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Silencing of long non-coding RNA FOXD2-AS1 inhibits the progression of gallbladder cancer by mediating methylation of MLH1

Gene Therapy volume 28, pages 306–318 (2021)Cite this article

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Abstract

Evidence has documented the tumor-promoting properties of long non-coding RNA (lncRNA) FOXD2 adjacent opposite strand RNA 1 (FOXD2-AS1) in many cancers. However, little is known about its role in gallbladder cancer. Here, we aimed to characterize the functional relevance of lncRNA FOXD2-AS1 in gallbladder cancer and the possible mechanisms associated with methylation of MutL homolog-1 (MLH1). Initially, microarray-based gene expression profiling of gallbladder cancer was employed to identify differentially expressed lncRNAs. Next, the expression of lncRNA FOXD2-AS1 was examined, and the cell line presenting with the highest lncRNA FOXD2-AS1 expression was selected for subsequent experimentation. Then, the interaction between lncRNA FOXD2-AS1 and MLH1 was identified. The effect of lncRNA FOXD2-AS1 on proliferation, migration, invasion, and apoptosis as well as tumorigenicity of transfected GBC-SD cells was examined with gain- and loss-of-function experiments. We found that lncRNA FOXD2-AS1 was highly expressed, while MLH1 was poorly expressed in gallbladder cancer cells. Besides, lncRNA FOXD2-AS1 could promote MLH1 methylation by recruiting DNMT1 to the MLH1 promoter, and consequently inhibit MLH1 transcription. Silencing of lncRNA FOXD2-AS1 or overexpression of MLH1 inhibited gallbladder cancer cell proliferation, invasion, and migration, while facilitating cell apoptosis in vitro as well as retarding tumor growth in vivo. Thus, silencing of lncRNA FOXD2-AS1 suppressed the progression of gallbladder cancer via upregulation of MLH1 by inhibiting MLH1 promoter methylation. These findings present lncRNA FOXD2-AS1 knockdown as a potential candidate for the treatment of gallbladder cancer.

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Fig. 1: LncRNA FOXD2-AS1 is highly expressed in gallbladder cancer tissues and cells.
Fig. 2: Silencing of lncRNA FOXD2-AS1 suppresses gallbladder cancer GBC-SD cell proliferation, migration, and invasion but enhances cell apoptosis and senescence in vitro.
Fig. 3: Silencing of lncRNA FOXD2-AS1 increases MLH1 expression, thus attenuating cell proliferation, migration, and invasion but promoting cell apoptosis and senescence in gallbladder cancer GBC-SD cells.
Fig. 4: LncRNA FOXD2-AS1 enhances MLH1 methylation by recruiting DNMT1 in vitro.
Fig. 5: Overexpressing of MLH1 reverses the simulative role of lncRNA FOXD2-AS1 in tumor growth in vivo.
Fig. 6: The graphical depiction of a mechanism regulatory network involving lncRNA FOXD2-AS1 in gallbladder cancer.

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The datasets generated/analyzed during the current study are available.

References

  1. Rakic MPL, Kopljar M, Klicek R, Kolovrat M, Loncar B, Busic Z. Gallbladder cancer. Hepatobiliary Surg Nutr. 2014;3:221–6.

    PubMed  PubMed Central  Google Scholar 

  2. Wernberg JALD. Gallbladder cancer. Surg Clin N Am. 2014;94:343–60.

    Article  Google Scholar 

  3. Hundal R, Shaffer EA. Gallbladder cancer: epidemiology and outcome. Clin Epidemiol. 2014;6:99–109.

    PubMed  Google Scholar 

  4. Ma MZ, Chu BF, Zhang Y, Weng MZ, Qin YY, Gong W, et al. Long non-coding RNA CCAT1 promotes gallbladder cancer development via negative modulation of miRNA-218-5p. Cell Death Dis. 2015;6:e1583.

    Article  CAS  Google Scholar 

  5. Ma F, Wang SH, Cai Q, Zhang MD, Yang Y, Ding J. Overexpression of LncRNA AFAP1-AS1 predicts poor prognosis and promotes cells proliferation and invasion in gallbladder cancer. Biomed Pharmacother. 2016;84:1249–55.

    Article  CAS  Google Scholar 

  6. Ma MZKX, Weng MZ, Zhang MD, Qin YY, Gong W, Zhang WJ, et al. Long non-coding RNA-LET is a positive prognostic factor and exhibits tumor-suppressive activity in gallbladder cancer. Mol Carcinog. 2015;54:1397–406.

    Article  CAS  Google Scholar 

  7. Wang SH, Zhang MD, Wu XC, Weng MZ, Zhou D, Quan ZW. Overexpression of LncRNA-ROR predicts a poor outcome in gallbladder cancer patients and promotes the tumor cells proliferation, migration, and invasion. Tumour Biol. 2016;37:12867–75.

