# Evaluation of lncRNA FOXD3-AS1 as a Biomarker for Early-Stage Lung Cancer Diagnosis and Subtype Identification

Authors: Xiaofeng Liu; Wenyan Chen; Yu Qi; Yongqian Zhu
Journal: Evidence-Based Complementary and Alternative Medicine
 (2022)

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## Abstract

Purpose. Lung cancer (LC) is the most commonly diagnosed cancer and the leading cause of cancer-related deaths. More and more long noncoding RNA (lncRNA) are associated with cancer. This study aimed to assess whether plasma lncRNA could be used to diagnose early-stage LC and identify subtypes of LC. Methods. For bioinformatic analysis, we used genetic data from the Cancer Genome Atlas, lung adenocarcinoma (LUAD), and lung squamous cell carcinoma (LUSC) datasets and a small cell lung cancer (SCLC) dataset from the Gene Expression Omnibus. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to examine the relative expression of lncRNA in LC tissues and plasma samples. The patients’ clinical information was obtained at the time of sample collection. Results. According to public datasets, the lncRNA forkhead box D3 antisense 1 (FOXD3-AS1) was significantly upregulated in LUAD, LUSC, and SCLC tissues over controls. RT-qPCR assays confirmed this finding in LUAD, LUSC, and SCLC tissues and plasma samples. Even early-stage receiver operating characteristic analysis showed that plasma FOXD3-AS1 could be used to discriminate LUAD, LUSC, and SCLC from normal controls and identify LC subtypes SCLC. Conclusion. FOXD3-AS1 is significantly upregulated in LC tissues and plasma. FOXD3-AS1 could be a potential biomarker for LC subtype identification and early diagnosis.


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## Body

## 1. Introduction

Lung cancer (LC) is the leading cause of cancer-related morbidity and mortality in China, accounting for 36.98% of cases and 39.21% of deaths worldwide, posing a significant challenge to the medical system and research [1, 2]. Nonsmall cell lung cancer (NSCLC) is the most common histological subtype of LC, accounting for 85% of diagnosed LCs [3]. It has always progressed to unresectable stage III or IV at the time of diagnosis. The most common types of NSCLC are lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), which arise from alveolar cells in the smaller airway epithelium and cells in the airway epithelium, respectively, and have divergent oncogenic driver mutations and pathways [4, 5]. Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumor that arises as a perihilar mass from the airway submucosa. It is usually diagnosed at metastasis extensive-stage or unresectable limited-stage [6, 7]. Early LC screening with low-dose computed tomography is most effective in reducing mortality, but it has a high false-positive rate. Many types of imaging, particularly high-resolution computed tomography and preoperative pathology, aid in the clinical diagnosis and treatment planning of LC [8–10]. However, repeated ionizing radiation is expensive and may increase cancer risk. As a result, finding novel biomarkers for early and accurate LC detection is critical. Furthermore, a noninvasive and sensitive method to identify LC histological subtypes would greatly improve LC therapy.Long noncoding RNAs (lncRNAs) are transcripts with a length of more than 200 nucleotides and no open reading frame or protein-coding function. They encompass a large and diverse group of transcripts with varying biogenesis and biological functions [11]. LncRNAs could induce chromatin remodeling and histone modification by invading chromatin structure or regulating chromatin/histone accessibility of the regulator/modifier; regulate transcription by gene-dosage compensation, acting as enhancers, or lncRNAs regulatory networks; regulate splicing, degradation, or sponging absorbing of transcription products; and act in cellular organelles function, structural functions, and genome integrity primarily through protein interactions [12, 13].LncRNA forkhead box D3 antisense 1 (FOXD3-AS1) is a 963 bp antisense transcript of the gene FOXD3, whose abnormal expression has been linked to pathophysiological characteristics of several diseases [14]. FOXD3-AS1, for example, affects the development of breast cancer, nasopharyngeal carcinoma, colon adenocarcinoma, and hepatocellular carcinoma via competing for the endogenous RNAs (ceRNAs) mechanism [15–17]. FOXD3-AS1 binds to poly (ADP-ribose) polymerase 1 in neuroblastoma, preventing the CCCTC-binding factor from being PARylated and thus derepressing the production of downstream tumor-suppressive genes [18]. According to Yang’s research, FOXD3-AS1 binds to Y-box binding protein 1 to mediate the H3K27ac enrichment in the promoter region in nasopharyngeal carcinoma [19]. Through the PI3K/Akt pathway, FOXD3-AS1 interacts with and activates RNA-binding protein ELAV-like RNA-binding protein 1, causing LC cell proliferation, invasion, and 5-fluorouracil resistance [20]. FOXD3-AS1 has also been shown to keep mediator subunit 28, murine double minute 2, and cyclin-dependent kinase 6 expressions in NSCLC by sequestering miRNAs [21–23]. We confirmed that FOXD3-AS1 is overexpressed in both blood and tissues of LUAD, LUSC, and SCLC samples when compared to control in the current study, based on hints from the Cancer Genome Atlas (TCGA) lung adenocarcinoma projects, lung squamous carcinoma projects, and Gene Expression Omnibus (GEO) datasets (GSE60052) as well as our RT-qPCR assays. Furthermore, FOXD3-AS1 has the potential to aid in the early detection of LC and identifying SCLC subtypes.


