Hsa_circ_0000520 Promotes Non-Small Cell Lung Cancer Progression through the miR-1258/AKT3 Axis

Background There are several previous studies suggesting that circular RNAs (circRNAs) are involved in tumorigenesis of non-small cell lung cancer (NSCLC). Nevertheless, the role of circRNA_0000520 (circ_0000520) in this disease has not yet been studied. Methods circ_0000520, microRNA (miR)-1258, and AKT serine/threonine kinase 3 (AKT3) mRNA expression levels were detected by qPCR. CCK-8, EdU, and Transwell assays were utilized to detect NSCLC cells' malignant biological behaviors. The targeted relationship between miR-1258 and AKT3 3′-UTR or circ_0000520 was verified through the dual-luciferase reporter gene assay. Western blotting was utilized to measure the AKT3 expression after circ_0000520 and miR-1258 were selectively regulated. Results circ_0000520 was upregulated in NSCLC. Highly expressed circ_0000520 is linked to the NSCLC patient's advanced TNM stage and lymph node metastasis. circ_0000520 overexpression facilitated NSCLC cell growth, migration, and invasion. miR-1258 was identified as the downstream target of circ_0000520. miR-1258 overexpression weakened the effect of circ_0000520 overexpression on NSCLC cells. miR-1258 targeted and inhibited AKT3. circ_0000520 positively regulated the AKT3 expression in NSCLC cells by sponging miR-1258. Conclusion circ_0000520 upregulates AKT3 by competitively binding with miR-1258 to facilitate NSCLC progression.


Introduction
Globally, lung carcinoma is the biggest cause of cancerrelated deaths [1]. Non-small cell lung cancer (NSCLC) makes up over 80% of lung cancer cases [2]. At present, the common treatment strategies are surgery, chemotherapy, radiotherapy, targeted therapy, and immunotherapy [3]. Despite recent improvements in its diagnosis and therapy strategies, NSCLC patients' prognosis is still adverse [4,5].
In the last several decades, more and more non-coding RNAs (ncRNAs) have been discovered and investigated [6,7]. With a covalently closed-loop structure, circular RNAs (circRNAs) have neither a 5′ end cap nor a 3′ end poly(A) tail [8]. circRNAs are very stable and are transcribed in a tissue-specifc manner [9]. Besides, circRNAs participate in tumorigenesis and may act as biomarkers [10]. Reportedly, multiple circRNAs are dysregulated in NSCLC and can promote or inhibit NSCLC progression. For instance, circ_100395 overexpression suppresses the malignancy of NSCLC cells [11]. In NSCLC tissues, circ_POLA2 is highly expressed and high circ_POLA2 expression is associated with NSCLC patients' poor prognosis [12]. circ_0000520 is downregulated in gastric carcinoma and breast carcinoma and can act as a new biomarker [13,14]. Nevertheless, the functions of circ_0000520 in NSCLC tumorigenesis warrant further elucidation.
Known as highly conserved small ncRNAs, microRNAs (miRNAs) bind to mRNA 3′-UTR to result in mRNA degradation or translation inhibition, thus modulating posttranscriptional gene expression [15]. A lot of miRNAs serve as tumor-suppressive factors or oncogenic factors to partake in tumorigenesis, development, recurrence, and metastasis [16]. Tere are increasing studies showing that miRNAs participate in NSCLC tumorigenesis and development [17][18][19][20][21]. For instance, miR-451a targets ATF2 to repress the aggressiveness of NSCLC cells [20]. In NSCLC tissues and cells, miR-1258 is downregulated and it suppresses NSCLC progression via the GRB2/Ras/Erk axis [22]. Nonetheless, the mechanism of miR-1258 dysregulation in NSCLC is yet to be clarifed.
Te present work investigated the expression pattern of circ_0000520 and subsequently explored the exact role of the circ_0000520/miR-1258/AKT serine/threonine kinase 3 (AKT3) regulatory axis in NSCLC. Our work broadens our understanding of NSCLC's pathogenesis and provides potential biomarkers for the disease.

