CARMA3 Transcriptional Regulation of STMN1 by NF-κB Promotes Renal Cell Carcinoma Proliferation and Invasion

Abstract

CARD-containing MAGUK protein 3 (CARMA3) is associated with tumor occurrence and progression. However, the signaling pathways involved in CARMA3 function remain unclear. We aimed to analyze the association between CARMA3 and stathmin (STMN1) through the NF-κB pathway, which is associated with cell proliferation and invasion, in clear cell renal cell carcinoma (ccRCC). We evaluated the effects of CARMA3 and STMN1 expression on cell migration, proliferation, and invasion in various cell lines, and their expression in tissue samples from patients with ccRCC. CARMA3 was highly expressed in ccRCC tissues and cell lines. Moreover, CARMA3 promoted the proliferation and invasion of RCC cells by activating the NF-κB pathway to transcribe STMN1. Stathmin exhibited a consistent profile with CARMA3 in ccRCC tissue, and could be an effector for CARMA3-activated cell proliferation and invasion of ccRCC cells. In summary, CARMA3 may serve as a promising target for ccRCC treatment.

Keywords: CARMA3; STMN1; renal cell carcinoma.

Figure 1.

Expression of CARMA3 in clear cell renal cell carcinoma (ccRCC) samples and cells. A, Reverse-transcription polymerase chain reaction (RT-PCR) analysis of the CARMA3 expression level in 45 ccRCC samples paired with normal tissues. B, Western blot analysis of the CARMA3 expression level in 45 ccRCC samples paired with normal tissues, shown in partial examples and statistical plots. C, RT-PCR analysis of the CARMA3 expression level in ccRCC cell lines and normal renal tubular epithelial cells. D, RT-PCR and western blot analyses of the CARMA3 expression level in CARMA3-shRNA-knockdown ccRCC cells. *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 2.

CARMA3 promoted the growth and invasion of clear cell renal cell carcinoma (ccRCC) cells. A, Proliferation ability of CARMA3-knockdown cells measured using 5-ethynyl-2′-deoxyuridine (EdU) assays, normalized to the negative control group. B, Migration and invasion ability of CARMA3-knockdown cells measured using transwell assays, normalized to the negative control group. *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 3.

CARMA3 regulated the expression of STMN1 via the NF-κB pathway. A, Reverse-transcription polymerase chain reaction (RT-PCR) analysis of the STMN1 expression level in CARMA3-shRNA-knockdown clear cell renal cell carcinoma (ccRCC) cells. B, Western blot analysis of the IκBα phosphorylation level in CARMA3-shRNA-knockdown ccRCC cells. C, RT-PCR analysis of the STMN1 expression level after application of the pyrrolidinedithiocarbamic acid NF-κB inhibitor. D, Luciferase assays of the NF-κB1 transcription activity between the wild-type and mutated STMN1 promotors. *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 4.

Highly expressed STMN1 promoted the growth and invasion of clear cell renal cell carcinoma (ccRCC) cells. A, Reverse-transcription polymerase chain reaction (RT-PCR) analysis of the STMN1 expression level in 45 ccRCC samples paired with normal tissues. B, The Pearson correlation coefficient calculated between the expression levels of CARMA3 and STMN1 in ccRCC samples. C, RT-PCR analysis of the STMN1 expression level in STMN1-shRNA-knockdown ccRCC cells. D, Western blot analysis of the STMN1 expression level in STMN1-shRNA-knockdown ccRCC cells. E, Proliferation ability of STMN1-knockdown cells measured using 5-ethynyl-2′-deoxyuridine (EdU) assays, normalized to the negative control group. F, Migration and invasion ability of STMN1-knockdown cells measured using transwell assays, normalized to the negative control group. *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 5.

STMN1 restored the proliferation and invasion effects caused by the knockdown of CARMA3 in clear cell renal cell carcinoma (ccRCC) cells. A, Western blot analysis of the expression levels of CARMA3 and STMN1 in stable CARMA3-shRNA-knockdown and STMN1-overexpressing ccRCC cells. B, Proliferation ability of stable CARMA3-shRNA-knockdown and STMN1-overexpressing cells measured using 5-ethynyl-2′-deoxyuridine (EdU) assays, normalized to the control group. C, Migration and invasion ability of stable CARMA3-shRNA-knockdown and STMN1-overexpressing cells measured using transwell assays, normalized to the control group. *P < 0.05, **P < 0.01 and ***P < 0.001.