Hsa_circ_0006427 Suppresses Multiplication, Migration and Invasion of Non-Small Cell Lung Cancer Cells through miR-346/VGLL4 Pathway.

Objective: Circular RNAs (circRNAs) are identified as key modulators in cancer biology. Nonetheless, the role of circ_0006427 in non-small cell lung cancer (NSCLC) and its modulatory mechanism are undefined. This study aimed to investigate the potential function and mechanism of circ_0006427 in NSCLC. Materials and
Methods: In this experimental study, circ_0006427, miR-346 and vestigial like family member 4 (VGLL4) mRNA expressions were analyzed in NSCLC tissues and cells, using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Multiplication, migration and invasion of NSCLC cells were examined using the CCK-8 method and Transwell experiment, respectively. Dual-luciferase reporter gene experiments were conducted to identify the paring relationship between circ_0006427 and miR-346. Western blot was employed to determine expressions of VGLL4 and epithelial-mesenchymal transition (EMT) markers on protein levels. Immuno-histochemistry (IHC) method was adopted to assess VGLL4 protein expression in NSCLC tissues.
Results: Circ_0006427 expression was down-regulated in NSCLC tissues and cells, and circ_0006427 suppressed multiplication, migration, invasion and EMT of NSCLC cells. miR-346 expression was upregulated in NSCLC tissues and cells, and miR-346 worked as a target of circ_0006427. VGLL4 was down-regulated in NSCLC tissues and cells, and knockdown of VGLL4 accelerated multiplication, migration, invasion and EMT of NSCLC cells. Circ_0006427 enhanced VGLL4 expression by competitively binding with miR-346.
Conclusion: Circ_0006427/miR-346/VGLL4 axis regulated NSCLC progression. Copyright© by Royan Institute. All rights reserved.

Introduction

Lung cancer takes up approximately 20% of all cancerrelated deaths (1). Non-small cell lung cancer (NSCLC) is the main pathological type of lung cancer, composed of about 85% of all lung cancer cases (2). Despite the significant diagnostic and therapeutic progress, 5-year overall survival rate of NSCLC patients is still below 20%, due to metastasis and recurrence of malignancy (3). Therefore, it is imperative to better comprehend the underlying molecular mechanisms involved in NSCLC progression to develop novel and effective therapies and to improve prognosis.

Circular RNA (circRNA) is a kind of non-coding RNA (ncRNA) widely found in eukaryocytes (4). Unlike linear RNAs, circRNAs are covalently closed loop molecules that do not have a 5’ or 3’ end; so they are resistant to degradation and more stable than linear RNAs (5). circRNAs not only function as competitive endogenous RNAs (ceRNAs) to modulate the availability of microRNAs (miRNAs), but also modulate gene transcription and translation via interacting with proteins (6-8). Reportedly, circRNA partakes in regulating multiplication, cell cycle, apoptosis and metastasis of cancer cells (9-11). Circ_0006427 is a newly discovered circRNA, linked to tumorigenesis. Its expression is downregulated in lung adenocarcinoma tissues and cell lines, and it can impede disease progression by modulating miR-6783-3p/DKK1 axis (12). Nevertheless, function of circ_0006427 in NSCLC progression and the potential mechanisms remain to be further elucidated.

miRNAs are crucial regulators in cancer biology (13, 14). miR-346 is reported to accelerate hepatocellular carcinoma progression by suppressing BRMS1 expression (15). Furthermore, expression of miR-346 is validated to be upregulated in NSCLC, and miR-346 facilitates growth and metastasis of NSCLC cells by modulating the XPC/ERK/Snail/E-cadherin pathway (16). Vestigiallike family member 4 (VGLL4) is a tumor suppressor that competitively binds to TEA domain-containing transcription factors (TEADs) to block its interaction with Yes kinase-associated protein (YAP), and suppresses lung cancer progression (17, 18). In this study, we aimed to investigate the effect of circ_0006427 on NSCLC cell proliferation, migration and invasion, and to explore the mechanism of its interaction with the miR-346/VGLL4 axis in NSCLC.

