Identification and validation of mRNA 3'untranslated regions of DNMT3B and TET3 as novel competing endogenous RNAs of the tumor suppressor PTEN

Roquid,Kenneth Anthony  R.; Alcantara,Krizelle Mae  M.; Garcia,Reynaldo  L.

doi:10.3892/ijo.2019.4947

Identification and validation of mRNA 3'untranslated regions of DNMT3B and TET3 as novel competing endogenous RNAs of the tumor suppressor PTEN

Authors:
- Kenneth Anthony R. Roquid
- Krizelle Mae M. Alcantara
- Reynaldo L. Garcia
View Affiliations

Affiliations: Disease Molecular Biology and Epigenetics Laboratory, National Institute of Molecular Biology and Biotechnology, National Science Complex, University of the Philippines Diliman, Quezon City, Metro Manila 1101, Philippines
Published online on: December 20, 2019 https://doi.org/10.3892/ijo.2019.4947
Pages: 544-558
Copyright: © Roquid et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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

PTEN inactivation is a frequent event in oncogenesis. Multiple regulatory mechanisms such as promoter hypermethylation, antisense regulation, histone modifications, targeting by microRNAs (miRNAs/miRs) and regulation by transcription factors have all been shown to affect the tumor suppressor functions of PTEN. More recently, the functional involvement of competing endogenous RNAs (ceRNAs) in miRNA‑dependent and coding‑independent regulation of genes shed light on the highly nuanced control of PTEN expression. The present study has identified and validated DNA methyltransferase 3β (DNMT3B) and TET methylcytosine dioxygenase 3 (TET3) as novel ceRNAs of PTEN, with which they share multiple miRNAs, in HCT116 colorectal cancer cells. miR‑4465 was identified and characterized as a miRNA that directly targets and regulates all 3 transcripts via their 3'untranslated regions (3'UTRs) through a combination of luciferase reporter assays, abrogation of miRNA response elements (MREs) via site‑directed mutagenesis, target protection of MREs with locked nucleic acids, RT‑qPCR assays and western blot analysis. Competitive miRNA sequestration was demonstrated upon reciprocal 3'UTR overexpression and siRNA‑mediated knockdown of their respective transcripts. Overexpression of DNMT3B or TET3 3'UTR promoted apoptosis and decreased migratory capacity, potentially because of shared miRNA sequestration and subsequent activation of PTEN expression. Knockdown of TET3 and DNMT3B decoupled their protein‑coding from miRNA‑dependent, coding‑independent functions. Furthermore, the findings suggested that the phenotypic outcome of ceRNAs is dictated largely by the number of shared miRNAs, and predictably, by the existence of other ceRNA networks in which they participate. Taken together, the findings of the present study identified DNMT3B and TET3 as novel ceRNAs of PTEN that may impact its dose‑sensitive tumor suppressive function.

