Circular RNA has circ 0014130 inhibits apoptosis in non ... · Circular RNA has_circ_0014130...

Circular RNA has_circ_0014130 inhibits apoptosis in non-small cell lung

cancer by sponging miR-136-5p and upregulating BCL2

Ying Geng*,1, Yongxia Bao*,1, Wei Zhang#,2, Lili Deng3, Dongju Su1, Hongyan

Zheng4

1 Department of Respiratory Medicine, The Second Affiliated Hospital, Harbin

Medical University, Harbin, Heilongjiang 150000, P. R. China.

2 Department of Respiratory Medicine, The First Affiliated Hospital, Harbin

Medical University, Harbin, Heilongjiang 150001, P. R. China.

3 Department of Oncology, The Second Affiliated Hospital of Harbin Medical

University, Harbin, Heilongjiang 150000, P. R. China.

4 Department of Respiratory Medicine, The Third Affiliated Hospital, Qiqihar

Medical University, Qiqihar, Heilongjiang 161000, P. R. China.

*These authors contributed equally to this work and should be considered as

co-first authors.

#Wei Zhang, Department of Respiratory Medicine, The First Affiliated Hospital,

Harbin Medical University, No 23 Youzheng Street, Nangang District, Harbin,

Heilongjiang 150001, P. R. TEL: 86-0451-85555788. China. Email:

[email protected]

Running title: ceRNA regulation of BCL2 expression in NSCLC

Conflicts of interest

The authors declare no competing financial interests.

Abstract

Previous studies indicated that circular RNAs (circRNAs) played vital roles in

on July 25, 2020. © 2020 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 14, 2020; DOI: 10.1158/1541-7786.MCR-19-0998





http://mcr.aacrjournals.org/



the development of non-small-cell lung cancer (NSCLC). Although

hsa_circ_0014130 might be a potential NSCLC biomarker, its function in

NSCLC remains unknown. Thus, this study aimed to investigate the role of

hsa_circ_0014130 in the progression of NSCLC. The levels of

hsa_circ_0014130 in NSCLC tissues and adjacent normal tissues were

determined by RT-qPCR. In addition, the expressions of Bcl-2, cleaved

caspase 3 in A549 cells were detected with western blot. Meanwhile, the dual

luciferase reporter system assay was used to determine the interaction of

hsa_circ_0014130 and miR-136-5p, or Bcl-2 and miR-136-5p in NSCLC

respectively. The level of hsa_circ_0014130 was significantly upregulated in

NSCLC tissues. Downregulation of hsa_circ_0014130 markedly inhibited the

proliferation and invasion of A549 cells via inducing apoptosis. In addition,

downregulation of hsa_circ_0014130 inhibited the tumorigenesis of

subcutaneous A549 xenograft in mice in vivo. Meanwhile, mechanistic

analysis indicated that downregulation of hsa_circ_0014130 decreased the

expression of miR‐136‐5p targeted gene Bcl-2 via acting as a competitive

‘sponge’ of miR‐136-5p. In this study, we found that hsa_circ_0014130 was

upregulated in NSCLC tissues. In addition, hsa_circ_0014130 functions as a

tumor promoter in NSCLC to promote tumor growth through up‐regulating

Bcl-2 partially via ‘sponging’ miR‐136-5p.

Implications statement

In conclusion, hsa_circ_0014130 might function as a prognostic factor for

patients with NSCLC and might be a therapeutic target for the treatment of

NSCLC in future.

Keywords: Non-small-cell lung cancer, apoptosis, hsa_circ_0014130,

miR-136-5p, Bcl-2.

Introduction

Non-small-cell lung cancer (NSCLC) is the main cause of death in human




worldwide, which accounts for more than 80% of patients with lung cancer (1).

Adenocarcinoma, squamous cell carcinoma, and large-cell lung cancer are the

major histological subtypes of NSCLC (2). At present, the overall 5-year

survival rate of patients with NSCLC was only about 10% (3). Despite

enormously advances in diagnostic technologies and treatment methods, the

survival rate and prognosis of patients with NSCLC is generally considered

poor (3,4). One of the key causes of high morbidity and mortality rate was the

low rate of early detection (5). Therefore, it is important to explore novel

therapeutic targets for NSCLC.

Circular RNAs (circRNAs) are a class of non-coding RNA molecules, which

was formed by exon back-splicing (6). CircRNAs extensively exist in

mammalian cells, which have a closed loop structure without 5’ cap and 3’ tail

(7). Previous studies indicated that circRNAs are closely associated with the

development of human cancer, such as NSCLC (8,9). Zhang et al found that

the level of hsa_circ_0014130 was upregulated in NSCLC tissues, and might

be a potential NSCLC biomarker (10). However, the role of hsa_circ_0014130

in NSCLC remains unclear and need further investigation.

MicroRNAs (miRNA) are another group of endogenous non-coding RNA,

approximately 21 to 23 nucleotides in length (11). MiRNAs negatively regulate

gene expression via binding to the complementary sequence in the

3'-untranslated region (3'- UTR) of target genes (12). Previous study indicated

that circRNAs regulated the expression of the target genes via acting as a

microRNA sponge (13). Xue et al found that circAGFG1 promoted metastasis

in NSCLC cells by sponging miR-203 (14).