Materials and Methods

Clinical specimens

In this experimental study, NSCLC tissues and matched paracancerous tissues (n=50) were obtained from Zhejiang hospital (Hangzhou, China). All subjects were pathologically diagnosed after surgery and all of the patients signed the informed consent form. This study was endorsed by the Research Ethics Committee of Zhejiang Hospital (ZJH20170133), and the procedures were designed according to the guidance of the Declaration of Helsinki. Tissue samples were obtained during surgery and preserved in liquid nitrogen until RNA extraction.

Cell culture and transfection

Normal human bronchial epithelial cell (HNBEC) line, BEAS-2B, and lung cancer cell lines (L9981, A549, H292, NCI-H460, and H460) were obtained from the China Center for Type Culture Collection (CCTCC, Wuhan, China). All cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Hyclone, USA) containing 10% fetal bovine serum (FBS, Sijiqing, China), 100 U/ml penicillin and 100 U/ml streptomycin (Hyclone, USA) at 37˚C in 5% CO2.

For circ_0006427 overexpression, the pcDNA 3.1 vectors (pcDNA, Invitrogen, USA), containing the fragment of circ_0006427, were constructed by RiboBio (Guangzhou, China), namely circ_0006427/pcDNA 3.1 (circ_0006427). shRNA against VGLL4 (VGLL4 shRNA, sh-VGLL4) and the corresponding negative control (sh-NC), as well as miR-346 mimic and the negative control (NC-mimic) were available from GenePharma (Shanghai, China). NSCLC cells were transfected using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s instructions.

Quantitative reverse transcription polymerase chain reaction

Total RNA was extracted from NSCLC tissues and cells using TRIzol reagent (Takara, Japan). Next, 1 µg total RNA was reversely transcribed into cDNA using PrimeScript RT Master Mix (Takara, Japan). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was executed on ABI 7500 system (Applied Biosystems, USA) employing TransStart Tip Green qPCRSuperMix (Transgen Biotech, China). β-ACTIN was the endogenous control for circ_0006427 and VGLL4, while U6 was the endogenous control for miR-346. 2-ΔΔCt method was utilized to calculate relative expression of the genes. The specific primer sequences were as follows:

circ_0006427-

F: 5ˊ-ACAGCTACCGGATGAATGCT-3ˊ

R: 5ˊ-AGGGCTTCAGCTGTTTCTCA-3ˊ

VGLL4-

F: 5ˊ-AACTGCAACCTCTCGCACTG-3′

R: 5ˊ-GCTCGGGCTCCTTGTAATTCT-3′

miR-346-

F: 5ˊ-TGTCTGCCCGCATGCCT-3′

R: 5ˊ-AGTGCAGGGTCCGAGGTATT-3′

β-ACTIN-

F: 5ˊ-CTGTCACCTTCACCGTTCCAGTTT-3ʹ

R: 5ˊ-AGGGGCCATCCACAGTCTTC-3ʹ

U6-

F: 5ˊ-GCTTCGGCAGCACATATACTAAAAT-3ʹ

R: 5ˊ-CGCTTCACGAATTTGCGTGTCAT-3ʹ

Subcellular fractionation assay

Cytoplasmic and nuclear RNA purification kit (Thermo Fisher Scientific, USA) was used to isolate and extract cytoplasmic and nuclear RNA from A549 and H292 cells. Then, expression of circ_0006427 in the cytoplasm and nucleus of A549 and H292 cells was measured by qRTPCR. β-ACTIN and U6 were used as cytoplasmic and nuclear controls, respectively.

Cell multiplication experiment

NSCLC cells were transfected for 48 hours, and then their cell viability was detected by cell counting kit-8 (CCK-8, Beyotime, China) assay. In brief, NSCLC cells were inoculated in 96-well plates (3×103 cells/well). After 12 hours, 24 hours, 48 hours, 72 hours and 96 hours of culture, 10 μl CCK-8 solution was supplemented to each well and incubated at 37°C for 2 hours. Cell viability of NSCLC cells was subsequently assessed at 450 nm using a spectrophotometer (Bio-Rad, USA).