View Figures

View References

1	Carnero A, Blanco-Aparicio C, Renner O, Link W and Leal J: The PTEN/PI3K/AKT signalling pathway in cancer, therapeutic implications. Curr Cancer Drug Targets. 8:187–198. 2008. View Article : Google Scholar : PubMed/NCBI
2	Sun Z, Huang C, He J, Lamb KL, Kang X, Gu T, Shen WH and Yin Y: PTEN C-terminal deletion causes genomic instability and tumor development. Cell Rep. 6:844–854. 2014. View Article : Google Scholar : PubMed/NCBI
3	Murphy SJ, Karnes RJ, Kosari F, Castellar BE, Kipp BR, Johnson SH, Terra S, Harris FR, Halling GC, Klein JL, et al: Integrated analysis of the genomic instability of PTEN in clinically insignificant and significant prostate cancer. Mod Pathol. 29:143–156. 2016. View Article : Google Scholar
4	Molinari F and Frattini M: Functions and regulation of the PTEN gene in colorectal cancer. Front Oncol. 3:3262014. View Article : Google Scholar : PubMed/NCBI
5	Berger AH, Knudson AG and Pandolfi PP: A continuum model for tumour suppression. Nature. 476:163–169. 2011. View Article : Google Scholar : PubMed/NCBI
6	Alimonti A, Carracedo A, Clohessy JG, Trotman LC, Nardella C, Egia A, Salmena L, Sampieri K, Haveman WJ, Brogi E, et al: Subtle variations in Pten dose determine cancer susceptibility. Nat Genet. 42:454–458. 2010. View Article : Google Scholar : PubMed/NCBI
7	Trotman LC, Niki M, Dotan ZA, Koutcher JA, Di Cristofano A, Xiao A, Khoo AS, Roy-Burman P, Greenberg NM, Van Dyke T, et al: Pten dose dictates cancer progression in the prostate. PLoS Biol. 1:E592003. View Article : Google Scholar : PubMed/NCBI
8	Garofalo M, Di Leva G, Romano G, Nuovo G, Suh SS, Ngankeu A, Taccioli C, Pichiorri F, Alder H, Secchiero P, et al: miR-221&222 regulate TRAIL resistance and enhance tumorigenicity through PTEN and TIMP3 downregulation. Cancer Cell. 16:498–509. 2009. View Article : Google Scholar : PubMed/NCBI
9	Huse JT, Brennan C, Hambardzumyan D, Wee B, Pena J, Rouhanifard SH, Sohn-Lee C, le Sage C, Agami R, Tuschl T and Holland EC: The PTEN-regulating microRNA miR-26a is amplified in high-grade glioma and facilitates gliomagenesis in vivo. Genes Dev. 23:1327–1337. 2009. View Article : Google Scholar : PubMed/NCBI
10	Tabach Y, Billi AC, Hayes GD, Newman MA, Zuk O, Gabel H, Kamath R, Yacoby K, Chapman B, Garcia SM, et al: Identification of small RNA pathway genes using patterns of phylogenetic conservation and divergence. Nature. 493:694–698. 2013. View Article : Google Scholar : PubMed/NCBI
11	Mullany LE, Herrick JS, Wolff RK, Stevens JR, Samowitz W and Slattery ML: MicroRNA-transcription factor interactions and their combined effect on target gene expression in colon cancer cases. Genes, Chromosom Cancer. 57:192–202. 2018. View Article : Google Scholar
12	Volinia S, Calin GA, Liu CG, Ambs S, Cimmino A, Petrocca F, Visone R, Iorio M, Roldo C, Ferracin M, et al: A microRNA expression signature of human solid tumors defines cancer gene targets. Proc Natl Acad Sci USA. 103:2257–2261. 2006. View Article : Google Scholar : PubMed/NCBI
13	Frankel LB, Christoffersen NR, Jacobsen A, Lindow M, Krogh A and Lund AH: Programmed cell death 4 (PDCD4) is an important functional target of the microRNA miR-21 in breast cancer cells. J Biol Chem. 283:1026–1033. 2008. View Article : Google Scholar
14	Zhu S, Si ML, Wu H and Mo YY: MicroRNA-21 targets the tumor suppressor gene tropomyosin 1 (TPM1). J Biol Chem. 282:14328–14336. 2007. View Article : Google Scholar : PubMed/NCBI
15	Chan JA, Krichevsky AM and Kosik KS: MicroRNA-21 Is an antiapoptotic factor in human glioblastoma cells. Cancer Res. 65:6029–6033. 2005. View Article : Google Scholar : PubMed/NCBI
16	Tay Y, Rinn J and Pandolfi PP: The multilayered complexity of ceRNA crosstalk and competition. Nature. 505:344–352. 2014. View Article : Google Scholar : PubMed/NCBI