Ethical Statement and Patient
Tissues. Tirty-seven pairs of tumorous tissues and para-tumorous tissues of NSCLC patients surgically resected at Xiangyang Central Hospital were collected. None of the patients received neoadjuvant therapy. Tis study was performed with each patient's informed consent. Te procedures of the present work were approved by the Ethics Committee of Xiangyang Central Hospital. NSCLC patients' clinical features are described in Table 1.

qPCR.
TRIzol reagent (Invitrogen) was adopted to isolate the total RNA. A PrimeScript RT kit (TaKaRa) was used for the reverse transcription. On an ABI 7900 fast realtime PCR system (Applied Biosystems), qRT-PCR was conducted utilizing a SYBR Green Master Mix II kit (TaKaRa). Te expression of GAPDH was adopted to normalize the expression levels of mRNA and circ_0000520, and the expression of miRNA was normalized with small RNA RNU6B (U6). Te relative expression of genes was quantifed through the 2 −ΔΔCT method. Check Table 2 for the sequences of the primers.

Nucleocytoplasmic Separation Experiment.
A PARIS ™ kit (TermoFisher) was applied for carrying out the nucleocytoplasmic separation experiment. Te TRIzol method was used to extract the cytoplasmic and nuclear RNA, and then, qPCR was conducted to examine the circ_0000520 expression in the nucleus and cytoplasm, respectively. U6 and GAPDH functioned as the controls of subcellular localization.  Assay. Te NSCLC cells were inoculated into 96-well plates (2 × 10 3 cells/well). Ten, 10 μL of CCK-8 solution (MedChemExpress) was supplemented into each well at diferent time points, and the cells were incubated at 37°C for another 2 h. After terminating the culture, the absorbance values were measured at a wavelength of 450 nm. 2.6. 5-Ethynyl-2′-Deoxyuridine (EdU) Assay. An EdU detection kit (RiboBio) was used to detect cell proliferation. H460 and A549 cells were cultured for 24 h. Te cells were then treated with 50 μM EdU at 37°C for 2 h. Next, the culture solution was discarded, and subsequently, the A549 and H460 cells were fxed for 30 min with 4% paraformaldehyde. Next, 0.5% Triton X-100 was applied to increase the permeability, and then, the A549 and H460 cells were incubated with Apollo fuorescence staining reaction solution for 30 min in a dark place. Ten, the cells were stained for 15 min with DAPI staining solution. Finally, the cells were placed under a fuorescence microscope (magnifcation of 200) (Olympus), and the EdU-positive cells were counted.

Transwell
Assays. A549 and H460 cells, after being digested with 0.25% trypsin, were centrifuged and resuspended in a serum-free medium. Matrigel (pore size: 8 µm; 1 : 10; BD Biosciences) was only used for invasion assay. A549 and H460 cells (5 × 10 4 ) were added to the top compartment of Transwell, and 10% FBS-containing DMEM was added to the bottom chamber, and the cells were cultured for 24 h at 37°C. Subsequently, the cells that had failed to migrate were discarded; the cells that had migrated were fxed for 10 min with 4% paraformaldehyde and subsequently stained with 0.5% crystal violet solution. Under an inverted microscope (Olympus), the cells were counted.

