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. 2023 Jan 21;14(7):683–699. doi: 10.1111/1759-7714.14794

Circ_0043256 upregulates KLF2 expression by absorbing miR‐1206 to suppress the tumorigenesis of lung cancer

Ying Zhou 1, Hongliu Liu 2, Rui Wang 2, Mingtao Zhang 2,✉
PMCID: PMC9981313  PMID: 36680456

Abstract

Background

Circular RNAs (circRNAs) have been reported to play roles in lung cancer development. The purpose of this work was to explore the function and mechanism of circ_0043256 in lung cancer tumorigenesis.

Methods

Quantitative real‐time polymerase chain reaction (qRT‐PCR) and western blot were used for the detection of the levels of genes and proteins. Cell growth, angiogenesis ability, migration, and invasion were analyzed by using 5‐ethynyl‐2′‐deoxyuridine (EdU) assay, flow cytometry, tube formation assay, transwell assay, and murine xenograft model, respectively. The target between miR‐1206 and circ_0043256 or Krüppel‐like factor 2 (KLF2) was verified by dual‐luciferase reporter assay.

Results

Circ_0043256 was a stable circRNA, which was found to be decreased in lung cancer tissues and cells. Functionally, forced expression of circ_0043256 suppressed lung cancer cell growth, angiopoiesis, migration, and invasion. Mechanistically, circ_0043256 directly bound to miR‐1206 and miR‐1206 targeted KLF2, circ_0043256 could regulate KLF2 expression via absorbing miR‐1206. Rescue assay showed that miR‐1206 overexpression reversed the anticancer effects of circ_0043256 on lung cancer cells. Moreover, inhibition of miR‐1206 could suppress the malignant phenotypes of lung cancer cells, which was attenuated by KLF2 knockdown. Pre‐clinically, lentivirus‐mediated circ_0043256 overexpression impeded lung cancer growth in nude mice.

Conclusion

Forced expression of circ_0043256 could impede the tumorigenesis of lung cancer via miR‐1206/KLF2 axis, indicating a potential therapeutic approach for lung cancer.

Keywords: Angiogenesis, circ_0043256, KLF2, lung cancer, miR‐1206

circ_0043256 acted as a tumor suppressor to restrain lung cancer growth and tumorigenesis by miR‐1206/KLF2 axis, providing the molecular theoretical basis for subsequent clinical treatment of lung cancer.

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INTRODUCTION

Globally, lung cancer is still one of the most fatal malignancies, accounting for the leading cause of cancer death. 1 The morbidity of lung cancer is higher; there are ~1.76 million deaths and 2.09 million new cases in 2018. 1 , 2 Although progress has been made in treatment, the prognosis of lung cancer shows an unsatisfactory 5‐year survival rate (16%) in patients who are diagnosed at late stage and metastasis. 3 Therefore, an in‐depth understanding on the carcinogenesis of lung cancer is necessary for developing novel and effective therapeutic strategy.

Circular RNAs (circRNAs) are a class of conserved RNA molecules that are resistant to the degradation by exonuclease. 4 , 5 CircRNAs are implicated in regulating various cellular crucial biological processes linked with carcinogenesis, differentiation, growth, apoptosis, and drug resistance. 6 , 7 , 8 Moreover, deregulated circRNAs have been identified in lung cancer, and some circRNAs have crucial regulatory roles in the tumorigenesis of lung cancer. 9 For example, circRNA‐002178 was confirmed to act as an oncogene in lung adenocarcinoma to stimulate T‐cell exhaustion by elevating programmed death‐ligand 1 (PDL1) expression. 10 Zhang et al. 11 showed that highly expressed hsa_circRNA_101237 was linked to poor outcome in lung cancer patients and knockdown of hsa_circRNA_101237 repressed cancer cell proliferative, migratory and invasive abilities. Moreover, circ_0007385 was also confirmed to promote lung cancer growth and cisplatin resistance. 12 In addition, a decreased circ_0043256 was found in lung cancer. 13 However, the action of circ_0043256 in lung cancer carcinogenesis remains unclear.

Here, this study focused on exploring the role of circ_0043256 in lung cancer cell malignant phenotypes and growth. It has been proposed that circRNAs can exert their functions by acting as competitive endogenous RNAs (ceRNAs), namely, circRNAs sequester microRNAs (miRNAs) to modulate the target genes. 14 , 15 Furthermore, a ceRNA network was constructed to elucidate the potential mechanism of circ_0043256 in lung cancer tumorigenesis.

MATERIAL AND METHODS

Human samples

In total, 36 pairs of tumor tissues and adjacent para‐tumor tissues were collected from lung cancer patients at Jingmen No.1 People's Hospital. All cases did not obtain preoperative therapy. All samples were stored at −80°C. Informed consent was collected from all patients. This study was approved by the Ethics Committee of Jingmen No.1 People's Hospital according to the Declaration of Helsinki.

