Elsevier

Life Sciences

Volume 261, 15 November 2020, 118311
Life Sciences

Silencing of miR-152 contributes to DNMT1-mediated CpG methylation of the PTEN promoter in bladder cancer

https://doi.org/10.1016/j.lfs.2020.118311Get rights and content

Abstract

Aim

Bladder cancer (BCa) is one of the most commonly occurring urological malignancy. DNA methylation mediated by DNA methyltransferase 1 (DNMT1) plays a crucial role in the physiological and pathological processes of cancer. However, the role of upstream regulatory factors and downstream target genes of DNA methylation mediated by DNMT1 needs further study in BCa. We aim to discover the upstream regulatory factor and downstream target gene of DNMT1, which form a signaling pathway to regulate the progression of BCa.

Main methods

DNMT1 expression in BCa tissues and cells was detected by qPCR and Western Blot. Balbc/nu/nu mice were used to determine the relationship between DNMT1 expression and tumor growth. CCK8, EdU, and transwell assays were employed to measure cell viability, proliferation, and migration respectively. RNA immunoprecipitation (RIP) assays and dual luciferase reporter assays were applied to determine the relationships among DNMT1, miR-152-3p and PTEN.

Key findings

A significant up-regulation of DNMT1 in BCa tissues and cells, and silencing of DNMT1 expression inhibited the tumor growth in vivo. Knockdown of DNMT1 inhibited the cell growth and migration of BCa cells. miR-152-3p inhibited the DNMT1 and over-expression of DNMT1 restored the cellular function of miR-152-3p in BCa cells. DNMT1 regulated the phosphatase and tensin homolog (PTEN) expression via modulating the status of DNA methylation in the promoter of PTEN.

Significance

This study confirmed the role and underlying mechanism of DNMT1-mediated DNA methylation and displayed a novel regulatory pathway miR-152/DNMT1/PTEN in BCa, thus, providing a potential diagnostic and therapeutic targets for BCa.

Introduction

As one of the most commonly occurring urological malignancy with approximately 440,000 new cases and 130,000 deaths every year worldwide, urinary bladder cancer (BCa), is the ninth most prevalent cancers [1]. To date, the main treatments for BCa patients are surgery, radiation and chemotherapy [1,2]. Among the BCa patients, approximately 75% are non-muscle-invasive, followed by high recurrence rate, poor prognosis and a low 5-year cancer-specific survival rate [3]. Presently, BCa has become the highest incident urinary system tumor in China [4]. As a result, a better understanding of a detailed molecular mechanisms is beneficial for the interventions to control progression and metastasis of BCa. However, the biomarkers and underlying mechanism in growth, metastasis and progression of BCa remains vague.

As a mainly epigenetic modification, DNA methylation adds the methyl group at the carbon 5 position of the cytosine ring in CpG islands which are commonly located at the promoters and the first exon of genes [5,6]. There are three main DNA methyltransferases that can catalyze the DNA methylation in mammals: DNA methyltransferase 1(DNMT1, the maintenance methyltransferase); DNMT3A and DNMT3B (de novo methyltransferases) [7]. DNA methylation results in gene repression via modulating the genomic stability, chromatin remodeling and transcriptional regulation [7]. DNA methylation plays its role by affecting a variety of cellular progression, including cell growth, metastasis, cycle, apoptosis, development, and tumorigenesis [8]. Aberrant DNA methylation and the accompanying dis-regulation of DNMT1 expression is correlated with different diseases [9]. In human cancers, DNMT1 is usually up-regulated in cancer tissues, when compared to the paired adjacent normal tissues; thus, hypermethylating the CpG islands in the promoter of the tumor suppressor gene which leads to its inhibition, resulting in the promotion of the occurrence and development of tumor [10].DNA methylation is involved in the physiological and pathological process of BCa [11]. Antoine G van der Heijden and colleagues applied a three-gene methylation classifier to analyze the DNA methylation of genes in urine cells from 626 urine samples of BCa patients for the purpose of monitoring the disease [12]. However, the DNA methylation and the accompanying regulatory mechanisms in physiological and pathological progression of BCa are still largely unclear.