## 2. Materials and Methods

### 2.1. RNA Sequencing (RNA-Seq) Data Collection from TCGA and GEO Databases

The gene expression matrix and clinical data for LUAC projects were downloaded from TCGA’s official website (527 tumor vs. 58 normal,https://xenabrowser.net/datapages/?cohort=GDC%20TCGA%20Lung%20Adenocarcinoma%20(LUAD)&removeHub=https%3A%2F%2Fxena.treehouse.gi.ucsc.edu%3A443), LUSC (501 tumor vs. 49 normal, https://xenabrowser.net/datapages/?cohort=GDC%20TCGA%20Lung%20Squamous%20Cell%20Carcinoma%20(LUSC)&removeHub=https%3A%2F%2Fxena.treehouse.gi.ucsc.edu%3A443), and GEO official website for SCLC (79 tumor vs. 7 normal, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE60052).


### 2.2. Bioinformatic Analysis

Gene categories lncRNA was screened based on human genome annotation files (gif files) downloaded from the Gencode database. On the lncRNA expression matrices, differential expression analysis of tumor versus normal was performed for each of the three datasets using theR package DESeq2. |log2 fold change (FC)|>1 and adjusted P value <0.05 were used to screen differentially expressed lncRNAs (DELs). To visualize the findings, volcano plots were created. The R package heatmap was used to plot heat maps of differential expressed lncRNAs. The results of the up and downregulated genes of the three subtypes of tumors were analyzed separately and presented in a Venn diagram. The R package ggplot2 was used to plot the expression Boxplot.


### 2.3. Patients and Clinical Samples

This study included 150 patients with LC (50 with LUAD, 50 with LUSC, and 50 with SCLC) and 50 health subjects (HS) of similar sex and age. All surgical specimens were obtained between January 2020 and December 2021, and the clinical data were collected at the same time (Table1). Three months prior to surgery, no patients received chemotherapy or radiotherapy. The Ethics Committee of Traditional Chinese Medicine Hospital of LuAn approved the study protocol on scientific research, and all participants provided written informed consent. The tissues were immediately frozen in liquid nitrogen and then stored at −80°C for RNA extraction. Before the operation, blood samples were collected in a vacutainer with an anticoagulant. The whole blood samples were centrifuged at 3000 rpm at 4°C for 10 min, and the supernatant plasma was frozen at −80°C.Table 1
The clinicopathological parameters of patients with lung cancer.
CharacteristicsHS (n = 50)LCP value (HS vs. LC)SCLC (n = 50)LUAD (n = 50)LUSC (n = 50)Age in years median (range)69.98 ± 11.2570.26 ± 12.0574.14 ± 8.7671.96 ± 11.720.237Gender0.280Male33352823Female17152227Smoking status0.612Yes30363228No20141822Stage—I-II—191618III-IV—313432Lymph metastasisNo—161415—Yes—343635Distal metastasis—Yes—161814No—343236HS, healthy subjects; LC, lung cancer; SCLC, small cell lung cancer; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma.


### 2.4. RNA Isolation and Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR)

TRIzol reagent (Invitrogen) and QIAGEN RNeasy Serum/Plasma Maxi Kit (QIAGEN) were used to extract total RNA from tissues and plasma, respectively, according to the manufacturer’s instructions. The Prime ScriptTM RT reagent kit (Takara) was used for reverse transcription, which was carried out according to the manufacturer’s protocol. For RT-qPCR, the SYBR Premix Ex Taq kit (Takara) was used. The endogenous reference glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to calibrate the initial mRNA concentrations. The 2−ΔΔCT method [24] was used to determine relative gene expression. The primer sequences were as follows: FOXD3-AS1, forward 5′-GAATAGTTGCCGAGAGAAA-3′ and reverse 5′-GACAGACAGGGATTGGGTT-3′; GAPDH, forward 5′-GGGGCTCTCCAGAACATC-3′ and reverse 5′-TGACACGTTGGCAGTGG-3′.


### 2.5. Statistical Analysis

R (v.4.1.0) and SPSS (IBM Corp., Armonk, USA) were used to conduct all statistical analyses. Thet-test was used to compare the two groups of continuous data. The difference between the two groups was analyzed using the chi-square test, which was used to express the enumeration data in a fourfold table. The receiver operating characteristic curve (ROC) and the area under the curve (AUC) were performed to evaluate the diagnostic value of FOXD3-AS1. P<0.05 was considered statistically significant.


## 2.1. RNA Sequencing (RNA-Seq) Data Collection from TCGA and GEO Databases

The gene expression matrix and clinical data for LUAC projects were downloaded from TCGA’s official website (527 tumor vs. 58 normal,https://xenabrowser.net/datapages/?cohort=GDC%20TCGA%20Lung%20Adenocarcinoma%20(LUAD)&removeHub=https%3A%2F%2Fxena.treehouse.gi.ucsc.edu%3A443), LUSC (501 tumor vs. 49 normal, https://xenabrowser.net/datapages/?cohort=GDC%20TCGA%20Lung%20Squamous%20Cell%20Carcinoma%20(LUSC)&removeHub=https%3A%2F%2Fxena.treehouse.gi.ucsc.edu%3A443), and GEO official website for SCLC (79 tumor vs. 7 normal, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE60052).


## 2.2. Bioinformatic Analysis

Gene categories lncRNA was screened based on human genome annotation files (gif files) downloaded from the Gencode database. On the lncRNA expression matrices, differential expression analysis of tumor versus normal was performed for each of the three datasets using theR package DESeq2. |log2 fold change (FC)|>1 and adjusted P value <0.05 were used to screen differentially expressed lncRNAs (DELs). To visualize the findings, volcano plots were created. The R package heatmap was used to plot heat maps of differential expressed lncRNAs. The results of the up and downregulated genes of the three subtypes of tumors were analyzed separately and presented in a Venn diagram. The R package ggplot2 was used to plot the expression Boxplot.


