
==== Front
Heliyon
Heliyon
Heliyon
2405-8440
Elsevier

S2405-8440(24)12588-1
10.1016/j.heliyon.2024.e36557
e36557
Research Article
CDT1, transcriptionally regulated by E2F2, promotes lung adenocarcinoma progression
Lin Bao-Quan a
Chen Feng b
Gu Lei c
Wu Zai-Xin d
Ye Jia ef
Zhang Lei ef
Huang Bing-jing g
Yu Zong-yang ef
Lai Guo-Xiang ef
Lan Xiao-Peng h
Zhao Hu zhaohubear@163.com
ij⁎⁎
Liu Wei ivyliu2012@163.com
ef⁎
a Cardio-Thoracic Surgery Department, Fuzong Clinical Medical College of Fujian Medical University, The 900th Hospital of the Joint Logistic Support Force, People's Liberation Army, Fuzhou, Fujian, 350025, China
b Fuzong Teaching Hospital of Fujian University of Traditional Chinese Medicine (The 900th Hospital of the Joint Logistic Support Force, People's Liberation Army), Fuzhou, Fujian, 350025, China
c Department of Pulmonary and Critical Care Medicine, The First Affiliated Hospital of Soochow University, Suzhou, China
d Medical Service Management Office, Fuzong Clinical Medical College of Fujian Medical University, The 900th Hospital of the Joint Logistic Support Force, People's Liberation Army, Fuzhou, Fujian, 350025, China
e Department of Respiratory and Critical Care Medicine, Fuzong Teaching Hospital of Fujian University of Traditional Chinese Medicine (The 900th Hospital of the Joint Logistic Support Force, People's Liberation Army), Fuzhou, Fujian, 350025, China
f Department of Respiratory and Critical Care Medicine, Fuzong Clinical Medical College of Fujian Medical University, The 900th Hospital of the Joint Logistic Support Force, People's Liberation Army, Fuzhou, Fujian, 350025, China
g Department of Respiratory and Critical Care Medicine, Xiamen Haicang Hospital, No. 89 Haiyu Road, Xiamen, Fujian, 361026, China
h Institute for Laboratory Medicine, The 900th Hospital of the Joint Logistic Support Force, People's Liberation Army, Fujian Medical University, Fuzhou, Fujian, 350025, China
i Department of General Surgery, Fuzong Clinical Medical College of Fujian Medical University, 900TH Hospital of Joint Logistics Support Force, Fuzhou, Fujian, 350025, China
j Department of General Surgery, Dongfang Hospital of Xiamen University, School of Medicine, Xiamen University, 900TH Hospital of Joint Logistics Support Force, Fuzhou, Fujian, 350025, China
⁎ Corresponding author. Department of Respiratory and Critical Care Medicine, Fuzong Teaching Hospital of Fujian University of Traditional Chinese Medicine (The 900th Hospital of the Joint Logistic Support Force, People's Liberation Army), Fuzhou, Fujian, 350025, China. ivyliu2012@163.com
⁎⁎ Corresponding author. Department of General Surgery, Fuzong Clinical Medical College of Fujian Medical University, Department of General Surgery, 900TH Hospital of Joint Logistics Support Force, Fuzhou, Fujian, 350025, China. zhaohubear@163.com
22 8 2024
30 8 2024
22 8 2024
10 16 e3655720 1 2024
18 8 2024
19 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).
CDT1, a gene that shows excessive expression in various malignancies, functions as a pivotal regulator of replication licensing. In this study, we observed a positive correlation in expression between CDT1 and E2F2 among patients with lung adenocarcinoma (LUAD). Our findings substantiated that E2F2 directly interacted with the promoter region of CDT1, as confirmed by ChIP-qPCR assays, and depletion of E2F2 resulted in a downregulation of CDT1 expression in LUAD cell lines by gene interference technology. Furthermore, we identified an upregulation of CDT1 mRNA level in Chinese LUAD samples. Notably, in the loss-of-function assays, depletion of CDT1 in LUAD cell lines inhibited cell proliferation, migration, and invasion. Concurrently, it promoted cell apoptosis and induced G0/G1 phase arrest using MTT, flow cytometry, and Transwell assays, reinforcing its role as an oncogene.Furthermore, enhanced tumor ablation was determined in a CDT1-downregulated LUAD tumor-bearing nude mouse model. Collectively, our results strongly suggest that E2F2 positively regulates CDT1 expression and actively participates in the progression of lung adenocarcinoma, thereby providing valuable insights into identifying novel therapeutic targets for LUAD treatment.