    Article  CAS  Google Scholar 

  8. Wang J, Li B, Wang C, Luo Y, Zhao M, Chen P. Long noncoding RNA FOXD2-AS1 promotes glioma cell cycle progression and proliferation through the FOXD2-AS1/miR-31/CDK1 pathway. J Cell Biochem. 2019;120:19784–95.

    Article  CAS  Google Scholar 

  9. An Q, Zhou L, Xu N. Long noncoding RNA FOXD2-AS1 accelerates the gemcitabine-resistance of bladder cancer by sponging miR-143. Biomed Pharmacother. 2018;103:415–20.

    Article  CAS  Google Scholar 

  10. Yang XDB, Zhou X. Long non-coding RNA FOXD2-AS1 functions as a tumor promoter in colorectal cancer by regulating EMT and Notch signaling pathway. Eur Rev Med Pharmacol Sci. 2017;21:3586–91.

    CAS  PubMed  Google Scholar 

  11. Xu LFL, Changning L, Liangcai Z, Yinghui L, Yu L, Shanguang C, et al. Comparison of the prognostic utility of the diverse molecular data among lncRNA, DNA methylation, microRNA, and mRNA across five human cancers. PLoS ONE. 2015;10:e0142433.

    Article  Google Scholar 

  12. Zhao YSH, Wang H. Long noncoding RNAs in DNA methylation: new players stepping into the old game. Cell Biosci. 2016;6:45.

    Article  Google Scholar 

  13. Takahashi T, Shivapurkar N, Riquelme E, Shigematsu H, Reddy J, Suzuki M, et al. Aberrant promoter hypermethylation of multiple genes in gallbladder carcinoma and chronic cholecystitis. Clin Cancer Res. 2004;10:6126–33.

    Article  CAS  Google Scholar 

  14. Letelier PBP, Tapia O, Roa JC. DNA promoter methylation as a diagnostic and therapeutic biomarker in gallbladder cancer. Clin Epigenetics. 2012;4:11.

    Article  CAS  Google Scholar 

  15. Koda M, Yashima K, Kawaguchi K, Andachi H, Hosoda A, Shiota G, et al. Expression of Fhit, Mlh1, and P53 protein in human gallbladder carcinoma. Cancer Lett. 2003;199:131–8.

    Article  CAS  Google Scholar 

  16. Roa JC, Anabalon L, Roa I, Melo A, Araya JC, Tapia O, et al. Promoter methylation profile in gallbladder cancer. J Gastroenterol. 2006;41:269–75.

    Article  CAS  Google Scholar 

  17. Pinto D, Pinto C, Guerra J, Pinheiro M, Santos R, Vedeld HM, et al. Contribution of MLH1 constitutional methylation for Lynch syndrome diagnosis in patients with tumor MLH1 downregulation. Cancer Med. 2018;7:433–44.

    Article  CAS  Google Scholar 

  18. Rao B, Gao Y, Zhou Q, Xiao P, Xia S, Ma J, et al. A recombinant adenovirus vector encoding the light chain of human coagulation factor VII and IgG1 Fc fragment to targeting tissue factor for colorectal cancer immunotherapy in the mouse model. J Cancer Res Clin Oncol. 2013;139:1015–23.

    Article  CAS  Google Scholar 

  19. Ayuk SM, Abrahamse H, Houreld NN. The role of photobiomodulation on gene expression of cell adhesion molecules in diabetic wounded fibroblasts in vitro. J Photochem Photobiol B. 2016;161:368–74.

    Article  CAS  Google Scholar 

  20. Dunagin M, Cabili MN, Rinn J, Raj A. Visualization of lncRNA by single-molecule fluorescence in situ hybridization. Methods Mol Biol. 2015;1262:3–19.

    Article  CAS  Google Scholar 

  21. Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB, Methylation-specific PCR. a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA. 1996;93:9821–6.

    Article  CAS  Google Scholar 

  22. Ning H, Albersen M, Lin G, Lue TF, Lin CS. Effects of EdU labeling on mesenchymal stem cells. Cytotherapy. 2013;15:57–63.

    Article  CAS  Google Scholar 

  23. Zhu YQL, Zhou Y, Ma N, Wang C, Zhou J. Long non-coding RNA FOXD2-AS1 contributes to colorectal cancer proliferation through its interaction with microRNA-185-5p. Cancer Sci. 2018;109:2235–42.

    Article  CAS  Google Scholar 

  24. Xiao YH, Yi H, Tan T, Liang T, Chen ZC, Xiao ZQ. Analysis of in vitro anti-leukemia effect of 5-aza-2′-deoxycitydine. Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2008;33:344–52.