## 2.3. Patients and Clinical Samples

This study included 150 patients with LC (50 with LUAD, 50 with LUSC, and 50 with SCLC) and 50 health subjects (HS) of similar sex and age. All surgical specimens were obtained between January 2020 and December 2021, and the clinical data were collected at the same time (Table1). Three months prior to surgery, no patients received chemotherapy or radiotherapy. The Ethics Committee of Traditional Chinese Medicine Hospital of LuAn approved the study protocol on scientific research, and all participants provided written informed consent. The tissues were immediately frozen in liquid nitrogen and then stored at −80°C for RNA extraction. Before the operation, blood samples were collected in a vacutainer with an anticoagulant. The whole blood samples were centrifuged at 3000 rpm at 4°C for 10 min, and the supernatant plasma was frozen at −80°C.Table 1
The clinicopathological parameters of patients with lung cancer.
CharacteristicsHS (n = 50)LCP value (HS vs. LC)SCLC (n = 50)LUAD (n = 50)LUSC (n = 50)Age in years median (range)69.98 ± 11.2570.26 ± 12.0574.14 ± 8.7671.96 ± 11.720.237Gender0.280Male33352823Female17152227Smoking status0.612Yes30363228No20141822Stage—I-II—191618III-IV—313432Lymph metastasisNo—161415—Yes—343635Distal metastasis—Yes—161814No—343236HS, healthy subjects; LC, lung cancer; SCLC, small cell lung cancer; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma.


## 2.4. RNA Isolation and Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR)

TRIzol reagent (Invitrogen) and QIAGEN RNeasy Serum/Plasma Maxi Kit (QIAGEN) were used to extract total RNA from tissues and plasma, respectively, according to the manufacturer’s instructions. The Prime ScriptTM RT reagent kit (Takara) was used for reverse transcription, which was carried out according to the manufacturer’s protocol. For RT-qPCR, the SYBR Premix Ex Taq kit (Takara) was used. The endogenous reference glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to calibrate the initial mRNA concentrations. The 2−ΔΔCT method [24] was used to determine relative gene expression. The primer sequences were as follows: FOXD3-AS1, forward 5′-GAATAGTTGCCGAGAGAAA-3′ and reverse 5′-GACAGACAGGGATTGGGTT-3′; GAPDH, forward 5′-GGGGCTCTCCAGAACATC-3′ and reverse 5′-TGACACGTTGGCAGTGG-3′.


## 2.5. Statistical Analysis

R (v.4.1.0) and SPSS (IBM Corp., Armonk, USA) were used to conduct all statistical analyses. Thet-test was used to compare the two groups of continuous data. The difference between the two groups was analyzed using the chi-square test, which was used to express the enumeration data in a fourfold table. The receiver operating characteristic curve (ROC) and the area under the curve (AUC) were performed to evaluate the diagnostic value of FOXD3-AS1. P<0.05 was considered statistically significant.


## 3. Results

### 3.1. Identification of Key lncRNAs from TCGA and the GEO RNA-Seq Data

Mining of TCGA, LUAD, and LUSC RNA sequencing (RNA-seq) datasets, as well as GEO, SCLC, and RNA-seq datasets (GSE60052) was performed to find lncRNAs as diagnostic biomarkers in patients with LC. The LUAD cohort, LUSC cohort, and SCLC cohort each had 527 LUAD samples vs. 58 paracancer tissue samples, 501 LUSC samples vs. 49 paracancer tissue samples, and 79 SCLC tumors vs. 7 normal lung tissue samples. We found 1368, 1436, and 524 significantly upregulated lncRNAs and 755, 1386, and 153 downregulated lncRNAs (|log2 FC|>1 and adjustedP<0.001 value <0.05), associated with the histological subtypes of LUAD, LUSC, and SCLC, respectively (Tables S1–S3). Heatmaps were created using differentially expressed lncRNAs (DELs) (Figures 1(a)–1(c)). Volcano maps were used to show the two-dimensional distribution of −log10 (adjusted P value) and log2 (FC) of three sets of DELs (Figures 1(d)–1(f)). Among these DELs, 66 upregulated lncRNAs coexisted in all three LC subtypes (Figure 1(g) and Table S4), while 10 lncRNAs were downregulated (Figure 1(h) and Table S5).Figure 1
Bioinformatic analysis of dysregulated lncRNAs in different subtypes of LC. (a)–(c) Heatmap and (d)–(f) volcano plot of differentially expressed lncRNAs (DELs) between paracancer tissues and cancer tissues in TCGA LUAD project (a and d), TCGA LUSC project (b and e), and GEO SCLC dataset (c and f). (g)-(h) A Venn diagram of the upregulated (g) and downregulated (h) DELs overlapping between LUAD, LUSC, and SCLC.
(a)(b)(c)(d)(e)(f)(g)(h)We ranked the DELs of each comparison by FC or adjustedP value. We performed a Venn diagram analysis using the top 10 lncRNAs of each comparison to find the most significant DELs. Finally, FOXD3-AS1 was identified using FC and adjusted P value screening. In contrast, HOXC-AS2 was identified using adjusted P value screening (Figure 2(a) and 2(b)). When compared to normal controls (P<0.0001), the expression of FOXD3-AS1 was significantly higher in lung tumors, regardless of whether they were LUAD, LUSC, or SCLC (Figures 2(c)–2(e)). FOXD3-AS1 was selected for further research.Figure 2
FOXD3-AS1 was the most significantly upregulated lncRNA in LUAD, LUSC, and SCLC according to public datasets. Venn diagram of the intersection of top 10 DELs ranked by FC (a) and adjustedP value (b). FOXD3-AS1 expression determined in cancer specimens (tumor) and paracancer specimens (normal) using TCGA LUAD dataset (c), TCGA LUSC dataset (d), and GEO SCLC dataset (e). The t-test was used to calculated P value.
(a)(b)(c)(d)(e)


### 3.2. Participants and Clinical Characteristics of the Validation Phase

A total of 50 healthy candidates and 150 patients with LC (50 SCLC, 50 LUAD, and 50 LUSC) were enrolled in the study to verify the diagnostic potential of FOXD3-AS1. Table1 summarizes the demographic and clinical parameters. In the healthy and LC groups, sex ratios (males: females) and smoking rates were similar. According to disease characteristics, there were 53 patients with stages I-II and 97 with stages III-IV.