Dual-Luciferase Reporter Gene Assay.
From Promega (Madison), all luciferase reporter vectors circ_0000520 mutant (MUT), circ_0000520 wild type (WT), AKT3 MUT, and AKT3 WT were obtained. Next, circ_0000520 WT/ MUT or AKT3 WT/MUT and miR-1258 mimics or its negative control were co-transfected into H460 and A549 cells. Te luciferase activity was measured at 48 h after the transfection.
2.9. Immunoblotting. Te transfected cells were lysed by RIPA bufer (Beyotime). After centrifugation, the cell supernatant was collected. Te supernatant was then heated in a 100°C water bath for 10 min for denaturing the protein.
Ten, the protein was isolated by SDS-PAGE and transferred to the polyvinylidene fuoride (PVDF) membrane (Millipore). Te membrane was blocked at room temperature with 5% skimmed milk for 1 h and subsequently rinsed with Tris-bufered saline with Tween-20 (TBST) 3 times. Subsequently, the membrane and primary antibodies (anti-AKT3 antibody (ab152157, 1 : 1000, Abcam) and anti-GAPDH antibody (ab245355, 1 : 1000, Abcam)) were incubated at 4°C overnight. After TBST rinsing, the membrane and goat anti-rabbit IgG H&L (ab205718, 1 : 5000, Abcam) were incubated for 1 h at room temperature. GAPDH acted as the internal control. Te ECL chemiluminescence kit (Promega) was utilized for developing the bands. Tree weeks later, mice were sacrifced with euthanasia and the volume of the tumor in the two groups was compared.

Statistical Analysis Technique.
Each assay was conducted in triplicates and repeated 3 times. SPSS21.0 (SPSS Inc.) was adopted to statistically analyze the data. "x ± s" was used to represent the data. t-test and one-way analysis of variance were performed to compare the means of 2 and more groups, respectively. Fisher's exact test was utilized to analyze the correlation of circ_0000520 expression with NSCLC patients' clinical parameters. Pearson correlation analysis was conducted to assess the correlation. A diference was of statistical signifcance when P < 0.05.

In NSCLC Tissues and Cells, circ_0000520 Is Upregulated.
Trough the analysis of the microarray data, the dataset GSE158695, it was revealed that circ_0000520 is upregulated in NSCLC tissues (Figure 1(a)). It was also revealed that as opposed to para-cancerous tissues or human immortalized bronchial epithelial cells (BEAS-2B), circ_0000520 was signifcantly upregulated in NSCLC (Figures 1(b) and 1(c)). Furthermore, circ_0000520 was primarily located in the cytoplasm (Figure 1(d)). To assess the correlation of 37 NSCLC patients' clinicopathological features with circ_0000520 expression, the patients were divided into high (n � 18) and low (n � 19) expression groups. It was revealed that high circ_0000520 expression was strongly associated with the NSCLC patients' lymph node metastasis and a higher TNM stage (Table 1). Additionally, a high circ_0000520 expression was positively correlated with low overall survival rate of the patients (Figure 1(e)).

Impacts of the Overexpression of circ_0000520 on NSCLC Cells.
To study circ_0000520s role in NSCLC, circ_0000520 overexpression plasmids were transfected into A549 and H460 cells (Figure 2(a)). circ_0000520 overexpression  (Figures 2(b) and 2(c)). Additionally, circ_0000520 overexpression can remarkably promote A549 and H460 cell migration and invasion (Figure 2(d)). Additionally, in vivo experiments suggested that high expression of circ_0000520 promoted the growth of tumor cells which were transplanted into the nude mice (Figure 2(e)). Te aforementioned fndings indicate that circ_0000520 might be an oncogene in NSCLC progression.

circ_0000520
Adsorbs miR-1258. Next, we searched CircInteractome online website to predict circ_0000520s potential target miRNAs, and observed that there was a binding sequence between circ_0000520 and miR-1258 (Figure 3(a)). Overexpression of miR-1258 markedly repressed circ_0000520 WT's luciferase activity in H460 and A549 cells, with no obvious infuence on that of circ_0000520 MUT (Figure 3(b)). Next, qPCR indicated that unlike para-cancerous tissues or BEAS-2B cells, miR-1258 expression was underexpressed in the cancer group (Figures 3(c) and 3(d)). Notably, circ_0000520 expression and miR-1258 expression were in negative correlation in the tissue samples (Figure 3(e)).

circ_0000520 Regulates the AKT3 Expression in NSCLC
Cells by Repressing miR-1258. miR-1258's downstream target genes were predicted utilizing the TargetScan database, and it was revealed that AKT3 has a binding site to miR-1258 ( Figure 5(a)). Overexpression of miR-1258 inhibited AKT3 WT's luciferase activity ( Figure 5(b)). Next, we found that upregulating circ_0000520 promoted the AKT3 protein   expression, while upregulating miR-1258 could reduce this efect (Figures 5(c) and 5(d)). Furthermore, the AKT3 mRNA expression was markedly elevated in NSCLC ( Figure 5(e)). Te expression levels of miR-1258 and AKT3 mRNA were in negative correlation ( Figure 5(f)) and those of AKT3 mRNA and circ_0000520 expression were in positive correlation ( Figure 5(g)). Unlike BEAS-2B cells, AKT3 mRNA expression was enhanced in NSCLC cell lines ( Figure 5(h)).