Cell culture

Human lung cancer cells (PC9 and A549) and normal human bronchial epithelial cell (Beas‐2B) were obtained from Procell and cultured in Dulbecco's modified Eagle medium (Procell) harboring 1% antibiotics (Invitrogen) and 10% fetal bovine serum (FBS) (Procell) with 5% CO2 at 37°C.

Cell transfection

The pCD5‐ciR/circ_0043256 overexpression plasmids (oe‐circ_0043256), Krüppel‐like factor 2 (KLF2) specific small interference RNA (si‐KLF2), miR‐1206 mimic or inhibitor were constructed by GenePharma with empty pCD5‐ciR plasmids (vector), non‐target siRNA (si‐NC), miR‐NC, or miR‐NC inhibitor as the contrasts. Thereafter, transient transfection was conducted using Lipofectamine 2000 (Invitrogen).

The overexpression plasmids of circ_0043256 were subcloned into a lentiviral vector (GenePharma) to establish a Lenti‐oe‐circ_0043256. The puromycin (2–5 μg/mL) was applied to select stable expressing lentiviral particles, which were then transfected into A549 cells for in vivo assay.

Subcellular fractionation and quantitative real‐time polymerase chain reaction

The PARIS kit (Life Technologies) was applied to determine the cytoplasmic and nuclear circ_0043256 as per the instruction. Total RNA was extracted by using TRIzol reagent (Invitrogen). Approximately 3 μg isolated RNAs were treated with mock without the enzyme or 5 U/μg RNase R at 37°C for 20 minutes to assess the stability of circ_0043256. The complementary DNA (cDNA) was synthesized by using the Prime Script RT Reagent Kit (Takara), followed by quantitative real‐time polymerase chain reaction (qRT‐PCR) analysis with SYBR Green kit (Takara). The relative fold changes were calculated by 2−ΔΔCt method and GAPDH or U6 was used as the internal reference. The primers are presented in Table 1.

TABLE 1.

Primers sequences used for PCR

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5‐Ethynyl‐2′‐deoxyuridine assay

After transfection, PC9 and A549 cells were placed into a 96‐well plate containing 50 μM 5‐Ethynyl‐2′‐deoxyuridine (EdU) labeling solution (RiboBio) and incubated for 3 hours. Following reacting with click reaction solution for 30 minutes, DAPI was used for cell nuclei staining. Finally, EdU‐positive cells were detect to assess cell proliferation.

Colony formation assay

Following different transfection, PC9 and A549 cells (500/well) were added into a six‐well plate with complete medium and incubated for 2 weeks. After being fixed by methanol and stained with 0.1% crystal violet (Sigma‐Aldrich), visible colonies were imaged (100×) and counted using a microscope.

Flow cytometry

After transfection, PC9 and A549 cells were dyed with 10 μL Annexin V‐FITC and 10 μL propiduim iodide (BD Biosciences) in the darkness for 15 minutes. Last, the FACSCanto II flow cytometer (BD Biosciences) was used to assay cell apoptosis.

Western blotting

Total protein was isolated by using RIPA buffer (Beyotime) containing protease inhibitors and the concentration of proteins was qualified by BCA detecting kit (Keygen). Next, protein electrophoresis membrane was transferred. Next, the membrane was incubated with primary antibodies against Bax (ab32503, 1:2000, Abcam), Bcl‐2 (ab692, 1:2000, Abcam), KLF2 (ab194486, 1:2000, Abcam), and β‐actin (ab6276, 1:1000, Abcam) overnight at 4°C. After incubating with horseradish peroxidase (HRP)‐conjugated secondary antibodies at 37°C for 2 hours, the chemiluminescent (ECL) detection reagent (Beyotime) was applied to assay the protein bands.

Tube formation assay

Each well of the 96‐well plate was pre‐coated with 50 μL Matrigel for 1 hour. The conditioned medium of transfected PC9 and A549 cells was collected. Human umbilical vein endothelial cells were starved for 24 hours and resuspended in 100 μL cell conditioned medium (4 × 105 cells/mL), and then seeded onto the Matrigel gel. Forty eight hours later, the number of tubes was detected by a microscope.

Transwell assay

Transwell chambers (8.0 μm pore size) (Corning) pre‐coated with or without Matrigel (BD Biosciences) were used for cell invasion and migration detection. Transfected PC9 and A549 cells resuspended in serum‐free medium were added into the upper chambers. Then, 500 μL of medium containing 10% FBS was filled into the lower chambers. Twenty‐four hours later, migrated and invaded cells were fixed by 4% formalin and stained with crystal violet, and the number of cells were then counted using a microscope.