### 3.3. FOXD3-AS1 Expression in Tissues and Plasma Was Correlated with the LC Progression

In all LUAD, LUSC, and SCLC patients, FOXD3-AS1 expression in tumor tissues (CA) was much higher than in paracancer tissues (PC) (P<0.001, Figure 3(a)). We investigated whether FOXD3-AS1 could be secreted into circulating plasma by tumor cells because it has been shown to stimulate invasion in NSCLC [23]. As we can see, lncRNA FOXD3-AS1 was found in plasma and expressed significantly higher in LUAD, LUSC, and SCLC blood samples than in HS (P<0.001, Figure 3(b)). We also looked at how the expression of FOXD3-AS1 changed as LC progressed. In all three subtypes, FOXD3-AS1 production in blood was significantly higher in stages III-IV than in stages I-II (P<0.001, Figure 3(c)).Figure 3
FOXD3-AS1 was verified to be overexpressed in SCLC, LUAD, and LUSC tissues and plasma comparing with healthy subjects. (a) RT-qPCR assay performed to detect the expression of FOXD3-AS1 in cancer tissues and paracancer tissues from patients with SCLC, LUAD, and LUSC.P value was obtained by the t-test. ∗∗∗P<0.001. (b) RT-qPCR analysis of FOXD3-AS1 in the plasma of healthy candidates (HS) and patients with SCLC, LUAD, and LUSC. The asterisk sign indicated the comparison with the HS group, while the hash sign represented the SCLC group as the control. P value was obtained by the t-test. ∗∗∗P<0.001. ###P<0.001. (c) RT-qPCR analysis of FOXD3-AS1 in the plasma samples of patients with SCLC, LUAD, and LUSC at stages I-II (I/II) and stages III-IV (III/IV). P value was obtained by the t-test. ∗∗∗P<0.001.
(a)(b)(c)Based on the median FOXD3-AS1 expression threshold (5.045 for SCLC, 3.52 for LUAD, and 4.04 for LUSC), we divided the cancer plasma samples into low and high-FOXD3-AS1 subgroups (L-lncRNA and H-lncRNA for short). We analyzed their relationship with the clinical characteristics of the patients. As shown in Table2, there were no significant differences in gender or smoking history between the H-lncRNA and L-lncRNA groups, but the patients who expressed high levels of FOXD3-AS1 were more likely older than 65 in LUAD (P=0.047) and LUSC (P=0.022). Furthermore, a high level of FOXD3-AS1 indicated a more advanced stage of LUAD (P=0.012) and LUSC (P=0.010), a greater probability of lymph metastasis in LUAD (P=0.025) and LUSC (P<0.001), and a greater probability of distal metastasis in SCLC (P=0.032). These findings showed that the level of FOXD3-AS1 increased as LC progressed and that the fluctuating levels of FOXD3-AS1 in tissues were primarily related to the presence of disease lesions rather than factors such as gender or smoking.Table 2
Relationship between lncRNA FOXD3-AS1 expression in serum and clinicopathological parameters of patients with lung cancer.
CharacteristicsSCLCLUADLUSCH-lncRNA (n = 25)L-lncRNA (n = 25)P valueH-lncRNA (n = 25)L-lncRNA (n = 25)P valueH-lncRNA (n = 25)L-lncRNA (n = 25)P valueGender (male/female)17/818/7113/1215/100.77614/1118/70.377Age (<65 y/≥65 y)7/1814/11/0.0854/2115/200.0477/1816/90.022Smoking story (yes/no)19/617/80.75417/815/100.76913/1215/100.776Stage (I-II/III-IV)19/613/120.14022/313/120.01225/018/70.010Lymph metastasis (yes/no)20/514/110.12822/314/110.02525/010/15<0.001Distal metastasis (yes/no)12/134/210.03210/158/170.76910/154/210.114