Transwell experiment

NSCLC cells were resuspended with fresh medium containing 1% FBS. To detect cell migration, NSCLC cells were inoculated in the upper compartment of the Transwell system (Costar, USA). DMEM containing 10% FBS was replenished to the bottom compartment. After culturing the cells were for 24 hours, the remained NSCLC cells on the top surface of filter were removed by a cotton swab. The cells on the below surface of filter were fixed and stained, followed by observing under a microscope. To detect cell invasion, the filters were pre-coated with Matrigel (Millipore, USA) before the inoculation of cells, and the other procedures were performed the same as the migration assay.

Western blot

NSCLC cells were lysed in RIPA lysis buffer (Beyotime, China). The proteins were separated via SDS-PAGE and electrophoretically transferred to PVDF membranes (Millipore, USA). The membranes were then blocked with 5% skimmed milk for 2 hours and they were incubated with the primary antibodies including anti-VGLL4 (Abcam, China, 1:1000), E-cadherin (Abcam, China, 1:1000), Vimentin (Abcam, China, 1:1000), and β-ACTIN (Abcam, China, 1:1000) at 4°C overnight, respectively. Following that, the membranes were rinsed with TBST buffer and incubated with horseradish peroxidase (HRP)- coupled secondary antibodies (Beyotime, China) for 2 hours. Ultimately, development of protein bands was performed with ECL kit (Amersham Pharmacia Biotech, UK), and the grayscale values of each protein were analyzed using Image J.

Bioinformatics analysis

The binding sites between circ_0006427 and miR-346, or miR-346 and VGLL4 were predicted using CircInteractome database (https://circinteractome.nia. nih.gov/) and StarBase (http://starbase.sysu.edu.cn/) database.

Dual-luciferase reporter experiment

The wild-type (WT) or mutant (MUT) circ_0006427 and VGLL4 3ˊUTR sequences containing the binding site for miR-346 were cloned into the pGL3 vector (Promega, USA) to construct the reporter vectors: circ_0006427-WT, VGLL4-WT, circ_0006427-MUT and VGLL4-MUT. A549 and H292 cells were co-transfected with miR-346 mimics or NC-mimics with the reporter vectors, respectively. 48 hours after the transfection, luciferase activity of the cells in each group was examined with a dual-luciferase reporter gene reporter system (Promega, USA).

IHC experiment

In brief, samples were fixed in formalin and embedded in paraffin. Xylene was used for paraffin section dewaxing. After rehydrating the sections, antigen repair was performed. Next, the sections were incubated with anti-VGLL4 antibody (Abcam, China, 1:100) overnight at 4°C. After rinsing with PBS 5 times, secondary antibody IgG (Beyotime, China, 1:400) was supplemented, with which the sections were incubated for 30 minutes. Then, the sections were rinsed with PBS three times. Next, the tissue sections were stained with DAB solution. Finally, staining intensity was photographed using an Olympus BX51 microscope (Olympus, Japan) and scored by two independent pathologists. The scoring system was based on the staining intensity and staining extent. The staining intensity was classified as 0 (negative), 1 (weak), 2 (medium) and 3 (strong). The degree of staining extent depended on the percentage of positive cells, classified into 0 (<5%), 1 (5-25%), 2 (26-50%), 3 (51-75%) and 4 (>75%). Then, these two scores were multiplied and IHC results were classified as 0-3 (negative) and 4-12 (positive).

Statistical analysis

Each experiment was conducted in triplicate. Statistical analysis was implemented using SPSS (version 25.0, SPSS Inc., USA). All data were expressed as mean ± standard deviation (SD). Student’s t tests or one-way ANOVA and post-hoc Dunnett’s test were applied to analyze the differences. Pearson’s correlation analysis was employed to analyze the correlations among circ_0006427, miR-346 and VGLL4 in NSCLC samples. Correlations between circ_0006427 expression and clinicopathological parameters were analyzed using the Chi-square test. P<0.05 was considered to be statistically significant.

