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

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 CENPFKnockdown+Fulvestrant group was lower than CENPFNegative 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 CENPFNegative 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.


Introduction
Lung cancer is one of the most common and a leading cause of death in patients with malignant tumors 1 . The incidence and mortality of lung cancer is on a significant increase worldwide 2 . In addition to the risk factors including smoke and environment, growth factor pathway and hormonal regulation also play important roles in the pathogenesis of lung cancer 3 .
Estrogen receptors (ER) belong to the nuclear receptor steroid superfamily, and are closely related with the imbalances of hormone regulation. ER is classified into two subtypes, ERα and ERβ, which have different tissue distributions and biological effects in different types of tumors 4 . Previous studies have revealed that estrogen activates the transcription of target genes by binding to ERα and ERβ directly, which is referred to as the classical gene signaling pathway 5 . Numerous studies have found that ER also interacts with other transcription factors such as activator protein 1 (AP-1), specificity protein 1 (SP-1), interleukin 6 (IL-6) and epidermal growth factor (EGF) through protein-protein interactions in the gene signaling pathway 6 . By analyzing four lung cancer gene chips, we found that the nuclear protein gene, human centromere protein F (CENPF), is highly expressed in lung adenocarcinoma (LUAD).
Furthermore, the expression of CENPF and ERβ2/5 in the LUAD patients showed association with TNM staging, providing a basis for exploring the interaction between CENPF and ERβ2/5. Among them, CENPF protein, discovered by Rattner et al., is involved in mitosis and infinite proliferation of tumor cells 7 . The full-length molecular weight of CENPF is 367 KDa and contains 3,210 amino acids 8 . It is involved in a variety of cancer diseases, especially in the development of prostate cancer, predicting the survival rate and tumor metastasis 9 . CENPF is directly associated with the disease outcomes after undergoing gene amplification 10 . However, the role of CENPF in the progression of LUAD still remained unclear.
In addition, a large number of studies including ours have shown that E2 promotes the progression of lung cancer by binding to nuclear ER 11 . Our previous related studies have found that only ERβ1/2/5 are expressed in the lung cancer tissues among five subtypes 12,13 . As a full-length fragment of ERβ subtype, ERβ1 forms the basis for the action of other subtypes 14 , and so the role of ERβ2/5 in the progression of LUAD remained the focus of our current research.
Based on estrogen gene signaling pathway, a high co-expression of CENPF and ERβ2/5 that is associated with the clinicopathological features and prognosis of LUAD patients. CENPF is probably one of the key nuclear proteins in estrogen gene signaling pathway. Therefore, this study mainly explored the relationship between CENPF and ERβ2/5, and also explored their role alone in the development of LUAD.
This study likely provides a better understanding of ER gene signaling pathway and improves the prognosis of LUAD patients.

Tissue specimens of patient and cell culture
This study was approved by the Ethics Review Committee of Wuhan University (no. 2018096). Fifty-six cases of LUAD and ten cases of benign pulmonary lesions (BPL) tissue specimens who underwent surgery from April 2014 to July 2017 for tissue chip, one pair of LUAD and paracancerous tissue for RNA-Seq and three pairs of tissues for profiling protein were obtained from the Department of Thoracic and Cardiovascular Surgery, Zhongnan Hospital of Wuhan University. The tissue chip was customized by Shanghai Core Biotech Co., Ltd. 11 . The isolated tissue samples were immediately stored in liquid nitrogen and sent to Huada Gene (Beijing) for RNA-Seq

Lung cancer patient data set
Training data sets (GSE19804, GSE30219, GSE32863, GSE63459) based on the Affymetrix platform (Affymetrix HG-U133 Plus 2.0 array and HG-U133A array) and corresponding clinical information were retrieved from the Gene Expression Omnibus.
Two NSCLC genome-wide expression profiles were extracted from GSE19804 (including 60 paired tumors and normal tissues) and GSE30219 (including 293 tumors and 14 non-tumor tissues). Two LUAD genome-wide expression profiles were extracted from GSE32863 (including 58 paired tumor and normal tissues) and GSE63459 (including 65 tumor tissues).

Analysis and verification of hub gene
The data sets of GSE19804, GSE30219, GSE32863 and GSE63459 were used to construct co-expression networks and clinical function related modules. The criteria for screening genes was performed according to the false discovery rate (FDR) <0.05 and | log2 fold change (FC) |> 1.5. Next, a weighted gene co-expression network analysis (WGCNA) package was used to construct a co-expression network 15 16 .
Finally, the hub gene was selected based on the degree of centrality using the Venn diagram to obtain key genes.
The raw data of RNA-Seq was subjected to quality control, and then mapped with STAR 17 to obtain differential genes. The screening criteria for differential genes were abs(log2FC) > 1 and p value < 0.05.
The Oncomine, Gene Expression Profiling Interactive Analysis (GEPIA) and clinical data from The Cancer Genome Atlas (TCGA) database were used to verify the expression, progression and prognosis of hub gene.
The specificity of the above antibodies were confirmed by numerous laboratories including ours 11,18 . Immunohistochemical method to analyze the optical density was calculated by Image-Pro Plus software.