### 3.4. Role of Plasma FOXD3-AS1 in LC Diagnosis and Identifying LC Subtypes

The diagnostic efficacy of plasma FOXD3-AS1 in three subtypes of LC was assessed using ROC analysis. FOXD3-AS1 had an AUC of 0.959 (95% confidence interval (CI): 0.926–0.992) in identifying patients with SCLC out of HS (Figure4(a) and Table 3), an AUC of 0.763 (95% CI: 0.670–0.855) in identifying patients with LUAD out of HS (Figure 4(b) and Table 3), an AUC of 0.854 (95% CI: 0.782–0.927) in identifying patients with LUSC out of HS (Figure 4(c) and Table 3), and an AUC of 0.859 (95% CI: 0.802–0.915) in identifying patients with LC out of HS (Figure 4(d) and Table 3).Figure 4
The efficiency of plasma FOXD3-AS1 in cancer diagnosis and SCLC identification. (a)–(g) ROC curves used to validate the discrimination efficiency of plasma FOXD3-AS1 for SCLC vs. HS (a), LUAD vs. HS (b), LUSC vs. HS (c), LC vs. HS (d), SCLC vs. LUAD (e), SCLC vs. LUSC (f), and LUAD vs. LUSC (g).
(a)(b)(c)(d)(e)(f)(g)Table 3
The diagnostic values of lncRNA FOXD3-AS1 in discriminating lung cancer subtypes.
IndexAUC95% CIP valueSensitivitySpecificityCritical valueYoden indexDownUpSCLC vs. HS0.9590.9260.992<0.00188.00092.0000.8003.940LUAD vs. HS0.7630.6700.855<0.00178.00064.0000.4203.195LUSC vs. HS0.8540.7820.927<0.00182.00078.0000.6003.465LC vs. HS0.8590.8020.915<0.00178.00078.0000.5603.465SCLC vs. LUAD0.8880.8220.953<0.00178.00092.0000.7004.375SCLC vs. LUSC0.8210.7370.905<0.00178.00080.0000.5804.380LUAD vs. LUSC0.6580.5520.7650.00680.00052.0000.3203.535We further looked at the discrimination efficiency of FOXD3-AS1 between them. With a sensitivity of 78% and a specificity of 92%, it created an AUC value of 0.888 (95% CI: 0.822–0.953) in distinguishing patients with SCLC from patients with LUAD (Figure4(e) and Table 3). With a sensitivity of 78% and a specificity of 80%, it created an AUC value of 0.821 (95% CI: 0.737–0.905) in distinguishing patients with SCLC from patients with LUSC (Figure 4(f) and Table 3). In contrast, with a sensitivity of 80% and a specificity of 52%, it created an AUC value of 0.658 (95% CI: 0.552–0.765) in determining patients with LUAD from patients with LUSC (Figure 4(g) and Table 3).


### 3.5. Diagnostic Accuracy of FOXD3-AS1 as Diagnostic Biomarkers to Detect Early-Stage LC

Because the expression of FOXD3-AS1 in LC was higher than in healthy people and increased as the disease progressed, we hypothesized that FOXD3-AS1 was activated early in the disease. We specifically analyzed stages I-II LC samples to estimate the efficiency of FOXD3-AS1 in the early detection of LC. FOXD3-AS1 yielded an AUC of 0.906 (95% CI: 0.831–0.982), a sensitivity of 73%, a specificity of 92%, and a Yoden index of 3.94 in distinguishing between SCLC and HS subjects (Figure5(a) and Table 4). In terms of LUAD early detection, the AUC value, sensitivity, specificity, and Yoden index was 0.591 (95% CI: 0.453–0.729), 100%, 30%, and 2.46, respectively (Figure 5(b) and Table 4). In LUSC early detection, the AUC value, sensitivity, specificity, and Yoden index was 0.686 (95% CI: 0.562–0.809), 100%, 42%, and 2.750, respectively (Figure 5(c) and Table 4). In total, FOXD3-AS1 produced an AUC value of 0.751 (95% CI: 0.658–0.845), a sensitivity of 73.6%, a specificity of 66%, and a Yoden index of 3.195 in discriminating early LC samples from HS (Figure 5(d) and Table 4).Figure 5
The efficiency of plasma FOXD3-AS1 in LC diagnosis and SCLC identification at early stage. (a)–(g) ROC curves used to validate the discrimination efficiency of plasma FOXD3-AS1 for early-stage SCLC vs. HS (a), early-stage LUAD vs. HS (b), early-stage LUSC vs. HS (c), early-stage LC vs. HS (d), early-stage SCLC vs. early-stage LUAD (e), early-stage SCLC vs. early-stage LUSC (f), and early-stage LUAD vs. early-stage LUSC (g).
(a)(b)(c)(d)(e)(f)(g)Table 4
The diagnostic values of lncRNA FOXD3-AS1 in discriminating early-stage lung cancer.
IndexAUC95% CIP valueSensitivitySpecificityCritical valueYoden indexDownUpSCLC vs. HS0.9060.8310.982<0.00173.70092.0000.6573.940LUAD vs. HS0.5910.4530.7290.275100.00030.0000.3002.460LUSC vs. HS0.6860.5620.8090.020100.00042.0000.4202.750LC vs. HS0.7510.6580.845<0.00173.60066.0000.3963.195SCLC vs. LUAD0.9010.8021.000<0.00173.700100.0000.7373.980SCLC vs. LUSC0.8570.7300.983<0.00173.700100.0000.7373.905LUAD vs. LUSC0.6740.4890.8580.08472.20062.5000.3473.240FOXD3-AS1 was also found to be effective in identifying LC subtypes SCLC. The AUC values for SCLC versus LUAD, SCLC versus LUSC, and LUAD versus LUSC were found to be 0.901 (95% CI: 0.802–1) (Figure5(e) and Table 4), 0.857 (95% CI: 0.730–0.983) (Figure 5(f) and Table 4), and 0.674 (95% CI: 0.489 0.858) (Figure 5(g) and Table 4), respectively.