However, the role of hsa_circ_0014130 and its binding miRNA in the

progression of NSCLC remains unclear. Therefore, the present study aimed to

investigate the function of hsa_circ_0014130 in NSCLC tumorigenesis.

Materials and methods

Patients and tissues samples




A total number of 30 paired NSCLC tissue samples and adjacent

noncancerous tissue samples were obtained from patients with NSCLC in the

First Affiliated Hospital, Harbin Medical University, between September 2017

and April 2018. None of patients had received any preoperative radiotherapy

and/or chemotherapy. This study was approved by the Institutional Ethical

Committee of the First Affiliated Hospital, Harbin Medical University. Written

informed consent was obtained from each patient.

Cell culture

Human NSCLC cell lines PC-9, and A549 were purchased from the Institute

of Biochemistry and Cell Biology of the Chinese Academy of Sciences

(Shanghai, China). Cells were cultured in Dulbecco’s Modified Eagle’s Medium

(DMEM, Thermo Fisher Scientific) supplied with 10% of fetal bovine serum

(FBS; Invitrogen, Carlsbad, CA, USA), 100 U/ml penicillin and 100 μg/ml

streptomycin at 37°C in a humidified incubator (5% CO2).

Quantitative real‐time polymerase chain reaction (RT‐qPCR)

TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) was used to

extract total RNA from the tissue samples and cells according to the standard

protocol. The complementary DNA (cDNA) was carried out using the

PrimeScript RT Master Mix (TaKaRa, Otsu, Shiga, Japan). After that, qRCR

was performed to detect the relative expression of circRNA and miRNA using

SYBR Green premix Ex TaqTM (TaKaRa) on an Applied Biosystems 7500 Real

Time PCR System (Applied Biosystems, Foster City, CA, USA). The

sequences of the primers were: hsa_circ_0014130, Forward: 5’-

AGAAGTTGGAGCACTCTTGGA-3’; Reverse: 5’-

AAAGTCCGAGGGTTCTGGTT-3’. GAPDH, Forward: 5’-

TCAAGAAGGTGGTGAAGCAGG-3’; Reverse: 5’-

TCAAAGGTGGAGGAGTGGGT-3’. The level of hsa_circ_0014130 was

normalized to the internal control GAPDH using the 2−ΔΔCT method.

Lentivirus production and cell transfection

The lentivirus short hairpin hsa_circ_0014130 (hsa_circ_0014130-shRNA1




and hsa_circ_0014130-shRNA2) plasmids were synthesized by GenePharma

(Shanghai, China). Empty pcDNA3.1 vector was used as negative control (NC).

The hsa_circ_0014130-shRNA1 and hsa_circ_0014130-shRNA2 plasmids

were infected into 293T cells respectively. Seventy-two hours later, the

supernatant was collected. PC-9 or A549 cells were then infected with NC or

hsa_circ_0014130-shRNAs supernatant for 72 h respectively. QRT-PCR was

used to detect the level of hsa_circ_0014130 in PC-9 and A549 cells.

MiR-136-5p mimics, miR-136-5p inhibitor, and negative control (NC) oligos

were purchased from Thermo Fisher Scientific. A549 cells were transfected

with miR-136-5p mimics or miR-136-5p inhibitor using Lipofectamine 2000

(Thermo Fisher Scientific) following the manufacturer’s protocol for 72 h.

CCK-8 assay

Cell Counting Kit (CCK)-8 (Beyotime, Shanghai, China) was applied to

detect the viability of NSCLC cells according to the manufacturer’s protocol.

PC-9 or A549 cells (5 × 103 cells/well) were plated onto 96-well plates

overnight at 37°C respectively. After that, cells were infected with

lenti-hsa_circ_0014130-siRNA1 for 24, 48 and 72 h, respectively. Later on, 10

μL of the CCK-8 solution was added to each well and incubated for another 2 h

at 37°C. The absorbance was measured using a microplate reader at 450 nm.

Flow cytometry

Annexin V-FITC apoptosis detection kit (Thermo Fisher Scientific) was used

to detect the number of apoptotic cells on a flow cytometer (FACScan™, BD

Biosciences, Franklin Lakes, NJ, USA). In detail, A549 cells were washed with

pre-cold PBS (Thermo Fisher Scientific) and resuspended in 100 μL binding

buffer (Thermo Fisher Scientific). After that, the cells were stained with 5 μL of

propidium iodide (PI) and 5 μL of Annexin V-FITC for 15 min in darkness at

room temperature. Then, the percentage of apoptotic cells was determined.

Transwell invasion assay

The A549 cells (5 × 104 cells suspended in 100 µL serum-free media) were

seeded into the upper chambers that covered with Matrigel (BD Biosciences,




NJ, USA). In addition, DMEM medium with 10% FBS (600 μL) was added into

the lower compartments as a chemoattractant. Twenty-four hours later, the

cells attached to the upper compartments were wiped using cotton swabs.

After that, cells on the lower surface of the inserts were fixed with 4%

paraformaldehyde, and then stained with 0.2% crystal violet. After washing

with PBS, cells were photographed using a laser confocal microscope

(Olympus Corp., Tokyo, Japan). Five independent fields of each membrane

were counted.

