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A Cancer Reprogramming Method Using MicroRNAs as a Novel Therapeutic Approach against Colon Cancer

Research for Reprogramming of Cancer Cells by MicroRNAs

  • Translational Research and Biomarkers
  • Published:
Annals of Surgical Oncology Aims and scope Submit manuscript

Abstract

Background

We previously generated induced pluripotent stem cells by reprograming adipose stem cells through the introduction of microRNAs targeting four transcription factors (Oct3/4, Sox2, c-Myc, and Klf4). In this study, we aimed to reprogram cancer cells using microRNAs to explore their therapeutic potential.

Methods

Mature microRNAs (mir-302a-d, 369-3p and 5p, and mir-200c, as needed) were introduced into colon cancer cells (DLD-1, RKO, and HCT116) using lipofection.

Results

The transfected cells exhibited an embryonic stem cell-like morphology and expressed the undifferentiated marker genes Nanog, Oct3/4, SOX2, and Klf4, as well as tumor-related antigen-1-60. These cells expressed neurogenic or adipogenic markers, indicating that reprogramming of the cancer cells was partially successful. Moreover, we found that miRNA-expressing DLD-1 cells showed low proliferative activity in vitro and in vivo, accompanied by increased expression of the tumor suppressor genes p16 ink4a and p21 waf1. miRNA-expressing DLD-1 cells also exhibited enhanced sensitivity to 5-fluorouracil, possibly through the downregulation of multidrug-resistant protein 8. The reprogrammed cells from DLD-1, RKO, and HCT116 cells exhibited reduced c-Myc expression, in contrast to the high c-Myc expression in the induced pluripotent cancer cells that were generated using four transcription factors.

Conclusions

Our cancer reprogramming method employing simple lipofection of mature microRNAs is safe and well suited for clinical application, because it avoids integration of exogenous genes into the host genome and allows escape from augmentation of c-Myc gene expression.

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Acknowledgement

This study was supported in part by a grant-in-aid from the Ministry of Health, Labour, and Welfare to M.M.

Conflict of interest

None.

Author information

Authors and Affiliations

  1. Department of Gastroenterological Surgery, Graduate School of Medicine, Osaka University, Osaka, Japan

    Susumu Miyazaki, Hirofumi Yamamoto MD, Xin Wu, Hisataka Ogawa, Mamoru Uemura, Junichi Nishimura, Taishi Hata, Ichiro Takemasa, Tsunekazu Mizushima, Yuichiro Doki & Masaki Mori

  2. Department of Surgery, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan

    Norikatsu Miyoshi

  3. Department of Frontier Science for Cancer and Chemotherapy, Graduate School of Medicine, Osaka University, Osaka, Japan

    Masamitsu Konno & Hideshi Ishii

  4. Department of Cancer Profiling Discovery, Graduate School of Medicine, Osaka University, Osaka, Japan

    Hideshi Ishii

Authors
  1. Susumu Miyazaki
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  2. Hirofumi Yamamoto MD
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  3. Norikatsu Miyoshi
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  4. Xin Wu
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  5. Hisataka Ogawa
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  6. Mamoru Uemura
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  7. Junichi Nishimura
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  8. Taishi Hata
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  9. Ichiro Takemasa
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  10. Tsunekazu Mizushima
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  11. Masamitsu Konno
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  12. Yuichiro Doki
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  13. Masaki Mori
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  14. Hideshi Ishii
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Corresponding author

Correspondence to Hirofumi Yamamoto MD.

Electronic supplementary material

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10434_2014_4217_MOESM1_ESM.pptx

Supplementary Fig. 1 Expression of endogenous and exogenous miRNAs in colon cancer cell lines. (A): qRT-PCR analysis of endogenous miRNAs in DLD-1, HCT116, Caco2, and RKO cells. miRNA expression was normalized against RNU48 expression, and the mean expression of each miRNA in human embryonic cells was set to 1. mir-200c expression was higher in the DLD-1 and HCT116 cell lines than in human embryonic stem (hES) cells. Expressions of mir-302s and mir-369s were almost undetectable in four colon cancer cell lines. (B): qRT-PCR analysis of miRNAs in DLD-1, pre-transfection and on days 3 and 5 after transfection. (C): qRT-PCR analysis of miRNAs in RKO, pre-transfection and on days 3 and 5 after transfection (PPTX 567 kb)

10434_2014_4217_MOESM2_ESM.pptx

Supplementary Fig. 2 Expression of endogenous and exogenous miRNAs in colon cancer cell lines. (A): On day 30, mRNAs of the colony cells from the RKO line were analyzed using qRT-PCR. Compared to the negative control and parental cells, the miRNA-induced colony cells showed higher levels of Nanog, Oct3/4, Sox2, and KLF4 (p < 0.05 for each). (B): miRNAs in the colony cells induced from the RKO line were analyzed using qRT-PCR. The expression of mir-369s remained high, and those of mir-200c, mir-302a, and mir-302c were increased to some extent, compared with the negative control cells (PPTX 223 kb)