    CAS  PubMed  Google Scholar 

  25. Diaz D, Prieto A, Reyes E, Barcenilla H, Monserrat J, Alvarez-Mon M. Flow cytometry enumeration of apoptotic cancer cells by apoptotic rate. Methods Mol Biol. 2015;1219:11–20.

    Article  CAS  Google Scholar 

  26. Burn SF. Detection of beta-galactosidase activity: X-gal staining. Methods Mol Biol. 2012;886:241–50.

    Article  CAS  Google Scholar 

  27. Nelson JD, Denisenko O, Sova P, Bomsztyk K. Fast chromatin immunoprecipitation assay. Nucleic Acids Res. 2006;34:e2.

    Article  Google Scholar 

  28. Alspach E, Stewart SA. RNA-binding protein immunoprecipitation (RIP) to examine AUF1 binding to senescence-associated secretory phenotype (SASP) factor mRNA. Bio Protoc. 2015;5:e1481.

    Article  Google Scholar 

  29. Wang K, Long B, Zhou LY, Liu F, Zhou QY, Liu CY, et al. CARL lncRNA inhibits anoxia-induced mitochondrial fission and apoptosis in cardiomyocytes by impairing miR-539-dependent PHB2 downregulation. Nat Commun. 2014;5:3596.

    Article  Google Scholar 

  30. Su FHW, Chen C, Liu M, Liu H, Xue F, Bi J, et al. The long non-coding RNA FOXD2-AS1 promotes bladder cancer progression and recurrence through a positive feedback loop with Akt and E2F1. Cell Death Dis. 2018;9:233.

    Article  Google Scholar 

  31. Rong L, Zhao R, Lu J. Highly expressed long non-coding RNA FOXD2-AS1 promotes non-small cell lung cancer progression via Wnt/beta-catenin signaling. Biochem Biophys Res Commun. 2017;484:586–91.

    Article  CAS  Google Scholar 

  32. Sun Q, Hao Q, Prasanth KV. Nuclear long noncoding RNAs: key regulators of gene expression. Trends Genet. 2018;34:142–57.

    Article  CAS  Google Scholar 

  33. Sharma P, Bhunia S, Poojary SS, Tekcham DS, Barbhuiya MA, Gupta S, et al. Global methylation profiling to identify epigenetic signature of gallbladder cancer and gallstone disease. Tumour Biol. 2016;37:14687–99.

    Article  CAS  Google Scholar 

  34. Sato KKY, Kohya N, Koga Y, Ohtaka K, Miyazaki K. CPT-11 (SN-38) chemotherapy may be selectively applicable to biliary tract cancer with low hMLH1 expression. Anticancer Res. 2007;27:865–72.

    CAS  PubMed  Google Scholar 

  35. Lu JY, Jin P, Gao W, Wang DZ, Sheng JQ. Estrogen enhances mismatch repair by induction of MLH1 expression via estrogen receptor-beta. Oncotarget. 2017;8:38767–79.

    Article  Google Scholar 

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Acknowledgements

The authors would like to acknowledge the helpful suggestions concerning this study received from their colleagues.

Funding

This study was supported by Key Foundation of Jiangxi Provincial Science and Technology Department (No. 20171ACB21064), Youth Science Foundation of Jiangxi Provincial Science and Technology Department (No. 20151BAB205105), and Science Planning Project of Jiangxi Provincial Education Department (No. GJJ14049).

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  1. These authors contributed equally: Jun Gao, Chao Dai

Authors and Affiliations

  1. Department of Hepatobiliary and Pancreatic Surgery, The Second Affiliated Hospital of Nanchang University, 330006, Nanchang, PR China

    Jun Gao, Chao Dai, Xin Yu, Xiang-Bao Yin, Wen-Jun Liao, Yong Huang & Fan Zhou

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Contributions

JG, CD, and XY designed the study. X-BY was involved in data collection. W-JL and YH performed the statistical analysis and preparation of figures. JG, CD, and FZ drafted the paper. All authors read and approved the final manuscript.

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Correspondence to Fan Zhou.

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The authors declare that they have no conflict of interest.

Ethical approval

The current study was conducted with the approval of the Ethics Committee of the Second Affiliated Hospital of Nanchang University, and all animal experiments were conducted in compliance with the recommendations of the Guide for the Care and Use of Laboratory Animal issued by the National Institutes of Health. Optimal measures were taken to minimize the number and suffering of the included animals.

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Gao, J., Dai, C., Yu, X. et al. Silencing of long non-coding RNA FOXD2-AS1 inhibits the progression of gallbladder cancer by mediating methylation of MLH1. Gene Ther 28, 306–318 (2021). https://doi.org/10.1038/s41434-020-00187-w

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