Overexpression of KLF4 promotes cell senescence through microRNA-203-survivin-p21 pathway

Abstract

Krüppel-like factor 4 (KLF4) is a transcription factor and functions as a tumor suppressor or tumor promoter in different cancer types. KLF4 regulates many gene expression, thus affects the process of cell proliferation, differentiation, and apoptosis. Recently, KLF4 was reported to induce senescence during the generation of induced pluripotent stem (iPS) cells, but the exact mechanism is still unclear. In this study, we constructed two doxycycline-inducing KLF4 cell models, and demonstrated overexpression of KLF4 could promote cell senescence, detected by senescence-associated β-galactosidase activity assay. Then we confirmed that p21, a key effector of senescence, was directly induced by KLF4. KLF4 could also inhibit survivin, which could indirectly induce p21. By miRNA microarray, we found a series of miRNAs regulated by KLF4 and involved in senescence. We demonstrated that KLF4 could upregulate miR-203, and miR-203 contributed to senescence through miR-203-survivin-p21 pathway. Our results suggest that KLF4 could promote cell senescence through a complex network: miR-203, survivin, and p21, which were all regulated by overexpression of KLF4 and contributed to cell senescence.

Keywords: KLF4; miR-203; p21; senescence; survivin.

Figure 1. Overexpression of KLF4 induced cellular senescence in epithelial cells

(A) KLF4 expression in KLF4 inducible expression system. KLF4 inducible cell lines were treated with 2 μg/ml DOX for 24 hours, and KLF4 expression was detected by Western blotting. (B) Growth curves and (C) MTT assay of T-REx-293 KLF4 and T-REx-293 PC4 cells after DOX treatment for o to 6 days. Results represent means ± s.d (n = 3). (D) Colony formation assay of T-REx-293 KLF4 cells. Representative clone formation photos were presented and colony number was counted. Bars represent the mean ± SD (n = 3). **p < 0.01. (E) BrdU incorporation assay of T-REx-293 KLF4 cells. (F) Flow cytometry assay of T-REx-293 KLF4 cells with or without DOX for 72hrs. (G) Detection of senescence in KLF4 inducible cells. T-REx-293 KLF4 and T-REx-HeLa KLF4 cells were seeded into 6-well plates, three days after DOX treatment, cellular senescence was detected by SA-β-Gal staining assay. Bars represent the mean ± SD of three independent experiments. (H) Western blotting analysis of senescence related protein in T-REx-293 KLF4 cells with or without DOX treatment for 3 days.

Figure 2. p21 expression increased in KLF4-induced senescence

(A) p21 mRNA detected by Real-time PCR. T-REx-293 KLF4 cells were treated with DOX for 72 h and harvested for RNA extraction. Bars represent the mean ± SD (n = 3). **p < 0.01. (B) PCR result of KLF4 binding site of p21 gene promoter pulled down by ChIP. T-REx-293 KLF4 treated with DOX for 72 h were harvested and subjected to immunoprecipitation with either anti-KLF4 antibody or mouse IgG. Input DNA was applied as a positive control, and immunoprecipitation of IgG as a negative control. (C) p21 expression detected by Western blotting. T-REx-293 cells were transfected with shp21 and shCtrl plasmids and harvested after 72 h. (D) Representative SA-β-gal staining photos(magnification 100×) and percentage of senescence cells. Bars represent the mean ± SD (n = 3). **p < 0.01.

Figure 3. Survivin was involved in KLF4-induced senescence

(A) Expression of survivin protein and (B) mRNA with or without DOX treatment of T-REx-293 KLF4 cells. Bars represent the mean ± SD (n = 3). *p < 0.05.(C) Expression of exogenous survivin detected by Western blotting. (D) Representative SA-β-gal staining photos and percentage of senescence cells. Bars represent the mean ± SD (n = 3). **p < 0.01. (E) Western blotting analysis of p21 in T-REx-293 KLF4 Survivin and T-REx-293 KLF4 PC3 cells. (F) Quantitative PCR analysis of direct binding of survivin to p53 distal (left) or proximal (right) binding site of p21 gene promoter. Anti-survivin antibodies were used for immunoprecipitation. Input DNA was used as internal control for quantification, and immunoprecipitation of IgG as negative control. Bars represent the mean ± SD of two independent experiments. *p < 0.05, **p < 0.01. (G) Reporter assay of p21 promoter region. T-REx-293 cells were transfected with pcDNA3.0 HA-Survivin or pcDNA 3.0 plasmids, pGL3 Basic, pGL3 p21 plasmids, and pRL-TK plasmids and assayed for p21 luciferase activities. Firefly/Renilla luciferase ratios were used to calculate fold induction. All experiments were performed in triplicated. **p < 0.01.

Figure 4. Mir-203 could directly be upregulated by KLF4 and inhibit survivin expression

(A) Real-time PCR analysis of relative miR-203 expression upon DOX treatment of T-REx-293 KLF4 cells. Bars represent the mean ± SD (n = 3). **p < 0.01. (B) PCR analysis of direct binding of KLF4 to KLF4 binding sites of miR-203 promoter in T-REx-293 KLF4 cells with/without DOX treatment. Anti-KLF4 antibody or mouse IgG were used for immunoprecipitation. Input DNA was used as positive control, and immunoprecipitation of IgG as negative control. (C) Detection of miR-203 in T-REx-293 cells transfected with pre-miR-203 precursor or miR-203 inhibitor by qRT-PCR. Bars represent the mean ± SD (n = 3). **p < 0.01. (D) Real-time PCR analysis of survivin mRNA regulated by miR-203. Bars represent the mean ± SD (n = 3). *p < 0.05,**p < 0.01. (E) Western blotting analysis of protein expression of survivin. (F) Expression of miR-203.T-REx-293 KLF4 cells were pre-transfected with miR-203 inhibitor or its scrambled control for 24 h, and then treated with DOX for 24 h. Bars represent the mean ± SD (n = 3). **p < 0.01. (G) mRNA level of survivin detected by qRT-PCR. Bars represent the mean ± SD (n = 3). *p < 0.05,**p < 0.01. (H) Survivin and p21 protein expression analyzed through Western blotting. (I) Representative SA-β-gal staining photos(magnification 100×) and percentage of senescence cells. T-REx-293 KLF4 cells were pre-transfected with miR-203 inhibitor or its scrambled control for 24 h, and then induced by DOX for 72 h. Bars represent the mean ± SD (n = 3).

Figure 5. KLF4 expression predicted better prognosis of ESCC and CRC patients

(A) Representive immunohistochemical staining of KLF4 in normal squamous epithelium and squamous cell carcinoma of esophagus. (magnification 100×) a, b, in normal squamous epithelium, the staining of KLF4 was intensely at cell nucleus and cytosol. c-f: grading of staining of KLF4 as 0,1,2,3. (B) Disease-free survival curves of KLF4 in 38 ESCC patients, analyzed by Kaplan-Meier method. (C) Disease-free survival curves of KLF4 analyzed in GEO database (GSE24551). (D) Correlations between KLF4 and Sur39H1 expression analyzed in GEO database (GSE24551).

Figure 6. The signaling network of KLF4-inducing senescence

KLF4 could induce senescence through a complex regulatory network: direct induction of p21, inhibition of survivin that indirectly induced p21, and induction of miR-203, which inhibited survivin and induced p21 overexpression.