Skip to main content
SpringerLink
Log in

LncRNA IGF2-AS promotes endometriosis progression through targeting miR-370-3p/IGF2 axis and activating PI3K/AKT/mTOR signaling pathway

  • Reproductive physiology and disease
  • Published:
Journal of Assisted Reproduction and Genetics Aims and scope Submit manuscript

Abstract

Purpose

Endometriosis, a gynecological disease, is difficult to be cured. Currently, to identify more potential biomarkers for the early diagnosis of endometriosis is urgently needed. Insulin like growth factor 2 (IGF2) has been revealed to correlate with endometriosis. This research aimed to further explore the role of IGF2 and its up-stream mechanism in endometriosis.

Methods

Primary ectopic endometrial stromal cells (EESCs) were extracted from ectopic endometrial tissues which were pathological endometrial tissues resected from three patients with II-III endometriosis. Primary normal endometrial stromal cells (NESCs) were extracted from normal endometrial tissues of two patients with grade III cervical dysplasia and one patient with uterine leiomyoma III. Four endometriotic cell lines (EEC145T, hEM15A, hEM5B2, and 12Z) and normal human endometrial epithelial cells (hEECs) were purchased. Cell proliferation, migration, and invasion were evaluated through functional assays. The molecular interaction between RNAs was investigated through mechanistic analyses.

Results

We discovered that IGF2 was upregulated in purchased endometriotic cells and primary EESC. Suppression of IGF2 hampered cell proliferation, migration, and invasion. Furthermore, insulin-like growth factor 2 antisense RNA (IGF2-AS) was uncovered to positively regulate IGF2 expression and enhanced proliferative, migratory, and invasive abilities of endometriotic cells. Mechanistically, miR-370-3p was found to bind with IGF2-AS and IGF2. IGF2-AS competitively bind with miR-370-3p to upregulate IGF2. Furthermore, IGF2-AS was revealed to activate the PI3K/AKT/mTOR signaling pathway through targeting miR-370-3p/IGF2 axis.

Conclusion

IGF2-AS promotes endometriotic cell growth via regulating IGF2/miR-370-3p axis and further activating PI3K/AKT/mTOR signaling pathway.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Data availability

Research data are not shared.

Abbreviations

lncRNA:

Long non-coding RNA

IGF2-AS:

Insulin like growth factor 2 antisense RNA

ceRNA:

Competing endogenous RNA

miRNA:

MicroRNA

IGF2:

Insulin like growth factor 2

qRT-PCR:

Quantitative real-time PCR

CCK-8:

Cell counting kit-8

EdU:

5-Ethynyl-2′-deoxyuridine

RIP:

RNA immunoprecipitation

shRNA:

Short hairpin RNA

FBS:

Fetal bovine serum

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

PVDF:

Polyvinylidene fluoride

DMEM:

Dulbecco’s modified Eagle’s medium

SD:

Standard deviation

NC:

Negative control

ANOVA:

Analysis of variance

ATCC:

American Type Culture Collection

OD:

Optical density

References

  1. Zondervan KT, Becker CM, Koga K, Missmer SA, Taylor RN, Viganò P. Endometriosis. Nat Rev Dis Primers. 2018;4(1):9. https://doi.org/10.1038/s41572-018-0008-5.

    Article  Google Scholar 

  2. Laganà AS, Salmeri FM, Ban Frangež H, Ghezzi F, Vrtačnik-Bokal E, Granese R. Evaluation of M1 and M2 macrophages in ovarian endometriomas from women affected by endometriosis at different stages of the disease. Gynecol Endocrinol. 2020;36(5):441–4. https://doi.org/10.1080/09513590.2019.1683821.

    Article  CAS  Google Scholar 

  3. Bulun SE, Yilmaz BD, Sison C, Miyazaki K, Bernardi L, Liu S, et al. Endometriosis. Endocr Rev. 2019;40(4):1048–79. https://doi.org/10.1210/er.2018-00242.