Luciferase reporter assay

Oligonucleotides containing the wild-type (WT) or mutant (MT) miR-136-5p

binding sites of the 3’-UTR of the Bcl-2 mRNA or hsa_circ_0014130 cDNA

fragment were ligated into the pmirGLO luciferase reporter vector (Promega,

Madison, WI, USA) respectively. For hsa_circ_0014130 reporter assay,

hsa_circ_0014130 reporter construct (WT or MT) was co-transfected with

miR-136-5p mimics and NC respectively using Lipofectamine 2000 (Invitrogen,

Carlsbad, CA, USA). For Bcl-2 reporter assay, Bcl-2 reporter construct (WT or

MT) was co-transfected with miR-136-5p mimics and NC respectively using

Lipofectamine 2000 (Invitrogen). Forty-eight hours later, the luciferase

activities were measured using a Dual-Luciferase Reporter Assay System

(Promega Madison, WI) with renilla luciferase activity as endogenous control.

Fluorescence in situ hybridization (FISH) analysis

Fluorescence in situ hybridization (FISH) analysis was performed as

previously describe (15,16). Alexa Fluor 555-labeled hsa_circ_0014130

oligonucleotide probes and Alexa Fluor 488-labeled miR-136-3p

oligonucleotide probes were synthesized by Geneseed Biotech (Guangzhou,

China). A549 cells were incubated with FISH probes in hybridization buffer at

37°C overnight in the dark. Fluorescent in Situ Hybridization Kit (RiboBio,

Guangzhou, China) was used to detect the probe signals according to the

manufacturer’s protocol. Cell nucleus were counterstained with DAPI (Thermo

Fisher Scientific) for 30 min. Images were captured with a fluorescence




microscope (Eclipse E600; Nikon Corporation, Tokyo, Japan).

Western blot

Total proteins were isolated from A549 cells using RIPA buffer (Thermo

Fisher Scientific) and then quantified using BCA method (Beyotime Institute of

Biotechnology). Appropriate amounts of proteins were separated by 10%

sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and

then transferred onto polyvinylidene difluoride (PVDF) membrane (Thermo

Fisher Scientific). After that, the membrane was blocked in 5% skim milk at

room temperature. Then, the membrane was incubated in primary antibodies

at 4°C overnight, including anti-Bcl-2 (1:1000, Abcam), anti-cleaved caspase 3

(1:1000, Abcam) and anti-β-actin (1:1000, Abcam). Later on, the membranes

were incubated with secondary antibodies (1:5000, Abcam) at the room

temperature for 1 h and then visualized using an ECL Chemiluminescent

Substrate Reagent Kit (Thermo Fisher Scientific). β-actin was acted as the

internal control.

Animal study

4-6-week-old BALB/c nude mice were purchased from the Shanghai SLAC

Animal Center (Shanghai, China) and animals were maintained following the

guidelines for use and care of laboratory animals. All animal experiments were

approved by the Institutional Ethical Committee of the First Affiliated Hospital,

Harbin Medical University. Animals were randomized into three groups: blank,

NC and hsa_circ_0014130-shRNA1 group. 1 × 107 A549 cells infected with

hsa_circ_0014130-shRNA1 were injected subcutaneously into the left flank of

nude mice (3 per group). Tumor volume was monitored every week with a

caliper and analyzed using the formula V = (length x width2)/2. All nude mice

were sacrificed after 4 weeks, and the entire tumors were dissected out and

weighed.

Statistical analysis

All data were repeated in triplicate. Data are presented as mean ± standard

error (S. D.). All statistical analyses were performed using GraphPad Prism




software (version 7.0, La Jolla, CA, USA). One-way analysis of variance

(ANOVA) and Tukey’s tests were carried out for multiple group comparisons. A

P-value < 0.05 was considered as a statistically significant.

Results

Hsa_circ_0014130 was upregulated in NSCLC tissues

To explore whether expression of hsa_circ_0014130 is dysregulated in

NSCLC, RT-qPCR was applied. As shown in Fig. 1A, the level of

hsa_circ_0014130 in NSCLC tissues was markedly higher than that in the

corresponding adjacent non-tumor tissues. In addition, to assess whether

hsa_circ_0014130 expression can discriminate between NSCLC tissues and

adjacent normal tissues, ROC analysis was used. The area under the ROC

curve (AUC) of 0.7486 indicated that the expression of hsa_circ_0014130

could discriminate between NSCLC tissues and adjacent normal tissues (Fig.

1B). As shown in Table 1, hsa_circ_0014130 expression correlated with

clinic-pathological parameters, including tumor size, lymphatic metastasis,

distant metastasis and TNM stage. These data indicated that the level of

hsa_circ_0014130 was upregulated in NSCLC tissues, and was associated

with a poor prognosis.

Downregulation of hsa_circ_0014130 inhibited the proliferation of

NSCLC cells

To investigate the biological function of hsa_circ_0014130 in NSCLC, we

used two shRNAs (hsa_circ_0014130-shRNA1 and

hsa_circ_0014130-shRNA2) to downregulate hsa_circ_0014130 in two

NSCLC cells, PC-9 and A549. RT-qPCR results indicated that

hsa_circ_0014130-shRNA1 downregulated the level of hsa_circ_0014130

more significantly than hsa_circ_0014130-shRNA2 in PC-9 and A549 cells

(Fig. 2A and 2B). CCK-8 assay indicated that downregulation of

hsa_circ_0014130 significantly inhibited the proliferation of NSCLC cells

compared with NC treatment (Fig. 2C and 2D). Hsa_circ_0014130-shRNA1




inhibited about 45% growth inhibitions in PC-9 cells, and inhibited about 52%

growth inhibitions in A549 cells. Therefore, A549 cells were utilized in the

following experiments. These results suggested that downregulation of

hsa_circ_0014130 could inhibit the proliferation of NSCLC cells.

