Small interference RNA targeting Krüppel-like factor 8 inhibits the renal carcinoma 786-0 cells growth in vitro and in vivo

Abstract

Purpose: Krüppel-like factor 8 (KLF8) plays an important role in oncogenic transformation and is highly overexpressed in several types of human cancer. We investigated the expression of KLF8 in renal cell carcinoma (RCC) tissues and the role of small interference RNA targeting KLF8 on growth, cell cycle, and apoptosis of human renal carcinoma cell line 786-0 in vitro and in vivo.

Methods: The expression of KLF8 protein and mRNA in human renal carcinoma samples was detected by immunochemistry and reverse transcription polymerase chain reaction (RT-PCR). The effects of small interference RNA (siRNA) targeting KLF8 on growth, invasiveness, cell cycle, and apoptosis of 786-0 cells were evaluated by MTT assay, Matrigel Invasion Assay, and flow cytometry in vitro. We also investigated effect of siRNA targeting KLF8 on growth of 786-0 cells in nude mice in vivo.

Results: Immunohistochemistry and RT-PCR results showed the expression of KLF8 protein and mRNA in RCC specimens was significantly higher than that in the adjacent non-tumorous renal tissues (P < 0.001). KLF8-siRNA depressed the cellular growth and invasion of 786-0 cells in vitro. The flow cytometry results revealed that KLF8-siRNA could induce an increase in G0/G1 phase cells and induce cell apoptosis. Intratumor injection of siRNA targeting KLF8 inhibited the growth of 786-0 cells in vivo in nude mice tumor model.

Conclusions: KLF8 possibly involved in regulating the cell growth, invasion, apoptosis, and proliferation of renal carcinoma cancer cells. Blocking the KLF8 channel might be a potential therapeutic strategy for RCC.

Fig. 1

Immunohistochemistry and RT-PCR analysis for KLF8 protein and mRNA expression in RCC tissues and adjacent normal renal tissues. a, b KLF8 protein expression in adjacent normal renal tissues and RCC tissues (200×). c KLF8 mRNA expression in adjacent normal renal tissues and RCC tissues. d RCC tissues had higher KLF8 mRNA level than adjacent normal renal tissues. ***P < 0.001, as compared with adjacent normal renal tissues

Fig. 2

The expression of KLF8 mRNA and protein in the renal cancer 786-0 cells after transfected with different KLF8-siRNA sequences. a RT-PCR analysis of KLF8 mRNA expression in different groups. GAPDH was shown as an internal control. b The relative expression of KLF8 mRNA in different groups. c Western blot analysis of KLF8 protein expression in different groups. GAPDH was shown as an internal control. d The relative expression of KLF8 protein in different groups. P > 0.05, ***P < 0.001, as compared with the blank control

Fig. 3

The effects of KLF8-siRNA2 on mRNA and protein expression of cyclin D1 and FAK in 786-0 cells. a The mRNA expression of cyclin D1 and FAK was analyzed by RT-PCR. b, c The relative mRNA expression of cyclin D1 and FAK in different groups. d The protein expression of cyclin D1 and FAK was analyzed by Western blot. e, f The relative protein expression of cyclin D1 and FAK in different groups. P > 0.05, ***P < 0.001, as compared with the blank control

Fig. 4

The effects of KLF8-siRNA2 on growth, invasion of 786-0 cells in vitro. a 786-0 cells were incubated with KLF8-siRNA2 or scrambled siRNA. 786-0 cells were harvested at indicated time points and analyzed for viability using the MTT Assay. b–d The 786-0 cells number of blank control group, scrambled siRNA group, and KLF8-siRNA2 group was calculated in Matrigel Invasion Assay. e KLF8-siRNA2 significantly inhibited invasion ability of 786-0 cells. P > 0.05, *P < 0.05, ***P < 0.001, as compared with the blank control

Fig. 5

The effects of KLF8-siRNA2 on cell cycle and cell apoptosis. a–c Flow cytometric analysis showed that knockdown of KLF8 expression induced G0/G1 arrest to disrupt cell cycle progression and increased cell apoptosis. P > 0.05, ***P < 0.001, as compared with the blank control

Fig. 6

Effects of KLF8-siRNA on tumor growth in nude mice. a–c The tumor size and weight of KLF8-siRNA2 group was significantly decreased than that of blank control group and scrambled siRNA group. d Tumor growth curve showed a significant growth tendency in blank control group and in scrambled siRNA group, while the tumor growth in KLF8-siRNA2 group was obviously inhibited. P > 0.05, *P < 0.05, ***P < 0.001, as compared with the blank control group

Fig. 7

Effects of KLF8-siRNA2 on KLF8 expression in vivo as determined by RT-PCR and Western blot. a KLF8 mRNA expression in different groups. b The relative mRNA expression of KLF8 in different groups. c KLF8 protein expression in different groups. d The relative protein expression of KLF8 in different groups. P > 0.05, ***P < 0.001, as compared with the blank control group