Aberrant expression of HIF3A in plasma of patients with non‐small cell lung cancer and its clinical significance

Abstract Background Hypoxia‐inducible factors (HIFs) have been evaluated in various cancers and diseases. However, the specific role of hypoxia‐inducible factor 3 alpha (HIF3A) in non‐small cell lung cancer (NSCLC) remains controversial. Materials and Methods We investigated HIF3A mRNA expression in the plasma and tumor tissues of patients with NSCLC and explored its clinical significance. Plasma samples from 103 cases of lung adenocarcinoma (LUAD) and 96 cases of lung squamous cell carcinoma (LUSC), and tumor‐adjacent normal tissues from 58 LUAD and 62 LUSC cases were retrospectively evaluated at the No.8 People's Hospital of Qing Dao. HIF3A expression was explored using RT‐qPCR. The clinical significance of HIF3A was evaluated in the plasma and tumor tissues using the receiver operating curve (ROC) and the area under the curve (AUC). Results Hypoxia‐inducible factor 3 alpha expression was notably downregulated in the plasma or tumor tissues of patients with LUAD and LUSC, compared with the healthy control group or adjacent normal tissues. Furthermore, HIF3A expression had a significant positive correlation in the plasma and tumor tissues of LUAD and LUSC patients. Meanwhile, the ROC‐AUCs achieved a significantly higher range, from 0.84 to 0.93, with the plasma or tumor tissues of NSCLC patients. Thus, HIF3A expression was not only correlated with plasma and tumor tissues, but also showed potential significance in NSCLC. Conclusion Hypoxia‐inducible factor 3 alpha is aberrantly detectable in NSCLC patients in the plasma and tumor tissues. HIF3A may be involved in hypoxic responses during the development and occurrence of NSCLC.

HIF3A expression was explored using RT-qPCR. The clinical significance of HIF3A was evaluated in the plasma and tumor tissues using the receiver operating curve (ROC) and the area under the curve (AUC).
Results: Hypoxia-inducible factor 3 alpha expression was notably downregulated in the plasma or tumor tissues of patients with LUAD and LUSC, compared with the healthy control group or adjacent normal tissues. Furthermore, HIF3A expression had a significant positive correlation in the plasma and tumor tissues of LUAD and LUSC patients. Meanwhile, the ROC-AUCs achieved a significantly higher range, from 0.84 to 0.93, with the plasma or tumor tissues of NSCLC patients. Thus, HIF3A expression was not only correlated with plasma and tumor tissues, but also showed potential significance in NSCLC.
Conclusion: Hypoxia-inducible factor 3 alpha is aberrantly detectable in NSCLC patients in the plasma and tumor tissues. HIF3A may be involved in hypoxic responses during the development and occurrence of NSCLC.

K E Y W O R D S
HIF3A, hypoxia, mRNA gene, non-small cell lung cancer, plasma persists due to the inability to diagnose to condition at early stages or treat advanced conditions surgically. 2,3 Consequently, research into novel diagnostic biomarkers, target genes, and therapeutic target structures of NSCLC has attracted the attention of researchers.
Hypoxia-inducible factors (HIFs) have been evaluated in various cancers and diseases. 4 HIFs are heterodimers that include HIF α (oxygen-labile) and HIF β subunits. 4,5 Three distinct HIF genes are found in humans and other vertebrates, but HIF signaling is acquired from variants of HIF-1 α and HIF-2 β. [6][7][8][9][10] Meanwhile, the functions of the HIF-3 complex are because of existing alternative spliced variants of HIF3A (hypoxia-inducible factor 3 alpha, also known as HIF-3 α), which result in four dissimilar variants of mRNA that code for additional isoforms by using different promoters. 11 The effect of suppression on HIF-1 and HIF-2 activity is represented by the factors embracing the α-3 subunit, which are anti-regulatory for hypoxia-induced expression of genes. 12,13 There is much to discover about HIF3A compared with HIF1A and HIF2A. Previous studies have discovered the tumor-suppressive activities of HIF1A and HIF2A in renal cell carcinoma and lung cancer. 4 However, the specific role of HIF3 in NSCLC remains controversial. The current study aimed to evaluate HIF3A mRNA expression in the plasma and tumor tissues of patients with LUAD and LUSC. Furthermore, this study aimed to evaluate the diagnostic value of HIF3A in NSCLC.

| RNA isolation
Total RNA was extracted using phenol-chloroform solutions and then homogenized using guanidine isothiocyanate (Trizol RNA Preparation kit; Invitrogen). RNA concentration was evaluated using a NanoDrop spectrophotometer ND1000 (NanoDrop Technologies Inc.).

| RT-qPCR
The total RNA in the plasma and tissue samples after transfection was extracted with TRIzol reagent, according to the manufacturer's instructions. Thereafter, the total RNA was reversetranscribed into cDNA using a reverse transcription kit (Shanghai Sangon Biological Engineering Co., Ltd.). The primers used were as follows: HIFA3,5′-F: AGGCGCCAGAGGCACCATGGAC-3′, R: 5′-CATCCTGTGCGTTGGCTGCC-3′,U6 F: 5′-GAAGGTGAAG GTCGGAGTC-3′, R: 5′-GAAGATGGTGATGGGATTT-3′. The PCR reaction conditions were as follows: A: pre-denaturation at 95°C for 10 min, B: denaturation at 95°C for 15 s, annealing at 60°C for 15 s, and elongation at 72°C for 20 s, for a total of 40 cycles; and C: 72°C for 15 min. The reaction was terminated at 4°C. Three replicates were set for each sample, and 2 −△△Ct was used for the relative quantitative analysis of the data.

| Bioinformatics
Bioinformatics were performed using GEPIA 2(http://gepia2.cance r-pku.cn/). The data used were from TCGA normal and GTEx data. SPSS 20.0 (SPSS Inc.) was used for all statistical analyses in the study, and data were expressed as mean ± SD. Clinical information of the patients in Tables 1 and 2 was analyzed by chi-square test.

| Statistical analysis
A t test was performed to compare the two groups, and one-way ANOVA was used to compare multiple groups. Pearson's correlation was used for the correlation analysis. The clinical significance of HIF3A was evaluated in plasma and tumor tissues using the receiver operating curve (ROC) and the area under the curve (AUC).
Statistical significance was set at p < 0.05.  (Table 1 and Table 2).

| HIF3A expression in the tumor tissues and plasma of NSCLC patients
Bioinformatics tools were used to analyze HIF3A expression be-   Figure 2A), and HIF3A mRNA expression was significantly lower in plasma samples from 103 LUAD patients and 96 LUSC patients compared with healthy recruits (Figure 3A,B). Thus, HIF3A expression was significantly downregulated in the tumor tissues or plasma samples of NSCLC patients.

| Correlation of HIF3A between plasma and tumor tissues
The correlation between plasma and tumor tissues with respect to Therefore, the expression level of HIF3A was directly correlated between the plasma and tumor tissues of NSCLC patients.

| Efficacy of HIF3A in NSCLC
The efficacy of detection of HIF3A was reported using the ROC-

| CON CLUS ION
Hypoxia-inducible factor 3 alpha is aberrantly detectable in plasma and tumor tissues of NSCLC patients and is a potentially discoverable target gene that might play an effective role in LUAD and LUSC. HIF3A expression was notably downregulated in the plasma or tumor tissues of LUAD and LUSC patients compared with the plasma of the healthy control group or adjacent tumor tissues. The present study validated the expression of HIF3A and its clinical significance in NSCLC. HIF3A may be involved in hypoxic responses during tumor occurrence and development.

ACK N OWLED G M ENTS
Nothing to declare.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no potential conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.