The interaction between miR‑148a and DNMT1 suppresses cell migration and invasion by reactivating tumor suppressor genes in pancreatic cancer

Hong,Le; Sun,Gen; Peng,Long; Tu,Yi; Wan,Zhen; Xiong,Haiwei; Li,Yong; Xiao,Weidong

doi:10.3892/or.2018.6700

The interaction between miR‑148a and DNMT1 suppresses cell migration and invasion by reactivating tumor suppressor genes in pancreatic cancer

Authors:
- Le Hong
- Gen Sun
- Long Peng
- Yi Tu
- Zhen Wan
- Haiwei Xiong
- Yong Li
- Weidong Xiao
View Affiliations

Affiliations: Department of General Surgery, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330006, P.R. China, Department of General Surgery, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330006, P.R. China, Department of General Surgery, The Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330006, P.R. China, Department of Pathology,The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330006, P.R. China
Published online on: September 12, 2018 https://doi.org/10.3892/or.2018.6700
Pages: 2916-2925

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

DNA methylation is an epigenetic mechanism that cells use to control gene expression, which serves an important role in tumorigenesis. DNA methyltransferase 1 (DNMT1) is responsible for the maintenance of the pattern of DNA methylation. Overexpression of DNMT1 is observed in numerous malignant tumors, including pancreatic cancer, and results in silencing of several key tumor suppressor genes (TSGs). Recent studies have suggested that microRNAs (miRNAs/miRs) contribute to the regulation of DNMT1 expression, and promoter hypermethylation caused by DNMT1 overexpression is associated with the dysfunction of some miRNAs. The present study aimed to reveal the interaction between miR‑148a and DNMT1, and its effects on cell proliferation, migration and invasion of pancreatic cancer cells. Initially, the expression levels of DNMT1 and miR‑148a were detected in pancreatic cancer tissues and AsPC‑1 cells by reverse transcription‑quantitative polymerase chain reaction (PCR). Secondly, the regulatory effects of DNMT1 on miR‑148a were evaluated using methylation‑specific PCR. Furthermore, bioinformatics analysis and dual luciferase reporter assay were used to verify the target relationship between miR‑148a and DNMT1. Finally, in vitro rescue experiments were conducted to evaluate the effects of miR‑148a on the expression of TSGs and the malignant phenotype in AsPC‑1 cells. The results demonstrated that DNMT1 was aberrantly upregulated in pancreatic cancer, and was responsible for hypermethylation of the miR‑148a promoter. Furthermore, DNMT1 was revealed as a direct target of miR‑148a by dual luciferase reporter assay, and restoration of miR‑148a could reactivate TSGs, such as p16, preproenkephalin and Ras association domain family member 1 by targeting DNMT1 in the AsPC‑1 pancreatic cancer cell line. These results indicated that an interaction exists between miR‑148a and DNMT1 in pancreatic cancer. Notably, miR‑148a overexpression significantly inhibited cell proliferation, migration and invasion in AsPC‑1 cells. Therefore, miR‑148a may serve as a novel therapeutic target for the treatment of pancreatic cancer.

