KLF15 promotes the proliferation and metastasis of lung adenocarcinoma cells and has potential as a cancer prognostic marker

Abstract

Lung adenocarcinoma (LADC)is a general form of non-small cell lung cancer that represents a significant threat to public health worldwide. The 5-year-survival rate for LADC is currently below 15%. The transcription factor KLF15, also called kidney-enriched KLF (KKLF), has been proven to play a role in inhibiting proliferation and diversification of carcinoma cells, including those of the endometrium, pancreas and breast, but the involvement of KLF15 in LADC has not previously been studied. In this study, we compared the in vitro expression of KLF15 in human LADC tissues and adjacent normal lung tissues. Expression of KLF15 was found to be abnormally high in LADC tissues and cells compared with adjacent non-tumorous tissues, and was correlated with tumor TNM stage and tumor differentiation (P = 0.003, P = 0.001, respectively). The effect of KLF15 on cell growth and migration were explored in vitro by Western Blotting, CCK8 and colony formation assays, flow cytometry analysis and transwell migration assays, and in vivo by analysis of tumorigenesis in 5-week old BALB/c nude mice. Knockdown of KLF15 significantly upregulated the protein levels of cleaved caspase-3, caspase-7, caspase-8 and PARP, thereby inducing apoptosis. Downregulation of KLF15 in A549 and NCI-H1650 cell lines resulted in these cell lines exhibiting markedly slower growth rates when injected subcutaneously into the flank of nude mice, compared with the comparator control groups (P < 0.05). Collectively, our findings suggest that KLF15 may have a significant effect on LADC cell survival, and that it represents a potential therapeutic and preventive biomarker for LADC prognosis and treatment.

Keywords: KLF15; cell migration; cell proliferation; lung adenocarcinoma; prognosis.

Figure 1. Upregulation of KLF15 in clinical specimens and LADC derived cell lines

(A) The expression of KLF15 mRNA in 60 paired LADC and adjacent non-tumorous tissue specimens, as analyzed by qRT-PCR. (B) Representative results of the upregulation of KLF15in LADC specimens by Western Blotting analysis. (C) mRNA expression of KLF15 in five LADC cell lines and in normal human bronchial epithelial cells. (D) Representative results of the upregulation of KLF15 in LADC specimens as determined by immunohistochemistry analyses. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Figure 2. Kaplan–Meier overall survival curve of LADC patients correlated with KLF15 expression

Figure 3. The effect of KLF15inLADC on cell proliferation, tumor growth, apoptosis and the expression of EMT related proteins

(A) The expression of KLF15in KLF15-depletedA549 and NCI-H1650 cell lines. (B) Cellular proliferation of treated LADC cells as measured by CCK8 cell proliferation assay at various time points. (C) Proliferating colonies in incubated A549 and NCI-H1650 cells as determined by the colony formation assay. Statistical significance was determined on the basis of the numbers of identified colonies, as identified by light microscopy. The numbers of colonies are shown in the column charts. (D) LADC Tumor sizes in nude BALB/c male mice measured every fifth day after inoculation with KLF15 transfected or controlA549 and NCI-H1650 cells. (E) The metastatic condition of treated A549 and NCI-H1650 cells as measured by cell migration assays. (F) Western Blotting analysis of the expression levels of Vimentin, N-cadherin and E-cadherin in KLF15 transfected and control A549 and NCI-H1650 cells. (G) Cell apoptosis in KLF15 transfected and control A549 and NCI-H1650 cells, as determined by annexin V-FITC and PI staining using flow cytometry. The representative apoptosis pattern is shown, and the apoptotic cells are indicated in the UR and LR quadrants. (H) Knockdown of KLF15 in A549 and NCI-H1650 cells induced expression of the cleaved forms of caspase-3, caspase-7, caspase-8 and PARP, as determined by Western Blot analysis. All data shown are means ± SD. *P < 0.05. β-actin was used as the loading control.