Propofol-mediated circ_0000735 downregulation restrains tumor growth by decreasing integrin-β1 expression in non-small cell lung cancer

Abstract Propofol, an intravenous anesthetic agent, exerts an anti-tumor peculiarity in multifarious tumors. Circular RNA hsa_circ_0000735 (circ_0000735) is involved in non-small cell lung cancer (NSCLC) progression. The purpose of this study is to investigate whether propofol can curb NSCLC progression via regulating circ_0000735 expression. Cell viability, proliferation, apoptosis, and invasion were detected using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5-ethynyl-2′-deoxyuridine, flow cytometry, and transwell assays. Evaluation of protein levels was performed using western blotting or immunohistochemistry. Detection of circ_0000735 in tissue samples and cells was carried out using a real-time quantitative polymerase chain reaction. The molecular mechanisms associated with circ_0000735 were predicted by bioinformatics analysis and verified by dual-luciferase reporter assays. The relationship between propofol and circ_0000735 in vivo was verified by xenograft models. The results showed that circ_0000735 was overexpressed in NSCLC samples and cells. Propofol treatment overtly decreased circ_0000735 expression in NSCLC cells and repressed NSCLC cell viability, proliferation, invasion, and facilitated NSCLC cell apoptosis, but these effects mediated by propofol were counteracted by circ_0000735 overexpression. Circ_0000735 functioned as a miR-153-3p sponge and regulated integrin-β1 (ITGB1) expression via adsorbing miR-153-3p. ITGB1 overexpression reversed circ_0000735 silencing-mediated effects on NSCLC cell viability, proliferation, invasion, and apoptosis. In conclusion, propofol restrained NSCLC growth by downregulating circ_0000735, which functioned as a miR-153-3p sponge and regulated ITGB1 expression via adsorbing miR-153-3p. This study provides evidence to support that propofol curbs NSCLC progression by regulating circRNA expression.


Introduction
Currently, surgery is still the main treatment for cancer. A series of studies uncover that the management of anesthetics during surgery is related to the prognosis of cancer [1,2]. Retrospective clinical studies manifest that the prognosis of cancer surgery with intravenous anesthesia is better than that with inhalation anesthesia [3,4]. Propofol (2,6-diisopropylphenol), an intravenous anesthetic agent, is the most extensively used for induction and maintenance of anesthesia [5]. Recently, many studies have uncovered that propofol plays an anti-tumor peculiarity in multifarious tumors [6]. Also, propofol anesthesia is implicated in better survival in stage I non-small cell lung cancer (NSCLC) patients who underwent radical surgery [7]. However, the mechanisms by which propofol regulates NSCLC progression are unclear.
Circular RNAs (circRNAs), endogenous biomolecules, show differential expression among different species, developmental stages, and pathologies. They are characterized by a covalently closed loop structure produced by a special type of alternative splicing [8]. Most circRNAs have known functions to sequester proteins or microRNAs (miRs), translate to produce polypeptides, regulate transcription, and interfere with splicing [9]. Also, the deregulation of circRNAs is associated with the tumorigenesis of many cancers [10]. Recently, several studies have revealed that circRNAs participate in the repressive effect of propofol on tumor growth. For instance, propofol restrained tumorigenesis through downregulation of circ_PVT1 in gastric cancer [11], circ_TADA2A and circ_ERBB2 in lung cancer [12,13], and circ_VPS13C in ovarian cancer [14]. In contrast, propofol postponed tumor progression via upregulation of circ_0026344 in colorectal cancer [15]. CircRNA hsa_circ_ 0000735 (circ_0000735), located on chr17: 3802927-3808661, is generated from the P2RX1 gene. A previous study manifested that circ_0000735 exerted a tumor-promoting activity in NSCLC [16]. However, whether propofol can restrain progression by changing circ_0000735 expression is unclear.
Accordingly, the purpose of the research was to check whether propofol can curb NSCLC progression via regulating circ_0000735 expression.

Surgical specimens
Thirty-two NSCLC patients were enrolled in the research after obtaining written informed consent. Lung tumors and paired non-tumor adjacent samples were collected during surgery at Hulunbeier Municipal People's Hospital. The patients did not receive any preoperative treatment.
Ethics approval and consent to participate: Written informed consent was obtained from patients with approval by the Institutional Review Board in Hulunbeier Municipal People's Hospital.