Results

circ_0006427 was downregulated in NSCLC tissues and cells

To assess expression of circ_0006427 in NSCLC tissues and cells, qRT-PCR was employed. The data displayed that circ_0006427 was markedly downregulated in NSCLC tissues relative to that in paracancerous tissues (Fig .1A). Similarly, circ_0006427 expression was unveiled to be downregulated in NSCLC cell lines compared to that in HNBEC line BEAS-2B (Fig .1B). Furthermore, the subcellular distribution of circ_0006427 was analyzed. Findings showed that circ_0006427 was mainly located in the cytoplasm of A549 and H292 cells (Fig .1C). Additionally, the relationship between circ_0006427 expression and clinicopathologic indicators of NSCLC was analyzed by the Chi-square test. The data manifested that reduced level of circ_0006427 in NSCLC tissues was associated with larger tumor size and positive lymph node metastasis (Table S1, See Supplementary Online Information at www.celljournal.org).

Fig.1

circ_0006427 was down-regulated in NSCLC. A. circ_0006427 expression in NSCLC tissues and paracancerous tissues was examined by qRT-PCR. B. circ_0006427 expression in NSCLC cells and BEAS-2B cells was detected by qRT-PCR. C. Subcellular fractionation assay was used to measure the expression levels of circ_0006427, U6 and β-ACTIN in the nuclear and cytoplasm of A549 and H292 cells. All experiments were repeated thrice with triplicate samples in each experiment. *; P<0.05, NSCLC; Non-small cell lung cancer, and qRT-PCR; Quantitative reverse transcription polymerase chain reaction.

Upregulation of circ_0006427 expression repressed multiplication, migration, invasion and EMT of NSCLC cells

To elucidate the biological function of circ_0006427 in NSCLC, A549 and H292 cells with the circ_0006427 lowest expression were transfected with over-expressed circ_0006427 plasmid to construct an overexpressed model of circ_0006427 (Fig .2A). The multiplication of NSCLC cells was examined by CCK-8 method, and cell migration and invasion were detected by Transwell experiment. The data implied that upregulation of circ_0006427 remarkably impeded the multiplication, migration and invasion of NSCLC cells (Fig .2B-F). Moreover, EMT-related protein expression in NSCLC cells was assessed by Western blot, and the data unmasked that upregulation of circ_0006427 markedly augmented E-cadherin protein expression and inhibited Vimentin protein expression (Fig .2G, H). These data suggested that circ_0006427 overexpression impeded multiplication, migration, invasion and EMT process of NSCLC cells.

Fig.2

circ_0006427 overexpression restrained multiplication, migration, invasion and EMT of NSCLC cells. A. circ_0006427 expression in A549 and H292 cells after circ_0006427 overexpression was detected using qRT-PCR. B. The CCK-8 method was employed to detect NSCLC cell multiplication. C-F. Transwell experiments were utilized to detect NSCLC cell migration and invasion. G and H. Western blot was performed to determine E-cadherin and Vimentin protein expressions in NSCLC cells. Original blots are shown in Figure S2 (See Supplementary Online Information at www.celljournal.org), Figure 2G. All experiments were repeated thrice with triplicate samples in each experiment. *; P<0.05, EMT; Epithelial-mesenchymal transition, NSCLC; Non-small cell lung cancer, and NC; Negative control.

circ_0006427 acted as a molecular sponge for miR-346 in NSCLC cells

The potential binding sites between circ_0006427 and miR-346 were projected by bioinformatics analysis (Fig .3A). Subsequently, the relationship between circ_0006427 and miR-346 was examined by dualluciferase reporter gene experiments, the data of which demonstrated that miR-346 mimics substantially diminished the luciferase activity of circ_0006427 WT reporter, while did not notably repress the luciferase activity of circ_0006427 MUT reporter (Fig .3B). Moreover, miR-346 was remarkably overexpressed in NSCLC tissues and cell lines (Fig .3C, D). Findings of qRT-PCR uncovered that miR-346 expression was markedly downregulated by circ_0006427 overexpression in NSCLC tissues (Fig .3E). Additionally, circ_0006427 expression in NSCLC tissues was negatively associated with miR-346 expression (Fig .3F). Collectively, these data implied that circ_0006427 negatively regulated miR-346 in NSCLC.