    Article  Google Scholar 

  4. Laganà AS, Salmeri FM, Vitale SG, Triolo O, Götte M. Stem cell trafficking during endometriosis: may epigenetics play a pivotal role? Reproductive sciences (Thousand Oaks, Calif). 2018;25(7):978–9. https://doi.org/10.1177/1933719116687661.

    Article  Google Scholar 

  5. Giudice LC, Kao LC. Endometriosis. Lancet (London, England). 2004;364(9447):1789–99. https://doi.org/10.1016/s0140-6736(04)17403-5.

    Article  Google Scholar 

  6. Benagiano G, Guo SW, Puttemans P, Gordts S, Brosens I. Progress in the diagnosis and management of adolescent endometriosis: an opinion. Reprod Biomed Online. 2018;36(1):102–14. https://doi.org/10.1016/j.rbmo.2017.09.015.

    Article  Google Scholar 

  7. Vercellini P, Vigano P, Somigliana E, Fedele L. Endometriosis: pathogenesis and treatment. Nat Rev Endocrinol. 2014;10(5):261–75. https://doi.org/10.1038/nrendo.2013.255.

    Article  CAS  Google Scholar 

  8. Czyzyk A, Podfigurna A, Szeliga A, Meczekalski B. Update on endometriosis pathogenesis. Minerva Ginecol. 2017;69(5):447–61. https://doi.org/10.23736/s0026-4784.17.04048-5.

    Article  Google Scholar 

  9. Kobayashi H. Imprinting genes associated with endometriosis. EXCLI J. 2014;13:252–64.

    Google Scholar 

  10. Kim H, Park JH, Ku SY, Kim SH, Choi YM, Kim JG. Association between endometriosis and polymorphisms in insulin-like growth factors (IGFs) and IGF-I receptor genes in Korean women. Eur J Obstet Gynecol Reprod Biol. 2011;156(1):87–90. https://doi.org/10.1016/j.ejogrb.2010.12.018.

    Article  CAS  Google Scholar 

  11. Wang J, Su Z, Lu S, Fu W, Liu Z, Jiang X, et al. LncRNA HOXA-AS2 and its molecular mechanisms in human cancer. Clin Chim Acta. 2018;485:229–33. https://doi.org/10.1016/j.cca.2018.07.004.

    Article  CAS  Google Scholar 

  12. Huang Y. The novel regulatory role of lncRNA-miRNA-mRNA axis in cardiovascular diseases. J Cell Mol Med. 2018;22(12):5768–75. https://doi.org/10.1111/jcmm.13866.

    Article  CAS  Google Scholar 

  13. Xing C, Sun SG, Yue ZQ, Bai F. Role of lncRNA LUCAT1 in cancer. Biomed Pharmacother. 2021;134:111158. https://doi.org/10.1016/j.biopha.2020.111158.

    Article  CAS  Google Scholar 

  14. Wang L, Xing Q, Feng T, He M, Yu W, Chen H. SNP rs710886 A>G in long noncoding RNA PCAT1 is associated with the risk of endometriosis by modulating expression of multiple stemness-related genes via microRNA-145 signaling pathway. J Cell Biochem. 2019. https://doi.org/10.1002/jcb.29406.

    Article  Google Scholar 

  15. Yu J, Chen LH, Zhang B, Zheng QM. The modulation of endometriosis by lncRNA MALAT1 via NF-kappaB/iNOS. Eur Rev Med Pharmacol Sci. 2019;23(10):4073–80. https://doi.org/10.26355/eurrev_201905_17908.

    Article  CAS  Google Scholar 

  16. Zhang C, Wu W, Ye X, Ma R, Luo J, Zhu H, et al. Aberrant expression of CHL1 gene and long non-coding RNA CHL1-AS1, CHL1-AS2 in ovarian endometriosis. Eur J Obstet Gynecol Reprod Biol. 2019;236:177–82. https://doi.org/10.1016/j.ejogrb.2019.03.020.