Propofol treatment
Dissolution of propofol (Sigma, St Louis, MO, USA) was performed using 10% intralipid (Astra-Zeneca, London, UK), followed by diluting with serum-free medium to a stock concentration of 0.4 mg/mL. For propofol treatment, the cells were cultured in the complete medium containing diverse doses of propofol (5, 10, and 15 μg/mL).

Detection of cell viability
After transfection, the cells were cultured in the complete medium containing diverse doses of propofol for 48 h. Following this, 10 μL 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) solution (Roche, Basel, Switzerland) was added. Then, 100 μL of dimethyl sulfoxide was used to dissolve the purple crystals. The optical density was evaluated using a microplate reader VarioSkan Flash (Thermo Scientific).

5-Ethynyl-2′-deoxyuridine (EdU) assay
Detection of cell proliferation was executed using an EdU Detection Kit (Ribobio, Guangzhou, China) according to the manufacturer's protocol. Staining of the nucleus was performed with the 4',6-diamidino-2-phenylindole solution (Thermo Fisher), and the proportion of EdU-positive cells was observed using fluorescence microscopy (Olympus, Tokyo, Japan).

Flow cytometry assay
Assessment of cell apoptosis was carried out using the Annexin V-FITC Apoptosis Detection Kit (Sigma). Briefly, the cells were washed and re-suspended in binding buffer. Next, the cells were incubated in the dark with Annexin V-FITC solution and propidium iodide solution, followed by flow cytometry analysis with an LSRII Fortessa flow cytometer (BD Biosciences, San Jose, CA, USA). Apoptotic rate was the sum of the early and late apoptotic rates.

Transwell invasion assay
Invasion capacity was evaluated using the CHEMICON Cell Invasion Assay Kit (ECM550, Sigma). In short, about 5 × 10 5 cells were seeded on the top of the invasion chamber after 24 h of transfection. The cells were stained with 0.1% crystal violet (Sigma) after another 24 h of incubation. The number of invading cells was counted using an inverted microscope (Olympus, Tokyo, Japan).

In vivo experiments
The animal experiments were conducted with the approval of the Animal Ethics Committee of Hulunbeier Municipal People's Hospital. Thirty-two BALB/c nude mice (Vital River Laboratory, Beijing, China) were randomly divided into four groups (eight mice in each group). Then, 16 mice in two groups were injected with A549 cells transduced with sh-NC and treated with intralipid or propofol. Analogously, 16 mice in two groups were injected with A549 cells transduced with sh-circ_0000735 and treated with intralipid or propofol. From Day 7, mice were treated with propofol (45 mg/kg) every 3 days via tail vein injection, and intralipid acted as vehicle control. Tumor volume was measured every 3 days (volume = (length × width 2 )/2) from propofol administration. Mice were maintained for 22 days before being sacrificed, following the appropriate protocols. Xenograft tumors were excised for subsequent analysis.

Statistical analysis
All experiments were performed with a minimum of n = 3 biological replicates and n = 3 technical replicates. Results were represented as mean ± standard deviation using GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). Significance was assessed using Student's t-tests (comparing data between two variances) or analysis of variance (comparing data with three or more variances) with Tukey's post hoc test. Asterisks indicate significant differences between experimental groups (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).

Propofol restrained proliferation, invasion, and induced apoptosis of NSCLC cells
To evaluate the changes in NSCLC cell viability in response to anesthesia by propofol, we treated NSCLC cells with drug concentration gradients. Data in Figure 1a-c present that propofol restrained NSCLC cell viability in a concentrationdependent manner, but it did not affect the viability of HBE cells. Next, we assessed the impacts of propofol on NSCLC cell proliferation, apoptosis, and invasion. EdU assays exhibited that propofol significantly repressed NSCLC cell proliferation as its dose increases ( Figure  1d). Flow cytometry assays showed that propofol led to a prominent increase in the proportion of apoptotic cells as its concentration increases (Figure 1e). Transwell invasion assays displayed that propofol resulted in a concentration-dependent suppression of NSCLC cell invasion ( Figure 1f). We then assessed the protein levels of Cleaved-caspase3 and MMP9 in NSCLC cells in response to anesthesia by propofol. As shown in Figure 1g, propofol elevated the Cleaved-caspase3 protein level in NSCLC cells in a concentration-dependent manner. In contrast, propofol caused a marked decrease in the MMP9 protein level in a concentration-dependent manner ( Figure 1h). Collectively, these results manifested that propofol restrained proliferation, invasion, and induced apoptosis of NSCLC cells as its concentration increases.

