CircZNF652 accelerates the proliferation and migration of primary lung carcinoma cells by downregulating miR-766

Purpose: To explore the biological functions and molecular mechanism of circZNF652 involvement in primary lung carcinoma. Methods: CircZNF652 levels in primary lung carcinoma cases and controls were determined using quantitative real-time polymerase chain reaction (qRT-PCR). Its prognostic value in primary lung carcinoma was examined by depicting it with Kaplan-Meier curves. The biological functions of circZNF652 in regulating proliferative and migratory capacities in A549 and SPC-A-1 cells were analyzed from the curves. Interaction between circZNF652 and its downstream gene, miR-766, was assessed, and their co-regulation on primary lung carcinoma was determined by rescue experiments. Results: CircZNF652 was abnormally and significantly upregulated in primary lung carcinoma cases (p < 0.05), resulting in a poor prognosis. The knockdown effect of circZNF652 attenuated the proliferative and migratory capacities of A549 and SPC-A-1 cells, and downregulated epithelial-mesenchymal transition (EMT)-associated genes. CircZNF652 bound and negatively regulated miR-766, a key downstream gene involved in circZNF652-induced aggravation of primary lung carcinoma. Conclusion: CircZNF652 serves as an oncogene, triggering the aggravation of primary lung carcinoma by negatively regulating miR-766. The results of this study may provide new insights into the treatment of lung carcinoma.


INTRODUCTION
Primary lung carcinoma is one of the leading cause of cancer death in the world, with the number of diagnosed cases growing rapidly. [1]. Non-small cell lung cancer (NSCLC) is a major histological subtype of lung cancer, accounting for 80 % of primary lung carcinoma cases [2]. Surgery, chemotherapy and radiotherapy are the main therapeutic strategies for managing primary lung carcinoma [3]. To improve the clinical outcomes in NSCLC patients, novel molecular targeted therapy has been utilized [4]. Individualized therapy is conducted in order to prolong the survival chances of advanced NSCLC patients [5]. Nevertheless, sensitive biomarkers which support individualized therapy for primary lung carcinoma are limited. Circular RNAs (CircRNAs) are a type of unique noncoding RNAs and they have been highlighted in recent research. They have a closed loop structure, and are resistant to RNA exonucleases [6]. CircRNAs are enriched by miRNA binding sites, which serve miRNA sponge effect as ceRNAs. In recent times, research has shown the close relationship between abnormally expressed circRNAs and cancer. CircRNAs exert vital regulations on malignant phenotypes of cancer cells and cancer metabolism. It has been reported that circLONP2 triggers invasiveness and metastasis of colorectal carcinoma by regulating the maturation and secretion of miRNAs [7]. Circ_0032821 serves as an oncogene that mediates growth, metastasis and autophagy of gastric cancer cells via the MEK1/ERK1/2 signaling [8].
Malignant gastric cancer can be alleviated by circMRPS35-induced histone modification on KAT7 [9]. Previous studies have demonstrated the oncogenic role of circZNF652 in renal carcinoma and hepatocellular carcinoma through the targeting of EMT and corresponding miRNAs [10]. In this study, circZNF652 demonstrated abnormal expression in primary lung carcinoma cases. Its potential influence on the malignant process of primary lung carcinoma and the underlying mechanism were explored in detail.

Subjects and samples
Twenty-three primary lung carcinoma patients who were admitted in our hospital from May 2012 to December 2013 year were recruited for retrospective analysis. They did not have preoperative chemotherapy or radiotherapy. Cancer and paracancerous tissues that were 3 cm away from cancerous lesions were sampled and pathologically confirmed. Recruited patients were followed up for 5 years, up till December 2018. Tumor, Node, Metastasis (TNM) staging of primary lung carcinoma was as defined by UICC criteria. This study was approved by the research ethics committee of Taizhou Hospital of traditional Chinese Medicine (approval no. JS-652-TZ-02-193). Informed consent was obtained from the patients. This study was conducted in accordance with the guidelines of the Declaration of Helsinki [11].

