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Interaction of 14-3-3? with KCMF1 suppresses the proliferation and colony formation of human colon cancer stem cells.

ABSTRACT: To investigate the biological function of 14-3-3? protein and to look for proteins that interact with 14-3-3? protein in colon cancer stem cells.Reverse transcription polymerase chain reaction was performed to amplify the 14-3-3? gene from the mRNA of colon cancer stem cells. The gene was then cloned into the pGEM-T vector. After being sequenced, the target gene 14-3-3? was cut from the pGEM-T vector and cloned into the pGBKT7 yeast expression plasmid. Then, the bait plasmid pGBKT7-14-3-3? was transformed into the yeast strain AH109. After the expression of the pGBKT7-14-3-3? fusion protein in the AH109 yeast strain was accomplished, a yeast two-hybrid screening assay was performed by mating AH109 with Y187 that contained a HeLa cDNA library plasmid. The interaction between the 14-3-3? protein and the proteins obtained from positive colonies was further confirmed by repeating the yeast two-hybrid screen. After extracting and sequencing the plasmids from the positive colonies, we performed a bioinformatics analysis. A coimmunoprecipitation assay was performed to confirm the interaction between 14-3-3? and the proteins obtained from the positive colonies. Finally, we constructed 14-3-3? and potassium channel modulatory factor 1 (KCMF1) siRNA expression plasmids and transfected them into colon cancer stem cells.The bait plasmid pGBKT7-14-3-3? was constructed successfully, and the 14-3-3? protein had no toxic or autonomous activation effect on the yeast. Nineteen true-positive colonies were selected and sequenced, and their full-length sequences were obtained. We searched for homologous DNA sequences for these sequences from GenBank. Among the positive colonies, four coding genes with known functions were obtained, including KCMF1, quinone oxidoreductase (NQO2), hydroxyisobutyrate dehydrogenase (HIBADH) and 14-3-3?. For the subsequent coimmunoprecipitation assay, the plasmids PCDEF-Flag-14-3-3?, PCDEF-Myc-KCMF1, PCDEF-Myc-NQO2 and PCDEF-Myc-HIBADH were successfully constructed, and the sequences were further confirmed by DNA sequencing. The Fugene 6 reagent was used to transfect the plasmids, and fluorescence-activated cell sorting analysis showed the transfection efficiency was 97.8% after 48 h. The HEK 293FT cells showed the stable expression of the PCDEF-Flag-14-3-3?, PCDEF-Myc-KCMF1, PCDEF-Myc-NQO2 and PCDEF-Myc-HIBADH plasmids. After anti-Myc antibody immunoprecipitation with Myc-KCMF1, Myc-NQO2 and Myc-HIBADH from cell lysates, the presence of Flag-14-3-3? protein in the immunoprecipitated complex was determined by western blot analysis. The knock-down expression of the 14-3-3? and KCMF1 proteins significantly inhibited cell proliferation and colony formation of SW1116csc.Genes of the proteins that interacted with 14-3-3? were successfully screened from a HeLa cDNA library. KCMF1 and 14-3-3? protein may affect the proliferation and colony formation of human colon cancer stem cells.

SUBMITTER: Zou J

PROVIDER: S-EPMC3703518 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Interaction of 14-3-3σ with KCMF1 suppresses the proliferation and colony formation of human colon cancer stem cells.

Zou Jian J Mi Lin L Yu Xiao-Feng XF Dong Jie J

World journal of gastroenterology 20130601 24

<h4>Aim</h4>To investigate the biological function of 14-3-3σ protein and to look for proteins that interact with 14-3-3σ protein in colon cancer stem cells.<h4>Methods</h4>Reverse transcription polymerase chain reaction was performed to amplify the 14-3-3σ gene from the mRNA of colon cancer stem cells. The gene was then cloned into the pGEM-T vector. After being sequenced, the target gene 14-3-3σ was cut from the pGEM-T vector and cloned into the pGBKT7 yeast expression plasmid. Then, the bait ...[more]

PMID: 23840115

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Project description:BACKGROUND:Phospholipases D1 and D2 (PLD1/2) hydrolyse cell membrane glycerophospholipids to generate phosphatidic acid, a signalling lipid, which regulates cell growth and cancer progression through effects on mTOR and PKB/Akt. PLD expression and/or activity is raised in breast, colorectal, gastric, kidney and thyroid carcinomas but its role in prostate cancer (PCa), the major cancer of men in the western world, is unclear. METHODS:PLD1 protein expression in cultured PNT2C2, PNT1A, P4E6, LNCaP, PC3, PC3M, VCaP, 22RV1 cell lines and patient-derived PCa cells was analysed by western blotting. PLD1 protein localisation in normal, benign prostatic hyperplasia (BPH), and castrate-resistant prostate cancer (CRPC) tissue sections and in a PCa tissue microarray (TMA) was examined by immunohistochemistry. PLD activity in PCa tissue was assayed using an Amplex Red method. The effect of PLD inhibitors on PCa cell viability was measured using MTS and colony forming assays. RESULTS:PLD1 protein expression was low in the luminal prostate cell lines (LNCaP, VCaP, 22RV1) compared with basal lines (PC3 and PC3M). PLD1 protein expression was elevated in BPH biopsy tissue relative to normal and PCa samples. In normal and BPH tissue, PLD1 was predominantly detected in basal cells as well in some stromal cells, rather than in luminal cells. In PCa tissue, luminal cells expressed PLD1. In a PCa TMA, the mean peroxidase intensity per DAB-stained Gleason 6 and 7 tissue section was significantly higher than in sections graded Gleason 9. In CRPC tissue, PLD1 was expressed prominently in the stromal compartment, in luminal cells in occasional glands and in an expanding population of cells that co-expressed chromogranin A and neurone-specific enolase. Levels of PLD activity in normal and PCa tissue samples were similar. A specific PLD1 inhibitor markedly reduced the survival of both prostate cell lines and patient-derived PCa cells compared with two dual PLD1/PLD2 inhibitors. Short-term exposure of PCa cells to the same specific PLD1 inhibitor significantly reduced colony formation. CONCLUSIONS:A new specific inhibitor of PLD1, which is well tolerated in mice, reduces PCa cell survival and thus has potential as a novel therapeutic agent to reduce prostate cancer progression. Increased PLD1 expression may contribute to the hyperplasia characteristic of BPH and in the progression of castrate-resistant PCa, where an expanding population of neuroendocrine-like cells express PLD1.

Dataset Information

Interaction of 14-3-3? with KCMF1 suppresses the proliferation and colony formation of human colon cancer stem cells.

Publications

Interaction of 14-3-3σ with KCMF1 suppresses the proliferation and colony formation of human colon cancer stem cells.

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