RPIA regulates colon cell proliferation through β-catenin expression in HCT116 cells (original) (raw)

2018

Abstract

(A) Knockdown of RPIA significantly reduced cell proliferation and RPIA overexpression enhanced cell proliferation in HCT116 cells. Co-treatment of si-RPIA and pcDNA-RPIA rescued the reduction of cellular proliferation upon knockdown of RPIA in HCT116. Cell viability assays were performed by measuring the cells at the second, third, fourth, and fifth days and the proliferation fold is compared to control cell at the first day. Control: Co-transfect with scramble RNA and pcDNA empty vector as negative control. (B) RPIA knockdown significantly reduced colony formation ability, and RPIA overexpression enhanced colony formation ability in HCT116 cells. si-NC: Transfect with scramble siRNA as negative control. Representative images of colonies were shown on top of the quantification result. (C) Knockdown of RPIA reduced β-catenin protein levels as measured by western blotting (left panel) and quantified using Image J (middle panel) but did not significantly alter mRNA levels of β-catenin as measured by qPCR (right panel) in HCT116 cells. (D) RPIA overexpression increased β-catenin protein levels (left and middle panels) but did not affect β-catenin mRNA levels (right panel) in HCT116 cells. (E) Knock down of RPIA reduced the β-catenin/TCF luciferase reporter activity in HCT116 cells. (F) Overexpression of RPIA induced the β-catenin/TCF luciferase reporter activity in HCT116 cells. (G) Knockdown of RPIA decreased the mRNA levels of the β-catenin target genes CCND1, CCNE2, and AXIN2 in HCT116 cells. (H) Overexpression of RPIA increased the mRNA levels of the β-catenin target genes CCND1, CCNE2, and AXIN2 in HCT116 cells. The statistical significance was calculated with Student t test (* 0.01 < P < 0.05, ** 0.001 < P < 0.01, and *** P < 0.001). Data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003714#pbio.2003714.s010" target="_blank">S2 Data</a>. AXIN2, Axis inhibition protein 2; CCND1, Cyclin D1; CCNE2, Cyclin E2; CTNNB1, CATENIN BETA 1; pcDNA, pcDNA3 vector control; qPCR, quantitative PCR; RPIA, ribose-5-phosphate isomerase A; si-NC, negative control small interfering RNA; si-RPIA, RPIA small interfering RNA; TCF, T-cell transcription factor.</p

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