Quantitative real-time polymerase chain reaction (qRT-PCR)
Qualified RNA isolated from cells or tissues was reversely transcribed into cDNA using the PrimeScript RT reagent Kit (TaKaRa, Dalian, China). The cDNA was subjected to qRT-PCR using the SYBR Green Master Mix (Applied Biosystems, San Diego, CA, USA). Each sample was made in triplicate, and the relative level was calculated by 2 -ΔΔCt . The primers used are shown in Table 1.

Cell Counting Kit (CCK-8) assay
The cells were inoculated in a 96-well plate at a density of 2 × 10 3 cells/well. At 0, 24, 48 and 72 h, optical density at 450 nm of each sample was recorded using the CCK-8 kit (Dojindo Laboratories, Kumamoto, Japan) for plotting the viability curves.
Transwell assay 5 × 10 4 cells were implanted on the top chamber, while 500 μl of medium was applied on the bottom of a Transwell chamber. One hour later, migratory cells on the bottom were reacted with 15-min methanol and 20-min crystal violet, and then captured using a microscope. Migratory cells were counted in 10 randomly selected fields in each sample.

Wound healing assay
Cells were prepared into suspension with 1 × 10 5 cells/ml, and implanted in 6-well plates. When 80 % of the cells had attached, an artificial scratch was made using a sterilized pipette tip. The cells were washed in phosphate buffered saline (PBS) 2-3 times and cultured in the medium containing 1 % FBS. 24 hours later, the percentage of wound healing was calculated based on the migrated distance.

Dual-luciferase reporter assay
Sequences containing wild-type or mutant-type binding sites between circZNF652 and miR-766 were inserted in pmiR-RB-REPORT for synthesizing circZNF652-WT and circZNF652-MUT, respectively. They were co-transfected in cells with miR-766 NC or miR-766 mimics for 48 h, followed by measure of assay luciferase activity.

Western blot
Cellular proteins were isolated using RIPA lysis buffer (Beyotime, Haimen, China), which were separated by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) and loaded on polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After incubation in 5 % skim milk for 2 h, membranes were washed and cultured with primary and secondary antibodies. Band exposure was conducted using electrochemiluminescence (ECL) (Pierce, Rockford, IL, USA).

Statistical analysis
Data are expressed as mean ± standard deviation, and processed using GraphPad Prism 6.0 (La Jolla, CA, USA). Differences between two groups were analyzed using the Student's t-test. Comparison between multiple groups was done using One-way ANOVA followed by Post-hoc test (Least Significant Difference). Kaplan-Meier curves were plotted for survival analysis. P < 0.05 was considered indicative of statistically significant difference.

CircZNF652 was upregulated in primary lung carcinoma cases
Compared with normal tissues, circZNF652 was upregulated in primary lung carcinoma ones (Figure 1 A). As expected, circZNF652 was highly expressed in lung carcinoma cell lines (Figure 1 B). According to the follow-up data of recruited patients, Kaplan-Meier curves presented a poor prognosis in primary lung carcinoma patients expressing high level of circZNF652, and showing a certain prognostic value ( Figure 1C).

CircZNF652 stimulated proliferative and migratory capacities in primary lung carcinoma
i-circZNF652 was synthesized and transfected in A549 and SPC-A-1 cells for silencing circZNF652 (Figure 2 A). The knockdown of circZNF652 remarkably reduced viability in A549 and SPC-A-1 cells, indicating the inhibited proliferative capacity (Figure 2 B and C). Moreover, wound healing assay and Transwell assay yielded the conclusion that the knockdown of circZNF652 suppressed migratory capacity in primary lung carcinoma ( Figure 2 D and E). In addition, protein levels of N-cadherin and Vimentin were downregulated, whereas E-cadherin was upregulated in A549 and SPC-A-1 cells transfected with si-circZNF652 ( Figure 2 F and G). This indicated that circZNF652 was able to affect EMT in primary lung carcinoma, which may be a key mechanism for the oncogenic role of circZNF652.

