Skip to main content

Multiple transcriptome analysis of Piwil2-induced cancer stem cells, including piRNAs, mRNAs and miRNAs reveals the mechanism of tumorigenesis and development



Cancer stem cells play important roles in the process of tumorigenesis. Our research group obtained cancer stem cell-like cells named Piwil2-iCSCs by reprogramming human preputial fibroblasts (FBs) with the PIWIL2 gene, but the mechanism of Piwil2-iCSCs is still unclear.


We sequenced the piRNAs, miRNAs and mRNAs of Piwil2-iCSCs and FBs, and analyzed the differences. Gene Ontology (GO) and, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses and gene set enrichment analysis (GSEA) were performed on the differentially expressed (DE) mRNAs. In addition, we analyzed the variable shear events and fusion genes in the Piwil2-iCSCs. Target gene prediction and functional enrichment analysis were performed for the DE miRNAs.


A total of 1119 DE mRNAs, 220 DE piRNAs, and 440 DE miRNAs were obtained between the Piwil2-iCSCs and FBs. Functional enrichment analysis showed that the genes with upregulated expression were mainly involved in DNA repair, mismatch repair, base excision repair, and nucleotide excision repair. Genes with downregulated expression were mainly involved in the TGF-β receptor signaling pathway, senescence and autophagy in cancer. More frequent shear events occurred in Piwil2-iCSCs and FBs, especially in intron retention (IR) events. We also identified three fusion genes MCM3AP-C21orf58, LRRFIP2-CAV3 and TMEM184B-DMC1. Enrichment analysis of DE miRNAs showed that they were associated with apoptosis, the TGF-β signaling pathway, and the stem cell regulatory signaling pathway. In particular, target gene prediction of the top three miRNAs with upregulated expression showed that they targeted SMAD, GREM1 and other genes to participate in the regulation of TGF-β and other pathways.


PIWIL2-induced cancer stem cells have significantly altered levels of miRNAs, piRNAs and mRNAs.TGF-β, autophagy, apoptosis and other pathways may play an important role in stem cell development. The occurrence of alternative splicing and fusion genes may be related to the occurrence of cancer stem cells.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Data availability

The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.


  1. Hewitt HB (1958) Studies of the dissemination and quantitative transplantation of a lymphocytic leukaemia of CBA mice. Br J Cancer 12(3):378–401

    Article  CAS  Google Scholar 

  2. Hamburger AW, Salmon SE (1977) Primary bioassay of human tumor stem cells. Science (New York, NY) 197(4302):461–463

    Article  CAS  Google Scholar 

  3. Pierce GB, Wallace C (1971) Differentiation of malignant to benign cells. Can Res 31(2):127–134

    CAS  Google Scholar 

  4. Pierce GB, Speers WC (1988) Tumors as caricatures of the process of tissue renewal: prospects for therapy by directing differentiation. Can Res 48(8):1996–2004

    CAS  Google Scholar 

  5. Bussolati B, Bruno S, Grange C, Ferrando U, Camussi G (2008) Identification of a tumor-initiating stem cell population in human renal carcinomas. FASEB J 22(10):3696–3705

    Article  CAS  Google Scholar 

  6. Yan Y, Zuo X, Wei D (2015) Concise review: Emerging role of CD44 in cancer stem cells: a promising biomarker and therapeutic target. Stem Cells Transl Med 4(9):1033–1043

    Article  CAS  Google Scholar 

  7. Weng Z, Lin J, He J, Gao L, Lin S, Tsang LL et al (2021) Human embryonic stem cell-derived neural crest model unveils CD55 as a cancer stem cell regulator for therapeutic targeting in MYCN-amplified neuroblastoma. Neuro Oncol.