Keywords

CDT1
E2F2
Lung adenocarcinoma
Oncogene
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pmc1 Introduction

CDT1 is a critical component of the pre-replication complex (pre-RC), responsible for DNA replication licensing and maintaining genetic stability [[1], [2], [3]]. Elevated levels of CDT1 could lead to re-replication, genomic instability, and promote malignant transformation [[3], [4], [5]]. Overexpression of CDT1 were observed in various cancers and is associated with cancer progression and poor prognosis [[6], [7], [8], [9], [10], [11]]. E2F2, a member of the E2F transcription factor family, regulates the expression of cell cycle genes and is overexpressed in lung adenocarcinoma (LUAD) [12]. However, it remains unclear whether E2F2 regulated the CDT1 gene. Research on CDT1 expression in LUAD, especially in China, is limited. This study aimed to investigate the expression and interaction of CDT1 and E2F2 in LUAD using bioinformatics and molecular biology methods. We also assessed CDT1 expression in Chinese LUAD patients, examined the effects of CDT1 downregulation on LUAD cell proliferation, apoptosis, migration, and invasion, and evaluate tumor formation in nude mice transplanted with CDT1 knockdown LUAD cell lines.

2 MATERIALS and METHODS

2.1 Bioinformatics analysis

The expression and interaction of the CDT1 and E2F2 genes in LUAD and normal tissues were analyzed using the Gene Expression Profiling Interactive Analysis (GEPIA) platform (http://gepia.cancer-pku.cn) [13]. The correlation between CDT1 expression and survival rates, as well as pathological stages in LUAD patients, was also investigated. Additionally, CDT1 expression in LUAD cell lines was queried using the Cancer Cell Line Encyclopedia (CCLE, www.broadinstitute.org/ccle) database.

2.2 Collection of LUAD samples and adjacent normal tissues

Nineteen pairs of lung adenocarcinoma and their adjacent normal tissues were obtained from patients undergoing tumor resection at the 900 th Hospital of Joint Logistics Support Force. The study was approved by the Ethics Committee of the 900 th hospital of Joint Logistics Support Force.

2.3 Cell culture

The A549 and H2228 cell lines were obtained from the cell bank of Shanghai Biology Institute, Chinese Academy of Sciences and cultured in RPMI 1640 medium (Gibco, USA) with 10 % fetal bovine serum(Gibco, USA) at 37 °C in a 5 % CO2 environment. The medium was refreshed every 1–2 days until confluency.

2.4 siRNA transfections

Three siRNAs targeting CDT1 and E2F2 were designed and synthesized by RiboBio Co., Ltd. (Guangzhou, China) and Hippo Bio Co., Ltd. (Huzhou, China), with the most effective sequences chosen based on RT-qPCR and Western blot analysis. Targeting CDT1, the siRNA sequences were: 5′-GCACCAGGAGGUCAGAUUA-3' (active) and 5′-UAAUCUGACCUCCUGGUGC-3' (control). For E2F2, the sequences were: 5′-UCCGUGCUGUUGGCAACUUUATT-3' (active) and 5′-UAAAGUUGCCAACAGCACGGATT-3' (control). A549 and H2228 cells were cultured and transfected with siRNAs by using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). The sequence exhibiting the most remarkable efficacy in down-regulating CDT1 expression, as confirmed by quantitative real-time PCR (RT-qPCR) and Western blot (WB) analysis, was meticulously selected for subsequent experiments.