Dual‐luciferase reporter assay

The wild‐type (WT) fragments and the mutated (MUT) seed sequences of miR‐1206 on circ_0043256 and KLF2 were inserted into the psiCHECK2 plasmids (Promega). Next, 200 ng recombinant psiCHECK2 plasmids and 50 nM miR‐1206 or the controls were co‐transfected into PC9 and A549 cells for 48 hours. The luciferase activity was assayed by a Dual‐luciferase Reporter Assay System (Promega).

Xenograft models

The nude mice were divided into three groups (vector, oe‐circ_0043256, or oe‐circ_0043256 + miR‐1206). A549 cells infected with lentiviruses carrying oe‐circ_0043256 or vector were subcutaneously vaccinated into the right back flank of blindly randomized nude mice (n = 5/group; 6 weeks). When the tumor grew to 100 mm3, miR‐1206 agonist was directly administered via intra‐tumor injection into each mouse once a week for 4 weeks. The size of tumors was monitored every 7 days, and the volume was assessed as the formula: volume = length × width2 × 0.5. Four weeks later, the tumors were excised, weighed, and divided either for molecule detection or fixed in formalin for immunohistochemistry (IHC) analysis as described previously. 16 The experiments abided the supervision of the Animal Care and Use Committee of Jingmen No. 1 People's Hospital.

Statistical assay

All experiments were repeated three times. Data are manifested as mean ± standard deviation (SD). The difference was evaluated by the Student's t‐test (two groups) and analysis of variance with Turkey test hoc post (multiple groups). p < 0.05 indicated significant differences.

RESULTS

Circ_0043256 is decreased in lung cancer tissues and cells

Circ_0043256 is produced by back‐splicing of exon 15–18 of the ACACA gene, it is located at chr17: 35604934–35 609 962 and finally forms a circular transcript of 483 nt (Figure 1(a)). Circ_0043256 was found to be decreased in lung cancer tissues compared with the normal control (Figure 1(b)). Moreover, its expression was also lower in lung cancer cell lines (PC9 and A549) than those in normal Bease‐2B cells (Figure 1(c)). The nuclear‐cytoplasmic fractionation assay showed that circ_0043256 was mainly distributed in the cytoplasm of A549 and PC9 cells (Figure 1(d),(e)). In addition, it was observed that circ_0043256 was resistant to the degradation by RNase R in A549 and PC9, whereas RNase R treatment digested linear acetyl‐CoA carboxylase α (ACACA) messenger RNA (mRNA) (Figure 1(f),(g)), implying that circ_0043256 was a stable circRNA.

FIGURE 1.

FIGURE 1

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Circ_0043256 is decreased in lung cancer tissues and cells. (a) The genomic locus of circ_0043256 and the back‐spliced junction of circ_0043256 were indicated. (b), (c) Detection of circ_0043256 expression in lung cancer tissues and normal tissues, as well as in lung cancer cell lines (PC9 and A549) and normal Bease‐2B cells by quantitative real‐time polymerase chain reaction (qRT‐PCR). (d), (e) Nuclear‐cytoplasmic fractionation assay for the distribution of circ_0043256 in PC9 and A549 cells. (f), (g) Stability analysis of circ_0043256 by RNase R treatment. ****p < 0.0001

Overexpression of circ_0043256 suppresses lung cancer cell malignant phenotypes in vitro

Next, the clinical value of circ_0043256 on lung cancer was investigated using in vitro assays. Circ_0043256 overexpression plasmids were constructed and transduced into A549 and PC9 cells. As expected, circ_0043256 expression was significantly elevated after oe‐circ_0043256 transfection compared with vector transfection (Figure 2(a)). Functionally, forced expression of circ_0043256 suppressed the proliferation of A549 and PC9 cells, evidenced by decreased EdU‐positive cells (Figure 2(b)). Moreover, colony formation assay also demonstrated that cell cloning capabilities of A549 and PC9 cells were suppressed after circ_0043256 overexpression (Figure S1(a)). Conversely, overexpression of circ_0043256 induced apoptosis in A549 and PC9 cells, which was accompanied with decreased Bcl‐2 level and increased Bax level (Figure 2(c)–(e)). Moreover, circ_0043256 upregulation could impair tubule formation ability of A549 and PC9 cells (Figure 2(f)). In transwell assay, it was found that the migration and invasion abilities of A549 and PC9 cells were repressed after circ_0043256 upregulation (Figure 2(g),(h)). Taken together, circ_0043256 acted a tumor suppressor to inhibit lung cancer cell growth, angiopoiesis, migration, and invasion.

FIGURE 2.