    Article  PubMed Central  Google Scholar 

  8. Majeti R (2011) Monoclonal antibody therapy directed against human acute myeloid leukemia stem cells. Oncogene 30(9):1009–1019

    Article  CAS  Google Scholar 

  9. Lee JW, Lee HY (2021) Targeting cancer stem cell markers or pathways: a potential therapeutic strategy for oral cancer treatment. Int J Stem Cells 14(4):386

    Article  CAS  Google Scholar 

  10. Kuramochi-Miyagawa S, Kimura T, Ijiri TW, Isobe T, Asada N, Fujita Y et al (2004) Mili, a mammalian member of piwi family gene, is essential for spermatogenesis. Development (Cambridge, England) 131(4):839–849

    Article  CAS  Google Scholar 

  11. Sasaki T, Shiohama A, Minoshima S, Shimizu N (2003) Identification of eight members of the Argonaute family in the human genome. Genomics 82(3):323–330

    Article  CAS  Google Scholar 

  12. Doxzen KW, Doudna JA (2017) DNA recognition by an RNA-guided bacterial Argonaute. PLoS ONE 12(5):e0177097

    Article  Google Scholar 

  13. Azlan A, Dzaki N, Azzam G (2016) Argonaute: the executor of small RNA function. J Genet Genomics 43(8):481–494

    Article  Google Scholar 

  14. Wang S, Li F, Fan H, Xu J, Hu Z (2019) Expression of PIWIL2 in oral cancer and leukoplakia: prognostic implications and insights from tumors. Cancer Biomark 26(1):11–20

    Article  CAS  Google Scholar 

  15. Feng D, Yan K, Liang H, Liang J, Wang W, Yu H et al (2021) CBP-mediated Wnt3a/β-catenin signaling promotes cervical oncogenesis initiated by Piwil2. Neoplasia (New York, NY) 23(1):1–11

    Article  CAS  Google Scholar 

  16. Zhao X, Huang L, Lu Y, Jiang W, Song Y, Qiu B et al (2021) PIWIL2 interacting with IKK to regulate autophagy and apoptosis in esophageal squamous cell carcinoma. Cell Death Differ 28(6):1941–1954

    Article  CAS  Google Scholar 

  17. Li J, Xu L, Bao Z, Xu P, Chang H, Wu J et al (2017) High expression of PIWIL2 promotes tumor cell proliferation, migration and predicts a poor prognosis in glioma. Oncol Rep 38(1):183–192

    Article  Google Scholar 

  18. Qu X, Liu J, Zhong X, Li X, Zhang Q (2015) PIWIL2 promotes progression of non-small cell lung cancer by inducing CDK2 and Cyclin A expression. J Transl Med 13:301

    Article  Google Scholar 

  19. Zhang D, Wu X, Liu X, Cai C, Zeng G, Rohozinski J et al (2017) Piwil2-transfected human fibroblasts are cancer stem cell-like and genetically unstable. Oncotarget 8(7):12259–12271

    Article  Google Scholar 

  20. Bonnet D, Dick JE (1997) Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 3(7):730–737

    Article  CAS  Google Scholar 

  21. Lee J, Kotliarova S, Kotliarov Y, Li A, Su Q, Donin NM et al (2006) Tumor stem cells derived from glioblastomas cultured in bFGF and EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines. Cancer Cell 9(5):391–403

    Article  CAS  Google Scholar 

  22. Adikrisna R, Tanaka S, Muramatsu S, Aihara A, Ban D, Ochiai T et al (2012) Identification of pancreatic cancer stem cells and selective toxicity of chemotherapeutic agents. Gastroenterology 143(1):234–245

    Article  CAS  Google Scholar 

  23. Abbaszadegan MR, Bagheri V, Razavi MS, Momtazi AA, Sahebkar A, Gholamin M (2017) Isolation, identification, and characterization of cancer stem cells: a review. J Cell Physiol 232(8):2008–2018

    Article  CAS  Google Scholar 

  24. Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126(4):663–676

    Article  CAS  Google Scholar 

  25. Shahali M, Kabir-Salmani M, Nayernia K, Soleimanpour-Lichaei HR, Vasei M, Mowla SJ et al (2013) A novel in vitro model for cancer stem cell culture using ectopically expressed piwil2 stable cell line. Cell J 15(3):250–257

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Valencia-González HA, Ruíz G, Ortiz-Sánchez E, García-Carrancá A (2019) Cancer stem cells from tumor cell lines activate the DNA damage response pathway after ionizing radiation more efficiently than noncancer stem cells. Stem Cells Int 2019:7038953