circ_0000735 was overexpressed in NSCLC
To validate the changes in circ_0000735 expression in NSCLC tissues and cells, we carried out RT-qPCR analysis with divergent primers. The results showed a particularly strong enhancement of circ_0000735 in NSCLC tissues and cells compared to their respective controls (Figure 2a and b). Resistance to the RNase R exonuclease was used to test the form of circ_0000735. We observed a prominent decrease in linear P2RX1 after RNase R treatment, but circ_0000735 was resistant to RNase R digestion, manifesting that circ_0000735 was more stable than its linear cognate mRNA (Figure 2c and d). A nuclear mass separation assay exhibited a dominantly cytoplasmic distribution of circ_0000735 (Figure 2e and f). Collectively, these results suggested that circ_0000735 expression was elevated in NSCLC.

Propofol controlled NSCLC cell proliferation, invasion, and apoptosis by regulating circ_0000735 expression
To verify the involvement of propofol and circ_0000735 in NSCLC cell proliferation, invasion, and apoptosis, we detected circ_0000735 expression in propofol-treated NSCLC cells. The results exhibited that propofol caused a distinct decrease in circ_0000735 expression, and 10 μg/mL of propofol was used for subsequent analysis (Figure 3a). Next, the circ_0000735 overexpression plasmid was constructed to explore the effects of circ_0000735 overexpression on proliferation, invasion, and apoptosis of propofol-treated NSCLC cells. After transfection, circ_0000735 was highly expressed in NSCLC cells (Figure 3b). Also, the downregulation of circ_0000735 in NSCLC cells caused by propofol was reversed after circ_0000735 introduction (Figure 3c). Also, exogenous circ_0000735 overturned propofol-mediated impacts on NSCLC cell proliferation, apoptosis, and invasion (Figure 3d-g). As expected, circ_0000735 overexpression reversed the elevation of Cleaved-caspase3 and the decrease of MMP9 in propofol-treated NSCLC cells (Figure 3h-j). Together, these results indicated that propofol curbed NSCLC cell proliferation, invasion, and induced NSCLC cell apoptosis via repressing circ_0000735 expression.

circ_0000735 was identified as a miR-153-3p decoy
To further investigate the function of circ_0000735, we searched for miRs that might interact with circ_0000735. Through software prediction (Starbase2.0), we discovered that circ_0000735 had a complementary sequence to the miR-153-3p seed region (Figure 4a). To verify this hypothesis, NSCLC cells were overexpressed with miR-153-3p (Figure 4b). Dual-luciferase reporter assay revealed a marked decrease in the luciferase activity in NSCLC cells co-transfected with the WT-circ_0000735 reporter and miR-153-3p mimic, but there was no difference change in NSCLC cells co-transfected with the MUT-circ_0000735 reporter and miR-153-3p mimic (Figure 4c and d). As expected, miR-153-3p was lowly expressed in NSCLC tis-sues and it was negatively correlated with circ_0000735 expression (Figure 4e and f). Similar results were obtained with miR-153-3p in NSCLC cells (Figure 4g). MiR-153-3p overexpression decreased NSCLC cell viability, proliferation, elevated NSCLC cell apoptosis, and repressed NSCLC cell invasion ( Figure A1a-d). Additionally, miR-153-3p was highly expressed in propofol-treated NSCLC cells as propofol dose increases ( Figure A2a). The transfection efficiency of miR-153-3p inhibitor is presented in Figure A2b. Moreover, miR-153-3p inhibitor impaired the elevation of miR-153-3p in NSCLC cells mediated by propofol ( Figure A2c). Also, miR-153-3p silencing reversed the impacts of propofol on NSCLC cell proliferation, apoptosis, and invasion ( Figure  A2d-g). And miR-153-3p knockdown overturned propofolmediated effects on protein levels of Cleaved-caspase3 and MMP9 ( Figure A2h-j). To assess the effect of circ_0000735 on miR-153-3p expression, we then designed a siRNA against circ_0000735. The interference efficiency of si-circ_0000735 is presented in Figure 4h. The exogenous circ_0000735 decreased miR-153-3p expression in NSCLC cells. In contrast, circ_0000735 silencing elevated miR-153-3p expression in NSCLC cells (Figure 4i). These results indicated that circ_0000735 acted as a miR-153-3p decoy. .
(e and f) After cell fractionation, the level of circ_0000735 in the nuclear and cytoplasmic fractions was analyzed using RT-qPCR (n = 3). U6 and GAPDH served as a positive control for nuclear and cytoplasmic fractions, respectively. ***P < 0.001 and ****P < 0.0001.