2.5 Construction of stable transfected cells

A shRNA targeting CDT1 and a GFP reporter gene were inserted into a lentiviral vector and transfected into A549 cells (Anti-hela Biological Technology Trade Co. Ltd, Xiamen, China). Puromycin selection was used until 85 % of the cells exhibited GFP expression. The lentiviral vectors carrying the shRNA (Lv-shCDT1) or the control vectors containing a control shRNA (Lv-shctrl) were then transfected into A549 cell lines. Following the addition of the virus to the culture medium, A549 cells were subjected to puromycin selection and examined under a fluorescence microscope until 85 % of the cells exhibited positive GFP expression.

2.6 Quantitative real-time PCR (qPCR)

RNA was extracted from cryopreserved tissues/cells using TRIzol (Invitrogen, Carlsbad, CA, USA), and reverse transcribed into cDNA the Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland). Real-time PCR was performed with primers listed in Supplementary Table S1, and detected by SYBR Green PCR Master Mix(Thermo Fisher Scientific, Waltham, MA, USA). Expression values were determined by normalizing 18S rRNA gene using the comparative CT method.

2.7 Western blot

Proteins were extracted and incubated with primary antibodies against GAPDH (1:1000, CTS, #2118) and CDT1 (1:1000, CTS, #8064). The secondary antibody used was HRP-labeled goat anti-rabbit IgG (1:2,000, CTS, #7074). Protein detection was performed using enhanced chemiluminescence reagents, and the relative protein level was determined by calculating. The relative level of protein was determined by calculating the ratio of the gray value of the target gene to that of the internal reference, with GAPDH serving as the internal control.

2.8 ChIP-qPCR assay

A549 cells were cultured in RPMI 1640 medium (10 % FBS) at 37 °C with 5 % CO2. ChIP-qPCR was performed using a standard protocol [14], immunoprecipitating E2F2-bound chromatins with an anti-Flag antibody (1:1000, 20543-1-AP, Proteintech). Primer pairs for detecting E2F2 binding in CDT1 ChIP 1 were: Forward 5′-GAGACGGAGTTTCTCGCT-3′, Reverse 5′-CCAGCACTTTGAAAGGCC-3'; For ChIP 2: Forward 5′-TTCACATATCAAATCCAC-3′, Reverse 5′-AAAATACAAAAAATTAGC-3'.

2.9 Cell proliferation assay

The MTT assay kit (#C0009, Beyotime) was used. Cells were seeded in 96-well plates (2 × 103/well) and MTT reagent added at 24, 48, 72 h. Absorbance at 570 nm was measured using a Microplate Spectrophotometer(BioTek, Winooski, VT, USA), repeated thrice.

2.10 Cell cycle analysis

Transfected A549 (siCtrl & siCDT1) and H2228 (siCtrl & siCDT1) cells were seeded in triplicate in six plates (1 × 105 cells/well). After 48 h, cells were collected, washed twice with PBS, resuspended, and fixed in 70 % ethanol at 4 °C overnight. Cells were then stained with PI (50 μg/mL, Sigma) at 25 °C for 30 min for cell cycle analysis using a flow cytometer(FACS Calibur, BD Biosciences, USA).

2.11 Cell apoptosis analysis

Apoptosis detection assays followed the Annexin V-FITC/PI kit protocol (Keygen Biotechnology). Transfected A549 (siCtrl & siCDT1) and H2228 (siCtrl & siCDT1) cells were seeded with cisplatin for 24 h. Cells were dissociated with trypsin, rinsed with PBS, and resuspended in binding buffer (0.25–1.0 × 107 cells/ml). FITC Annexin V (5 μl) and PI (5 μl) (BD Biosciences, NJ, USA) were added to 100 μl of cells, vortexed, and incubated for 15 min at RT in dark. Flow cytometry (FACS Calibur, BD Biosciences, USA) detected apoptosis. Experiment repeated thrice.