FIGURE 2

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Overexpression of circ_0043256 suppresses lung cancer cell malignant phenotypes in vitro. (a)–(h) Oe‐circ_0043256 or vector was constructed and transduced into A549 and PC9 cells. (a) Confirmation of the transfection efficiency by quantitative real‐time polymerase chain reaction (qRT‐PCR). (b) 5‐Ethynyl‐2′‐deoxyuridine (EdU) assay for cell proliferation. (c) Flow cytometry for cell apoptosis. (d), (e) Western blotting analysis for the protein levels of Bax and Bcl‐2. (f) Tube formation assay for cell tubule formation ability. (g), (h) Transwell assay for cell migration and invasion. ****p < 0.0001

MiR‐1206 is a target of circ_0043256

Given the cytoplasm distribution of circ_0043256, the downstream targets of circ_0043256 were investigated. According to the prediction of circinteractome, miR‐1206 was found to have complementary sequences on circ_0043256 (Figure 3(a)). qRT‐PCR showed that miR‐1206 mimic could significantly elevate miR‐1206 expression in A549 and PC9 cells (Figure 3(b)). Next, dual‐luciferase reporter assay was conducted. The results showed that miR‐1206 mimic notably led to a decrease of the luciferase activity of WT‐circ_0043256 vector, but bot the mutated one in A549 and PC9 cells (Figure 3(c),(d)). Moreover, miR‐1206 expression was higher in lung cancer tissues and cell lines than those in normal controls (Figure 3(e),(f)). In all, circ_0043256 directly targeted miR‐1206.

FIGURE 3.

FIGURE 3

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MiR‐1206 is a target of circ_0043256. (a) The complementary sequences of miR‐1206 on circ_0043256. (b) Confirmation of the transfection efficiency of the miR‐1206 mimic or miR‐NC by quantitative real‐time polymerase chain reaction (qRT‐PCR). (c), (d) Dual‐luciferase reporter assay was used to verify the binding between miR‐1206 and circ_0043256. (e), (f) Detection of miR‐1206 expression in lung cancer tissues and normal tissues, as well as in lung cancer cell lines (PC9 and A549) and normal Bease‐2B cells by qRT‐PCR. ****p < 0.0001

Overexpression of circ_0043256 suppresses malignant phenotypes mediated by miR‐1206 in lung cancer cells

Next, we explored whether the anticancer effects of circ_0043256 was mediated by miR‐1206. A549 and PC9 cells were transfected with oe‐circ_0043256 alone or co‐transfected with oe‐circ_0043256 and miR‐1206. qRT‐PCR showed that oe‐circ_0043256 transfection suppressed miR‐1206 expression level in A549 and PC9 cells, which was rescued by the introduction of miR‐1206 (Figure 4(a)). Thereafter, it was found that miR‐1206 up‐regulation attenuated circ_0043256 overexpression‐induced proliferation inhibition (Figure 4(b),(c) and Figure S1(b)) and apoptosis enhancement (Figure 4(d)–(g)). Moreover, the suppression of cell angiopoiesis, migration, and invasion abilities caused by circ_0043256 was also reversed by miR‐1206 upregulation in A549 and PC9 cells (Figure 4(h)–(j)). These data confirmed that circ_0043256 affected lung cancer cell tumorigenesis via regulating miR‐1206.

FIGURE 4.

FIGURE 4

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Overexpression of circ_0043256 suppresses malignant phenotypes mediated by miR‐1206 in lung cancer cells. (a)–(j) A549 and PC9 cells were transfected with oe‐circ_0043256 alone or co‐transfected with oe‐circ_0043256 and miR‐1206. (a) Quantitative real‐time polymerase chain reaction (qRT‐PCR) for miR‐1206 levels in cells. (b) (c) 5‐Ethynyl‐2′‐deoxyuridine (EdU) assay for cell proliferation. (d) Flow cytometry for cell apoptosis. (e)–(g) Western blotting analysis for the protein levels of Bax and Bcl‐2. (h) Tube formation assay for cell tubule formation ability. (i) and (j) Transwell assay for cell migration and invasion. ***p < 0.001, ****p < 0.0001

KLF2 is a target of miR‐1206, and circ_0043256 can regulate KLF2 by sponging miR‐1206