10434_2014_4217_MOESM3_ESM.pptx

Supplementary Fig. 3 Lineage-directed differentiation of miRNA-induced cells. (A): The colonies from the DLD-1 line were grown in standard medium after EB formation, and the cells were isolated on day 15. (B): qRT-PCR analyses showed that the mir-302s level in miRNA-induced cells was reduced to control levels, while the expression of mir-369s remained high. miRNA expression was normalized against RNU48 expression, and the mean expression of each miRNA from human embryonic cells was set as 1. (C): qRT-PCR analysis of mRNAs in miRNA-induced DLD-1 cells. The levels of the differentiation marker mRNAs, including FABP, PPARγ, PAX6, MAP2, albumin, and vimentin, increased. The mRNA expression levels were normalized against GAPDH mRNA expression. *p < 0.05 (PPTX 60 kb)

10434_2014_4217_MOESM4_ESM.pptx

Supplementary Fig. 4 Adipogenic or neurogenic differentiation. (A): ES-like colonies were cultured in a floating state for five days in EB-forming medium composed of DMEM/F12 with 20% knockout-certified serum replacement (KSR; Invitrogen), 2 mM L-glutamine, 100 μM nonessential amino acids, and 100 μM 2-mercaptoethanol (Invitrogen). After five days of EB formation, the miRNA-induced colony-derived cells from the DLD-1 line were cultured in NH AdipoDiff or NDiff N2B27 medium for 16 days, and the cells were examined for differentiation to the adipocyte or neurocyte lineage, respectively. Negative control cells transfected with negative control miRNA were produced under the same culture conditions and treatment procedures. (B): Cells positively stained with Oil Red O. Scale bar: 200 μm. (C): qRT-PCR results indicated approximately 8.3-fold and 6.9-fold increases in the adipogenic marker genes FABP4 and PPARγ in the miRNA-induced cells compared with NC (p < 0.05 for each). The mRNA expression levels were normalized to GAPDH mRNA expression (PPTX 636 kb)

10434_2014_4217_MOESM5_ESM.pptx

Supplementary Fig. 5 (A): Investigations of the neurocyte lineage showed that the cells tended to exhibit an elongated axis foot. They also positively stained for the neurogenic markers (II) GFAP and (IV) TUBB3. (I, III) Phase contrast images. Scale bar: 200 μm. (B): qRT-PCR results indicated 3.3-fold and at 130.4-fold increases in the expression of MAP2 and PAX6, respectively, compared with the negative control. The mRNA expression levels were normalized to GAPDH mRNA expression. p < 0.05 for each (PPTX 125 kb)

10434_2014_4217_MOESM6_ESM.pptx

Supplementary Fig. 6 Proliferation activity and chemosensitivity to 5-FU in miPC cells from RKO. (A): In vitro cell proliferation assays showed significant differences in growth at 72 h and 96 h in miPC cells from RKO, compared to in NC and parental cells (p < 0.01). (B): MTT assays showed that miPC cells from RKO acquired enhanced sensitivity to 5-FU at the indicated concentrations as compared to NC. IC50 values were 0.157 μg/ml in miPC, 0.676 μg/ml in NC, and 0.295 μg/ml in parental cells. p < 0.05 for each (PPTX 288 kb)

10434_2014_4217_MOESM7_ESM.pptx

Supplementary Fig. 7 The mRNA expression levels were normalized to GAPDH mRNA expression. (A): In vivo tumor growth. The miPC cells and DLD-1 parental cells were subcutaneously transplanted into NOD/SCID mice (n = 5). The tumor growth of miPC cells was significantly suppressed compared with that of parental cells on day 21 (p = 0.0339) and 28 (p = 0.018). The negative control tumors grew rapidly, and the mice (n=3) died on day 21 (p<0.05). (B): H&E-stained sections of parental and miPC tumors. No histological difference was noted. Scale Bars: 100 μm (PPTX 139 kb)

10434_2014_4217_MOESM8_ESM.pptx

Supplementary Fig. 8 During adipocyte differentiation, mir-369s levels returned to normal. After five days of EB formation, the miRNA-induced colony-derived cells from DLD-1 were cultured in NH AdipoDiff medium for 16 days. qRT-PCR analyses showed that mir-369s levels, which remained high when cells were cultured with DMEM, decreased during adipocyte differentiation. miRNA expressions were normalized against RNU48 expression. The mean expression of each miRNA in human embryonic cells was set as 1 (PPTX 213 kb)

Supplementary Table 1 (PPTX 73 kb)

Supplementary Table 2 (PPTX 112 kb)

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Miyazaki, S., Yamamoto, H., Miyoshi, N. et al. A Cancer Reprogramming Method Using MicroRNAs as a Novel Therapeutic Approach against Colon Cancer. Ann Surg Oncol 22 (Suppl 3), 1394–1401 (2015). https://doi.org/10.1245/s10434-014-4217-1

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  • DOI: https://doi.org/10.1245/s10434-014-4217-1

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