2.12 Cell invasion and migration analysis

For cell invasion assay, 8-μm transwell chambers (Corning, Corning, NY, USA) were coated with 12 μl ice-cold Matrigel (Becton-Dickinson Labware, Bedford, MA, USA). A549 (siCtrl & siCDT1) and H2228 (siCtrl & siCDT1) cells were cultured in serum-free RPMI 1640 for 12 h, washed, resuspended in serum-free RPMI 1640 + 0.5%–1.0 % BSA (1 × 105 cells/ml). 100 μl cell suspension was added to upper chamber of Matrigel-coated transwell in 24-well plates with 10 % FBS RPMI 1640. Residual cells removed. Cells fixed with 4 % paraformaldehyde, stained with crystal violet, counted under microscope. Assays duplicated, results averaged. For migration assay, procedure was same, excluding Matrigel coating.

2.13 Animal experiment

Log-phase A549/shCtrl and A549/shCDT1 cells were dissociated, rinsed with PBS, and adjusted to 2 × 107/ml. 14 nude mice (shctrl & shCDT1) were anesthetized with 1 % pentobarbital sodium. Under sterile conditions, 2x106 cells/0.1 ml were injected into each mouse's right axillary region. Tumor growth was monitored, and volumes were calculated using the formula: length x width2 x 0.5. On day 38, mice were euthanized, tumors excised and weighed. Tumor growth inhibition rate was determined by dividing the average shCDT1 tumor weight by the shctrl weight.

2.14 Statistical analysis

Data reported as mean ± SD. Analyses performed using SPSS 22.0 (IBM SPSS, Armonk, NY, USA). Differential expression assessed by Mann-Whitney test (for non-parametric data or when parametric assumptions were not met). For parametric data, Student's t-test was used to examine the differences between the two groups of data. Specifically, Unpaired t-test used for group comparison. A two-tailed P-value of <0.05 was considered significant.

3 Results

3.1 Overexpressed CDT1 correlates with prognosis in LUAD samples

RNA sequencing expression data from 9736 tumors and 8587 normal samples from the TCGA and GTEx projects were analyzed using a standardized processing pipeline. Initially, it was observed that the expression of the CDT1 gene was elevated in LUAD compared to normal tissues in the analysis of tumor/normal differential expression (p < 0.001, Fig. 1-A and 1-B). To further validate CDT1 expression in Chinese LUAD patients (Supplementary Table S2), RNA levels of CDT1 were examined in samples obtained. The results showed a significant upregulation of the CDT1 gene in LUAD samples when compared to adjacent normal tissues (p ＜0.05, Fig. 1-C), which aligned with the findings of the bioinformatics analysis. CDT1 expression in LUAD tumor tissues was also consistently higher than that in normal tissues across all stages of T (Fig. 1-D), N (Fig. 1-E), and M (Fig. 1-F) (*p ＜0.05, **p ＜0.01, ***p ＜0.001). And the CDT1 high expression group exhibited a lower overall survival probability (Fig. 1-G, p = 0.013) and disease-specific survival probability (Fig. 1-H, p = 0.004) compared to the CDT1 low expression group. But there was no difference in progress free interval between the two groups (Fig. 1-I, p = 0.461).Fig. 1 Upregulation of CDT1 expression in lung cancer and expression level correlates with prognosis. In both unpaired (A) and paired sample data (B), compared with normal tissues, the expression of CDT1 in lung adenocarcinoma tissues was increased significantly. (C) In Chinese Han LUAD patients, CDT1 expression in tumor tissues was higher than adjacent normal tissues. CDT1 expression in LUAD tumor tissues was also higher than that in normal tissues at each stage of T (D), N (E) and M (F). Compared with LUAD patients with low CDT1 expression, those with high CDT1 expression had a lower overall survival probability (G) and disease specific survival probability (H), but there was no difference in progress free interval between the two groups (I). *p ＜0.05, **p ＜0.01, ***p ＜0.001.

Fig. 1

3.2 LUAD cell lines selection

Based on CCLE data mining, a total of 26 LUAD cell lines were found to express CDT1 (Fig. 2-A). Among them, the cell lines H2228 and A549 were selected as representatives, with H2228 showing the highest expression and A549 demonstrating medium expression levels. Additionally, the elevated expression of CDT1 at RNA levels was validated in these two cell lines (Fig. 2-B). To clarify the role of CDT1 in LUAD, CDT1 siRNA transfected A549 and H2228 were also constructed as described previously (Fig. 2-C and D).Fig. 2 LUAD cell lines selection. (A) CDT1 mRNA expressions in all CCLE LUAD cell lines. (B) The expression of CDT1 mRNA in LUAD cell lines H2228 and A549 detected by qPCR. The mRNA expression of CDT1 in H2228 (C) and A549 (D) cells transfected with CDT1 siRNA or siCtrl detected by qPCR. ***p < 0.001.