The targets of miR‐1206 were also explored. Targetscan database predicted that miR‐1206 had the binding site on KLF2 3'UTR (Figure 5(a)). The results of dual‐luciferase reporter assay further exhibited that miR‐1206 overexpression declined the luciferase activity of WT‐KLF2 3'UTR vector, but bot affected the luciferase activity of MUT‐KLF2 3'UTR in A549 and PC9 cells (Figure 5(b)). After confirming the knockdown efficiency of miR‐1206 inhibitor (Figure 5(c)), it was confirmed that KLF2 expression level was decreased by miR‐1206 mimic and increased by miR‐1206 inhibitor (Figure 5(d)). The mRNA content of KLF2 was found to be decreased in lung cancer tissues and was negatively correlated with miR‐1206 expression in cancer tissues (Figure 5(e),(f)). Western blotting analysis also showed the decreased KLF2 protein level in lung cancer tissues (Figure 5(g)) and cell lines (Figure 5(h)). Interestingly, we also showed that oe‐circ_0043256 introduction was accompanied with increased KLF2, which was subsequently reduced in response to miR‐1206 mimic in A549 and PC9 cells (Figure 5(i)). Collectively, KLF2 was a target of miR‐1206, and circ_0043256/miR‐1206/KLF2 formed an axis.

FIGURE 5.

FIGURE 5

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Krüppel‐like factor 2 (KLF2) is a target of miR‐1206, and circ_0043256 can regulate KLF2 by sponging miR‐1206. (a) The binding site of miR‐1206 on KLF2 3'UTR. (b) Dual‐luciferase reporter assay was used to verify the binding between miR‐1206 and KLF2. (c) Confirmation of the interference efficiency of miR‐1206 inhibitor or miR‐NC inhibitor by quantitative real‐time polymerase chain reaction (qRT‐PCR). (d) Western blot analysis of KLF2 expression in cells after miR‐1206 increase or decrease. (e) Detection of KLF2 mRNA expression in lung cancer tissues and normal tissues by qRT‐PCR. (f) The negative correlation between KLF2 mRNA expression and miR‐1206 expression in cancer tissues. (g), (h) Detection of KLF2 protein expression in lung cancer tissues and normal tissues as well as in lung cancer cell lines (PC9 and A549) and normal Bease‐2B cells by western blotting. (i) The effects of circ_0043256/miR‐1206 axis on KLF2 expression. ****p < 0.0001

Knockdown of miR‐1206 inhibits lung cancer cell malignant phenotypes by KLF2

To evaluate the functions of miR‐1206/KLF2 axis on lung cancer cell malignant phenotypes. The rescue experiments were performed. A549 and PC9 cells were transfected with miR‐1206 inhibitor alone or co‐transfected with miR‐1206 inhibitor and KLF2 siRNA (si‐KLF2). The western blotting displayed that si‐KLF2 introduction reversed miR‐1206 inhibitor‐induced elevation of KLF2 level in A549 and PC9 cells (Figure 6(a)). Functionally, we demonstrated that miR‐1206 inhibitor could suppress cell proliferation (Figure 6(b)–(d) and Figure S1(c)) and stimulate apoptosis (Figure 6(e)–(g)) in A549 and PC9 cells, whereas these effects were reversed after KLF2 knockdown (Figure 6(b)–(g)). In addition, the angiopoiesis, migration, and invasion abilities of A549 and PC9 cells were markedly suppressed by miR‐1206 inhibitor, which were then abolished by KLF2 silencing (Figure 6(h)–(j)). Altogether, miR‐1206/KLF2 axis was engaged in regulating the tumorigenesis of lung cancer.

FIGURE 6.

FIGURE 6

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Knockdown of miR‐1206 inhibits lung cancer cell malignant phenotypes by Krüppel‐like factor 2 (KLF2). (a)–(j) A549 and PC9 cells were transfected with miR‐1206 inhibitor alone or co‐transfected with miR‐1206 inhibitor and si‐KLF2. (a) Western blotting for KLF2 levels in cells. (b)–(d) 5‐Ethynyl‐2′‐deoxyuridine (EdU) assay for cell proliferation. (e) Flow cytometry for cell apoptosis. (f), (g) Western blotting analysis for the protein levels of Bax and Bcl‐2. (h) Tube formation assay for cell tubule formation ability. (i), (j) Transwell assay for cell migration and invasion. **p < 0.01, ****p < 0.0001

Overexpression of circ_0043256 suppresses lung cancer growth in vivo

Subsequently, we explored whether circ_0043256 affected the growth of lung cancer in vivo. Consistent with the results of in vitro experiments, overexpression of circ_0043256 hindered the growth of lung cancer in nude mice, reflected by the smaller tumor size and lighter tumor weigh in xenografts of oe‐circ_0043256 group (Figure 7(a),(b)). Furthermore, compared with the oe‐circ_0043256 group, the oe‐circ_0043256 group + miR‐1206 group showed bigger tumor size and heavier tumor weigh in xenografts (Figure 7(a),(b)). Moreover, the expression levels of circ_0043256 were higher in xenografts of oe‐circ_0043256 group, whereas there was no change in the expression of circ_0043256 between oe‐circ_0043256 group and oe‐circ_0043256 group + miR‐1206 group (Figure 7(c)). In addition, miR‐1206 expression was decreased and KLF2 expression was increased in xenografts isolated from oe‐circ_0043256 group, whereas these effects were attenuated after miR‐1206 overexpression (Figure 7(c),(d)). Next, IHC analysis showed that the contents of ki67 and Bcl‐2 were decreased, and Bax content was increased in xenografts with circ_0043256 overexpression, whereas oe‐circ_0043256 group + miR‐1206 group showed the opposite trend on their expression (Figure 7(e)). In all, circ_0043256 could suppress lung cancer growth in vivo.