Western blotting
Detailed western blotting analysis has been described previously 19 . E-cadherin and GADPH (1E6D9) were obtained from Proteintech. The specificity of the above antibodies was verified by numerous laboratories including ours 11, 20, 21 .

Reverse transcription and quantitative real-time PCR (RT-qPCR)
Specific experimental methods were found in our previous published article 6 .

Cell culture experiment
The sense sequence of CENPF knockdown (KD) and negative control (NC) were integrated into the pWSLV-sh08-GFP vector for transfecting of A549 and H1299 cells.
The stable CENPF-deficient A549 and H1299 cells were immunofluorescently labeled with anti-CENPF and anti-ERβ 22 . Cell apoptosis assay was measured using TdT-mediated dUTP-biotin nick end labeling test (TUNEL, Roche Applied Science, Germany) according to the manufacturer's instructions 23 . MTT 24 , invasion, migration, scratch and cell cycle experiments were used to evaluate the effect of LV-CENPF sh and LV-NC on the biological function of A549 and H1299 cells 25, 26 .

Xenograft mouse model
Male nude mice were obtained from Beijing HFK Bioscience Co., Ltd., China.
Mice were housed in specific-pathogen free (SPF) environment for one week, and then were subcutaneously injected with 100 μL of 8x10 6 LV-CENPF sh or LV-NC cells. Tumor size was measured with an interval of three days (tumor size = length × width 2 × 0.5 mm 3 ). When the tumor size has reached 80-120 mm 3 , the mice were injected with E2 (0.036 mg/ml, purity 98%, Sigma) or Fulvestrant (Ful, 0.800 mg/ml, Sigma) subcutaneously twice a week (6 weeks) 11 . All mice were then sacrificed on day 45, wherein the xenograft tumors were harvested and the tumor weight and size were measured 27 . Finally, the tumor tissues were divided into two parts, one part was fixed with 4% paraformaldehyde for undergoing immunohistochemistry, and another part was quickly frozen with liquid nitrogen and stored at -80°C to perform western blotting.

Data Analysis
Data are expressed as mean ± SD. All analyses were performed at least thrice and the representative data were obtained from three independent experiments. Two-tailed Student's t-test was used to assess significance differences between the groups. The effect of LV-CENPF sh and LV-NC on the biological function of A549 and H1299 cells, the expression of key signaling molecules, immunohistochemistry results in tissue microarray and in vivo experiments were analyzed by one-way analysis of variance. Statistical analysis was performed using SPSS 22.0 software (SPSS Inc., Chicago, IL). P<0.05 was considered to be statistically significant.

Bioinformatics analysis of lung cancer datasets and the determination of CENPF
Differential genes with similar expression pattern in each dataset (GSE19804, GSE30219, GSE32863, GSE63459) were used ( Figure 1A-C, Figure S2A-I, β=5, 6, 5, 7) 28 . The four datasets obtained the module genes that are significantly associated with TNM staging of lung cancer, which included the brown module of GSE19804 (n=185), the green module of GSE30219 (n=413), the yellow module of GSE32863 (n=63) and the yellow module of GSE63459 (n=160) ( Figure 1B; Figure S2B, E, H; Table S1). By overlapping the module genes, five key genes including CENPF, CDC20, TOP2A, CCNB2 and BIRC5 were obtained ( Figure 1D-G). Finally, based on the central degree of the five key genes in different datasets and relevant literature 9, 29 , the hub gene was identified as CENPF.

CENPF is highly expressed in LUAD and negatively correlated with the prognosis of LUAD patients
The results revealed that CENPF was highly expressed in LUAD when compared to the normal lung tissues (Figure 2A-H). The expression of CENPF showed a positive correlation with TNM staging of LUAD (P<0.01, Figure 2I-M). Additionally, high expression of CENPF was negatively correlated with overall survival and disease-free survival in patients with LUAD (P=0.01, 0.027, 0.0267, Figure 2N-P).
The resulting RNA-Seq included 1515 high expressed genes and 1370 low expressed genes in LUAD patients ( Figure 2Q). CENPF was included in high expressed genes ( Figure 2R, P < 0.05) and was associated with poor prognosis of LUAD patients ( Figure 2S, T).

relationship with TNM staging and prognosis of LUAD patients
The expression of CENPF, ERβ, ERβ1, ERβ2 and ERβ5 in different TNM stages of LUAD and benign primary lesions (BPL) was examined ( Figure S1A, Figure S3A-D).
The results revealed that CENPF, ERβ, ERβ2 and ERβ5 were highly expressed in LUAD and showed a positive correlation with TNM staging and T grade of LUAD patients (P<0.001, Figure S1B, C), but showed no association with N grade ( Figure   S1D). Meanwhile, high expression of CENPF was significantly associated with shorter survival by analyzing the high expression of CENPF in LUAD patients (Table   1).