Downregulation of hsa_circ_0014130 suppressed the invasion of A549

cells via inducing apoptosis

To investigate the role of hsa_circ_0014130 on the apoptosis of A549 cells,

flow cytometry assay was used. As shown in Fig. 3A and 3B, downregulation

of hsa_circ_0014130 obviously induced the apoptosis of A549 cells. In

addition, the invasive ability of A549 cells decreased notably after the cells

were infected with hsa_circ_0014130-shRNA1 or hsa_circ_0014130-shRNA2

plasmids (Fig. 3C and 3D). A549 cells infected with

hsa_circ_0014130-shRNA1 exhibited higher decrease in cell viability and

invasion, compared with that of in hsa_circ_0014130-shRNA2 group.

Therefore, hsa_circ_0014130-shRNA1 cells were utilized in the following

experiments. These data illustrated that downregulation of hsa_circ_0014130

could suppress the invasion of A549 cells via inducing apoptosis.

Hsa_circ_0014130 functions as a ceRNA of miR-136-5p in NSCLC

Evidence has been shown that circRNAs contain multiple binding sites for

miRNAs, which could act as miRNA sponge (17,18). Online bioinformatics tool

circular RNA interactome (https://circinteractome.nia.nih.gov) was used to

determine the potential targets of hsa_circ_0014130. 32 potential miRNA

targets of hsa_circ_0014130 were identified. The data indicated that four

miRNAs, miR-136-5p, miR-127-5p, miR-197 and miR-142-5p were closely

associated with hsa_circ_0014130. Meanwhile, miR-136-5p, miR-127-5p,

miR-197 and miR-142-5p have been found to play important roles in the

progression of human cancers (4,19-21). These data indicated that

miR-136-5p, miR-127-5p, miR-197 and miR-142-5p might be the potential

targets of hsa_circ_0014130 (Fig. 4A, Supplementary Figure 1A, 1B and 1C).

In addition, dual luciferase reporter assay was applied to validate the potential




targets of hsa_circ_0014130. The data indicated that miR-136-5p mimics

significantly inhibited the luciferase activity of

psiCHECK-2-hsa_circ_0014130-WT, but it did not affect the luciferase activity

of psiCHECK-2-hsa_circ_0014130-MT (Fig. 4B). However, limited

relationships between hsa_circ_0014130 and miRNAs (miR-127-5p, miR-197

and miR-142-5p) were assessed using the dual luciferase reporter assay

(Supplementary Fig. 1A, 1B and 1C). These data indicated that miR-136-5p

was a binding target of hsa_circ_0014130. In addition, online bioinformatics

tool targetScan indicated that Bcl-2 might be a potential target of miR-136-5p

(Fig. 4C). Dual luciferase reporter assay illustrated that miR-136-5p mimics

significantly suppressed the luciferase activity of psiCHECK-2-Bcl-2-WT (Fig.

4D). These data indicated that Bcl-2 was a binding target of miR-136-5p.

Meanwhile, the FISH assay indicated that hsa_circ_0014130 and miR-136-5p

were partially co-localized in the cytoplasm (Fig. 4E), suggesting the direct

interaction of hsa_circ_0014130 with miR-136-5p. These data indicated that

hsa_circ_0014130 functions as a ceRNA of miR-136-5p in NSCLC.

Hsa_circ_0014130’s oncogenic roles are partially via sponging

miR-136-5p, and then activating Bcl-2

Having verified hsa_circ_0014130 was a target of miR-136-5p, the

mechanism of miR-136-5p in hsa_circ_0014130-shRNA1-induced apoptosis

on A549 cells was still unclear. As shown in Fig. 5A, Supplementary Fig. 2A,

the level of miR-136-5p was markedly increased in A549 and PC-9 cells

following transfection with miR-136-5p mimics respectively, whereas the level

of miR-136-5p was significantly decreased following transfection with

miR-136-5p inhibitor. In addition, the level of Bcl-2 was decreased, and the

expression of cleaved caspase 3 was increased in A549 and PC-9 cells

following infection with hsa_circ_0014130, which were obviously reversed in

the presence of miR-136-5p inhibitor (Fig. 5B, 5C and 5D, Supplementary Fig.

2B, 2C and 2D). These data indicated that hsa_circ_0014130 act as an

oncogene via hsa_circ_0014130/miR-136-5p/Bcl-2 axis.




Downregulation of hsa_circ_0014130 inhibited the tumorigenesis of

A549 subcutaneous xenograft in vivo

We established a NSCLC xenograft model in mice to further explore the role

of hsa_circ_0014130 on tumor growth in vivo. As shown in Fig. 6A, 6B and 6C,

downregulation of hsa_circ_0014130 significantly inhibited xenograft tumor

volume and tumor weight compared with NC group. In addition, we tested the

expressions of Bcl-2 and cleaved caspase 3 in tumor tissues in vivo. The

results indicated that silencing of hsa_circ_0014130 markedly downregulated

the protein level of Bcl-2, and upregulated the protein level of cleaved caspase

3 in tumor tissues (Fig. 6D, 6E, 6F). These data illustrated that downregulation

of hsa_circ_0014130 could inhibit the tumorigenesis of A549 subcutaneous

xenograft in vivo.