CircZNF652 binding negatively regulated miR-766
To elucidate on how circZNF652 exerts oncogenic role in primary lung carcinoma, its potential target was predicted using online software. Binding sites were identified in the 3'UTR of circZNF652 and miR-766 (Figure 3 A). Subsequently, dual-luciferase reporter assay confirmed the binding relationship between circZNF652 and miR-766 (Figure 3 B and C). Transfection of si-circZNF652 markedly upregulated miR-766 level in A549 and SPC-A-1 cells (Figure 3 D). Consistently, a negative correlation was identified between circZNF652 and miR-766 in primary lung carcinoma tissues (Figure 3 E).

MiR-766 was the key downstream gene for the oncogenic role of circZNF652
Focus was thereafter shifted to investigating the involvement of miR-766 in primary lung carcinoma. CCK-8 assay showed a higher viability in A549 and SPC-A-1 cells, with cosilence of circZNF652 and miR-766, than in those with knockdown of circZNF652 (Figure 4 A  and B). Besides, knockdown of miR-766 partially reversed the declined migratory cell number and percentage of wound healing closure of lung carcinoma cells with circZNF652 knockdown (Figure 4 C -F). Taken together, knockdown of miR-766 abolished the role of circZNF652 in regulating primary lung carcinoma phenotypes.

DISCUSSION
Lung carcinoma is a highly lethal cancer of the respiratory system, and its casualty numbers rank amongst the highest when it comes to malignancies [12]. For a long period of time, anticancer treatments for primary lung carcinoma were limited, and a variety of effective strategies have now been applied in clinical diagnosis and treatment, including immunotherapy and targeted therapy. The pathogenesis of lung carcinoma is complicated, and it includes EMT, disorder of intracellular environment, and reprogramming of cell metabolism [13]. With the process of genomics, genetic heterogeneity has been identified as vital in carcinogenesis, including abnormally activated tumor-associated genes and epigenetic modification [14]. Recently, circRNAs are considered as novel biomarker for cancers because of their high conservatism, stability and specificity. They contribute to form a regulatory network involved in the regulation of cancer epigenetic modification [15,16].
Previous findings have demonstrated that circZNF652 is a vital oncogene [17,18]. Consistently, this study showed that circZNF652 was upregulated in primary lung carcinoma cases and highly expressed circZNF652 was unfavorable to the prognosis of the patients. A series of functional experiments verified that circZNF652 stimulated proliferative and migratory capacities, as well as EMT in A549 and SPC-A-1 cells.
To further clarify the molecular mechanism of circZNF652 in regulating primary lung carcinoma, its molecular target was determined using an online software (https://circinteractome.nia.nih. gov/), and miR-766 was selected. The MiR-766 is confirmed as a tumor-suppressor gene, and it was reported that it alleviates the process of hepatocellular carcinoma by directly targeting the Wnt signaling [19].
MiR-766 prevents the clinical progression of colorectal carcinoma by limiting cancer cell metastasis [20]. Besides, through the activation of the well-known p53, miR-766 participated in cell cycle progression of cancer cells [21]. In lung adenocarcinoma and NSCLC, miR-766 is a potential regulator that influences cancer progression [22,23]. It induces the biological function of noncoding RNAs and thus determines lung carcinoma aggravation [24]. In this study, miR-766 was the downstream gene of circZNF652, presenting a negative correlation between each other. Notably also, the knockdown of miR-766 was able to abolish the inhibited proliferative and migratory capacities in A549 and SPC-A-1 cells with circZNF652 knockdown. We have demonstrated a negative axis circZNF652/miR-766 that was responsible for stimulating the malignant process of primary lung carcinoma.

CONCLUSION
The results obtained indicate that CircZNF652 serves as an oncogene that triggers the aggravation of primary lung carcinoma by negatively regulating miR-766.