Panoply™ Human NR1H4 Knockdown Stable Cell Line

Nuclear receptor subfamily 1H group 1, member 4 (NR1H4) has been reported in various cancer types, but little is known about its clinical value and biological functions in clear cell renal cell carcinoma (ccRCC). Here, researchers found that NR1H4 is highly expressed in ccRCC tissues and ccRCC cell lines. Knockdown of NR1H4 significantly inhibited cancer cell proliferation, migration, and invasion. Mechanistically, tumor-related signaling pathways were enriched in the NR1H4-overexpressing group, and si-NR1H4 induced downregulation of Cyclin E2 (CCNE2). Bioinformatics analysis identified NR1H4 as highly expressed in stage I ccRCC with high diagnostic accuracy (area under the receiver operating characteristic curve > 0.8). Genetic alterations and DNA methylation of NR1H4 were significantly associated with the prognosis of ccRCC patients. Furthermore, NR1H4 expression correlated with the level of immune cell infiltration in ccRCC, providing new insights for immunotherapy.

The cell cycle signaling pathway was enriched in the NR1H4 high-expression group and had the largest expression (Figure 1A). Cell cycle proteins regulate multiple cellular functions, and inhibition of cell cycle proteins may aid cancer therapy. Researchers analyzed the TCGA dataset to further evaluate the correlation between NR1H4 expression and cell cycle proteins. Figure 1B shows that NR1H4 expression was correlated with the expression of CCNB3, CCND1, CCND2, and CCNE2. qRT-PCR results showed that si-NR1H4 induced downregulation of CCNE2 expression, while downregulation of other cell cycle proteins was not significant (Figure 1C). Western blot and immunofluorescence analysis further demonstrated that NR1H4 knockdown was associated with downregulation of cyclin E2 expression (Figure 1D, E). The primary function of cyclin E2 is to facilitate cell transition from G0/G1 to S phase by binding to CDK2 (cyclin E2's catalytic partner). Flow cytometry revealed that NR1H4 knockdown slowed cell cycle progression, with an increase in cells in the G0/G1 phase and a decrease in cells in the S phase (Figure 1F). Furthermore, NR1H4 expression in the TCGA dataset was positively correlated with CDK2 expression (Figure 1G). qRT-PCR and Western blot analysis revealed a significant decrease in CDK2 expression in NR1H4-knockdown cells (Figures 1H, I). Therefore, the researchers hypothesized that si-NR1H4 suppresses the malignant phenotype of ccRCC cells through downregulation of CCNE2/CDK2, thereby promoting the transition of cancer cells from the G0/G1 phase to the S phase.

Figure 1. NR1H4 regulates the expression of CCNE2, CDK2. (Huang S, et al., 2022)