ITGB1 was a target of miR-153-3p transfected
We further explored the downstream mechanism of miR-153-3p. Based on bioinformatics analysis and preliminary experiments, ITGB1 was selected as a candidate gene. Sequence alignment of wild-type and mutant 3′-UTR of ITGB1 with miR-153-3p are shown in Figure 5a. Also, miR-153-3p mimic strongly decreased the luciferase activity of the WT-ITGB1 3′-UTR reporter but not the MUT-ITGB1 3′-UTR reporter (Figure 5b and c). Furthermore, the level of ITGB1 mRNA was upregulated in NSCLC tissues and it was negatively correlated with miR-153-3p (Figure 5d and e). Also, there was a prominent increase in the ITGB1 protein level in NSCLC tissues and cells (Figure 5f and g). In addition, propofol caused an obvious decrease in the ITGB1 protein level as its dose increases ( Figure A3a). We then constructed the ITGB1 overexpression plasmid to explore the effects of ITGB1 overexpression on propofol-treated NSCLC cells. The transfection efficiency of the ITGB1 overexpression plasmid is shown in Figure A3b. Also, ectopic expression of ITGB1 rescued the downregulation of ITGB1 in propofol-treated NSCLC cells ( Figure A3c). Moreover, upregulation of ITGB1 rescued propofol-mediated effects on NSCLC cell proliferation, apoptosis, and invasion ( Figure A3d-g). In addition, the changes in the protein levels of Cleaved-caspase-3 and MMP9 in NSCLC cells mediated by propofol were overturned after ITGB1 overexpression ( Figure  A3h-j). As expected, miR-153-3p mimic repressed the ITGB1 protein level in NSCLC cells, while miR-153-3p inhibitor exerted an opposing effect (Figure 3h). Accordingly, these results indicated that ITGB1 acted as a miR-153-3p target.

circ_0000735 controlled NSCLC cell proliferation, apoptosis, and invasion via ITGB1
We then conducted rescue experiments to identify whether circ_0000735 modulated NSCLC cell proliferation, apoptosis, and invasion via ITGB1. The results exhibited that transfection with si-circ_0000735 repressed cell viability, proliferation, and promoted cell apoptosis in NSCLC cells, but these effects caused by circ_0000735 knockdown were reversed by overexpression of ITGB1 (Figure 6a-c). Also, ectopic expression of ITGB1 overturned the repressive effect of circ_0000735 silencing on NSCLC cell invasion ( Figure  6d). We also observed an overt increase in the Cleavedcaspase-3 protein level and a distinct reduction in the MMP9 protein level in si-circ_0000735-transfected NSCLC cells, but these trends were rescued by ITGB1 overexpression (Figure 6e and f). Collectively, these results manifested that circ_0000735 modulated NSCLC cell proliferation, apoptosis, and invasion via ITGB1.

Propofol modulated ITGB1 expression via the circ_0000735/miR-153-3p axis
Considering the above findings, we further explored whether circ_0000735 regulated ITGB1 expression via adsorbing miR-153-3p. Data in Figure 7a display that transection with miR-153-3p inhibitor rescued the   downregulation of ITGB1 in NSCLC cells caused by circ_ 0000735 inhibition. Also, ectopic expression of circ_ 0000735 overturned the decrease in the ITGB1 protein level in NSCLC cells mediated by propofol (Figure 7b). Together, these results suggested that propofol regulated ITGB1 expression via the circ_0000735/miR-153-3p axis.