## 3.1. Identification of Key lncRNAs from TCGA and the GEO RNA-Seq Data

Mining of TCGA, LUAD, and LUSC RNA sequencing (RNA-seq) datasets, as well as GEO, SCLC, and RNA-seq datasets (GSE60052) was performed to find lncRNAs as diagnostic biomarkers in patients with LC. The LUAD cohort, LUSC cohort, and SCLC cohort each had 527 LUAD samples vs. 58 paracancer tissue samples, 501 LUSC samples vs. 49 paracancer tissue samples, and 79 SCLC tumors vs. 7 normal lung tissue samples. We found 1368, 1436, and 524 significantly upregulated lncRNAs and 755, 1386, and 153 downregulated lncRNAs (|log2 FC|>1 and adjustedP<0.001 value <0.05), associated with the histological subtypes of LUAD, LUSC, and SCLC, respectively (Tables S1–S3). Heatmaps were created using differentially expressed lncRNAs (DELs) (Figures 1(a)–1(c)). Volcano maps were used to show the two-dimensional distribution of −log10 (adjusted P value) and log2 (FC) of three sets of DELs (Figures 1(d)–1(f)). Among these DELs, 66 upregulated lncRNAs coexisted in all three LC subtypes (Figure 1(g) and Table S4), while 10 lncRNAs were downregulated (Figure 1(h) and Table S5).Figure 1
Bioinformatic analysis of dysregulated lncRNAs in different subtypes of LC. (a)–(c) Heatmap and (d)–(f) volcano plot of differentially expressed lncRNAs (DELs) between paracancer tissues and cancer tissues in TCGA LUAD project (a and d), TCGA LUSC project (b and e), and GEO SCLC dataset (c and f). (g)-(h) A Venn diagram of the upregulated (g) and downregulated (h) DELs overlapping between LUAD, LUSC, and SCLC.
(a)(b)(c)(d)(e)(f)(g)(h)We ranked the DELs of each comparison by FC or adjustedP value. We performed a Venn diagram analysis using the top 10 lncRNAs of each comparison to find the most significant DELs. Finally, FOXD3-AS1 was identified using FC and adjusted P value screening. In contrast, HOXC-AS2 was identified using adjusted P value screening (Figure 2(a) and 2(b)). When compared to normal controls (P<0.0001), the expression of FOXD3-AS1 was significantly higher in lung tumors, regardless of whether they were LUAD, LUSC, or SCLC (Figures 2(c)–2(e)). FOXD3-AS1 was selected for further research.Figure 2
FOXD3-AS1 was the most significantly upregulated lncRNA in LUAD, LUSC, and SCLC according to public datasets. Venn diagram of the intersection of top 10 DELs ranked by FC (a) and adjustedP value (b). FOXD3-AS1 expression determined in cancer specimens (tumor) and paracancer specimens (normal) using TCGA LUAD dataset (c), TCGA LUSC dataset (d), and GEO SCLC dataset (e). The t-test was used to calculated P value.
(a)(b)(c)(d)(e)


## 3.2. Participants and Clinical Characteristics of the Validation Phase

A total of 50 healthy candidates and 150 patients with LC (50 SCLC, 50 LUAD, and 50 LUSC) were enrolled in the study to verify the diagnostic potential of FOXD3-AS1. Table1 summarizes the demographic and clinical parameters. In the healthy and LC groups, sex ratios (males: females) and smoking rates were similar. According to disease characteristics, there were 53 patients with stages I-II and 97 with stages III-IV.


## 3.3. FOXD3-AS1 Expression in Tissues and Plasma Was Correlated with the LC Progression

In all LUAD, LUSC, and SCLC patients, FOXD3-AS1 expression in tumor tissues (CA) was much higher than in paracancer tissues (PC) (P<0.001, Figure 3(a)). We investigated whether FOXD3-AS1 could be secreted into circulating plasma by tumor cells because it has been shown to stimulate invasion in NSCLC [23]. As we can see, lncRNA FOXD3-AS1 was found in plasma and expressed significantly higher in LUAD, LUSC, and SCLC blood samples than in HS (P<0.001, Figure 3(b)). We also looked at how the expression of FOXD3-AS1 changed as LC progressed. In all three subtypes, FOXD3-AS1 production in blood was significantly higher in stages III-IV than in stages I-II (P<0.001, Figure 3(c)).Figure 3
FOXD3-AS1 was verified to be overexpressed in SCLC, LUAD, and LUSC tissues and plasma comparing with healthy subjects. (a) RT-qPCR assay performed to detect the expression of FOXD3-AS1 in cancer tissues and paracancer tissues from patients with SCLC, LUAD, and LUSC.P value was obtained by the t-test. ∗∗∗P<0.001. (b) RT-qPCR analysis of FOXD3-AS1 in the plasma of healthy candidates (HS) and patients with SCLC, LUAD, and LUSC. The asterisk sign indicated the comparison with the HS group, while the hash sign represented the SCLC group as the control. P value was obtained by the t-test. ∗∗∗P<0.001. ###P<0.001. (c) RT-qPCR analysis of FOXD3-AS1 in the plasma samples of patients with SCLC, LUAD, and LUSC at stages I-II (I/II) and stages III-IV (III/IV). P value was obtained by the t-test. ∗∗∗P<0.001.
(a)(b)(c)Based on the median FOXD3-AS1 expression threshold (5.045 for SCLC, 3.52 for LUAD, and 4.04 for LUSC), we divided the cancer plasma samples into low and high-FOXD3-AS1 subgroups (L-lncRNA and H-lncRNA for short). We analyzed their relationship with the clinical characteristics of the patients. As shown in Table2, there were no significant differences in gender or smoking history between the H-lncRNA and L-lncRNA groups, but the patients who expressed high levels of FOXD3-AS1 were more likely older than 65 in LUAD (P=0.047) and LUSC (P=0.022). Furthermore, a high level of FOXD3-AS1 indicated a more advanced stage of LUAD (P=0.012) and LUSC (P=0.010), a greater probability of lymph metastasis in LUAD (P=0.025) and LUSC (P<0.001), and a greater probability of distal metastasis in SCLC (P=0.032). These findings showed that the level of FOXD3-AS1 increased as LC progressed and that the fluctuating levels of FOXD3-AS1 in tissues were primarily related to the presence of disease lesions rather than factors such as gender or smoking.Table 2
Relationship between lncRNA FOXD3-AS1 expression in serum and clinicopathological parameters of patients with lung cancer.
CharacteristicsSCLCLUADLUSCH-lncRNA (n = 25)L-lncRNA (n = 25)P valueH-lncRNA (n = 25)L-lncRNA (n = 25)P valueH-lncRNA (n = 25)L-lncRNA (n = 25)P valueGender (male/female)17/818/7113/1215/100.77614/1118/70.377Age (<65 y/≥65 y)7/1814/11/0.0854/2115/200.0477/1816/90.022Smoking story (yes/no)19/617/80.75417/815/100.76913/1215/100.776Stage (I-II/III-IV)19/613/120.14022/313/120.01225/018/70.010Lymph metastasis (yes/no)20/514/110.12822/314/110.02525/010/15<0.001Distal metastasis (yes/no)12/134/210.03210/158/170.76910/154/210.114