Discussion

Predictive and identify biomarkers are urgently needed for patients with

NSCLC. Previous studies indicated that some circRNAs are associated with

the development of NSCLC, which participated in multiple biological processes,

such as cell proliferation, apoptosis and metastasis (6,22,23). Zhang et al

found that hsa_circ_0014130 was aberrantly expressed in NSCLC tissues and

might be a new circular RNA biomarker in NSCLC (10). In this study, we

confirmed that hsa_circ_0014130 was upregulated in NSCLC tissues.

Meanwhile, hsa_circ_0014130 expression correlated with clinic-pathological

parameters, including tumor volume and distant metastasis, indicating that

hsa_circ_0014130 is associated with a poor prognosis (10). However, the

function of hsa_circ_0014130 in the occurrence and progression of NSCLC

remain unclear.

It has been shown that aberrantly expressed miRNAs are associated with

the progression and prognosis of human cancers (24). CircRNAs could bind to

miRNAs and regulate miRNAs function as microRNA sponges (25). Meanwhile,

circRNA-miRNA-mRNA axis might play an important role in cancer-related




pathways (25). Huang et al found that circRNA_001946 could promote EMT

and metastasis of NSCLC cells via a circAGFG1/miR-203/ZNF281 axis (14).

Based on this feature of circRNAs, we aimed to explore the underlying

mechanism of hsa_circ_0014130 on the progression of NSCLC, regulating

miRNAs function as microRNA sponges. In this study, online bioinformatics

analysis and dual luciferase reporter assay indicated that miR-136-5p might be

a potential target of hsa_circ_0014130. FISH assay indicated that

hsa_circ_0014130 and miR-136-5p were co-localized in the cytoplasm. These

data indicated that hsa_circ_0014130 might act as ceRNAs to regulate

miR-136-5p. In addition, Bcl-2 act as a pro-apoptotic protein, which plays an

important role in promoting cellular survival (26). Bioinformatics analysis and

dual luciferase reporter assay confirmed that miR-136-5p targeted Bcl-2

mRNA at its 3’-UTR, Bcl-2 might be a potential target of miR-136-5p.

Next, the role of hsa_circ_0014130 in proliferation of NSCLC cells was

explored. Downregulation of hsa_circ_0014130 significantly inhibited the

proliferation and invasion of A549 cells via inducing apoptosis. In addition,

hsa_circ_0014130-shRNA1 treatment significantly decreased the expression

of Bcl-2 in A549 cells. However, when treated A549 cells with

hsa_circ_0014130-shRNA1 plus miR-136-5p inhibitor, the unfavorable role of

hsa_circ_0014130 on protein expression of Bcl-2 was increased by

miR-136-5p overexpression. Our results indicated that miR-136-5p is sponged

by hsa_circ_0014130 and targets Bcl-2. Chi et al indicated that circRNA

circPIP5K1A (hsa_circ_0014130) promoted the proliferation of NSCLC cells

via sponging miR-600, and then promoting HIF-1α (27). In addition,

overexpression of circRNA circPIP5K1A could promote the metastasis of

NSCLC cells via upregulation HIF-1α (27). Our data demonstrated that

hsa_circ_0014130’s oncogenic functions are partially via sponging

miR-136-5p, and then activating Bcl-2. Bcl-2 is located the at outer

mitochondrial membrane, which could restrain the release of cytochrome C

and inhibit the activation of the caspase, and then exerts its anti-apoptotic




function (28). Therefore, via downregulation of Bcl‐2, knockdown of

hsa_circ_0014130 could inhibit cell apoptosis and suppress tumorigenesis in

NSCLC. The conclusions are consistent, while hsa_circ_0014130 has another

mechanism of action against human NSCLC.

This study had some limitations. It has been shown that oncogenic drivers

are important to cancer development and maintenance (29). Some of the

known oncogenic drivers have been identified, including ROS1, RET, EGFR,

ALK and KRAS, which have been found to play the potential prognostic or

predictive roles in the development of NSCLC (29). However, in the present

study, we have not come to conclusion that the relationship between

hsa_circ_0014130 and the oncogenic drivers in NSCLC. Therefore, in the

future, it will be important to clarify whether hsa_circ_0014130 could facilitate

the progression of NSCLC via regulating the oncogenic drivers.

Conclusion

In this study, our data revealed that high-expressed hsa_circ_0014130 is an

oncogenic circRNA that facilitates the progression of NSCLC via

miR-136-5p/Bcl-2 axis. In addition, hsa_circ_0014130 might function as a

prognostic factor in NSCLC. Therefore, hsa_circ_0014130 might be a potential

biomarker and therapeutic target for the treatment of NSCLC.

Reference

1. Zou A, Liu X, Mai Z, Zhang J, Liu Z, Huang Q, et al. LINC00472 Acts as a Tumor

Suppressor in NSCLC through KLLN-Mediated p53-Signaling Pathway via

MicroRNA-149-3p and MicroRNA-4270. Molecular therapy Nucleic acids

2019;17:563-77 doi 10.1016/j.omtn.2019.06.003.

2. Yang X, Zhang Q, Zhang M, Su W, Wang Z, Li Y, et al. Serum microRNA Signature Is

Capable of Early Diagnosis for Non-Small Cell Lung Cancer. International journal of




biological sciences 2019;15(8):1712-22 doi 10.7150/ijbs.33986.