FIGURE 7.

FIGURE 7

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Overexpression of circ_0043256 suppresses lung cancer growth in vivo. (a), (b) In vivo growth curve (a), the representative images and tumor weight at the end points (b) of xenografts formed by subcutaneous injection. (c), (d) The levels of circ_0043256, miR‐1206 and Krüppel‐like factor 2 (KLF2) in xenografts were detected by using quantitative real‐time polymerase chain reaction (qRT‐PCR) or western blotting. (e) The quantification and representative images of immunohistochemistry staining revealing the expression of ki67, Bax, and Bcl‐2 in subcutaneous xenografts. ****p < 0.0001

DISCUSSION

Currently, more and more proofs have revealed that circRNAs are functional molecules that can modulate cancer progression by involving in various cellular activities. For instance, Chen et al. 17 showed that circRNA_0000285 silencing could repress the migration and growth abilities of cervical cancer in vitro and in nude mice. High has_circ_104348 predicted poor prognosis in hepatocellular carcinoma (HCC), and has_circ_104348 deficiency negatively regulated the malignant phenotypes of HCC cells. 18 In addition, Yuan et al. showed that circRNA_102231 promoted gastric cancer cell invasion and proliferation both in vitro and in vivo. Therefore, targeting the deregulation of circRNAs may be a promising method for cancer prevention. In our work, a decreased circ_0043256 was observed in lung cancer tissues and cells. Functionally, forced expression of circ_0043256 restrained cancer cell proliferative, invasive, migratory, angiogenetic abilities and induced cell apoptosis in vitro. Pre‐clinically, lentivirus‐mediated circ_0043256 overexpression impeded lung cancer growth in nude mice, further indicting the anticancer potential of circ_0043256 in lung cancer.

This study verified the cytoplasmic distribution of circ_0043256 in lung cancer cells. Furthermore, the circRNA‐miRNA‐mRNA regulatory network has been identified to play crucial roles in the pathology of lung cancer. 19 , 20 Therefore, the potential miRNAs/mRNA axis underlying circ_0043256 was then explored. We confirmed that circ_0043256/miR‐1206/KLF2 formed an axis in lung cancer cells. MiRNAs have been confirmed to be implicated in the modulation of the pathogenesis of human diseases, including the cancer. 21 , 22 , 23 MiR‐1206 is a functional miRNA. It was found that could be used as the biomarker for ischemia reperfusion diagnosis. 24 MiR‐1206 variant was confirmed to be related to methotrexate‐induced oral mucositis in childhood acute lymphoblastic leukemia. 25 In cancer, Yu et al. 26 showed that circ_0092367 suppressed epithelial‐mesenchymal transition process and gemcitabine resistance via miR‐1206 in pancreatic cancer. Moreover, miR‐1206 overexpression could reverse the anticancer effects of circ_0129047 or BMPR2 on lung adenocarcinoma. 27 KLF2 belongs to the Krüppel‐like factor family transcription factors, which has been revealed to exert anticancer action in many types of cancers by impeding cancer cell malignant phenotypes. 28 , 29 , 30 In lung cancer, KLF2 expression was found to be decreased; restoration of KLF2 inhibited lung cancer cell proliferation. 31 In addition, KLF2 re‐expression disturbed the proliferation of lung cancer cells by disappearing energy metabolism through glutaminase inhibition. 32 In our study, we also observed an increase of miR‐1206 and a decrease of KLF2 in lung cancer tissues and cells. Moreover, miR‐1206 promoted cancer cell proliferative, invasive, migratory, angiogenetic abilities, which were reversed by KLF2. Additionally, miR‐1206 overexpression overturned the anticancer action of circ_0043256.

In all, we first confirmed that circ_0043256 acted as a tumor suppressor to restrain lung cancer growth and tumorigenesis by miR‐1206/KLF2 axis, providing the molecular theoretical basis for subsequent clinical treatment of lung cancer.

AUTHOR CONTRIBUTION

Ying Zhou conceived and designed the study, and drafted the first draft of the manuscript. All experiments were completed by all authors. Hongliu Liu, Rui Wang, Mingtao Zhang analyzed and collated the results. All authors reviewed and critiqued the manuscript, and agreed to the final submission of the manuscript. All authors read and approved the final manuscript.