    Article  CAS  Google Scholar 

  17. Sha L, Huang L, Luo X, Bao J, Gao L, Pan Q, et al. Long non-coding RNA LINC00261 inhibits cell growth and migration in endometriosis. J Obstet Gynaecol Res. 2017;43(10):1563–9. https://doi.org/10.1111/jog.13427.

    Article  CAS  Google Scholar 

  18. Liu J, Wang Y, Chen P, Ma Y, Wang S, Tian Y et al. AC002454.1 and CDK6 synergistically promote endometrial cell migration and invasion in endometriosis. Reproduction (Cambridge, England). 2019;157(6):535–43. https://doi.org/10.1530/rep-19-0005.

  19. Qiu JJ, Lin XJ, Zheng TT, Tang XY, Zhang Y, Hua KQ. The exosomal long noncoding RNA aHIF is upregulated in serum from patients with endometriosis and promotes angiogenesis in endometriosis. Reproductive sciences (Thousand Oaks, Calif). 2019;26(12):1590–1602. https://doi.org/10.1177/1933719119831775.

  20. Xu Z, Zhang L, Yu Q, Zhang Y, Yan L, Chen ZJ. The estrogen-regulated lncRNA H19/miR-216a-5p axis alters stromal cell invasion and migration via ACTA2 in endometriosis. Mol Hum Reprod. 2019;25(9):550–61. https://doi.org/10.1093/molehr/gaz040.

    Article  CAS  Google Scholar 

  21. Shi Y, Zha J, Zuo M, Yan Q, Song H. Long noncoding RNA CHL1-AS1 promotes cell proliferation and migration by sponging miR-6076 to regulate CHL1 expression in endometrial cancer. J Cell Biochem. 2019. https://doi.org/10.1002/jcb.29486.

    Article  Google Scholar 

  22. Wang Y, Huang T, Sun X, Wang Y. Identification of a potential prognostic lncRNA-miRNA-mRNA signature in endometrial cancer based on the competing endogenous RNA network. J Cell Biochem. 2019;120(11):18845–53. https://doi.org/10.1002/jcb.29200.

    Article  CAS  Google Scholar 

  23. Zhou CF, Liu MJ, Wang W, Wu S, Huang YX, Chen GB, et al. miR-205-5p inhibits human endometriosis progression by targeting ANGPT2 in endometrial stromal cells. Stem Cell Res Ther. 2019;10(1):287. https://doi.org/10.1186/s13287-019-1388-5.

    Article  CAS  Google Scholar 

  24. Wu W, Gao H, Li X, Zhu Y, Peng S, Yu J, et al. LncRNA TPT1-AS1 promotes tumorigenesis and metastasis in epithelial ovarian cancer by inducing TPT1 expression. Cancer Sci. 2019;110(5):1587–98. https://doi.org/10.1111/cas.14009.

    Article  CAS  Google Scholar 

  25. Long J, Bai Y, Yang X, Lin J, Yang X, Wang D, et al. Construction and comprehensive analysis of a ceRNA network to reveal potential prognostic biomarkers for hepatocellular carcinoma. Cancer Cell Int. 2019;19:90. https://doi.org/10.1186/s12935-019-0817-y.

    Article  Google Scholar 

  26. Li JH, Liu S, Zhou H, Qu LH, Yang JH. starBase v2.0: decoding miRNA-ceRNA, miRNA-ncRNA and protein-RNA interaction networks from large-scale CLIP-Seq data. Nucleic Acids Res. 2014;42(Database issue):D92-7. https://doi.org/10.1093/nar/gkt1248.

    Article  CAS  Google Scholar 

  27. Tian B, Zhao Y, Liang T, Ye X, Li Z, Yan D, et al. Curcumin inhibits urothelial tumor development by suppressing IGF2 and IGF2-mediated PI3K/AKT/mTOR signaling pathway. J Drug Target. 2017;25(7):626–36. https://doi.org/10.1080/1061186x.2017.1306535.

