Isoginkgetin inhibits lung cancer cell progression through miR-27a-5p/APEX1 axis

Purpose: To determine the influence of isoginkgetin on the progression of pulmonary carcinogenesis. Methods: A549 cells exposed to isoginkgetin were transfected with miR-27a-5p mimetic and suppressor, as well as short hairpin RNA against apurinic/apyrimidinic endo-deoxyribonuclease 1 (sh-APEX1) for 24 h. Then, the proliferative, migration and invasive potential of A549 cells were determined using CCK-8, wound healing and Transwell assays, respectively. The association between miR-27a-5p and APEX1 was determined by dual-luciferase reporter assay. Results: Isoginkgetin significantly suppressed A549 cell proliferative, migration and invasive activities (p < 0.05). Moreover, isoginkgetin significantly increased miR-27a-5p expression. Furthermore, miR-27a-5p suppressor annulled the influence of isoginkgetin on lung cancer progression. More importantly, the inhibitor directly targeted APEX1, and negatively modulated APEX1 expression (p < 0.05). However, APEX1 knockdown annulled the enhancing effect of miR-27a-5p inhibitor on A549 cell viability, migration and invasion (p < 0.05). Conclusion: Isoginkgetin exerts an anti-lung cancer effect via miR-27a-5p/APEX1 axis. Thus, it is a potential therapy for the management of lung cancer; however, clinical studies are required to validate these findings of this study.


INTRODUCTION
Pulmonary neoplasm is the most frequently diagnosed carcinoma worldwide, and it occupies the first position, both in new cancer cases and cancer-related deaths [1]. Earlystage lung cancer often presents no obvious symptoms. Since there are no obvious clinical features in the early stage, many cases are not diagnosed until the disease has advanced [2]. In addition, tumor cell migration and invasion are the main factors that affect the improvement of survival rate after lung cancer treatment [3]. Therefore, it is very important to identify and develop new anti-tumor drugs that inhibit lung cancer metastasis.
Several achievements have been made in the research and development of traditional Chinese medicines with anti-cancer potential [4]. However, at present, most of the studies are based on compound therapy. Although traditional Chinese medicines produce curative effects, the mechanisms involved in the use of traditional Chinese medicines for treating lung cancer are intricate. This limits their clinical application and promotion.
Ginkgo biloba is a traditional Chinese medicine which has been used for treating several cancers in China for thousands of years [5]. Evidence has shown that Ginkgo biloba exocarp extracts suppressed lung cancer growth and metastasis through induction of apoptosis and autophagy, and suppression of angiogenesis [6]. Isoginkgetin belongs to a family of flavonoids extracted from Ginkgo biloba leaves, and it exerts a variety of pleiotropic and pharmacological actions, including anti-cancer and anti-inflammatory effects [7]. However, the influence of isoginkgetin on pulmonary neoplasm has not been reported. The present research was focused on understanding the influence of isoginkgetin on growth, migration and spread of pulmonary carcinoma cells.

Cell treatment and transfection
To determine the effect of isoginkgetin on cell viability, graded doses of isoginkgetin (Thermo Fisher Scientific, USA) were applied in treating A549 cells for 24 h.

CCK-8 assay
Following plating of A549 cells for 72 h and 2-h incubation with CCK-8 solution (Beyotime, China), OD was read at 490 nm in a spectrophotometer (Bio-Rad, China).

Wound healing studies
The A549 cells were seeded at a density of 5 × 10 5 cells /well in 6-well plates. When the cells grew to 85 % confluence, a wound was made on the surface of the cells with a 200-µL sterile pipette tip (Axygen, USA). Then, the A549 cells were cultured without FBS for 48 h. The distance between both sides of the wound was recorded using an inverted microscope.

Transwell assay
In migration assay, A549 cells were plated in upper transwell chamber, and cultured in F-12K medium (ATCC, USA) without FBS. In invasion assay, A549 cells were plated in upper transwell compartment pre-surfaced with Matrigel. The lower transwell chamber contained F-12K medium (ATCC, USA) with 20 % FBS (Invitrogen, USA). Forty-eight hours later, the migrated and invaded cells were subjected to fixation in 4 % ρ-HCHO, and were stained using crystal violet (0.1 %).

Quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR)
Total RNA extraction from A549 cells was done with TRIzol. Then, the RNA was subjected to reverse-transcription to yield cDNA, followed by RT-qPCR assay with PCR kit (Qiagen, Germany). Relative expressions of genes were evaluated using the 2 −ΔΔCt procedure. The internal standards were U6 and GAPDH. Table 2 shows the primers used in qRT-PCR.

Immunoblot assay
Total proteins were isolated from A549 cells with RIPA buffer, and protein levels in samples were measured with BCA method. Then, the protein samples were subjected to 10 % SDS-PAGE, followed by transfer to PVDF membranes. Following membrane blocking with non-fat milk, the protein bands were subjected to 12-h incubation with 1 o antibodies for APEX1 (1:1000, ab189474, Abcam, USA) and GAPDH (1:5000, ab8245, Abcam, China) at 4 o C. Thereafter, the protein bands were incubated for 60 min with the related secondary antibodies, followed by visualization with ECL, and semi-quantification with ImageJ software. The internal reference was GAPDH.

Statistics
Data were analyzed with Prism GraphPad 5.0 and presented as mean ± SD. Statistically significant variations between two groups were assessed using t-test (two-tailed), while 1-way ANOVA and Tukey's post hoc test were employed for multi-group comparisons. Values of p < 0.05 were considered indicative of statistical significance.

Isoginkgetin suppressed cell proliferation and metastasis
Isoginkgetin treatment suppressed A549 cell proliferation, with 20 µM isoginkgetin producing the largest effect (Figure 1 A). Compared to the control group, 20 µM isoginkgetin group had marked inhibition of A549 cell migration (Figure 1 B and C). Transwell assay revealed that, compared to the control group, 20 µM isoginkgetin group had marked suppression of migratory and invasive potential of A549 cells ( Figure 1 D and E). The aforementioned results showed that isoginkgetin suppressed the worsening of lung cancer. An important target of traditional Chinese medicine is miRNA [8]. Therefore, this study determined whether isoginkgetin regulated lung cancer cell malignancy through regulation of miR-27a-5p. The results from RT-qPCR assay showed that, compared to lung epithelial cell line BEAS-2B, miR-27a-5p level was significantly decreased in pulmonary cancer cell line A549. Moreover, compared to untreated A549 group, isoginkgetin treatment suppressed miR-27a-5p level in A540 cells (Figure 2 A). Relative to control, there was significantly suppressed A549 cell viability in isoginkgetin + NC inhibitor group.  CTCGCTTCGGCAGCACA  AACGCTTCACGAATTTGCGT  APEX1  AGTAAGACGGCCGCAAAGAA  TTGCCACTGGGTGAGGTTTT  CLEC4E  CAACTCACAGGAGGAGCAGG  AGCTATGTTGTTGGGCTCCC  FSD2  AGCGACACTCACTTCACCAG  AGGAGAGGCAATTTGCTCCC  TMEM199  AACCCAGAACTAGTTGCCCG  CAGCAACCACCGTGACAATG  GAPDH GTCAAGGCTGAGAACGGGAA AAATGAGCCCCAGCCTTCTC In addition, relative to isoginkgetin + NC inhibitor group, isoginkgetin + miR-27a-5p inhibitor group had enhanced A549 cell viability (Figure 2 B). Moreover, miR-27a-5p inhibitor reversed the inhibitory influence of isoginkgetin on the migratory and invasive potential of A549 cells (Figure 2 C-F). Overall, isoginkgetin suppressed the deterioration of lung cancer through upregulation of miR-27a-5p.

MiR-27a-5p directly targeted APEX1 in lung cancer
Online databases miRDB, TargetScan and miRTarbase were employed for determination of intersecting target genes. Venn diagram showed 4 common elements (APEX1, CLEC4E, FSD2 and TMEM199) in online databases (Figure 3 A). The results of qRT-PCR assay showed that APEX1 level in A549 cells was upregulated, while isoginkgetin decreased APEX1 level, but had no effects on the expressions of CLEC4E, FSD2 and TMEM199 (Figure 3 B).