## 3.4. Role of Plasma FOXD3-AS1 in LC Diagnosis and Identifying LC Subtypes

The diagnostic efficacy of plasma FOXD3-AS1 in three subtypes of LC was assessed using ROC analysis. FOXD3-AS1 had an AUC of 0.959 (95% confidence interval (CI): 0.926–0.992) in identifying patients with SCLC out of HS (Figure4(a) and Table 3), an AUC of 0.763 (95% CI: 0.670–0.855) in identifying patients with LUAD out of HS (Figure 4(b) and Table 3), an AUC of 0.854 (95% CI: 0.782–0.927) in identifying patients with LUSC out of HS (Figure 4(c) and Table 3), and an AUC of 0.859 (95% CI: 0.802–0.915) in identifying patients with LC out of HS (Figure 4(d) and Table 3).Figure 4
The efficiency of plasma FOXD3-AS1 in cancer diagnosis and SCLC identification. (a)–(g) ROC curves used to validate the discrimination efficiency of plasma FOXD3-AS1 for SCLC vs. HS (a), LUAD vs. HS (b), LUSC vs. HS (c), LC vs. HS (d), SCLC vs. LUAD (e), SCLC vs. LUSC (f), and LUAD vs. LUSC (g).
(a)(b)(c)(d)(e)(f)(g)Table 3
The diagnostic values of lncRNA FOXD3-AS1 in discriminating lung cancer subtypes.
IndexAUC95% CIP valueSensitivitySpecificityCritical valueYoden indexDownUpSCLC vs. HS0.9590.9260.992<0.00188.00092.0000.8003.940LUAD vs. HS0.7630.6700.855<0.00178.00064.0000.4203.195LUSC vs. HS0.8540.7820.927<0.00182.00078.0000.6003.465LC vs. HS0.8590.8020.915<0.00178.00078.0000.5603.465SCLC vs. LUAD0.8880.8220.953<0.00178.00092.0000.7004.375SCLC vs. LUSC0.8210.7370.905<0.00178.00080.0000.5804.380LUAD vs. LUSC0.6580.5520.7650.00680.00052.0000.3203.535We further looked at the discrimination efficiency of FOXD3-AS1 between them. With a sensitivity of 78% and a specificity of 92%, it created an AUC value of 0.888 (95% CI: 0.822–0.953) in distinguishing patients with SCLC from patients with LUAD (Figure4(e) and Table 3). With a sensitivity of 78% and a specificity of 80%, it created an AUC value of 0.821 (95% CI: 0.737–0.905) in distinguishing patients with SCLC from patients with LUSC (Figure 4(f) and Table 3). In contrast, with a sensitivity of 80% and a specificity of 52%, it created an AUC value of 0.658 (95% CI: 0.552–0.765) in determining patients with LUAD from patients with LUSC (Figure 4(g) and Table 3).


## 3.5. Diagnostic Accuracy of FOXD3-AS1 as Diagnostic Biomarkers to Detect Early-Stage LC

Because the expression of FOXD3-AS1 in LC was higher than in healthy people and increased as the disease progressed, we hypothesized that FOXD3-AS1 was activated early in the disease. We specifically analyzed stages I-II LC samples to estimate the efficiency of FOXD3-AS1 in the early detection of LC. FOXD3-AS1 yielded an AUC of 0.906 (95% CI: 0.831–0.982), a sensitivity of 73%, a specificity of 92%, and a Yoden index of 3.94 in distinguishing between SCLC and HS subjects (Figure5(a) and Table 4). In terms of LUAD early detection, the AUC value, sensitivity, specificity, and Yoden index was 0.591 (95% CI: 0.453–0.729), 100%, 30%, and 2.46, respectively (Figure 5(b) and Table 4). In LUSC early detection, the AUC value, sensitivity, specificity, and Yoden index was 0.686 (95% CI: 0.562–0.809), 100%, 42%, and 2.750, respectively (Figure 5(c) and Table 4). In total, FOXD3-AS1 produced an AUC value of 0.751 (95% CI: 0.658–0.845), a sensitivity of 73.6%, a specificity of 66%, and a Yoden index of 3.195 in discriminating early LC samples from HS (Figure 5(d) and Table 4).Figure 5
The efficiency of plasma FOXD3-AS1 in LC diagnosis and SCLC identification at early stage. (a)–(g) ROC curves used to validate the discrimination efficiency of plasma FOXD3-AS1 for early-stage SCLC vs. HS (a), early-stage LUAD vs. HS (b), early-stage LUSC vs. HS (c), early-stage LC vs. HS (d), early-stage SCLC vs. early-stage LUAD (e), early-stage SCLC vs. early-stage LUSC (f), and early-stage LUAD vs. early-stage LUSC (g).
(a)(b)(c)(d)(e)(f)(g)Table 4
The diagnostic values of lncRNA FOXD3-AS1 in discriminating early-stage lung cancer.
IndexAUC95% CIP valueSensitivitySpecificityCritical valueYoden indexDownUpSCLC vs. HS0.9060.8310.982<0.00173.70092.0000.6573.940LUAD vs. HS0.5910.4530.7290.275100.00030.0000.3002.460LUSC vs. HS0.6860.5620.8090.020100.00042.0000.4202.750LC vs. HS0.7510.6580.845<0.00173.60066.0000.3963.195SCLC vs. LUAD0.9010.8021.000<0.00173.700100.0000.7373.980SCLC vs. LUSC0.8570.7300.983<0.00173.700100.0000.7373.905LUAD vs. LUSC0.6740.4890.8580.08472.20062.5000.3473.240FOXD3-AS1 was also found to be effective in identifying LC subtypes SCLC. The AUC values for SCLC versus LUAD, SCLC versus LUSC, and LUAD versus LUSC were found to be 0.901 (95% CI: 0.802–1) (Figure5(e) and Table 4), 0.857 (95% CI: 0.730–0.983) (Figure 5(f) and Table 4), and 0.674 (95% CI: 0.489 0.858) (Figure 5(g) and Table 4), respectively.