View Figures

View References

1	Siegel RL, Miller KD and Jemal A: Cancer statistics, 2018. CA Cancer J Clin. 68:7–30. 2018. View Article : Google Scholar : PubMed/NCBI
2	Rahib L, Smith BD, Aizenberg R, Rosenzweig AB, Fleshman JM and Matrisian LM: Projecting cancer incidence and deaths to 2030: The unexpected burden of thyroid, liver, and pancreas cancers in the United States. Cancer Res. 74:2913–2921. 2014. View Article : Google Scholar : PubMed/NCBI
3	Chen W, Zheng R, Baade PD, Zhang S, Zeng H, Bray F, Jemal A, Yu XQ and He J: Cancer statistics in China, 2015. CA Cancer J Clin. 66:115–132. 2016. View Article : Google Scholar : PubMed/NCBI
4	Chan SH and Wang LH: Regulation of cancer metastasis by microRNAs. J Biomed Sci. 22:92015. View Article : Google Scholar : PubMed/NCBI
5	Xu Q, Jiang Y, Yin Y, Li Q, He J, Jing Y, Qi YT, Xu Q, Li W, Lu B, et al: A regulatory circuit of miR-148a/152 and DNMT1 in modulating cell transformation and tumor angiogenesis through IGF-IR and IRS1. J Mol Cell Biol. 5:3–13. 2013. View Article : Google Scholar : PubMed/NCBI
6	Zhang H, Li Y, Huang Q, Ren X, Hu H, Sheng H and Lai M: MiR-148a promotes apoptosis by targeting Bcl-2 in colorectal cancer. Cell Death Differ. 18:1702–1710. 2011. View Article : Google Scholar : PubMed/NCBI
7	Sakamoto N, Naito Y, Oue N, Sentani K, Uraoka N, Oo Zarni H, Yanagihara K, Aoyagi K, Sasaki H and Yasui W: MicroRNA-148a is downregulated in gastric cancer, targets MMP7, and indicates tumor invasiveness and poor prognosis. Cancer Sci. 105:236–243. 2014. View Article : Google Scholar : PubMed/NCBI
8	Braconi C, Huang N and Patel T: MicroRNA-dependent regulation of DNA methyltransferase-1 and tumor suppressor gene expression by interleukin-6 in human malignant cholangiocytes. Hepatology. 51:881–890. 2010.PubMed/NCBI
9	Heo MJ, Kim YM, Koo JH, Yang YM, An J, Lee SK, Lee SJ, Kim KM, Park JW and Kim SG: microRNA-148a dysregulation discriminates poor prognosis of hepatocellular carcinoma in association with USP4 overexpression. Oncotarget. 5:2792–2806. 2014. View Article : Google Scholar : PubMed/NCBI
10	Bloomston M, Frankel WL, Petrocca F, Volinia S, Alder H, Hagan JP, Liu CG, Bhatt D, Taccioli C and Croce CM: MicroRNA expression patterns to differentiate pancreatic adenocarcinoma from normal pancreas and chronic pancreatitis. JAMA. 297:1901–1908. 2007. View Article : Google Scholar : PubMed/NCBI
11	Szafranska AE, Davison TS, John J, Cannon T, Sipos B, Maghnouj A, Labourier E and Haln SA: MicroRNA expression alterations are linked to tumorigenesis and non-neoplastic processes in pancreatic ductal adenocarcinoma. Oncogene. 26:4442–4452. 2007. View Article : Google Scholar : PubMed/NCBI
12	Zhan Q, Fang Y, Deng X, Chen H, Jin J, Lu X, Peng C, Li H and Shen B: The interplay between miR-148a and DNMT1 might be exploited for pancreatic cancer therapy. Cancer Invest. 33:267–275. 2015. View Article : Google Scholar : PubMed/NCBI
13	Zhang R, Li M, Zang W, Chen X, Wang Y, Li P, Du Y, Zhao G and Li L: MiR-148a regulates the growth and apoptosis in pancreatic cancer by targeting CCKBR and Bcl-2. Tumour Biol. 35:837–44. 2014. View Article : Google Scholar : PubMed/NCBI
14	Liffers ST, Munding JB, Vogt M, Kuhlmann JD, Verdoodt B, Nambiar S, Maghnouj A, Mimohammadsadegh A, Hahn SA and Tannapfel A: MicroRNA-148a is down-regulated in human pancreatic ductal adenocarcinomas and regulates cell survival by targeting CDC25B. Lab Invest. 91:1472–1479. 2011. View Article : Google Scholar : PubMed/NCBI
15	Feng H, Wang Y, Su J, Liang H, Zhang CY, Chen X and Yao W: MicroRNA-148a suppresses the proliferation and migration of pancreatic cancer cells by down-regulating ErbB3. Pancreas. 45:1263–1271. 2016. View Article : Google Scholar : PubMed/NCBI