Micro RNAs (miRNAs), which were first discovered in 1993, are a class of small non-coding RNAs with length range from 18 to 25-ribonucleotide [13]. miRNAs act by affecting the mRNAs degradation and through their direct binding, especially in the 3′- untranslated region (3′UTR) of mRNA targets [14,15]. miRNAs were identified as playing an important role in a vast number of biological functions, such as cell growth, metastasis, cycle, apoptosis, development, as well as tumorigenesis [16]. miRNAs could function both as oncogenes or tumor suppressors and there is increasing evidence that shows that abnormal expression of miRNAs corresponds with pathophysiological conditions; a growing list of functional miRNAs are identified in BCa [17]. Several studies demonstrated that miRNA plays its role by inhibiting the DNMT1 expression in cancers. Françoise Solly and colleagues revealed a miRNA-DNMT1 axis which is involved in Azacitidine resistance, prognosis of higher-risk myelodysplastic syndrome and low blast count acute myeloid leukemia [18]. Several miRNAs, including miR-185, miR-52 and miR-148a have been reported to regulate the DNMT1 expression [[19], [20], [21], [22]]. Also, Er-Bao Bian and colleagues have reported that DNMT1 decreases PTEN expression by methylating its promoter in liver fibrogenesis [23].

In this study, we aim to confirm the role of DNMT1 in BCa and further elucidate the regulatory factors on DNMT1 and the downstream target genes of DNMT1.We demonstrated that DNMT1 was increased due to the decreased miR-152-3p and thus, lead to the increase of DNA methylation in the promoter of PTEN, resulting in the inhibition of PTEN in BCa.

Section snippets

Tissue samples collection

All BCa and corresponding adjacent normal specimens were collected at Xiangya Hospital, Central South University. Written consents were obtained from all the patients and the research was approved and supervised by the Ethical Committee of Xiangya Hospital. All the tissues were immediately snap-frozen in liquid nitrogen and stored at −80 °C.

Cells and culture

Human normal bladder epithelial cells SVHUC-1 and human BCa cell line 5637, Ejm3, BIU-87, J82, T24 were routinely preserved in our lab. 5637, Ejm3, J82, T24

DNMT1 is up-regulated in BCa cancer tissues and cells

To measure the biological function of DNMT1 in BCa, we first employed qPCR and western blot assay to detect the DNMT1 expression in malignant and corresponding adjacent normal tissues. The results showed that the DNMT1 was up-regulated in BCa tissues, when compared to the corresponding adjacent normal tissues (Fig. 1A). We then tested the DNMT1 expression in SVHUC-1 and human BCa cell line 5637, Ejm3, BIU-87, J82 and T24. The results displayed that the DNMT1 expression was increased in BCa cell

Discussion

As one of the most prevalent cancer, BCa ranks as the ninth diagnosed urologic carcinomas worldwide and thus, causes severe burden to social health [27]. As we all known, better understanding of the progression and the underlying mechanism of cancer is beneficial for the research on tumor treatment methods. Researchers summarized the ten hallmarks of cancers, including resistance to cell death, tumor-promoting angiogenesis, avoidance of immune destruction, self-sufficient growth signals, tissue

Conclusion

In conclusion, this study displayed the oncogenic function of DNMT1, anti-tumor function of miR-152-3p and PTEN in BCa and further, showed that DNMT1 inhibited by miR-152-3p repressed the PTEN expression via modulation of the DNA methylation in the promoter of PTEN and thus, provided a pathway which might serve as diagnostic and therapeutic biomarkers for BCa.

CRediT authorship contribution statement

PH.L and LX.W were responsible for data curation. PH.L and LX.W designed the experiments. PH.L wrote the manuscript. H·C, C.L, XH·H and YL.L reviewed and approved the manuscript. YL.L was responsible for project administration. YL.L supervised the experiments.

Declaration of competing interest

The authors declare no competing financial interests.

Acknowledgements

This work was funded by The Youth Science Foundation of Xiangya Hospital, Central South University (2019Q09).

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