Fig. 2

3.3 CDT1 knockdown leads to LUAD cell viability inhibition

MTT assay was conducted to evaluate cell proliferation in CDT1 knockdown A549 and H2228 cell lines, with normal control siRNA transfected A549 and H2228 cells used as controls. The proliferation rate of CDT1 knockdown LUAD cell lines was significantly suppressed, particularly at 72 h (A549 p ＜0.0001; H2228 p ＜0.01, Fig. 3-A). These findings suggest that CDT1 may play a crucial role in LUAD cell proliferation.Fig. 3 Downregulation of CDT1 inhibited A549 and H2228 cells proliferation, migration and invasion. (A) Cell viability of H2228 and A549 cells transfected with CDT1 siRNA was measured by MTT assay (24, 48, 72 h time points). (B) Transwell of migration assay of H2228 and A549 cells transfected with CDT1 siRNA or siCtrl and the statistic results of the assay. (C) Transwell of invasion assay of H2228 and A549 cells transfected with CDT1 siRNA or siCtrl and the statistic results of the assay. *p ＜0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 3

3.4 CDT1 knockdown reduces the ability to migrate and metastasize LUAD cells

Cell migration plays a pivotal role in cancer metastasis, which is the primary cause of cancer-related mortality. To further elucidate the impact of CDT1 on the invasive ability of LUAD cells, transwell migration (Fig. 3-B) and invasion assays (Fig. 3-C) were performed. As anticipated, at 24 h, both CDT1 knockdown A549 and H2228 cells, as well as cells transfected with scramble control, were observed in the lower chamber. However, the number of CDT1 knockdown A549 and H2228 cells was reduced compared to cells transfected with control cells in both assays.

3.5 CDT1 knockdown induces cell apoptosis and cell cycle arrest of LUAD cells

CDT1 assumed a pivotal role in the licensing process of DNA replication, with its activity being meticulously regulated to prevent re-replication during the S and G2 phases of the cell cycle. The employment of a Fluorescence-Activated Cell Sorter (FACS) assay exhibited a significant increase in the apoptosis rate of A549 cells following CDT1 knockdown, reaching 20.75 %, as opposed to the 8.82 % observed in control cells (p ＜0.01, Fig. 4-A). Similarly, H2228 cells subjected to CDT1 knockdown displayed an apoptosis rate of 11.54 %, contrasting with the 6.83 % found in control cells (p ＜0.01, Fig. 4-A). These findings strongly suggest that the down-regulation of CDT1 induces apoptosis in LUAD cells. Furthermore, cell-cycle analysis conducted through flow cytometry revealed that 57.9 % of CDT1 knockdown A549 cells were arrested in the G0/G1 phase, while control cells exhibited a percentage of 44.1 %. In parallel, CDT1 knockdown H2228 cells displayed a G0/G1 phase population of 41.2 %, in comparison to the 36.3 % observed in control cells. Additionally, 28.2 % of CDT1 knockdown A549 cells and 33.3 % of CDT1 knockdown H2228 cells were found in the S phase, as opposed to 39.3 % and 37.2 % in control cells, respectively (Fig. 4-B). Collectively, these results indicate a G0/G1 phase cell cycle arrest induced by CDT1 knockdown.Fig. 4 Downregulation of CDT1 inhibits the proliferation and promotes apoptosis, and blocks cells at G0/G1 phase. (A) Effects of downregulation of CDT1 by siRNA on apoptosis of A549 cells and H2228 cells. (B) Downregulation of CDT1 by siRNA blocks A549 cells and H2228 cells at G0/G1 phase detected by flow cytometry. *p ＜0.05, **p < 0.01, ***p < 0.001.