FUNDING INFORMATION

None.

CONFLICTS OF INTEREST

The authors declare they have no conflicts of interest.

Supporting information

Figure S1. Cell proliferation detection by colony formation assay.

Click here for additional data file. (11.3MB, tif)

ACKNOWLEDGMENTS

None.

Zhou Y, Liu H, Wang R, Zhang M. Circ_0043256 upregulates KLF2 expression by absorbing miR‐1206 to suppress the tumorigenesis of lung cancer. Thorac Cancer. 2023;14(7):683–699. 10.1111/1759-7714.14794

Ying Zhou and Hongliu Liu contributed equally to this paper.

REFERENCES

  • 1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68:394–424. [DOI] [PubMed] [Google Scholar]
  • 2. Bade BC, Dela Cruz CS. Lung cancer 2020: epidemiology, etiology, and prevention. Clin Chest Med. 2020;41:1–24. [DOI] [PubMed] [Google Scholar]
  • 3. Mascaux C, Tomasini P, Greillier L, Barlesi F. Personalised medicine for nonsmall cell lung cancer. Eur Respir Rev. 2017;26:170066. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. Yu J, Xu QG, Wang ZG, Yang Y, Zhang L, Ma JZ, et al. Circular RNA cSMARCA5 inhibits growth and metastasis in hepatocellular carcinoma. J Hepatol. 2018;68:1214–27. [DOI] [PubMed] [Google Scholar]
  • 5. Chen LL, Yang L. Regulation of circRNA biogenesis. RNA Biol. 2015;12:381–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6. Kristensen LS, Andersen MS, Stagsted LVW, Ebbesen KK, Hansen TB, Kjems J. The biogenesis, biology and characterization of circular RNAs. Nat Rev Genet. 2019;20:675–91. [DOI] [PubMed] [Google Scholar]
  • 7. Wu J, Qi X, Liu L, Hu X, Liu J, Yang J, et al. Emerging epigenetic regulation of circular RNAs in human cancer. Mol Ther Nucleic Acids. 2019;16:589–96. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8. Hua X, Sun Y, Chen J, Wu Y, Sha J, Han S, et al. Circular RNAs in drug resistant tumors. Biomed Pharmacother. 2019;118:109233. [DOI] [PubMed] [Google Scholar]
  • 9. Wang C, Tan S, Li J, Liu WR, Peng Y, Li W. CircRNAs in lung cancer ‐ biogenesis, function and clinical implication. Cancer Lett. 2020;492:106–15. [DOI] [PubMed] [Google Scholar]
  • 10. Wang J, Zhao X, Wang Y, Ren FH, Sun DW, Yan YB, et al. circRNA‐002178 act as a ceRNA to promote PDL1/PD1 expression in lung adenocarcinoma. Cell Death Dis. 2020;11:32. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11. Zhang ZY, Gao XH, Ma MY, Zhao CL, Zhang YL, Guo SS. CircRNA_101237 promotes NSCLC progression via the miRNA‐490‐3p/MAPK1 axis. Sci Rep. 2020;10:9024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12. Ye Y, Zhao L, Li Q, Xi C, Li Y, Li Z. circ_0007385 served as competing endogenous RNA for miR‐519d‐3p to suppress malignant behaviors and cisplatin resistance of non‐small cell lung cancer cells. Thorac Cancer. 2020;11:2196–208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13. Li Y, Shi R, Zhu G, Chen C, Huang H, Gao M, et al. Construction of a circular RNA‐microRNA‐messenger RNA regulatory network of hsa_circ_0043256 in lung cancer by integrated analysis. Thorac Cancer. 2022;13:61–75. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14. Hansen TB, Jensen TI, Clausen BH, Bramsen JB, Finsen B, Damgaard CK, et al. Natural RNA circles function as efficient microRNA sponges. Nature. 2013;495:384–8. [DOI] [PubMed] [Google Scholar]
  • 15. Salmena L, Poliseno L, Tay Y, Kats L, Pandolfi PP. A ceRNA hypothesis: the Rosetta Stone of a hidden RNA language? Cell. 2011;146:353–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16. Zhou ZB, Huang GX, Fu Q, Han B, Lu JJ, Chen AM, et al. circRNA.33186 contributes to the pathogenesis of osteoarthritis by sponging miR‐127‐5p. Mol Ther. 2019;27:531–41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. Chen RX, Liu HL, Yang LL, Kang FH, Xin LP, Huang LR, et al. Circular RNA circRNA_0000285 promotes cervical cancer development by regulating FUS. Eur Rev Med Pharmacol Sci. 2019;23:8771–8. [DOI] [PubMed] [Google Scholar]
