Knockdown of CENPF inhibits the progression of lung adenocarcinoma mediated by ERβ2/5 pathway

Abstract

Many studies have reported that estrogen (E2) promotes lung cancer by binding to nuclear estrogen receptors (ER), and altering ER related nuclear protein expressions. With the GEO database analysis, Human centromere protein F (CENPF) is highly expressed in lung adenocarcinoma (LUAD), and the co-expression of CENPF and ERβ was found in the nucleus of LUAD cells through immunofluorescence. We identified the nuclear protein CENPF and explored its relationship with the ER pathway. CENPF and ERβ2/5 were related with T stage and poor prognosis (P<0.05). CENPF knockout significantly inhibited LUAD cell growth, the tumor growth of mice and the expression of ERβ2/5 (P<0.05). The protein expression of CENPF and ERβ2/5 in the CENPF-Knockdown+Fulvestrant group was lower than CENPF- Negative Control +Fulvestrant group (P=0.002, 0.004, 0.001) in A549 cells. The tumor size and weight of the CENPF-Knockdown+Fulvestrant group were significantly lower than CENPF- Negative Control +Fulvestrant group (P=0.001, 0.039) in nude mice. All the results indicated that both CENPF and ERβ2/5 play important roles in the progression of LUAD, and knockdown CENPF can inhibit the progression of LUAD by inhibiting the expression of ER2/5. Thus, the development of inhibitors against ERβ2/5 and CENPF remained more effective in improving the therapeutic effect of LUAD.

Keywords: WGCNA package; centromere protein F (CENPF); estrogen receptor beta; lung adenocarcinoma (LUAD); non-small cell lung cancer (NSCLC).

Figure 1

WGCNA analysis and determination of the CENPF gene. (A) Dendrogram of differentially expressed genes clustered based on a dissimilarity measure (1-TOM). (B) Heat map distribution histogram of differential genes for modules related to NSCLC staging in GSE19804 (The same results in the GSE30219, GSE32863, GSE63459 databases are shown in Supplementary Figure 1). (C) Analysis of the scale-free fit index for various soft-thresholding power (β) and analysis of the mean connectivity for various soft-thresholding power (GSE19804). (D) There are 14 gene differential expressions in the NSCLC staging modules. (E) There are 13 gene differential expressions in the LUAD staging modules. (F) In the four datasets, there were 5 overlapping genes that were significantly differentially expressed between NSCLC and LUAD. (G) The degree values of the five key genes in different datasets.

Figure 2

CENPF is upregulated in LUAD and is related with TNM staging and prognosis of LUAD patients. (A–F) Oncomine database results show that CENPF expression is significantly up-regulated in LUAD. (The corresponding P value and fold change are given above the picture). (G, H) The Human Protein Atlas database indicates that CENPF is strongly expressed in LUAD. G: LUAD (patient ID.4923, male, 57); H: normal lung tissue (patient ID. 4208, male, 75). (I–L) Analyzes the relationship between CENPF and LUAD staging based on four datasets. (M) Verify the correlation between the expression of CENPF and the pathological stage of LUAD (based on TCGA data in GEPIA). N-P: Survival analysis. (N, O) Kaplan Meier curves of OS (overall survival), DFS (Disease-free survival) in a cohort of LUAD stratified by CENPF expression. (P) Survival curves of CENPF gene in LUAD patients based on TCGA database. (Q) RNA sequencing analysis of volcano maps. (R) RNA sequencing results indicate that CENPF is highly expressed in LUAD tissues. Orange represents a high expression of the gene in LUAD, and blue represents a low expression of the gene in LUAD. (S, T) The CENPF gene was analyzed using Gene Set Enrichment Analysis (GSEA). The positive expression of the CENPF is related with a low prognosis in LUAD patients.

Figure 3

Expression of CENPF, ERβ, ERβ2 and ERβ5 are associated with T stage and TNM stage in LUAD patients. (A) Tissue microarray (TMA) was used to analyze the expression of CENPF in benign lung lesions and different TNM staging tissues of LUAD. The magnification of each slice is 40×, 100×, 200× in order. (B–D) Analysis of the relationship between the expression of CENPF, ERβ, ERβ1, ERβ2 and ERβ5 and the TNM staging or T stage or N stage of LUAD. The corresponding P value is marked above the picture.