3. Zhou W, Wang X, Yin D, Xue L, Ma Z, Wang Z, et al. Effect of miR-140-5p on the

regulation of proliferation and apoptosis in NSCLC and its underlying mechanism.

Experimental and therapeutic medicine 2019;18(2):1350-6 doi

10.3892/etm.2019.7701.

4. Sui A, Zhang X, Zhu Q. Diagnostic value of serum miR197 and miR145 in non-small

cell lung cancer. Oncology letters 2019;17(3):3247-52 doi 10.3892/ol.2019.9958.

5. Wood DE, Kazerooni EA, Baum SL, Eapen GA, Ettinger DS, Hou L, et al. Lung

Cancer Screening, Version 3.2018, NCCN Clinical Practice Guidelines in Oncology.

Journal of the National Comprehensive Cancer Network : JNCCN 2018;16(4):412-41

doi 10.6004/jnccn.2018.0020.

6. Liu YT, Han XH, Xing PY, Hu XS, Hao XZ, Wang Y, et al. Circular RNA profiling

identified as a biomarker for predicting the efficacy of Gefitinib therapy for non-small

cell lung cancer. Journal of thoracic disease 2019;11(5):1779-87 doi

10.21037/jtd.2019.05.22.

7. Wang J, Li H. CircRNA circ_0067934 silencing inhibits the proliferation, migration and

invasion of NSCLC cells and correlates with unfavorable prognosis in NSCLC.

European review for medical and pharmacological sciences 2018;22(10):3053-60 doi

10.26355/eurrev_201805_15063.

8. Wu K, Liao X, Gong Y, He J, Zhou JK, Tan S, et al. Circular RNA F-circSR derived

from SLC34A2-ROS1 fusion gene promotes cell migration in non-small cell lung

cancer. Molecular cancer 2019;18(1):98 doi 10.1186/s12943-019-1028-9.




9. Wei S, Zheng Y, Jiang Y, Li X, Geng J, Shen Y, et al. The circRNA circPTPRA

suppresses epithelial-mesenchymal transitioning and metastasis of NSCLC cells by

sponging miR-96-5p. EBioMedicine 2019;44:182-93 doi

10.1016/j.ebiom.2019.05.032.

10. Zhang S, Zeng X, Ding T, Guo L, Li Y, Ou S, et al. Microarray profile of circular RNAs

identifies hsa_circ_0014130 as a new circular RNA biomarker in non-small cell lung

cancer. Scientific reports 2018;8(1):2878 doi 10.1038/s41598-018-21300-5.

11. Vo DT, Karanam NK, Ding L, Saha D, Yordy JS, Giri U, et al. miR-125a-5p Functions

as Tumor Suppressor microRNA And Is a Marker of Locoregional Recurrence And

Poor prognosis in Head And Neck Cancer. Neoplasia (New York, NY)

2019;21(9):849-62 doi 10.1016/j.neo.2019.06.004.

12. Zhao F, Han Y, Liu Z, Zhao Z, Li Z, Jia K. circFADS2 regulates lung cancer cells

proliferation and invasion via acting as a sponge of miR-498. Bioscience reports

2018;38(4) doi 10.1042/bsr20180570.

13. Qu S, Zhong Y, Shang R, Zhang X, Song W, Kjems J, et al. The emerging landscape

of circular RNA in life processes. RNA biology 2017;14(8):992-9 doi

10.1080/15476286.2016.1220473.

14. Xue YB, Ding MQ, Xue L, Luo JH. CircAGFG1 sponges miR-203 to promote EMT and

metastasis of non-small-cell lung cancer by upregulating ZNF281 expression.

Thoracic cancer 2019;10(8):1692-701 doi 10.1111/1759-7714.13131.

15. Wu Y, Xie Z, Chen J, Chen J, Ni W, Ma Y, et al. Circular RNA circTADA2A promotes

osteosarcoma progression and metastasis by sponging miR-203a-3p and regulating




CREB3 expression. Molecular cancer 2019;18(1):73 doi 10.1186/s12943-019-1007-1.

16. Wang L, Tong X, Zhou Z, Wang S, Lei Z, Zhang T, et al. Circular RNA

hsa_circ_0008305 (circPTK2) inhibits TGF-beta-induced epithelial-mesenchymal

transition and metastasis by controlling TIF1gamma in non-small cell lung cancer.

Molecular cancer 2018;17(1):140 doi 10.1186/s12943-018-0889-7.

17. Cheng Z, Yu C, Cui S, Wang H, Jin H, Wang C, et al. circTP63 functions as a ceRNA

to promote lung squamous cell carcinoma progression by upregulating FOXM1.

Nature communications 2019;10(1):3200 doi 10.1038/s41467-019-11162-4.

18. Lee WJ, Moon J, Jeon D, Shin YW, Yoo JS, Park DK, et al. Possible epigenetic

regulatory effect of dysregulated circular RNAs in Alzheimer's disease model.

Scientific reports 2019;9(1):11956 doi 10.1038/s41598-019-48471-z.

19. Shen S, Yue H, Li Y, Qin J, Li K, Liu Y, et al. Upregulation of miR-136 in human

non-small cell lung cancer cells promotes Erk1/2 activation by targeting PPP2R2A.