Fig. 4

3.6 CDT1 silencing inhibits tumor proliferation in subcutaneously nude mice models

To elucidate this scientific inquiry, the lentivirus infection rate was demonstrated using the reporter gene GFP, and the knockdown rate was assessed via qPCR and Western blot analysis (p ＜0.01, Fig. 5-A). As previously mentioned, the establishment of stable CDT1 silencing cell lines in A549 was successfully accomplished. In comparison to the A549/shCtrl group, the A549/shCDT1 groups exhibited a decelerated tumor growth. Notably, there existed a significant disparity in both tumor volume growth (p < 0.001, Fig. 5-B) and tumor weight (p < 0.01, Fig. 5-B) between the two aforementioned groups.Fig. 5 CDT1 slencing inhibits tumor proliferation in vivo. (A) Downregulated CDT1 mRNA expression level in A549/shCDT1 cells tested by RT-qPCR. Downregulated CDT1 protein expression level in A549/shCDT1 cells tested by WB. Representative graphs of A549 cells infected with indicated lentivirus are shown by GFP. (B) CDT1 silence inhibited the growth of A549 xenograft, and on day 38 there was the largest volume and weight difference. ***p＜0.001，**p ＜0.01.

Fig. 5

3.7 E2F2 transcriptionally regulates CDT1 expression by directly binding to the CDT1 promoter region

Earlier studies have suggested that E2F2 possesses the ability to regulate CDT1 expression [15]. Subsequently, ChIP assays were conducted to investigate the binding site of E2F2 to the CDT1 promoter regions. The E2F2-bound chromatin was then subjected to qPCR analysis using primers designed for two regions within the CDT1 promoter that encompassed E2F2 binding sites. ChIP-qPCR results revealed a significantly higher binding level of E2F2 to the CDT1 promoter in the IP group compared to the IgG group, thereby suggesting that E2F2 could directly bind to the putative E2F2 binding site on the CDT1 promoter (Fig. 6-A, p < 0.001). To further substantiate the positive correlation between E2F2 expression and CDT1 expression, we employed gene knockdown techniques to suppress E2F2 gene expression in lung adenocarcinoma cells A549 and H2228. Subsequently, we assessed the impact of down-regulated E2F2 gene expression on CDT1 gene and protein levels. As anticipated, the expression of the CDT1 gene was significantly reduced in A549 cells and H2228 cells with E2F2 knockdown, in comparison to the mock group and blank transfection group (p < 0.001 and p < 0.0001, Fig. 6-B). Simultaneously, CDT1 protein expression was also markedly decreased (p < 0.05 and p < 0.001, Fig. 6-B). Biogenic analysis further confirmed a positive correlation between the expression levels of E2F2 and CDT1 in LUAD samples (p < 0.05, Fig. 6-C).Fig. 6 E2F2 transcriptionally regulates CDT1 expression by directly binding to the CDT1 promoter region. (A) Agarose gel electrophoresis analysis showed there were positive bands in the Input group, no bands in the IgG group, and positive bands in the CDT1 group. The above results indicated that the promoter sequence of CDT1 gene was enriched by immunoprecipitation, and E2F2 protein was bound to the promoter region of CDT1 in vivo. Sample in M group is DNA Marker; Sample in Input group is input DNA (positive control); Sample in IgG group is immunoprecipitated DNA of IgG antibody (negative control); Sample in CDT1 group is the DNA precipitated by FLAG antibody. ChlP-PCR found that the binding level of E2F2 to the CDT1 promoter in CDT1 group was significantly higher compared with the IgG group, ***p＜0.001 (B) Expression of CDT1 mRNA and protein in A549 and H2228 cells transfected with E2F2 siRNA was detected by RT-PCR and WB, ns indicated no statistically significant difference,*p < 0.05, ***p < 0.001, ***p < 0.0001. Biogenic analysis shows a positive correlation between the expression levels of E2F2 and CDT1 in LUAD samples, p < 0.001 (C).

