Krüppel-Like Factor 6 Downregulation Is Connected with a Poor Prognosis and Tumor Growth in Non-Small-Cell Lung Cancer

Abstract

Purpose: Research in this article was performed to explore the biological role and clinical significance of Krüppel-like transcription factor 6 (KLF6) in non-small-cell lung cancer (NSCLC).

Methods: KLF6 expression in NSCLC cell lines was analyzed using reverse transcription PCR and Western blot. The expressed KLF6 protein was examined in 50 surgical NSCLC tissues using immunohistochemistry. Statistical analyses were employed for clinical association examinations. CCK8 assay and Annexin V/PI analysis were used to execute cell proliferation and apoptosis in KLF6-overexpression cell lines and the control groups. Cleaved caspase-3 expression was also detected in KLF6-overexpression cells and NSCLC tissues. KLF6 expression correlation with cleaved caspase-3 was also examined.

Results: It was discovered that downregulation of KLF6 was seen in human NSCLC cell lines. Low KLF6 expression in NSCLC tissues was correlated with poor patient prognosis (P < 0.005); patients with less KLF6 expression possessed a lower cumulative 5-year survival rate. Multivariate analysis showed KLF6 expression as an independent prognostic indicator for NSCLC individuals. Expression levels of KLF6 were associated with NSCLC tumor size (P = 0.041). Overexpression of KLF6 inhibited cell proliferation and stimulated A549 and H322 cell line apoptosis. Cleaved caspase-3 protein had higher expression levels in KLF6-overexpressed cells than in the control group. The KLF6 expression levels were positively related to the cleaved caspase-3 protein expression in NSCLC tissues (r = 0.689, P = 0.001).

Conclusions: The results indicate that downregulation of KLF6 is a significant NSCLC progression marker. KLF6 prevents cell growth and promotes cell apoptosis, possibly caspase-3 activations.

Figure 1

KLF6 expression analysis in NHBE and human NSCLC cells. (a) RT-PCR examined the expression of KLF6 mRNA in NHBE and NSCLC cell lines (A549, H322, GLC-82, and PC-9). (b) The histogram showed downregulated KLF6 mRNA expression in the NSCLC cell lines (A549, H322, GLC-82, and PC-9). Loading control: β-actin. (c) Western blot analyzed the expression of KLF6 protein in NHBE and NSCLC cell lines (A549, H322, GLC-82, and PC-9). (d) The histogram showed that KLF6 protein expression was downregulated in the NSCLC cell lines (A549, H322, GLC-82, and PC-9). Loading control: GAPDH. The experiments were done in triplicate, and mean ± SD was used to represent the data ^∗P < 0.05 in contrast with NHBE.

Figure 2

KLF6 protein expression in NSCLC tissues was associated with patients' tumor size and overall survival time. (a–c) Representative images of 50 NSCLC cases evaluated by immunohistochemistry. The tissues display (a) negative staining, (b) moderate staining, and (c) strong staining (×400). (d) Considerably distinct overall survival time of NSCLC patients amongst the high and low KLF6 expression groups (P = 0.005). (e) The MTDs in NSCLC patients were substantially unique amongst the high and low KLF6 expression groups (P = 0.041). Each dot signifies a single patient's MTD. The low KLF6 expression group median MTD was 3.55 cm (range: 1.5-14.0 cm) and 2.65 cm (range: 1.8-6.5 cm) in the high KLF6 expression group.

Figure 3

KLF6 overexpression in A549 or H322 cells inhibited cell proliferation. (a–d) RT-PCR and WB showed enhanced KLF6 mRNA and protein expression in KLF6-overexpression A549 and H322 cells compared to vector control-transfected cells.

Figure 4

(a, b) The growth curves of the A549 and H322 cells overexpressing KLF6 were lower than those of the vector control cells. Mean ± SD of three experiments was applied to express the results. P = 0.0216 and P = 0.0253, respectively.

Figure 5

KLF6 overexpression induced cell apoptosis, possibly by activating caspase-3. (a) The Annexin V/PI staining results were analyzed with flow cytometry to assess cell apoptosis in KLF6-overexpression or vector control A549 and H322 cells. (b) The histogram exhibited knowingly enhanced that apoptotic A549 and H322 cell percentages in the KLF6-overexpression groups when compared to the control groups with KLF6 overexpression, ^∗#P < 0.05. (c) WB analysis revealed an upregulation of cleaved caspase-3 protein expressed when KLF6 was overexpressed in A549 and H322 cells. Loading control: GAPDH. (d) Representative images from 10 NSCLC cases immunostained for cleaved caspase-3 or KLF6 (×400), scale bar: 50 μm. The two paraffin sections were adjacent sections from one patient. (e) Pearson correlation analysis showed a positive link with the expression of KLF6 and cleaved caspase-3 protein expression in lung cancer tissues, r = 0.689, P = 0.001.