Propofol repressed A549 cell growth via downregulating circ_0000735 in vivo
To validate the above findings, we constructed xenograft tumor models. A549 cells stably expressing sh-circ_0000735 or sh-NC were constructed and the knockdown efficiency of sh-circ_0000735 is displayed in Figure 8a. The volume and weight of xenograft tumors derived from the sh-circ_0000735 + intralipid group were smaller and lighter than those from the sh-NC + intralipid group, indicating that circ_0000735 silencing decreased tumor growth in vivo (Figure 8b and c). We also observed that propofol treatment reduced the tumor volume and weight of mice in the sh-NC group, and the volume and weight of these tumors were further reduced after the silence of circ_0000735 (Figure 8b and c). Both propofol treatment and circ_0000735 silencing decreased circ_0000735 and ITGB1 levels while elevated miR-153-3p levels in xenograft tumors, but these trends mediated by propofol treatment were further strengthened after circ_0000735 silencing (Figure 8d

Discussion
Recently, the anti-tumor activity of propofol in cancer has garnered significant interest [18]. Some researchers have revealed that propofol restrained cell malignant phenotypes via downregulating FOXM1 through regulating the circ_RHOT1/miR-326 axis in NSCLC [19] and the circ_TADA2A/ miR-155-3p axis [12] or circ_ERBB2/miR-7-5p axis [13] in lung cancer. Our work uncovered that propofol repressed NSCLC cell malignant phenotypes through downregulating ITGB1 by regulation of the circ_0000735/miR-153-3p axis, at least in part.
Previous studies have demonstrated that circ_0000735 exerts an oncogenic role in diverse cancers. Zheng et al. manifested that circ_0000735 facilitated cell invasion and proliferation via sequestration of miR-502-5p [20]. Also, upregulated circ_0000735 decreased cell sensitivity to docetaxel via functioning as a miR-7 sponge in prostate cancer [21]. Furthermore, high circ_0000735 expression could forecast the severity of NSCLC, and circ_0000735 upregulation promoted NSCLC cell malignant phenotypes via sponging miR-1182 and miR-1179 [22]. Also, circ_0000735 elevated BMPER or FAM83F expression via adsorbing miR-940 or miR-635, respectively, thus facilitating NSCLC progression [16,23]. Consistent with the previous studies [16,22,23], circ_0000735 was overexpressed in NSCLC in our study. Moreover, propofol caused a decrease in circ_0000735 expression in NSCLC cells. Upregulation of circ_0000735 reversed the suppressive impacts of propofol on NSCLC cell viability, proliferation, invasion, and the promoting influence of propofol on NSCLC cell apoptosis. Also, propofol decreased xenograft tumor growth, and circ_0000735 expression was lower in xenograft tumors derived from mice treated with propofol. Hence, we inferred that propofol curbed NSCLC cell malignant phenotypes by downregulating circ_0000735.
The integrin family includes a group of heterodimeric cell surface transmembrane proteins that can mediate cell-cell and cell-matrix interactions [29]. ITGB1, a subunit of the integrin family, is aberrantly overexpressed in solid tumors and is related to the poor prognosis of diverse cancers [30]. A series of studies have revealed that ITGB1 acted as an oncogene in NSCLC [31][32][33]. The downregulation of ITGB1 mediated by miR-374b [34], miR-134 [35], and miR-384 [36] repressed NSCLC cell malignant phenotypes. Here, ITGB1 served as a miR-153-3p target, and propofol led to a decrease in the ITGB1 protein level. Moreover, ITGB1 upregulation offset propofol-mediated impacts on NSCLC cell viability, proliferation, invasion, and apoptosis. Importantly, propofol regulated ITGB1 expression by regulation of the circ_0000735/miR-153-3p axis. Thus, we inferred that propofol regulated NSCLC progression through regulation of the circ_0000735/miR-153-3p/ITGB1 axis.