Fig. 6

4 Discussion

Primary lung cancer, the most prevalent malignant neoplasm worldwide and in China, exhibited the highest mortality rate among all malignant tumors in the country [16]. The prognosis for this disease was exceedingly grim, with a mere 16.1 % 5-year survival rate in China, imposing significant burdens and anguish upon both society and families [17,18]. It was widely recognized that enhanced cell proliferation constitutes a hallmark of cancer progression [19]. Perturbations in the cell cycle and genomic stability were commonly observed in cancer cells. Dysregulation in the initiation of gene replication played a pivotal role in the genesis of malignant tumors, including lung cancer. Thus, ensuring the stability and integrity of the genome during the process of cell proliferation becomes paramount, wherein DNA replication must occur precisely once in a cycle governed by the periodic assembly and disassembly of the Pre-RC complex [20].

During the S phase of the cell cycle, which was the phase where DNA replication occurrs, the initiation of cell replication was regulated by the Pre-RC complex [21]. This complex was composed of the origin recognition complex (ORC), the cell division cycle-6 protein (CDC-6), CDT1, and the mini chromosome maintenance helicases (MCM). The sequential recruitment of these components to the replication origin was essential for the licensing of DNA replication in each cycle. The regulation of CDT1 played a critical role in ensuring replication occurs only once per cell cycle and maintaining genetic stability. Overexpression of CDT1 in cell lines, animals, and various cancers [3,6,22] has been shown to lead to abnormal replication, activation of DNA damage checkpoints [23], and increased susceptibility to malignant transformation. In breast cancer [7], elevated CDT1 expression was associated with poor prognosis in patients, as well as in hepatocellular carcinoma [8]. Furthermore, in ovarian clear cell carcinoma (CCC) patients in Japan [24], those with high CDT1 expression had significantly worse overall survival compared to those with low CDT1 expression. While increased CDT1 expression has been observed in non-small-cell lung carcinomas (NSCLCs) [25] and considered to be a prognostic marker [11], its expression in Chinese LUAD and its role in cancer development remain less well understood.

In the current investigation, the expression of CDT1 was observed to be elevated in LUAD in both cancer tissues and cell lines. Furthermore, a correlation was established between higher CDT1 expression and lower survival rates in patient survival analysis, as well as poor pathological stage in differential gene expression analysis conducted through bioinformatic analysis. These findings align with the results obtained from 19 Chinese LUAD patients, where CDT1 gene expression in LUAD samples was found to be higher compared to adjacent normal tissues. CDT1 overexpression has been validated in diverse malignancies, including breast cancer, hepatocellular carcinoma, and non-small-cell lung carcinomas (NSCLC) [25]. Moreover, elevated RNA levels of CDT1 were also detected in numerous cell lines, prompting further examination of the H2228 and A549 cell lines.

To elucidate the role of CDT1 in LUAD cells, downregulated CDT1 LUAD cell lines, namely H2228/siCDT1 and A549/siCDT1, were generated. Notably, when compared to the shRNA NC, a significant reduction in proliferation rates was observed in both H2228/siCDT1 and A549/siCDT1 cells, as determined by MTT assay. This suggests an indispensable role for CDT1 in mediating the proliferation of LUAD cells. Additionally, transwell migration and invasion assays demonstrated a decrease in the migratory and invasive abilities of H2228/siCDT1 and A549/siCDT1, indicating that the downregulation of CDT1 expression attenuates the invasiveness of these cell lines.

Collectively, the data support the notion that knockdown of CDT1 inhibited the proliferation, migration, and invasion of LUAD cells. Furthermore, the downregulation of CDT1 was found to promote apoptosis and arrest cell cycle progression in LUAD cells, as confirmed by FACS analysis. The observed decrease in proliferation was attributed to CDT1-induced cell cycle arrest, which led to the arrest of LUAD cells in the G0/G1 phase of the cell cycle. To investigate the role of CDT1 in regulating LUAD cell tumorigenicity, a nude mouse model was also established. Downregulation of CDT1 inhibited the growth of LUAD xenografts in nude mice.

