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SDHB deficiency promotes TGFβ-mediated invasion and metastasis of colorectal cancer through transcriptional repression complex SNAIL1-SMAD3/4  Haiyu Wang,

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Presentation on theme: "SDHB deficiency promotes TGFβ-mediated invasion and metastasis of colorectal cancer through transcriptional repression complex SNAIL1-SMAD3/4  Haiyu Wang,"— Presentation transcript:

1 SDHB deficiency promotes TGFβ-mediated invasion and metastasis of colorectal cancer through transcriptional repression complex SNAIL1-SMAD3/4  Haiyu Wang, Yusheng Chen, Guohao Wu  Translational Oncology  Volume 9, Issue 6, Pages (December 2016) DOI: /j.tranon Copyright © 2016 The Authors Terms and Conditions

2 Figure 1 SDHB expression is low in CRC cell lines. (A) Quantitative RT-PCR analysis of SDHB expression in four CRC cell lines and normal colorectal epithelial cell line NCM460. (B) Semi- quantitative RT-PCR analysis of SDHB expression in four CRC cell lines and normal colorectal epithelial cell line NCM460. (C) Western blotting analysis of SDHB protein expression in four CRC cell lines and normal colorectal epithelial cell line NCM460. Translational Oncology 2016 9, DOI: ( /j.tranon ) Copyright © 2016 The Authors Terms and Conditions

3 Figure 2 SDHB expression is low in CRC tissues. (A) Quantitative RT-PCR analysis of SDHB expression in 29 pairs of colorectal tumor and its corresponding normal tissues. (B) Representative immunohistochemistry images showed varied but unanimously low expression of SDHB in 43 pair of CRC samples. (C) Succinate level was detected with colorimetric assay at 450 nm in 29 pairs of colorectal tumor and its corresponding normal tissues. Translational Oncology 2016 9, DOI: ( /j.tranon ) Copyright © 2016 The Authors Terms and Conditions

4 Figure 3 SDHB knockdown promoted invasion in CRC cell line HT-29. (A) Matrigel invasion assay showed that SDHB knockdown strengthened invasive capacity. (B) Absorbance at 570 nm showed quantitative matrigel invasion data after 24 hours. P<.05. (C) Quantitative RT-PCR analysis of knockdown efficiency in SDHB shRNA transfected stable cell lines. (D) Western blotting analysis of knockdown efficiency in SDHB shRNA transfected stable cell lines. (E) MTT assay showed SDHB knockdown had no remarkable effect on cell proliferation. Translational Oncology 2016 9, DOI: ( /j.tranon ) Copyright © 2016 The Authors Terms and Conditions

5 Figure 4 SDHB overexpression inhibited invasion in CRC cell line SW1116. (A) Matrigel invasion assay showed that SDHB overexpression decreased invasive capacity. (B) Absorbance at 570 nm showed quantitative matrigel invasion data after 24 hours. P<.05. (C) Quantitative RT-PCR analysis of overexpression efficiency in SDHB virus transfected stable cell lines. (D) Western blotting analysis of overexpression efficiency in SDHB virus transfected stable cell lines. (E) MTT assay showed SDHB overexpression had no remarkable effect on cell proliferation. Translational Oncology 2016 9, DOI: ( /j.tranon ) Copyright © 2016 The Authors Terms and Conditions

6 Figure 5 SDHB expression level regulated epithelial-mesenchymal transition (EMT) in CRC cell line. (A) Quantitative RT-PCR analysis showed that SDHB knockdown advanced EMT, as in down-regulation of epithelial markers and simultaneously up-regulation of mesenchymal markers. (B) Western blotting analysis showed that SDHB knockdown advanced EMT. (C) Quantitative RT-PCR analysis showed that SDHB overexpression suppressed EMT, as in up-regulation of epithelial markers and simultaneously down-regulation of mesenchymal markers. (D) Western blotting analysis showed that SDHB overexpression suppressed EMT. Translational Oncology 2016 9, DOI: ( /j.tranon ) Copyright © 2016 The Authors Terms and Conditions

7 Figure 6 SDHB knockdown promoted TGFβ signal pathway in CRC cells, which activated EMT through positive modulation of SNAIL1-SMAD3/4 complex. (A) Dual luciferase assay showed that SDHB knockdown promoted TGFβ signaling in HT-29 cells. CAGA and SBE are the promoter plasmids commonly used representing TGFβ signal pathway activity. (B) Dual luciferase assay showed that SDHB overexpression attenuated TGFβ signaling in HT-29 cells. (C) Quantitative RT-PCR assay showed that SDHB knockdown enhanced transcription factor SNAIL1 expression as well as TGFβ signaling marker gene SMAD3/4 expression in HT-29 cells. (D) Western blotting showed that SDHB knockdown enhanced transcription factor SNAIL1 expression as well as TGFβ signaling marker gene SMAD3/4 expression in HT-29 cells. (E) Matrigel invasion assay showed that the addition of murine SDHB, TGFβR1 inhibitor SB-431,542 and SMAD4shRNA could all rescue human SDHB knockdown phenotype, among which the last had the modest effect. Western blotting showed expression levels of SDHB and EMT marker genes along with SNAIL1-SMAD3/4 complex in the process accordingly. (F) Quantitative RT-PCR analysis showed that the expression of SDHB is significantly correlated with that of EMT marker genes and SNAIL1-SMAD3/4 complex in 29 pair of CRC tissues (P<.05, Student's t test). Translational Oncology 2016 9, DOI: ( /j.tranon ) Copyright © 2016 The Authors Terms and Conditions

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