Tumour biology : the journal of the International Society for Oncodevelopmental

Biology and Medicine 2014;35(1):631-40 doi 10.1007/s13277-013-1087-2.

20. Tan W, Gu J, Huang M, Wu X, Hildebrandt MA. Epigenetic analysis of microRNA

genes in tumors from surgically resected lung cancer patients and association with

survival. Molecular carcinogenesis 2015;54 Suppl 1:E45-51 doi 10.1002/mc.22149.

21. Wan J, Ling X, Peng B, Ding G. miR-142-5p regulates CD4+ T cells in human

non-small cell lung cancer through PD-L1 expression via the PTEN pathway.

Oncology reports 2018;40(1):272-82 doi 10.3892/or.2018.6439.

22. Zhang H, Wang X, Hu B, Zhang F, Wei H, Li L. Circular RNA ZFR accelerates




non-small cell lung cancer progression by acting as a miR-101-3p sponge to enhance

CUL4B expression. Artificial cells, nanomedicine, and biotechnology

2019;47(1):3410-6 doi 10.1080/21691401.2019.1652623.

23. Chen L, Nan A, Zhang N, Jia Y, Li X, Ling Y, et al. Circular RNA 100146 functions as

an oncogene through direct binding to miR-361-3p and miR-615-5p in non-small cell

lung cancer. Molecular cancer 2019;18(1):13 doi 10.1186/s12943-019-0943-0.

24. Lian S, Park JS, Xia Y, Nguyen TT, Joo YE, Kim KK, et al. MicroRNA-375 Functions

as a Tumor-Suppressor Gene in Gastric Cancer by Targeting Recepteur d'Origine

Nantais. International journal of molecular sciences 2016;17(10) doi

10.3390/ijms17101633.

25. Rong D, Sun H, Li Z, Liu S, Dong C, Fu K, et al. An emerging function of

circRNA-miRNAs-mRNA axis in human diseases. Oncotarget 2017;8(42):73271-81

doi 10.18632/oncotarget.19154.

26. Zhang H, Chen R, Wang X, Zhang H, Zhu X, Chen J. Lobaplatin-Induced Apoptosis

Requires p53-Mediated p38MAPK Activation Through ROS Generation in

Non-Small-Cell Lung Cancer. Frontiers in oncology 2019;9:538 doi

10.3389/fonc.2019.00538.

27. Chi Y, Luo Q, Song Y, Yang F, Wang Y, Jin M, et al. Circular RNA circPIP5K1A

promotes non-small cell lung cancer proliferation and metastasis through

miR-600/HIF-1alpha regulation. Journal of cellular biochemistry

2019;120(11):19019-30 doi 10.1002/jcb.29225.

28. Hu QL, Xu ZP, Lan YF, Li B. miR-636 represses cell survival by targeting CDK6/Bcl-2




in cervical cancer. The Kaohsiung journal of medical sciences 2019 doi

10.1002/kjm2.12181.

29. Gower A, Wang Y, Giaccone G. Oncogenic drivers, targeted therapies, and acquired

resistance in non-small-cell lung cancer. Journal of molecular medicine (Berlin,

Germany) 2014;92(7):697-707 doi 10.1007/s00109-014-1165-y.




Table 1. The relationship between hsa_circ_0014130 and

clinic-pathological parameters of patients with NSCLC

Parameters Number Relative

expression p value

Age (years)

0.541

≤ 50 12 1.112 ± 0.566

> 50 18 1.268 ± 0.677

Gender

0.468

Male 19 1.313 ± 0.668

Female 11 1.211 ± 0.612

Tumor size (cm)

≤ 5 17 1.078 ± 0.467 0.042*

> 5 13 1.561 ± 0.756

Lymphatic metastasis

0.048*

N0 12 0.971 ± 0.445

N1-N3 18 1.631 ± 0.611

Distant metastasis

0.033*

M0 13 0.846 ± 0.515

M1 17 1.795 ± 0.721

TNM stage

0.027*

I-II 10 0.772 ± 0.651

III- IV 20 1.861 ± 0.339

Student’s t-test, *P<0.05.

Figure legend

Figure 1 Hsa_circ_0014130 was upregulated in NSCLC tissues. (A) The

relative levels of hsa_circ_0014130 in NSCLC tissues and corresponding

adjacent non-tumor tissues were detected using RT-qPCR. (B) ROC analysis

was used to discriminate between NSCLC tissues and corresponding adjacent

non-tumor tissues. 0.8 > AUC > 0.7, excellent discrimination. **P < 0.01,

compared with the normal group.

Figure 2 Downregulation of hsa_circ_0014130 inhibited the proliferation

of NSCLC cells. (A) Hsa_circ_0014130 levels in PC-9 cells transfected with

the hsa_circ_0014130-shRNA1 or hsa_circ_0014130-shRNA2 were detected

by qRT-PCR. (B) Hsa_circ_0014130 levels in A549 cells infected with the

hsa_circ_0014130-shRNA1 or hsa_circ_0014130-shRNA2 were detected by




qRT-PCR. (C) CCK-8 assay was used to determine the cell viability. PC-9 cells


for 48 h. (D) A549 cells were infected with hsa_circ_0014130-shRNA1 or

hsa_circ_0014130-shRNA2 for 48 h. **P < 0.01, compared with the NC group.