E2F2 which was upregulated in LUAD and promotes LUAD progression [26] was an important transcriptional activator in the cell cycle regulation process. The bioinformation analysis in the early stage of this study suggested that the expression of E2F2 was positively correlated with CDT1 in LUAD. In the later stage, we confirmed the binding of E2F2 and CDT1 promoter by Chip-qPCR. In addition, knocking down the expression of E2F2 in LUAD cell lines confirmed that the expression of CDT1 was also down-regulated simultaneously, thus demonstrating the positive regulatory effect of E2F2 on CDT1 which suggested future therapeutic targets for the E2F2/CDT1 axis in LUAD. Although this study furnished compelling experimental evidence for elucidating the role of CDT1 in LUAD, certain limitations ought to be acknowledged. Firstly, the study was predominantly grounded in in vitro experiments and outcomes derived from animal models, which may not accurately replicate the intricate pathogenesis and complexity of human LUAD. Specifically, cell lines and animal models may fall short in reflecting the intricate diversity and complexity of the tumor microenvironment, thereby potentially influencing the expression and functionality of CDT1 and E2F2. Secondly, while an upregulation of CDT1 at the RNA level was identified in LUAD, an in-depth examination of its protein expression level and post-translational modifications was not conducted. The activity of CDT1 is potentially modulated by a diverse array of post-translational modifications, which may be pivotal in tumor initiation and progression. Furthermore, despite elucidating the direct regulatory impact of E2F2 on CDT1 expression, the study did not comprehensively delve into other potential regulators or signaling pathways that may contribute to CDT1 expression. The onset and progression of LUAD involve aberrations in numerous genes and pathways, and a sole focus on E2F2 and CDT1 may be insufficient for a comprehensive understanding of the molecular mechanisms underlying LUAD.

5 Conclusions

It has been demonstrated that CDT1 and E2F2 are upregulated in LUAD, and E2F2 positively regulats CDT1 expression by binding to its promoter region. CDT1 suppression hampers biological characteristics of LUAD, thereby suggesting that E2F2/CDT1 axis holds promise as a novel target for the diagnosis and therapy of LUAD.

Data availability statement

The original contributions presented in the study were included in the article, further inquiries can be directed to the corresponding author or access the website: https://doi.org/10.6084/m9.figshare.24461038.v.

Funding

This work was supported by grants from the General Project of 10.13039/501100003392 Fujian Natural Science Foundation Project (No. 2020J011129 ), Beijing 10.13039/100016966 Bethune Charitable Foundation (BJ-RW2020005J ) and key disciplines and specialties at Joint Logistic Support Force level (LQYZ-HX).

Ethics approval

All animals were kept in a pathogen-free environment and fed ad lib. The procedures for care and use of animals were approved by the Ethics Committee of the Ethics Review Committee of the 900th Hospital of the Joint Logistic Support Force and all applicable institutional and governmental regulations concerning the ethical use of animals were followed. Written informed consent was obtained from the patient for publication of this letter. This study was approved by the Ethics Review Committee of the 900th Hospital of the Joint Logistic Support Force, People's Liberation Army(approval number:2020034).

Consent to participate

Written informed consent was provided to all patients participating in the study.

CRediT authorship contribution statement

Bao-Quan Lin: Writing – original draft. Feng Chen: Formal analysis, Data curation. Lei Gu: Formal analysis, Data curation. Zai-Xin Wu: Resources. Jia Ye: Resources. Lei Zhang: Resources. Bing-jing Huang: Resources. Zong-yang Yu: Supervision. Guo-Xiang Lai: Supervision. Xiao-Peng Lan: Supervision. Hu Zhao: Visualization, Software. Wei Liu: Writing – review & editing, Writing – original draft, Funding acquisition.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Wei Liu reports financial support was provided by the General Project of 10.13039/501100003392 Fujian Natural Science Foundation Project:Wei Liu reports financial support was provided by Beijing 10.13039/100016966 Bethune Charitable Foundation . Wei Liu reports financial support was provided by key disciplines and specialties at Joint Logistic Support Force level. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A Supplementary data

The following are the Supplementary data to this article:Multimedia component 1

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Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.heliyon.2024.e36557.
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