16	Lopez-Serra P and Esteller M: DNA methylation-associated silencing of tumor-suppressor microRNAs in cancer. Oncogene. 31:1609–1622. 2012. View Article : Google Scholar : PubMed/NCBI
17	Xie K, Liu J, Chen J, Dong J, Ma H, Liu Y and Hu Z: Methylation-associated silencing of microRNA-34b in hepatocellular carcinoma cancer. Gene. 543:101–107. 2014. View Article : Google Scholar : PubMed/NCBI
18	Peng DF, Kanai Y, Sawada M, Ushijima S, Hiraoka N, Kosuge T and Hirohashi S: Increased DNA methyltransferase 1 (DNMT1) protein expression in precancerous conditions and ductal carcinomas of the pancreas. Cancer Sci. 96:403–408. 2005. View Article : Google Scholar : PubMed/NCBI
19	Li A, Omura N, Hong SM and Goggins M: Pancreatic cancer DNMT1 expression and sensitivity to DNMT1 inhibitors. Cancer Biol Ther. 9:321–329. 2010. View Article : Google Scholar : PubMed/NCBI
20	Zhu A, Xia J, Zuo J, Jin S, Zhou H, Yao L, Huang H and Han Z: MicroRNA-148a is silenced by hypermethylation and interacts with DNA methyltransferase 1 in gastric cancer. Med Oncol. 29:2701–2709. 2012. View Article : Google Scholar : PubMed/NCBI
21	Long XR, He Y, Huang C and Li J: MicroRNA-148a is silenced by hypermethylation and interacts with DNA methyltransferase 1 in hepatocellular carcinogenesis. Int J Oncol. 44:1915–1922. 2014. View Article : Google Scholar : PubMed/NCBI
22	Peng L, Liu Z, Xiao J, Tu Y, Wan Z, Xiong H, Li Y and Xiao W: MicroRNA-148a suppresses epithelial-mesenchymal transition and invasion of pancreatic cancer cells by targeting Wnt10b and inhibiting the Wnt/β-catenin signaling pathway. Oncol Rep. 38:301–308. 2017. View Article : Google Scholar : PubMed/NCBI
23	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2^−ΔΔCT method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
24	Sanger F, Nicklen S and Coulson AR: DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA. 74:5463–5467. 1977. View Article : Google Scholar : PubMed/NCBI
25	Kumaki Y, Oda M and Okano M: QUMA: Quantification tool for methylation analysis. Necleic Acids Res. 36:W170–W175. 2008. View Article : Google Scholar
26	Bock C, Reither S, Mikeska T, Paulsen M, Walter J and Lengauer T: BiQ Analyzer: Visualization and quality control for DNA methylation data from bisulfite sequencing. Bioinformatics. 21:4067–4068. 2005. View Article : Google Scholar : PubMed/NCBI
27	Lee J, Shin MK, Ryu DK, Kim S and Ryu WS: Insertion and deletion mutagenesis by overlap extension PCR. Methods Mol Biol. 634:137–146. 2010. View Article : Google Scholar : PubMed/NCBI
28	Bestor TH: The DNA methyltransferases of mammals. Hum Mol Genet. 9:2395–2402. 2000. View Article : Google Scholar : PubMed/NCBI
29	Cowan RW and Maitra A: Genetic progression of pancreatic cancer. Cancer J. 20:80–84. 2014. View Article : Google Scholar : PubMed/NCBI
30	Yonemori K, Kurahara H, Maemura K and Natsugoe S: MicroRNA in pancreatic cancer. J Hum Genet. 62:33–40. 2017. View Article : Google Scholar : PubMed/NCBI
31	Yang L, Luo P, Song Q and Fei X: DNMT1/miR-200a/GOLM1 signaling pathway regulates lung adenocarcinoma cells proliferation. Biomed Pharmacother. 99:839–847. 2018. View Article : Google Scholar : PubMed/NCBI
32	Ning X, Shi Z, Liu X, Zhang A, Han L, Jiang K, Kang C and Zhang Q: DNMT1 and EZH2 mediated methylation silences the microRNA-200b/a/429 gene and promotes tumor progression. Cancer Lett. 59:198–205. 2015. View Article : Google Scholar
33	Peng DF, Kanai Y, Sawada M, Ushijima S, Hiraoka N, Kitazawa S and Hirohashi S: DNA methylation of multiple tumor-related genes in association with overexpression of DNA methyltransferase 1 (DNMT1) during multistage carcinogenesis of the pancreas. Carcinogenesis. 27:1160–1168. 2006. View Article : Google Scholar : PubMed/NCBI