    Article  CAS  Google Scholar 

  28. Xu X, Yu Y, Zong K, Lv P, Gu Y. Up-regulation of IGF2BP2 by multiple mechanisms in pancreatic cancer promotes cancer proliferation by activating the PI3K/Akt signaling pathway. J Exp Clin Cancer Res. 2019;38(1):497. https://doi.org/10.1186/s13046-019-1470-y.

    Article  CAS  Google Scholar 

  29. Mu Q, Wang L, Yu F, Gao H, Lei T, Li P, et al. Imp2 regulates GBM progression by activating IGF2/PI3K/Akt pathway. Cancer Biol Ther. 2015;16(4):623–33. https://doi.org/10.1080/15384047.2015.1019185.

    Article  CAS  Google Scholar 

  30. Wang Y, Kuramitsu Y, Baron B, Kitagawa T, Tokuda K, Akada J, et al. PI3K inhibitor LY294002, as opposed to wortmannin, enhances AKT phosphorylation in gemcitabine-resistant pancreatic cancer cells. Int J Oncol. 2017;50(2):606–12. https://doi.org/10.3892/ijo.2016.3804.

    Article  CAS  Google Scholar 

  31. Angioni S. New insights on endometriosis. Minerva Ginecol. 2017;69(5):438–9. https://doi.org/10.23736/s0026-4784.17.04089-8.

    Article  Google Scholar 

  32. Borghese B, Santulli P, Marcellin L, Chapron C. Definition, description, clinicopathological features, pathogenesis and natural history of endometriosis: CNGOF-HAS Endometriosis Guidelines. Gynecologie, Obstetrique, Fertilite & Senologie. 2018;46(3):156–67. https://doi.org/10.1016/j.gofs.2018.02.017.

    Article  CAS  Google Scholar 

  33. Imesch P, Fink D. Endometriosis Update 2016. Praxis. 2016;105(5):253–7. https://doi.org/10.1024/1661-8157/a002295.

    Article  Google Scholar 

  34. Chen ZH, Hu HK, Zhang CR, Lu CY, Bao Y, Cai Z, et al. Down-regulation of long non-coding RNA FOXD3 antisense RNA 1 (FOXD3-AS1) inhibits cell proliferation, migration, and invasion in malignant glioma cells. Am J Transl Res. 2016;8(10):4106–19.

    CAS  Google Scholar 

  35. Song C, Song C, Chen K, Zhang X. Inhibition of long non-coding RNA IGF2AS protects apoptosis and neuronal loss in anesthetic-damaged mouse neural stem cell derived neurons. Biomed Pharmacother. 2017;85:218–24. https://doi.org/10.1016/j.biopha.2016.10.094.

    Article  CAS  Google Scholar 

  36. Hu Z, Mamillapalli R, Taylor HS. Increased circulating miR-370-3p regulates steroidogenic factor 1 in endometriosis. Am J Physiol Endocrinol Metab. 2019;316(3):E373–82. https://doi.org/10.1152/ajpendo.00244.2018.

    Article  CAS  Google Scholar 

  37. Hagstrom AD, Denham J. microRNAs in high and low responders to resistance training in breast cancer survivors. Int J Sports Med. 2018;39(6):482–9. https://doi.org/10.1055/a-0592-7691.

    Article  CAS  Google Scholar 

  38. Zhao JR, Cheng WW, Wang YX, Cai M, Wu WB, Zhang HJ. Identification of microRNA signature in the progression of gestational trophoblastic disease. Cell Death Dis. 2018;9(2):94. https://doi.org/10.1038/s41419-017-0108-2.