Knockdown CENPF inhibits the biological effects of LUAD cells and tumor growth in vivo
Compared with BEAS-2B and other LUAD cells, CENPF showed high expression in A549 and H1299 cells (P<0.05, Figure 3A; Figure S4A, B). RT-qPCR and cellular immunofluorescence confirmed that the efficiency of CENPF knockdown was above 70% in A549 and H1299 cells ( Figure 3B; Figure S4C). Cell proliferation of CENPF-KD group was significantly weaker than NC group from day 3 in stable CENPF-deficient cell lines A549 and H1299 (P=0.007, 0.000, Figure 3C, D). At the same time, cells in CENPF-KD group demonstrated less Ki67 staining than NC group ( Figure S4D). In addition, the invasion and migration of A549 cells in the CENPF-KD group were significantly decreased (P=0.000, 0.000, Figure 3E; Figure S4E), the related protein E-cadherin was significantly increased (P=0.009, Figure 3F; Figure  S4G), and N-cadherin and MMP2 were significantly decreased when compared with NC group (P=0.004, 0.012, Figure 3G; Figure S4H). In the stable CENPF-deficient cell line H1299, a similar trend was obtained (P<0.01, Figure S4E-H). Scratch experiment also demonstrated similar results (P=0.000, 0.000, Figure 3H; Figure S4I).
In A549 cells, cell percentage and DNA content were significantly increased in the G1 phase in the CENPF-KD group when compared with NC group (P=0.011, Figure 3I; Figure S4J). At the same time, the expression of CCND1, CDK2 and CDK4 was significantly lowered in CENPF-KD group (P=0.022, 0.001, 0.002, Figure 3J; Figure   S4M). In stable CENPF-deficient cell line H1299, similar results were obtained (P<0.05, Figure S4K-M). The CENPF-KD group also showed a significant increase in apoptosis when compared with NC group (P=0.001, 0.001, Figure 3K; Figure S4N).
The tumor weight and size in CENPF-KD group were lower than NC group after sacrificing in in vivo experiments (P=0.001, 0.001, Figure 3L-N). The results of immunohistochemistry also showed that the expression of CENPF and ERβ2/5 was significantly decreased in CENPF-KD group when compared with NC group (P=0.000, 0.000, 0.000, Figure S4O, P).

Knockdown of CENPF inhibits the expression of ERβ2/5 both in vitro and in vivo
The effect of CENPF knockdown on the expression of ERβ2/5 was examined in CENPF-NC and CENPF-KD groups both in vitro and in vivo. The protein expression of ERβ2/5 in CENPF-KD group was significantly lower than that in NC group (PA549=0.013, 0.000; PH1299=0.006, 0.002, Figure 5F; Figure S6A). The protein expression of CENPF and ERβ2/5 in CENPF-KD group was lower than NC group in vivo (P=0.024, 0.020, 0.003, Figure 5G; Figure S6B).
The effect of CENPF knockdown on the expression ERβ2/5 under the action of E2 and Ful was examined both in vitro and in vivo. The results revealed that the protein expression of CENPF and ERβ2/5 in CENPF-KD+Ful group was significantly lower than CENPF-NC+Ful group (P=0.002, 0.004, 0.001, Figure 5H; Figure S6C) in A549 cells. Similar results were obtained in stable CENPF-deficient cell line H1299 (P < 0.01, Figure 5H; Figure S6C). The protein expression of CENPF and ERβ2/5 was significantly decreased in CENPF-KD+Ful group when compared with CENPF-NC+Ful group in mice tumor tissues (P=0.020, 0.004, 0.002, Figure 5I; Figure S6D).

Discussion
Lung cancer remains one of the most common malignancies, posing a serious health problem worldwide. Targeted therapy for cell proliferation-related pathways and hormone therapy for lung cancer are considered to be important treatment strategies 30 . ER signaling pathways mainly included gene signaling pathways and non-gene signaling pathways 31 Figure S2, Table S1). Finally, CENPF was found by overlapping the key genes ( Figure 1D In addition, according to our previous study, E2 promoted the progression of lung cancer by binding to ERβ 35 . Significant differences in the biological functions were caused due to different subtypes of ERβ in E2 target organ tumors such as breast cancer, cervical cancer and prostate cancer 13,36 . Our previous findings indicated that ERβ2/5 are expressed in lung cancer 12 . Our study results revealed that ERβ2/5 are highly expressed in LUAD patients ( Figure S3B, D), and high expression of ERβ2/5 was positively associated with TNM staging of LUAD ( Figure S1B, C). It can be observed that ERβ2/5 also plays an important role in the occurrence of LUAD. Firstly, as the molecular weight of CENPF is too large and plasmid construction is easy to off target, no LUAD cell lines with high expression of CENPF can be constructed. Secondly, the number of specimens used for RNA-Seq is limited, as only specimens were used to validate the results of big database. These issues are key points that requires focus in our future research.