Fig.3

miR-346 was target of circ_0006427. A. Bioinformatics analysis predicted binding sequence between miR-346 and circ_0006427. B. Dual-luciferase reporter gene experiments were employed to verify binding relationship between miR-346 and circ_0006427. C, D. miR-346 expression in NSCLC tissues and cells was detected by qRT-PCR. E. miR-346 expression in NSCLC cells overexpressing circ_0006427 was detected by qRT-PCR. F. Pearson’s correlation analysis was adopted to analyze the correlation between miR-346 expression and circ_0006427 expression in NSCLC tissues. All experiments were repeated thrice with triplicate samples in each experiment. *; P<0.05, NSCLC; Non-small cell lung cancer, qRT-PCR; Quantitative reverse transcription polymerase chain reaction, WT; Wild type, MUT; Mutant, NC-mimics; Mimics negative control, and NC; Negative control.

miR-346 overexpression reversed the functions of circ_0006427 in NSCLC cells

To delve into whether circ_0006427 is involved in NSCLC progression through repressing miR-346 expression, miR-346 mimics or NC-mimics were transfected into overexpressed circ_0006427 cells, A549 and H292 cells respectively (Fig .4A). Cell multiplication, migration and invasion were further examined by CCK-8 method and Transwell experiment. Findings demonstrated that suppressing effects of circ_0006427 overexpression on cell multiplication, migration and invasion were partially attenuated by miR-346 mimics (Fig .4B-D). Furthermore, Western blot demonstrated that E-cadherin expression was decreased and Vimentin expression was increased in circ_0006427+NC-mimics group remarkably compared to those in circ_0006427+miR-346 group (Fig .4E, F). These results substantiated that circ_0006427 participated in modulating multiplication, migration, invasion and EMT of NSCLC cells through suppressing miR-346 expression.

Fig.4

circ_0006427 targeted miR-346 and modulated multiplication, migration, invasion and EMT of NSCLC cells. A. miR-346 expression in A549 and H292 cells transfected with circ_0006427 overexpression plasmid or co-transfected with miR-346 mimics was detected by qRT-PCR. B. CCK-8 assay was utilized to detect the multiplication of NSCLC cells after the transfection. C, D. Transwell experiments were exploited to detect migration and invasion of NSCLC cells after the transfection. E and F. Western blot was applied to determine E-cadherin and Vimentin protein expressions in NSCLC cells. Original blots are shown in Figure S2 (See Supplementary Online Information at www.celljournal.org), Figure 4E. All experiments were repeated thrice with triplicate samples in each experiment. *; P<0.05, EMT; Epithelial-mesenchymal transition, NSCLC; Non-small cell lung cancer, qRT-PCR; Quantitative reverse transcription PCR, NC; Negative control, and NC-mimics; Mimics negative control.

Knockdown of VGLL4 enhanced NSCLC cell multiplication, migration, invasion and EMT

As a tumor suppressor protein, VGLL4 associated with lung cancer progression (18). This work examined VGLL4 expression in NSCLC tissues and paracancerous tissues through IHC and qRT-PCR analyses. Consistently, our data unearthed that VGLL4 was markedly downregulated in NSCLC tissues (Fig .5A, B). Similarly, VGLL4 expression was remarkably reduced in NSCLC cell lines compared to the BESA-2B cells (Fig .5C, D). To fathom tumor-suppressive function of VGLL4 in NSCLC progression, sh-VGLL4 was transfected into A549 and H292 cells. As shown, VGLL4 expression was markedly repressed in NSCLC cells transfected with sh-VGLL4 (Fig .5E). Data of CCK-8 and Transwell experiments showed knockdown of VGLL4 remarkably enhanced multiplication, migration and invasion of A549 and H292 cells (Fig .5F-I). Moreover, Western blot revealed that knockdown of VGLL4 repressed E-cadherin expression in A549 and H292 cells, but upregulated Vimentin expression (Fig .5J, K). These findings elucidated that VGLL4 played a tumor-suppressive role in NSCLC.