  • 18. Huang G, Liang M, Liu H, Huang J, Li P, Wang C, et al. CircRNA hsa_circRNA_104348 promotes hepatocellular carcinoma progression through modulating miR‐187‐3p/RTKN2 axis and activating Wnt/β‐catenin pathway. Cell Death Dis. 2020;11:1065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19. Fan Z, Bai Y, Zhang Q, Qian P. CircRNA circ_POLA2 promotes lung cancer cell stemness via regulating the miR‐326/GNB1 axis. Environ Toxicol. 2020;35:1146–56. [DOI] [PubMed] [Google Scholar]
  • 20. Hong W, Xue M, Jiang J, Zhang Y, Gao X. Circular RNA circ‐CPA4/let‐7 miRNA/PD‐L1 axis regulates cell growth, stemness, drug resistance and immune evasion in non‐small cell lung cancer (NSCLC). J Exp Clin Cancer Res. 2020;39:149. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  • 21. Bernardo BC, Ooi JY, Lin RC, McMullen JR. miRNA therapeutics: a new class of drugs with potential therapeutic applications in the heart. Future Med Chem. 2015;7:1771–92. [DOI] [PubMed] [Google Scholar]
  • 22. Mishra S, Yadav T, Rani V. Exploring miRNA based approaches in cancer diagnostics and therapeutics. Crit Rev Oncol Hematol. 2016;98:12–23. [DOI] [PubMed] [Google Scholar]
  • 23. Rupaimoole R, Slack FJ. MicroRNA therapeutics: towards a new era for the management of cancer and other diseases. Nat Rev Drug Discov. 2017;16:203–22. [DOI] [PubMed] [Google Scholar]
  • 24. Abd El‐Aziz A, El‐Desouky MA, Shafei A, Elnakib M, Abdelmoniem AM. Influence of pentoxifylline on gene expression of PAG1/miR‐1206/ SNHG14 in ischemic heart disease. Biochem Biophys Rep. 2021;25:100911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25. Gutierrez‐Camino A, Oosterom N, den Hoed MAH, Lopez‐Lopez E, Martin‐Guerrero I, Pluijm SMF, et al. The miR‐1206 microRNA variant is associated with methotrexate‐induced oral mucositis in pediatric acute lymphoblastic leukemia. Pharmacogenet Genomics. 2017;27:303–6. [DOI] [PubMed] [Google Scholar]
  • 26. Yu S, Wang M, Zhang H, Guo X, Qin R. Circ_0092367 inhibits EMT and gemcitabine resistance in pancreatic cancer via regulating the miR‐1206/ESRP1 Axis. Genes (Basel). 2021;12:1701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27. Xue X, Chen Y. Circular RNA (circ)_0129047 upregulates bone morphogenetic protein receptor type 2 expression to inhibit lung adenocarcinoma progression by sponging microRNA (miR)‐1206. Bioengineered. 2022;13:12067–87. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28. Li Y, Tu S, Zeng Y, Zhang C, Deng T, Luo W, et al. KLF2 inhibits TGF‐β‐mediated cancer cell motility in hepatocellular carcinoma. Acta Biochim Biophys Sin (Shanghai). 2020;52:485–94. [DOI] [PubMed] [Google Scholar]
  • 29. Lu Y, Qin H, Jiang B, Lu W, Hao J, Cao W, et al. KLF2 inhibits cancer cell migration and invasion by regulating ferroptosis through GPX4 in clear cell renal cell carcinoma. Cancer Lett. 2021;522:1–13. [DOI] [PubMed] [Google Scholar]
  • 30. Yuedi D, Houbao L, Pinxiang L, Hui W, Min T, Dexiang Z. KLF2 induces the senescence of pancreatic cancer cells by cooperating with FOXO4 to upregulate p21. Exp Cell Res. 2020;388:111784. [DOI] [PubMed] [Google Scholar]
  • 31. Jiang W, Xu X, Deng S, Luo J, Xu H, Wang C, et al. Methylation of kruppel‐like factor 2 (KLF2) associates with its expression and non‐small cell lung cancer progression. Am J Transl Res. 2017;9:2024–37. [PMC free article] [PubMed] [Google Scholar]
  • 32. Xiao S, Jin‐Xiang Y, Long T, Xiu‐Rong L, Hong G, Jie‐Cheng Y, et al. Kruppel‐like factor 2 disturb non‐small cell lung cancer energy metabolism by inhibited glutamine consumption. J Pharm Pharmacol. 2020;72:843–51. [DOI] [PubMed] [Google Scholar]

Associated Data

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Supplementary Materials

Figure S1. Cell proliferation detection by colony formation assay.

Click here for additional data file. (11.3MB, tif)

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