Figure 4

Knockdown of CENPF inhibits cell proliferation, migration, invasion and increases apoptosis of LUAD cells. (A) The protein level of CENPF in A549 and H1299 cell lines were higher than in normal cell lines BEAS-2B and other LUAD cells. GAPDH served as the internal control. (The corresponding gray value are shown in Supplementary Figure 3). (B) The knockdown efficiency of LV-CENPF sh or LV-NC transfected with A549 and H1299 cells was verified by RT-qPCR. *P < 0.05 vs CENPF-KD. (C, D) MTT showed that CENPF knockdown suppressed the proliferative viability of cells in A549 and H1299 cells. *P < 0.05 vs CENPF-KD. (E) Migration assays and invasion assays revealed that CENPF-KD decreased cell migration and invasion abilities of A549. (F, G) The related protein E-cadherin was significantly increased (P=0.009, Figure 4F; The corresponding gray value are shown in Supplementary Figure 3G), and N-cadherin and MMP2 were significantly decreased when compared with NC group (P=0.004, 0.012; The corresponding gray value are shown in Supplementary Figure 3H). (H) Quantified histograms of scratch experiment of A549 and H1299. (I) The cell percentage and DNA content were significantly increased in the G1 phase in the CENPF-KD group(P=0.011). (J) The expression of CCND1, CDK2 and CDK4 was significantly lowered in CENPF-KD group (P=0.022, 0.001, 0.002; The corresponding gray value are shown in Supplementary Figure 3M). (K) CENPF knockdown increased apoptosis of A549 and H1299 cell lines (P=0.001, 0.001). Each experiment was performed in triplicate and repeated three times. P values were calculated with two-tailed unpaired Student’s t test.

Figure 5

Knockdown of CENPF inhibits proliferation, invasion and migration of LUAD cells via the ERβ2/5 pathway. (A) Immunofluorescence showed the co-localization of CENPF and ERβ in A549 and H1299 cells (400 x). (B) Cell proliferation assays of different grouped cells at specific times in A549 and H1299 cells. (C, D) Corresponding quantified histograms of migration and invasion in A549 and H1299 cells. The invasion and migration of cells in CENPF-KD+E2 group were significantly reduced when compared with CENPF-NC+E2 group. (E, F) Protein expression of MMP2, N-cadherin and E-cadherin in A549 and H1299 cells (The corresponding gray value are shown in Supplementary Figure 4B–4D): The expression of MMP2 and N-cadherin were significantly decreased in CENPF-KD+E2 group when compared with CENPF-NC+E2 group. (G) Scratch experiment showed that the migration of CENPF-KD+E2 group was significantly lower than CENPF-NC+E2 group in A549 and H1299 cells (P=0.000, 0.000). (H) Corresponding quantified histograms of the A549 cells at different stages of the cell cycle (G1, S and G2/M). (I) Protein expression of CCND1, CDK2 and CDK4 in A549 and H1299 cells (The corresponding gray value are shown in Supplementary Figure 4F). *P < 0.05. (J) Knockdown of CENPF inhibited the expression of ERβ2/5 in vitro. (The corresponding gray value are shown in Supplementary Figure 5A). (K) Protein expression of CENPF, ERβ, ERβ1, ERβ2 and ERβ5 in vitro experiment after treated with E2 and Ful treatment (The corresponding gray value are shown in Supplementary Figure 5C). *P < 0.05. P values were calculated with two-tailed unpaired Student’s t-test, or one-way analysis of variance.

Figure 6

Knockdown of CENPF can inhibit ERβ2/5 pathway-mediated tumor tissue growth in vivo. (A) Pictures of mice tumor tissues after resection. (B, C) Analysis of tumor size and tumor weight. *P < 0.05. (D) Tumor images of nude mice. (E, F) Statistical analysis of tumor size and tumor weight. (G, H) Immunohistochemical analysis of the expression of CENPF, ERβ, ERβ2 and ERβ5 in nude mice tumor tissues and corresponding quantified histograms. (I) Knockdown of CENPF inhibited the expression of ERβ2/5. (The corresponding gray value are shown in Supplementary Figure 5B). (J) Protein expression of CENPF, ERβ, ERβ1, ERβ2 and ERβ5 in vivo experiment after treated with E2 and Ful treatment (The corresponding gray value are shown in Supplementary Figure 5F). *P < 0.05. P values were calculated with two-tailed unpaired Student’s t-test, or one-way analysis of variance.