNA methylation can serve as markers for the diagnosis and prognosis of CRC [9‒11]. Therefore, gaining a deeper understanding of the role of DNA methylation in CRC not only helps unravel the molecular mechanisms of the disease but also provides crucial clues for the development of more effective treatment and diagnostic approaches.

DNA methyltransferase 3B (DNMT3B) is a DNA methyltransferase belonging to the DNA methyltransferase family. This enzyme is responsible for the addition of methyl groups to DNA molecules, thereby influencing gene expression and cellular genomic stability. Abnormal DNMT3B activity has been implicated in the occurrence and development of diverse cancers, specifically cervical cancer [12], breast cancer [13], endometrial cancer [14], and gastric cancer [15]. It induces abnormal DNA methylation, affecting the expression of tumor suppressor genes and oncogenes and consequently propelling tumor growth and advancement.