Fig.5

Knockdown of VGLL4 impeded multiplication, migration, invasion and EMT of NSCLC cells. A, B. IHC and qRT-PCR methods were employed to detect VGLL4 protein and mRNA expressions in NSCLC tissues and paracancerous tissues. C, D. Western blot and qRT-PCR were implemented to detect VGLL4 protein and mRNA expressions in NSCLC cells and BEAS2B cells. Original blots are shown in Figure S2 (See Supplementary Online Information at www.celljournal.org), Figure 5C. E. VGLL4 expression in NSCLC cells transfected with sh-VGLL4 was detected by qRT-PCR. F, G. CCK-8 assay was employed to monitor NSCLC cell multiplication. H, I. Transwell experiments were executed to detect NSCLC cell migration and invasion. J, K. Western blot was used to determine E-cadherin and Vimentin protein expressions in NSCLC cells transfected with sh-VGLL4. Original blots are shown in Figure S2 (See Supplementary Online Information at www.celljournal.org), Figure 5J. All experiments were repeated thrice with triplicate samples in each experiment. *; P<0.05, EMT; Epithelial-mesenchymal transition, NSCLC; Non-small cell lung cancer, IHC; Immunohistochemical, and qRT-PCR; Quantitative reverse transcription polymerase chain reaction.

Circ_0006427 elevated VGLL4 expression by competitively repressing miR-346 expression in NSCLC cells

Next, bioinformatics analysis indicated that VGLL4 3ˊUTR contained a binding site of miR-346 (Fig .6A). Subsequent dual-luciferase reporter gene experiments validated this binding relationship (Fig .6B). Effects of the circ_0006427/miR-346 axis on VGLL4 expression were then investigated. Data of qRT-PCR analysis and Western blot suggested that circ_0006427 overexpression up-regulated VGLL4 mRNA and protein expressions in NSCLC cells. This upregulation was remarkably reversed by the co-transfection of miR-346 mimics (Fig .6C, D). Furthermore, the expression of VGLL4 mRNA in NSCLC tissues was positively associated with circ_0006427 expression and negatively correlated with miR-346 expression (Fig .6E, F). From these data, we concluded that circ_0006427 could upregulate VGLL4 expression in NSCLC cells through repressing miR-346 expression.

Fig.6

VGLL4 was the target of miR-346 in NSCLC cells and positively regulated by circ_0006427. A. Bioinformatics analysis predicted binding sequence between miR-346 and VGLL4. B. Binding relationship between miR-346 and VGLL4 was verified using the dual-luciferase reporter gene assay. C, D. VGLL4 mRNA and protein expressions in A549 and H292 cells transfected with circ_0006427 overexpression plasmid or co-transfected with miR-346 mimics were detected by qRT-PCR and Western blot. Original blots are shown in Figure S2 (See Supplementary Online Information at www.celljournal.org), Figure 6D. E and F. Pearson’s correlation analysis was applied to determine correlation of VGLL4 mRNA with circ_0006427 or miR-346 expression in NSCLC tissues. All experiments were repeated thrice with triplicate samples in each experiment. *; P<0.05, NSCLC; Non-small cell lung cancer, qRT-PCR; Quantitative reverse transcription polymerase chain reaction, WT; Wild type, MUT; Mutant, NC-mimics; Mimics negative control, and NC; Negative control.