34	Liu B, Song J, Luan J, Sun X, Bai J, Wang H, Li A, Zhang L, Feng X and Du Z: Promoter methylation status of tumor suppressor genes and inhibition of expression of DNA methyltransferase 1 in non-small cell lung cancer. Exp Biol Med. 241:1531–1539. 2016. View Article : Google Scholar
35	Delpu Y, Lulka H, Sicard F, Saint-Laurent N, Lopez F, Hanoun N, Buscail L, Cordelier P and Torrisani J: The rescue of miR-148a expression in pancreatic cancer: An inappropriate therapeutic tool. PLoS One. 8:e555132013. View Article : Google Scholar : PubMed/NCBI
36	Pan FP, Zhou HK, Bu HQ, Chen ZQ, Zhang H, Xu LP, Tang J, Yu QJ, Chu YQ, Pan J, et al: Emodin enhances the demethylation by 5-AZa-CdR of pancreatic cancer tumor-suppressor genes P16, RASSF1A and ppENK. Oncol Rep. 35:1941–1949. 2016. View Article : Google Scholar : PubMed/NCBI
37	Witkiewicz AK, Knudsen KE, Dicker AP and Knudsen ES: The meaning of p16ink4a expression in tumors: Functional significance, clinical associations and future developments. Cell Cycle. 10:2497–2503. 2011. View Article : Google Scholar : PubMed/NCBI
38	Yang L, Yang H, Li J, Hao J and Qian J: ppENK gene methylation status in the development of pancreatic carcinoma. Gastroenterol Res Pract. 2013:1309272013. View Article : Google Scholar : PubMed/NCBI
39	Vos MD, Martinez A, Elam C, Dallol A, Taylor BJ, Latif F and Clark GJ: A role for the RASSF1A tumor suppressor in the regulation of tubulin polymerization and genomic stability. Cancer Res. 64:4244–4250. 2004. View Article : Google Scholar : PubMed/NCBI
40	Shivakumar L, Minna J, Sakamaki T, Pestell R and White MA: The RASSF1A tumor suppressor blocks cell cycle progression and inhibits cyclin D1 accumulation. Mol Cell Biol. 22:4309–4318. 2002. View Article : Google Scholar : PubMed/NCBI
41	Attri J, Srinivasan R, Majumdar S, Radotra BD and Wig J: Alterations of tumor suppressor gene p16INK4a in pancreatic ductal carcinoma. BMC Gastroenterol. 5:222005. View Article : Google Scholar : PubMed/NCBI
42	Dammann R, Schagdarsurengin U, Liu L, Otto N, Gimm O, Dralle H, Boehm BO, Pfeifer GP and Hoang-Vu C: Frequent RASSF1A promoter hypermethylation and K-ras mutations in pancreatic carcinoma. Oncogene. 22:3806–3812. 2003. View Article : Google Scholar : PubMed/NCBI
43	Fukushima N, Sato N, Ueki T, Rosty C, Walter KM, Wilentz RE, Yeo CJ, Hruban RH and Goggins M: Aberrant methylation of preproenkephalin and p16 genes in pancreatic intraepithelial neoplasia and pancreatic ductal adenocarcinoma. Am J Pathol. 160:1573–1581. 2002. View Article : Google Scholar : PubMed/NCBI
44	Xiao WD, Li Y, Li XM, Cai J, Zeng LS and Hu W: RNA interference-mediated silencing of the DNMT1 gene inhibits cell proliferation in human pancreatic carcinoma cell line BxPC-3. Shijie Huaren Xiaohua Zazhi. 19:3397–3401. 2011.(In Chinese).
45	Sun J, Song Y, Wang Z, Wang G, Gao P, Chen X, Gao Z and Xu H: Clinical significance of promoter region hypermethylation of microRNA-148a in gastrointestinal cancers. Onco Targets Ther. 7:853–863. 2014.PubMed/NCBI
46	Long XR, He Y, Huang C and Li J: MicroRNA-148a is silenced by hypermethylation and interacts with DNA methyltransferase in hepatocellular carcinogenesis. Int J Oncol. 44:1915–1922. 2014. View Article : Google Scholar : PubMed/NCBI
47	Li HP, Huang HY, Lai YR, Huang JX, Chang KP, Hsueh C and Chang YS: Silencing of miRNA-148a by hypermethylation activates the integrin-mediated signaling pathway in nasopharyngeal carcinoma. Oncotarget. 5:7610–7624. 2014. View Article : Google Scholar : PubMed/NCBI
48	Hanoun N, Delpu Y, Suriawinata AA, Bournet B, Bureau C, Selves J, Tsongalis GJ, Dufresne M, Buscail L, Cordelier P and Torrisani J: The silencing of microRNA 148a production by DNA hypermethylation is an early event in pancreatic carcinogenesis. Clin Chem. 56:1107–1118. 2010. View Article : Google Scholar : PubMed/NCBI