    Article  Google Scholar 

  27. Abad E, Civit L, Potesil D, Zdrahal Z, Lyakhovich A (2021) Enhanced DNA damage response through RAD50 in triple negative breast cancer resistant and cancer stem-like cells contributes to chemoresistance. FEBS J 288(7):2184–2202

    Article  CAS  Google Scholar 

  28. Yu Y, Liu D, Liu Z, Li S, Ge Y, Sun W et al (2018) The inhibitory effects of COL1A2 on colorectal cancer cell proliferation, migration, and invasion. J Cancer 9(16):2953–2962

    Article  Google Scholar 

  29. Wang L, Wang W, Xu Y, Wang Q (2020) Low levels of SPARC are associated with tumor progression and poor prognosis in human endometrial carcinoma. Onco Targets Ther 13:11549–11569

    Article  CAS  Google Scholar 

  30. Seoane J, Gomis RR (2017) TGF-β family signaling in tumor suppression and cancer progression. Cold Spring Harb Perspect Biol 9(12):a022277

    Article  Google Scholar 

  31. Colak S, Ten Dijke P (2017) Targeting TGF-β signaling in cancer. Trends Cancer 3(1):56–71

    Article  CAS  Google Scholar 

  32. Prieto F, Egozcue J, Forteza G, Marco F (1970) Identification of the Philadelphia (Ph-1) chromosome. Blood 35(1):23–27

    Article  CAS  Google Scholar 

  33. Zhang Y, Qian J, Gu C, Yang Y (2021) Alternative splicing and cancer: a systematic review. Signal Transduct Target Ther 6(1):78

    Article  CAS  Google Scholar 

  34. Pan Q, Shai O, Lee LJ, Frey BJ, Blencowe BJ (2008) Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nat Genet 40(12):1413–1415

    Article  CAS  Google Scholar 

  35. Yang L, Sun H, Guo L, Cao H (2021) MiR-10a-5p: a promising biomarker for early diagnosis and prognosis evaluation of bladder cancer. Cancer Manag Res 13:7841–7850

    Article  CAS  Google Scholar 

  36. Romero-Lorca A, Novillo A, Gaibar M, Gilsanz M, Galán M, Beltrán L et al (2021) miR-7, miR-10a and miR-143 expression may predict response to bevacizumab plus chemotherapy in patients with metastatic colorectal cancer. Pharmacogenomics Pers Med 14:1263–1273

    Google Scholar 

  37. Kang J, Huang X, Dong W, Zhu X, Li M, Cui N (2021) MicroRNA-1269b inhibits gastric cancer development through regulating methyltransferase-like 3 (METTL3). Bioengineered 12(1):1150–1160

    Article  CAS  Google Scholar 

Download references


That all authors are in agreement with the content of the manuscript.


The present study was supported by the Special Project of Science and Technology Innovation for Social Undertakings and Livelihood Guarantee of Chongqing (Grant Nos. cstc2019jscx-tjsbX0003 and cstc2017shmsA130103).

Author information

Authors and Affiliations



Conceptualization: [TM, XT, ZZ and DH]; Data curation: [TM, XT, ZZ and LJ]; Formal analysis: [TM, XT, JW and XW]; Funding acquisition: [DH]; Methodology: [TM, XT, ZW, ML and CZ]; Writing—original draft: [TM, XT and ZZ]; Resources: [DH]; Supervision: [DH].

Corresponding author

Correspondence to Dawei He.

Ethics declarations

Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

The Authors declare that the paper is being submitted for consideration for publication in Molecular Biology Reports, that the content has not been published or submitted for publication elsewhere.

Additional information

Publisher's Note

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

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (TIF 125 KB)

Fig. SF1 The PCA of miRNAs, piRNAs and mRNAs.

Supplementary file2 (CSV 61 KB)

Supplementary file3 (CSV 406 KB)

Supplementary file4 (CSV 172 KB)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Tan, X., Mi, T., Zhang, Z. et al. Multiple transcriptome analysis of Piwil2-induced cancer stem cells, including piRNAs, mRNAs and miRNAs reveals the mechanism of tumorigenesis and development. Mol Biol Rep 49, 6885–6898 (2022).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:


  • Piwil2-iCSCs
  • Transcriptome
  • PiRNAs
  • mRNAs
  • miRNAs