17	Zarringhalam K, Tay Y, Kulkarni P, Bester AC, Pandolfi PP and Kulkarni RV: Identification of competing endogenous RNAs of the tumor suppressor gene PTEN: A probabilistic approach. Sci Rep. 7:77552017. View Article : Google Scholar : PubMed/NCBI
18	Poliseno L and Pandolfi PP: PTEN ceRNA networks in human cancer. Methods. 77-78:41–50. 2015. View Article : Google Scholar : PubMed/NCBI
19	Karreth FA, Tay Y, Perna D, Ala U, Tan SM, Rust AG, DeNicola G, Webster KA, Weiss D, Perez-Mancera PA, et al: In vivo identification of tumor-suppressive PTEN ceRNAs in an oncogenic BRAF-induced mouse model of melanoma. Cell. 147:382–395. 2011. View Article : Google Scholar : PubMed/NCBI
20	Marone M, Mozzetti S, De Ritis D, Pierelli L and Scambia G: Semiquantitative RT-PCR analysis to assess the expression levels of multiple transcripts from the same sample. Biol Proced Online. 3:19–25. 2001. View Article : Google Scholar
21	Langlois MJ, Bergeron S, Bernatchez G, Boudreau F, Saucier C, Perreault N, Carrier JC and Rivard N: The PTEN phosphatase controls intestinal epithelial cell polarity and barrier function: Role in colorectal cancer progression. PLoS One. 5:e157422010. View Article : Google Scholar
22	Rhee I, Bachman KE, Park BH, Jair KW, Yen RW, Schuebel KE, Cui H, Feinberg AP, Lengauer C, Kinzler KW, et al: DNMT1 and DNMT3b cooperate to silence genes in human cancer cells. Nature. 416:552–556. 2002. View Article : Google Scholar : PubMed/NCBI
23	Uribe-Lewis S, Stark R, Carroll T, Dunning MJ, Bachman M, Ito Y, Stojic L, Halim S, Vowler SL, Lynch AG, et al: 5-hydroxy-methylcytosine marks promoters in colon that resist DNA hypermethylation in cancer. Genome Biol. 16:692015. View Article : Google Scholar
24	García JM, Silva J, Peña C, Garcia V, Rodríguez R, Cruz MA, Cantos B, Provencio M, España P and Bonilla F: Promoter methylation of the PTEN gene is a common molecular change in breast cancer. Genes Chromosom Cancer. 41:117–124. 2004. View Article : Google Scholar : PubMed/NCBI
25	Alvarez-Nuñez F, Bussaglia E, Mauricio D, Ybarra J, Vilar M, Lerma E, de Leiva A and Matias-Guiu X; Thyroid Neoplasia Study Group: PTEN promoter methylation in sporadic thyroid carcinomas. Thyroid. 16:17–23. 2006. View Article : Google Scholar : PubMed/NCBI
26	Lu J, Jeong H, Kong N, Yang Y, Carroll J, Luo HR, Silberstein LE, Yupoma and Chai L: Stem cell factor SALL4 represses the transcriptions of PTEN and SALL1 through an epigenetic repressor complex. PLoS One. 4:e55772009. View Article : Google Scholar : PubMed/NCBI
27	Hettinger K, Vikhanskaya F, Poh MK, Lee MK, de Belle I, Zhang JT, Reddy SA and Sabapathy K: c-Jun promotes cellular survival by suppression of PTEN. Cell Death Differ. 14:218–229. 2007. View Article : Google Scholar
28	Vasudevan KM, Gurumurthy S and Rangnekar VM: Suppression of PTEN expression by NF-kappa B prevents apoptosis. Mol Cell Biol. 24:1007–1021. 2004. View Article : Google Scholar : PubMed/NCBI
29	Meng F, Henson R, Wehbe-Janek H, Ghoshal K, Jacob ST and Patel T: MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer. Gastroenterology. 133:647–658. 2007. View Article : Google Scholar : PubMed/NCBI
30	Tay Y, Kats L, Salmena L, Weiss D, Tan SM, Ala U, Karreth F, Poliseno L, Provero P, Di Cunto F, et al: Coding-independent regulation of the tumor suppressor PTEN by competing endogenous mRNAs. Cell. 147:344–357. 2011. View Article : Google Scholar : PubMed/NCBI
31	Poliseno L, Salmena L, Zhang J, Carver B, Haveman WJ and Pandolfi PP: A coding-independent function of gene and pseudogene mRNAs regulates tumour biology. Nature. 465:1033–1038. 2010. View Article : Google Scholar : PubMed/NCBI
32	Salmena L, Poliseno L, Tay Y, Kats L and Pandolfi PP: A ceRNA Hypothesis: The rosetta stone of a hidden RNA language? Cell. 146:353–358. 2011. View Article : Google Scholar : PubMed/NCBI
