Tumor Suppressor DLEC1 can Stimulate the Proliferation of Cancer Cells When AP-2ɑ2 is Down-Regulated in HCT116

Abstract

Background: The molecular mechanisms of tumor suppressor gene DLEC1 are largely unknown.

Objectives: In this study, we established DLEC1 over-expression stable clones to study the cellular function of DLEC1 in the colorectal cancer cell line, HCT116.

Materials and methods: Stable clones with DLEC1 over-expression were first established by the transfection of DLEC1 expression construct pcDNA31DLEC1 in HCT116. On G418 selection, positive stable clones were screened for DLEC1 expression level by conventional reverse transcription-polymerase chain reaction (RT-PCR), and verified by real-time RT-PCR and Western blotting. Subsequently, these stable clones were subjected to colony formation and cell cycle analyses and identification of factors involved in G1 arrest. Lastly, three stable clones, DLEC1-7 (highest DLEC1 expression), DLEC1-3 (lowest expression) and pcDNA31 vector control, were employed to analyze cell proliferation and cell cycle after AP-2α2 knockdown by siRNAs.

Results: The DLEC1 over-expression was found to reduce the number of colonies in colony formation and to induce G1 arrest in seven clones, and apoptosis in one clone in the cell cycle analysis. Furthermore, regardless of the different cell cycle defects in all eight stable clones, the expression level of transcriptional factor AP-2α2 was found to be elevated. More interestingly, we found that when AP-2α2 was knocked down, DLEC1 over-expression neither suppressed cancer cell growth nor induced G1 arrest, yet, instead promoted cell growth and decreased cells in the G1 fraction. This promotion of cell proliferation and release of G1 cells also seemed to be proportional to DLEC1 expression levels in DLEC1 stable clones.

Conclusions: DLEC1 suppresses tumor cell growth the presence of AP-2α2 and stimulates cell proliferation in the down-regulation of AP-2α2 in DLEC1 over-expression stable clones of HTC116.

Keywords: AP-2ɑ; Cell Proliferation; DLEC1; Human; Promotion.

Figure 1.. Transcription Activities of DLEC1

A, Schematic illustration of the full-length of DLEC1 ORF and its four fragments used in this study. Numbers with arrows denote the boundary nucleotide position in DLEC1 DNA fragments (top); schematic representation of the four peptides encoded by DLEC1 DNA fragments. Numbers with bars depict boundary amino acid residues in DLEC1 protein fragments (bottom); B, Relative luciferase activities affected by DLEC1 and its fragments. HCT116 cells were transiently co-transfected with firefly luciferase reporter plasmid, GAL4-DLEC1 (or fragments) construct and Renilla luciferase plasmid. Relative luciferase activity of each sample was expressed as firefly luciferase activity divided by the corresponding Renilla luciferase activity to correct for transfection efficiency and subsequently standardized to that from the empty vector control (GAL4). All data are presented as mean ± Standard Error (SE) and are representative of one of at least two independent experiments performed in triplicates.

Figure 2.. Characterization of DLEC1 in Stable Clones

Stable clones of DLEC1 over-expression were generated by transfection of pcDNA31DLEC1 construct in HCT116. On G418 selection, positive stable clones were verified for DLEC1 expression level by A, Real-time RT-PCR and B, Western Blotting. The relative expression of DLEC1 was expressed as DLEC1 mRNA level divided by GAPDH mRNA level and subsequently normalized against that from the pcDNA31 vector control. DLEC1 over-expression was able to suppress cell proliferation as shown by C, representative colony formation; D, quantification of colony formation and E, MTT assay. The initial plated cell numbers are indicated below. All data are presented as mean ± SE and are one representative of at least two independent experiments performed in triplicates. *, P < 0.05.

Figure 3.. p21 Expression of DLEC1 Stable Clones

Cultured stable cells were harvested and RNA or protein was prepared for A, conventional RT-PCR; B, Real-time RT-PCR or C, Western blotting. GAPDH or β-actin was used as a loading control for PCR or Western blot analysis, respectively. The relative expression of p21 was expressed as the p21 mRNA level divided by GAPDH mRNA level and subsequently normalized against that from the pcDNA31 vector control.

Figure 4.. Identification of Factors Involved in G1 Arrest in Cell Cycle

Stable cells were cultured for extraction of RNA used in conventional RT-PCR analyses with indicated genes. GAPDH was used as an internal control for PCR.

Figure 5.. Effects of AP-2α2 Knockdown

A, Down-regulation of AP-2α2 by siRNAs against AP-2α2; B, relative cellular proliferation; C, cell cycle analyses; and D, alteration of cells in sub-G1 and G1 fractions after the down-regulation of AP-2α2. Stable cells of three clones were transfected with AP-2α2 siRNAs and cells were harvested for analysis after 72 hours (or as indicated). For fluorescence activated cell-sorting (FACS) analysis, transfected cells were harvested, fixed, treated with RNase, and stained by PI. Cells in the sub-G1 phase were regarded as apoptotic cells. The numbers in insets of C indicate sub-G1 (upper) or G1 (lower) percentage of cells. The numbers in D are the comparison results of sub-G1 or G1 in AP-2α2 knockdown samples with respective SCR samples in each stable clones. All data are presented as mean ± SE and are one representative of at least two independent experiments performed in triplicates. *, P < 0.05; **, P < 0.01.