Discussion

circRNAs figure prominently in the regulation of diverse biological processes (19，20). In recent years, circRNAs are proven to participate in the tumorigenesis and development of several cancers, including NSCLC (21). For example, circ_0008305 restrains TGF-βinduced NSCLC cell invasion and EMT by modulating TIF1γ expression (22); downregulation of circ_0067934 expression suppresses multiplication, migration and invasion of NSCLC cells. It is linked to unfavorable prognosis of patients (23). Notably, circ_0006427 expression is downregulated in lung adenocarcinoma, and circ_0006427 overexpression represses multiplication, migration, invasion and EMT of lung adenocarcinoma cells (12). Consistently, in this work, circ_0006427 was unearthed to be remarkably under-expressed in both NSCLC tissues and cell lines; circ_0006427 low expression was related to larger tumor size and positive LNM of NSCLC patients. Functionally, circ_0006427 was verified to be tumor-suppressive in NSCLC progression by restraining cell multiplication, migration, invasion and EMT.

The role of miRNAs in tumor progression is extensively studied (24, 25). For instance, miR-421 is highly expressed in NSCLC. It accelerates cell multiplication and cell cycle progression (26). Similarly, miR-619-5p in tumor-derived exosome facilitates growth and metastasis of NSCLC cells by impeding RCAN1.4 expression (27). Accumulating research demonstrated that circRNAs can play “molecular sponge” role by antagonizing miRNAs to modulate expression of the target genes, thus playing an essential regulatory role in tumor progression. For instance, circ-ZKSCAN1 facilitates NSCLC progression by targeting miR-330-5p to regulate FAM83A expression and inactivate the MAPK signaling pathway (28); circ_0000003 enhances multiplication and metastasis of NSCLC cells by regulating miR-338-3p/IRS2 axis (29). In this work, it was revealed that there was a pairing relationship between circ_0006427 and miR-346 in NSCLC progression and circ_0006427 could work as a molecular sponge for miR-346. Meanwhile, miR-346 expression was markedly upregulated in NSCLC tissues and cell lines, which is consistent with the previous reports (16). Additionally, miR-346 overexpression remarkably attenuated tumor-suppressive effect caused by circ_0006427 overexpression. These results implied that circ_0006427 could exert tumor-suppressive effects in NSCLC progression via sponging miR-346.

VGLL4, a member of the VGLL family, is an antagonist of the proto-oncogenic protein YAP (17). Unlike the other VGLL family proteins, VGLL4 has two TDU domains (18). The N-terminal of VGLL4 protein can bind to ubiquitin-specific protease 11, thus improving stability of the VGLL4 protein by facilitating its deubiquitination (30). The role of VGLL4 varies among cells and tissues. VGLL4 overexpression is reported to enhance the colony-formation ability of human embryonic stem cells (31). Absence of VGLL4 impedes PD-L1 expression and tumor immune escape (32). Additionally, aberrant expression of VGLL4 occurs frequently in diverse cancers and VGLL4 is closely related to cell multiplication, migration, invasion and EMT. This can suppress cancer progression by modulating multiple signaling pathways, such as YAP and Wnt/β-catenin (33, 34). Importantly, VGLL4 is tumor-suppressive in lung cancer, competing with YAP for binding to TEAD4 and repressing transcription of TEAD4 downstream genes (18, 35). In this work, we demonstrated that VGLL4 expression was remarkably downregulated in NSCLC tissues and cells. Simultaneously, knockdown of VGLL4 markedly enhanced multiplication, migration, invasion and EMT of NSCLC cells. Interestingly, VGLL4 expression is validated to be linked to multiple miRNAs, such as miR-130b, miR-222 and miR-130a (35-37). In this work, VGLL4 was uncovered to be a direct target of miR-346 in NSCLC cells. Further experimental data demonstrated that circ_0006427 could modulate VGLL4 expression by competitively binding to miR-346. Hence, we concluded that circ_0006427/miR-346/VGLL4 axis could probably take part in NSCLC development.

Conclusion

This work revealed that circ_0006427 was remarkably downregulated in NSCLC tissues and cells. Circ_0006427 overexpression restrains multiplication, migration, invasion and EMT of NSCLC cells. Mechanistically, circ_0006427 modulates VGLL4 expression via sponging miR-346. These data implicated that circ_0006427/miR-346/VGLL4 regulatory axis could be an essential factor in NSCLC progression.