    Article  CAS  Google Scholar 

  39. Li J, Huang Y, Deng X, Luo M, Wang X, Hu H, et al. Long noncoding RNA H19 promotes transforming growth factor-beta-induced epithelial-mesenchymal transition by acting as a competing endogenous RNA of miR-370-3p in ovarian cancer cells. Onco Targets Ther. 2018;11:427–40. https://doi.org/10.2147/ott.S149908.

    Article  Google Scholar 

  40. Liu S, Qiu J, Tang X, Cui H, Zhang Q, Yang Q. LncRNA-H19 regulates cell proliferation and invasion of ectopic endometrium by targeting ITGB3 via modulating miR-124-3p. Exp Cell Res. 2019;381(2):215–22. https://doi.org/10.1016/j.yexcr.2019.05.010.

    Article  CAS  Google Scholar 

  41. Liu Z, Liu L, Zhong Y, Cai M, Gao J, Tan C, et al. LncRNA H19 over-expression inhibited Th17 cell differentiation to relieve endometriosis through miR-342-3p/IER3 pathway. Cell Biosci. 2019;9:84. https://doi.org/10.1186/s13578-019-0346-3.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We appreciate the support of all lab members.

Author information

Authors and Affiliations

  1. VIP Ward, the First People’s Hospital of Wenling, No.333, South Chuan’an Road, Chengxi Street, Wenling, 317500, China

    Xiaoyan Jin & Xiao Cheng

  2. Department of Obstetrics and Gynecology, Nanjing Jiangbei People’s Hospital, Nanjing, 210044, Jiangsu, China

    Jingjing Feng

Authors
  1. Xiaoyan Jin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jingjing Feng
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xiao Cheng
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Jingjing Feng or Xiao Cheng.

Ethics declarations

Ethics approval and consent to participate

All clinical samples were acquired from the First People’s Hospital of Wenling following the ethical and legal standards. All patients have signed a statement of consent.

Consent for publication

The paper has been proofread by all authors enrolled in this study, and they have permitted the submission and publication of this paper.

Conflict of interest

The authors declare no competing interests.

Additional information

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

ESM 1 Figure S1. The influence of IGF2 knockdown on the proliferative capability of endometriotic cells was examined by CCK-8, EdU and colony formation assays. A. QRT-PCR was operated to test expression profile of IGF2 in endometriotic cells (EEC145T, hEM15A, hEM5B2, and 12Z) and human endometrial epithelial cells (hEEC). B. IGF2 knockdown efficiency was verified in endometriotic cells (hEM15A and hEM5B2) transfected with sh-NC, sh-IGF2#1, or sh-IGF2#2 by qRT-PCR. C. CCK-8 assays were utilized to test viability of hEM15A and hEM5B2 cells transfected with sh-IGF2#1/2. D-E. EdU and colony formation assays were utilized to demonstrate CC cell proliferation when IGF2 was inhibited in hEM15A and hEM5B2 cells. *P < 0.05, **P < 0.01.

(PNG 917 kb)

High resolution image (TIF 1306 kb)

ESM 2 Figure S2. The effects of IGF2 knockdown on the migration and invasion of endometriosis cells were detected through wound healing and transwell assays. A. Wound healing assays were operated to detect migration capacity of hEM15A and hEM5B2 cells transfected with sh-IGF2#1/2. B-C. Transwell assays were done to assess migratory and invasive capabilities of hEM15A and hEM5B2 cells after depletion of IGF2. D. Western blot assays were operated to measure MMP2 and MMP9 protein levels in IGF2-silenced hEM15A and hEM5B2 cells. **P < 0.01.

(JPG 1340 kb)

ESM 3 Figure S3. CCK-8, EdU and colony formation assays were conducted to determine the influences of IGF2-AS knockdown on proliferation of endometriotic cells. A. Transfection efficacy of sh-IGF2-AS#1/2/3 was verified with the use of qRT-PCR, and the expression of IGF2 was also quantified before and after IGF2-AS knockdown in hEM15A and hEM5B2 cells. B. Overexpression efficiency of pcDNA3.1-IGF2-AS was validated in qRT-PCR, and the influence of IGF2-AS overexpression on IGF2 expression was also examined in hEM15A and hEM5B2 cells. C-E. The effect of IGF2-AS depletion on proliferative ability of hEM15A and hEM5B2 cells was assessed by means of CCK-8, EdU and colony formation assays. **P < 0.01.