Isoginkgetin inhibited the proliferative and metastatic activities of A549 cells via the miR-27a-5p/APEX1 route
In order to determine whether isoginkgetin regulated lung cancer cell progression through miR-27a-5p/APEX1 route, cells were exposed to isoginkgetin and subjected to co-transfection with miR-27a-5p inhibitor and sh-APEX1 for 24 h. The inhibitor suppressed APEX1 expression, but knockdown of APEX1 increased APEX1 expression (Figure 4 A).
Functionally, knockdown of APEX1 reversed the miR-27a-5p inhibitor-induced enhancement of proliferative and invasive potential of A549 cells (Figures 4 B -F). Collectively, these data suggest that isoginkgetin suppressed lung carcinoma proliferation and metastasis through the miR-27a-5p/APEX1 axis.

DISCUSSION
Although significant advances have been made in lung cancer therapy, including surgery, radiotherapy, chemotherapy, molecular targeting and immunotherapy, the prognosis of lung cancer still remains poor [9]. Chemotherapy drugs are expensive and have very toxic side effects. In contrast, traditional Chinese medicines have the advantages of multiple targets, low toxic side effects, wide sources and reliable effects, and they are often used as adjuvant therapies for lung cancer [3].
The effects produced by monomers and derivatives of natural Chinese medicine in the prevention of lung cancer are gradually being recognized [10]. For instance, Lin et al found that resveratrol from the traditional Chinese medicine Polygonum cuspidatum increased the chemosensitivity of lung cells to cisplatin, and suppressed lung cancer progression through reduction in MUC5AC level [10]. Thus, it is crucial to study the molecular bases associated with the anti-cancer effects of bioactive components of traditional Chinese medicines. One of the fundamental measures for suppressing the incidence and spread of tumors involves controlling the growth, spread and invasion of lung carcinoma cells [11].
Isoginkgetin exerts anti-cancer and antiinflammation properties [12,13]. For instance, Yoon et al reported that isoginkgetin inhibited human fibrosarcoma cell growth by regulating the PI3K/Akt-dependent MMP-9 expression [12]. Besides, Tsalikis et al found that isoginkgetin sensitized cancer cells to apoptosis by disrupting lysosomal homeostasis and impairing protein clearance [13]. It has been reported that isoginkgetin enhanced immune-protective response against tumor antigens [14]. In the present study, isoginkgetin suppressed the deterioration of lung cancer. The miRNAs comprise small, highly conserved non-coding RNAs which bind to the 3'-UTR area of downstream mRNAs [15]. Many miRNAs with abnormal expressions in lung cancer participate in the occurrence and spread of lung cancer. It is well known that miRNA is an important target of traditional Chinese medicine [16]. High throughput sequencing has revealed several abnormally expressed miRNAs in curcumintreated lung cancer cells [16]. Therefore, the development of drugs targeting miRNAs is promising for the treatment of lung cancer through regulation of miRNA expressions. Studies have revealed that miR-27a-5p is downregulated in pulmonary neoplasms, a finding which suggests that it might be a promising biomarker for early diagnosis [17]. Furthermore, it has properties of a tumor suppressor gene: its overexpression significantly reduced the proliferation of pulmonary carcinoma cells [17]. In this work, the expression of miR-27a-5p was increased by isoginkgetin, but miR-27a-5p blocker reversed the inhibitory influence of isoginkgetin on lung cancer progression. These results indicate that miR-27a-5p is a potential target for isoginkgetin. The use of online databases for analysis of intersecting target genes revealed that miR-27a-5p directly targeted APEX1 and negatively modulated APEX1 expression. Apurinc/Apyrimidinic Endo nuclease 1 (APEX1) is associated with DNA damage response and redox reaction [18]. Inhibition of APEX1 expedited DNA damage response in lung cancer [18]. Previous studies demonstrated that APEX1 was abnormally expressed in a variety of tumors, and was associated with the pathogenesis of cancers [19,20]. For instance, Huang et al found that APEX1 level was increased in melanoma, and APEX1 overexpression enhanced the development of melanoma [19]. Wang et al showed that miR-296-3p inhibited lung cancer progression through down-regulation of APEX1 [20]. This research has demonstrated that APEX1 knockdown blocked miR-27a-5p suppressor-induced enhancement of the proliferative, migratory and invasive potential of the cells.

CONCLUSION
Isoginkgetin regulates miR-27a-5p/APEX1 axis, thereby suppressing lung cancer growth and metastasis. This finding indicates that miR-27a-5p/APEX1 axis may be an effective target for lung cancer treatment, thus providing a potential strategy for the treatment of lung cancer.

Acknowledgements
None provided.

Funding
None provided.

Ethical approval
None provided.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.