Discussion
A growing amount of evidence shows that the expression characteristics of circRNAs are closely associated with the adverse clinical parameters of patients, and circRNA dysregulation often promotes diferent malignant behaviors [23,24]. For instance, in NSCLC, circ_100395 is underexpressed, and circ_100395 overexpression represses the malignancy of NSCLC cells [25]. circ-ACACA is upregulated in NSCLC and knocking down circ-ACACA suppresses NSCLC development by sponging miR-1183 and regulating the PI3K/PKB axis [26]. Tis study reports that circ_000052 is dysregulated in NSCLC by analyzing the circRNA microarray. Furthermore, it is unveiled that high circ_0000520 expression is correlated with adverse prognosis of NSCLC patients. Moreover, we observe that circ_0000520 increases the malignancy of NSCLC cells. In a nutshell, the aforementioned evidence demonstrates that circ_0000520 may act as an oncogene in NSCLC. Te role of miR-1258 in tumorigenesis has been widely reported in recent years [27,28]. In breast carcinoma cells, miR-1258 expression is reduced and miR-1258 can downregulate E2F1 expression [29]. In cervical cancer cells, miR-1258 is downregulated and it inhibits the malignancy biological behaviors of cancer cells by modulating E2F1/P53 signaling [30]. circRNAs can function as miRNAs sponges in many cancers [31,32]. For instance, circ_0000326 accelerates lung adenocarcinoma progression via sponging miR-338-3p [33]. circ_0046264 suppresses the malignancy of lung carcinoma cell by acting on the miR-1245/BRCA2 axis [34]. Tis study revealed that circ_0000520 can sponge miR-1258 in NSCLC cells. Additionally, miR-1258 upregulation reversed the promotional impact that circ_0000520 overexpression had on the malignancy of NSCLC cells, suggesting circ_0000520 may regulate NSCLC development via sponging miR-1258. Previous studies have reported that some miRNAs are target miRNAs of circ_0000520, such as miR-512-5p, miR-1296, miR-556-5p, and so on [35][36][37]. Our study identifed a novel downstream miRNA target of circ_0000520 and miR-1258.
Known as a serine/threonine protein kinase, AKT3 is pivotal in modulating cell proliferation, diferentiation, apoptosis, and migration [38]. AKT3 is abnormally expressed in various cancers and afects cancer progression. For example, in papillary thyroid carcinoma tissues and cells, AKT3 expression is elevated; miR-203 represses the malignancy of papillary thyroid cancer cells by downregulating AKT3 [39]. Tere is a study showing that AKT3 can promote prostate cancer cell proliferation by regulating Akt, B-Raf, and TSC1/TSC2 [40]. Besides, miR-217 can suppress NSCLC development by reducing the AKT3 expression [41]. Tis study revealed that AKT3 is miR-1258's downstream target in NSCLC cells. Moreover, AKT3 expression is modulated by the circ_0000520/miR-1258 axis. Te abovementioned evidence demonstrates that circ_0000520 regulates NSCLC development through regulating the miR-1258/AKT3 axis.
To sum up, circ_0000520 expression in NSCLC is elevated, and it enhances the malignancy of cancer cells. In terms of mechanism, circ_0000520 increases AKT3 expression via absorbing miR-1258. Tese fndings may provide innovative ideas for NSCLC treatment.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.

Ethical Approval
Our study was approved by the Ethics Review Board of Xiangyang Central Hospital.