33	Yamakuchi M, Lotterman CD, Bao C, Hruban RH, Karim B, Mendell JT, Huso D and Lowenstein CJ: P53-induced microRNA-107 inhibits HIF-1 and tumor angiogenesis. Proc Natl Acad Sci USA. 107:6334–6339. 2010. View Article : Google Scholar : PubMed/NCBI
34	Wang B, Hsu SH, Wang X, Kutay H, Bid HK, Yu J, Ganju RK, Jacob ST, Yuneva M and Ghoshal K: Reciprocal regulation of microRNA-122 and c-Myc in hepatocellular cancer: Role of E2F1 and transcription factor dimerization partner 2. Hepatology. 59:555–566. 2014. View Article : Google Scholar
35	Sumazin P, Yang X, Chiu HS, Chung WJ, Iyer A, Llobet-Navas D, Rajbhandari P, Bansal M, Guarnieri P, Silva J and Califano A: An Extensive MicroRNA-mediated network of RNA-RNA interactions regulates established oncogenic pathways in glio-blastoma. Cell. 147:370–381. 2011. View Article : Google Scholar : PubMed/NCBI
36	Jeyapalan Z, Deng Z, Shatseva T, Fang L, He C and Yang BB: Expression of CD44 3'-untranslated region regulates endogenous microRNA functions in tumorigenesis and angiogenesis. Nucleic Acids Res. 39:3026–3041. 2011. View Article : Google Scholar
37	Rutnam ZJ, Du WW, Yang W, Yang X and Yang BB: The pseu-dogene TUSC2P promotes TUSC2 function by binding multiple microRNAs. Nat Commun. 5:29142014. View Article : Google Scholar
38	Karreth FA, Reschke M, Ruocco A, Ng C, Chapuy B, Léopold V, Sjoberg M, Keane TM, Verma A, Ala U, et al: The BRAF pseu-dogene functions as a competitive endogenous RNA and induces lymphoma in vivo. Cell. 161:319–332. 2015. View Article : Google Scholar : PubMed/NCBI
39	Wang L, Guo ZY, Zhang R, Xin B, Chen R, Zhao J, Wang T, Wen WH, Jia LT, Yao LB and Yang AG: Pseudogene OCT4-pg4 functions as a natural micro RNA sponge to regulate OCT4 expression by competing for miR-145 in hepatocellular carcinoma. Carcinogenesis. 34:1773–1781. 2013. View Article : Google Scholar : PubMed/NCBI
40	Sanchez-Mejias A and Tay Y: Competing endogenous RNA networks: Tying the essential knots for cancer biology and therapeutics. J Hematol Oncol. 8:302015. View Article : Google Scholar : PubMed/NCBI
41	Ergun S and Oztuzcu S: Oncocers: ceRNA-mediated cross-talk by sponging miRNAs in oncogenic pathways. Tumor Biol. 36:3129–3136. 2015. View Article : Google Scholar
42	Park SM, Gaur AB, Lengyel E and Peter ME: The miR-200 family determines the epithelial phenotype of cancer cells by targeting the E-cadherin repressors ZEB1 and ZEB2. Genes Dev. 22:894–907. 2008. View Article : Google Scholar : PubMed/NCBI
43	Xu J, Li Y, Lu J, Pan T, Ding N, Wang Z, Shao T, Zhang J, Wang L and Li X: The mRNA related ceRNA-ceRNA landscape and significance across 20 major cancer types. Nucleic Acids Res. 43:8169–8182. 2015. View Article : Google Scholar : PubMed/NCBI
44	Chen L, Zheng J, Zhang Y, Yang L, Wang J, Ni J, Cui D, Yu C and Cai Z: Tumor-specific Expression of MicroRNA-26a suppresses human hepatocellular carcinoma growth via cyclin-dependent and -independent pathways. Mol Ther. 19:1521–1528. 2011. View Article : Google Scholar : PubMed/NCBI
45	Sandhu R, Rivenbark AG and Coleman WB: Enhancement of chemotherapeutic efficacy in hypermethylator breast cancer cells through targeted and pharmacologic inhibition of DNMT3b. Breast Cancer Res Treat. 131:385–399. 2012. View Article : Google Scholar
46	Noack F and Calegari F: MicroRNAs meet epigenetics to make for better brains. EMBO Rep. 15:1224–1225. 2014. View Article : Google Scholar : PubMed/NCBI
47	Garcia DM, Baek D, Shin C, Bell GW, Grimson A and Bartel DP: Weak seed-pairing stability and high target-site abundance decrease the proficiency of lsy-6 and other miRNAs. Nat Struct Mol Biol. 18:1139–1146. 2011. View Article : Google Scholar : PubMed/NCBI
48	Friedman RC, Farh KK, Burge CB and Bartel DP: Most mammalian mRNAs are conserved targets of microRNAs. Genome Res. 19:92–105. 2009. View Article : Google Scholar :