## 4. Discussion

Despite the numerous diagnostic and treatment options available for LC [25, 26], it remains one of the most dangerous malignancies worldwide because it is often asymptomatic and detected late [1]. Early diagnosis is the most important factor in influencing the prognosis of patients with LC. We aimed to find an abnormally expressed lncRNA in patients with LC and investigate how effective this lncRNA is at diagnosing LC. Through bioinformatics analysis of public datasets, we discovered that FOXD3-AS1, a lncRNA, was significantly upregulated in three primary subtypes of LC, SCLC, LUAD, and LUSC. An RT-qPCR assay confirmed the finding in SCLC, LUAD, and LUSC tissues and plasma. Furthermore, ROC analysis revealed that plasma FOXD3-AS1 could be used to discriminate SCLC, LUAD, and LUSC from healthy subjects. On the other side, SCLC samples produced much more FOXD3-AS1 than LUAD or LUSC, and FOXD3-AS1 was more sensitive in discriminating SCLC from LUAD or LUSC by ROC analysis. Thus, FOXD3-AS1 would be an accurate molecular marker for the diagnosis of LC and SCLC subtype identification.Previous research found that the transcription factor specificity protein 1 promoted lymphatic invasion and distant metastasis in cervical cancer by inducing FOXD3-AS1 expression [27]. However, the factors that activate FOXD3-AS1 in LC are unknown, and more research studies are needed. FOXD3-AS1 has been shown to have predictive value and functional roles in various diseases, including breast cancer, cervical cancer, nasopharyngeal carcinoma, cerebral ischemia/reperfusion injury, cardiomyocyte injury, allergic rhinitis, and so on [15–17, 27, 28]. Furthermore, FOXD3-AS1 has been found in nuclear or cytoplasm fractions, and its molecular mechanisms include acting as competing endogenous RNA [15, 29], acting as mediators for gene promotor loci and histone modification factor interaction [19], stimulating proteins expression [20], interacting and sequestering proteins, and so on [14, 18]. In this study, we found that FOXD3-AS1 was upregulated in all primary subtypes of LC at an early stage and increased as the disease progressed. Furthermore, consistent trends in FOXD3-AS1 expression in tissues with disease progression suggest that plasma is partly derived from tumor tissues. Therefore, we have reason to believe that FOXD3-AS1 is a potential marker for the diagnosis of LC. On the contrary, Ji et al. discovered that FOXD3-AS1 expression was downregulated in NSCLC tissues and cell lines, linked to lymph node metastasis and a high tumor grade [29]. FOXD3-AS1 may thus play a variety of roles and be involved in multiple stages of LC development. Furthermore, the more aggressive expression of FOXD3-AS1 in SCLC over that in NSCLC (LUAD and LUSC) at early or all stages may be due to the fact that SCLC has a different pathological mechanism from NSCLC, such as a strong predilection for early metastasis [30].In our study, analysis displayed that FOXD3-AS1was significantly associated with tumor stage, lymph node metastasis, and distant metastasis. It is further suggested that FOXD3-AS1 may be helpful to distinguish LC subtypes. Interestingly, our results that not only did FOXD3-AS1 accurately distinguish LC patients from healthy subjects with the highest AUC but also distinguished LC subtypes very well, providing an objective basis for distinguishing among LC subtypes. Previous studies have shown that increased lncRNA levels can be used as a potential biomarker for LC detection, particularly at the early stage. For example, Li et al. reported that lncRNA GAS5 in exosomes may function as an ideal noninvasive marker for identifying patients with early NSCLC [31]. In this study, the results of ROC analysis demonstrated that FOXD3-AS1 had an advantage in the diagnostic efficiency of detecting early-stage SCLC patients. FOXD3-AS1 as a predictive biomarker in the clinical application might significantly enhance the efficacy of SCLC screening.Although the current study has improved our understanding of the relationship between FOXD3-AS1 and LC, it has some limitations. First, longitudinal cohort studies with large populations are required to determine the true value of FOXD3-AS1 in LC early diagnosis and subtype identification. Second, a logistic model based on multiple genes may be more efficient because LC is heterogeneous. Furthermore, evaluating a combination of biomarkers may offer more predictive value than assessing FOXD3-AS1 alone.


## 5. Conclusions

The overexpression of FOXD3-AS1 in LC tissues and plasmas was discovered in our study. Plasma FOXD3-AS1 has the potential to be a molecular biomarker for early diagnosis and identification SCLC subtype. Our study points to a new direction for early diagnosis of patients with LC, which may play an important role in diagnosing and treating patients with LC and prolonging their survival.



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