(JPG 1553 kb)

ESM 4 Figure S4. The influence of IGF2-AS knockdown on migration and invasion of endometriotic cells was evaluated through wound healing and transwell assays. A. Wound healing assays were operated to detect migration ability of hEM15A and hEM5B2 cells transfected with sh-IGF2-AS#1/2. B-C. Transwell assays were utilized to test migration and invasion ability of hEM15A and hEM5B2 cells after down-regulating IGF2-AS. D. Western blot experiments were performed to analyze MMP2 and MMP9 protein levels in IGF2-AS-silenced hEM15A and hEM5B2 cells. **P < 0.01.

(JPG 1396 kb)

ESM 5 Figure S5. The candidate miRNA of IGF2-AS was determined by using the bioinformatics tool, RNA pull-down and qRT-PCR assays. A. Subcellular fraction assay determined the subcellular location of IGF2-AS in endometriotic cells. B. Venn diagram revealed 10 candidate miRNAs binding to both IGF2-AS and IGF2 based on searching outcomes of starBase database. C. RNA pull-down assay was operated to examine the enrichment of 10 candidate miRNAs in biotin-labeled IGF2-AS. D-E. QRT-PCR examined overexpression efficiency of miR-370-3p or miR-491-5p. F. IGF2 expression was detected in cells transfected with miR-491-5p mimics. **P < 0.01, n.s.: no significance.

(JPG 857 kb)

ESM 6 Figure S6. Luciferase reporter and RIP assays were performed to examine if miR-370-3p could bind to both IGF2-AS and IGF2. A. QRT-PCR and western blot revealed the influence of miR-370-3p overexpression on IGF2 mRNA and protein levels in hEM15A and hEM5B2 cells. B. Binding sequences of wild/mutant IGF2-AS and miR-370-3p or wild/mutant IGF2 and miR-370-3p were obtained from starBase database. C. Luciferase reporter assay detected luciferase activity of wild and mutant IGF2-AS/IGF2 after miR-370-3p overexpression in hEM15A and hEM5B2 cells. D. RIP assay revealed the correlation of IGF2-AS, miR-370-3p and IGF2 in hEM15A and hEM5B2 cells. **P < 0.01.

(JPG 978 kb)

ESM 7 Figure S7. Rescue experiments were performed to assess regulation of IGF2-AS/miR-370-3p/IGF2 axis on proliferation, migration and invasion of endometriotic cells. A-C. CCK-8, EdU and colony formation assays were conducted to detect cell proliferation of hEM15A and hEM5B2 cells transfected with indicated plasmids. D-G. Migration and invasion of hEM15A and hEM5B2 cells in four groups were assessed through wound healing, transwell and western blot assays. **P < 0.01.

(JPG 1661 kb)

ESM 8 Figure S8. Western blot assays were conducted to determine whether IGF2-AS could activate PI3K/AKT/mTOR signaling pathway through targeting miR-370-3p/IGF2 axis. A. Western blot assays were utilized to detect the protein levels of IGF2, p-PI3K, PI3K, p-AKT, AKT, p-mTOR and mTOR in hEM15A and hEM5B2 cells under different conditions.

(JPG 671 kb)

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Jin, X., Feng, J. & Cheng, X. LncRNA IGF2-AS promotes endometriosis progression through targeting miR-370-3p/IGF2 axis and activating PI3K/AKT/mTOR signaling pathway. J Assist Reprod Genet 39, 2699–2710 (2022). https://doi.org/10.1007/s10815-022-02638-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10815-022-02638-2

Keywords

Search

Navigation