Figure 3 Downregulation of hsa_circ_0014130 suppressed the invasion

of A549 cells via inducing apoptosis. (A, B) A549 cells were infected with

hsa_circ_0014130-shRNA1 or hsa_circ_0014130-shRNA2 for 48 h. Apoptotic

cells were detected with Annexin V and PI double staining. (C, D) A549 cells


for 24 h. The invasive ability of A549 cells was detected using transwell

invasion assay. **P < 0.01, compared with the NC group.

Figure 4 Hsa_circ_0014130 functions as a ceRNA of miR-136-5p in

NSCLC. (A) Sequence alignment of miR-136-5p with the putative binding sites

within the WT or MT regions of hsa_circ_0014130. (B) The luciferase activity

in A549 cells following co-transfecting with hsa_circ_0014130-WT/MT 3’-UTR

plasmid and miR-136-5p mimics were detected using dual luciferase reporter

assay. (C) Sequence alignment of miR-136-5p with the putative binding sites

within the WT or MT regions of Bcl-2. (D) The luciferase activity in A549 cells

following co-transfecting with Bcl-2-WT/MT 3’-UTR plasmid and miR-136-5p

mimics were detected using dual luciferase reporter assay. (E) The cellular

localization of hsa_circ_0014130 and miR-136-5p in A549 cells was analyzed

by FISH assay. **P < 0.01, compared with the NC group.

Figure 5 Hsa_circ_0014130’s oncogenic roles are partially via sponging

miR-136-5p, and then activating Bcl-2 in A549 cells. (A) The level of

miR-136-5p in A549 cells transfected with the miR-136-5p mimics or inhibitor

was detected by qRT-PCR. (B) A549 cells were infected with

hsa_circ_0014130-shRNA1, or co-transfected with

hsa_circ_0014130-shRNA1 and miR-136-5p inhibitor. Expression levels of

Bcl-2 and cleaved caspase 3 in A549 cells were detected with western blotting.

(C, D) The relative expression of Bcl-2 and cleaved caspase 3 in A549 cells




were quantified via normalization to β-actin. **P < 0.01, compared with the NC

group. ##P < 0.01, compared with the shRNA1 group.

Figure 6 Downregulation of hsa_circ_0014130 inhibited the

tumorigenesis of A549 subcutaneous xenograft in vivo. (A) Mice were

subcutaneously implanted with hsa_circ_0014130-shRNA1-infected A549

cells. The xenograft tumor volumes were monitored weekly. (B) Xenografts

tumors were photographed after four weeks. (C) The weights of the tumors

were calculated. (D) Expression levels of Bcl-2 and cleaved caspase 3 in

tumor tissues were detected with western blotting. (E, F) The relative

expressions of Bcl-2 and cleaved caspase 3 in tumor tissues were quantified

via normalization to β-actin.




MOLECULAR CANCER RESEARCH | CORRECTION

Correction: Circular RNA hsa_circ_0014130Inhibits Apoptosis in Non–Small Cell LungCancer by Sponging miR-136-5p andUpregulating BCL2

In the original version of this article (1), the author order is incorrect for the final fourauthors. This error has been corrected in the latest online HTML and PDF versions of thearticle. The authors regret this error.

Reference1. Geng Y, Bao Y, Deng L, Su D, Zheng H, Zhang W. Circular RNA hsa_circ_0014130 inhibits apoptosis

in non–small cell lung cancer by sponging miR-136-5p and upregulating BCL2. Mol Cancer Res 2020;18:748–56.

Published online July 1, 2020.Mol Cancer Res 2020;18:1110doi: 10.1158/1541-7786.MCR-20-0499�2020 American Association for Cancer Research.

AACRJournals.org | 1110

http://crossmark.crossref.org/dialog/?doi=10.1158/1541-7786.MCR-20-0499&domain=pdf&date_stamp=2020-6-17

http://crossmark.crossref.org/dialog/?doi=10.1158/1541-7786.MCR-20-0499&domain=pdf&date_stamp=2020-6-17

Published OnlineFirst February 14, 2020.Mol Cancer Res Ying Geng, Yongxia Bao, Wei Zhang, et al. upregulating BCL2non-small cell lung cancer by sponging miR-136-5p and Circular RNA has_circ_0014130 inhibits apoptosis in

Updated version

10.1158/1541-7786.MCR-19-0998doi:

Access the most recent version of this article at:

Material

Supplementary

http://mcr.aacrjournals.org/content/suppl/2020/02/14/1541-7786.MCR-19-0998.DC1

Access the most recent supplemental material at:

Manuscript

Authorbeen edited. Author manuscripts have been peer reviewed and accepted for publication but have not yet

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mcr.aacrjournals.org/content/early/2020/02/14/1541-7786.MCR-19-0998To request permission to re-use all or part of this article, use this link



http://mcr.aacrjournals.org/lookup/doi/10.1158/1541-7786.MCR-19-0998

http://mcr.aacrjournals.org/content/suppl/2020/02/14/1541-7786.MCR-19-0998.DC1

http://mcr.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mcr.aacrjournals.org/content/early/2020/02/14/1541-7786.MCR-19-0998


Circular RNA has circ 0014130 inhibits apoptosis in non ... · Circular RNA has_circ_0014130...

Documents

Transcript of Circular RNA has circ 0014130 inhibits apoptosis in non ... · Circular RNA has_circ_0014130...