Propofol mediates miR-199a/PAK4 axis to regulate the proliferation, invasion and migration of non-small cell lung carcinoma cells

Propofol mediates miR-199a/PAK4 axis to regulate the proliferation, invasion and migration of non-small cell lung carcinoma cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Propofol mediates miR-199a/PAK4 axis to regulate the proliferation, invasion and migration of non-small cell lung carcinoma cells Rui-Long Yu, Kai Li, Jia-Jun Liu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2340235/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective To investigate if inhibitory effects of propofol on proliferation, invasion and migration of non-small cell lung carcinoma (NSCLC) cells was associated with the regulation of miR-199a/PAK4 axis. Methods Human NSCLC A549 and H1299 cells were treated with propofol of different concentrations at different time points. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed to evaluate miR-199a expression. A549 and H1299 cells were divided into Control, Propofol, Propofol + miR-NC and Propofol + miR-199a inhibitor groups. The proliferation, apoptosis, migration, and invasion were examined by CCK-8, flow cytometry, wound healing, and Transwell, respectively. Western blotting was used to measure the protein expression of PAK4. Xenograft model was established in nude mice to observe if propofol can mediate miR-199a expression to regulate the growth of subcutaneous xenograft tumor. Results Propofol can effectively enhance the expression of miR-199a in NSCLC. Compared with Control group, H1299 and A549 cells in Propofol group decreased in viability, invasion and migration, and increased in apoptosis. The inhibitory effect of propofol on NSCLC growth was reversed by miR-199a. In comparison with Propofol group, Propofol + miR-199a inhibitor group was declined in miR-199a expression and increased in PAK4 protein expression. According to dual-luciferase reporter assay, PAK4 was a target gene of miR-199a. Experiment in vivo revealed propofol can inhibit the growth and reduce the weight of xenograft tumor, which can be reversed by miR-199a inhibitor. Conclusion Propofol can suppress PAK4 expression by inducing miR-199a up-regulation, thereby inhibiting the proliferative, invasive and migrating abilities of NSCLC. Propofol miR-199a PAK4 Non-small cell lung carcinoma Proliferation Invasion Migration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Lung cancer is one of the most common malignant tumors with the highest incidence and mortality, becoming a serious risk to human health and quality of life (Bray et al. 2018 ). Histologically, it can divide into small-cell lung cancer and non-small cell lung carcinoma (NSLCC), with the latter accounting for more than 80% of all lung cancer patients (Osmani et al. 2018 ). Currently, the major treatment of NSCLC includes surgery, chemotherapy and radiotherapy, but the therapeutic outcome is far from satisfactory (Chen et al. 2016 ). Most patients already have distal metastasis at the time of diagnosis and cannot undertake surgical resection, and therapeutic methods like radiotherapy and chemotherapy used in middle- or late-stage usually have side effects, leading to a poor prognosis with the five-year survival rate of as low as only 15% (Chen et al. 2016 ; Hirsch et al. 2017 ). Therefore, it is urgent to develop new therapeutic strategies for NSCLC. As a common intravenous anesthetic, propofol can not only induce or maintain general anesthesia, but also have potential anti-tumor effects (Martin and Jiang 2010 ). Accumulating evidence has revealed that the anti-tumor effect of propofol is associated with the regulation of microRNAs (miRNAs) (Du et al. 2019 ; Yu et al. 2019 ). Coincidentally, Xiaoyu Zheng et al. also reported that propofol can inhibit the growth and accelerate the apoptosis of NSCLC by mediating miR-21/PTEN/AKT pathway (Martin and Jiang 2010 ). In the study by Xinhua Wu et al. , propofol down-regulated the miR-21-5p/MAPK10 axis, thus hindering the survival and promoting the apoptosis of NSCLC cells (Wu et al. 2020 ). However, it is still unclear if propofol can mediate other miRNAs to play its regulatory role in NSCLC. MiR-199a is a class of miRNA widely distributed in human tissues (Liu et al. 2017 ). As reported by many studies, miR-199a was abnormally expressed in many tumor cells and involved in cell proliferation, metabolism, angiogenesis and apoptosis (Fornari et al. 2010 ; Gu and Chan 2012 ). Of note, the study by Jian Zhang et al. proved that propofol can up-regulate miR-199a expression substantially and its effect on invasion and migration of hepatocellular carcinoma (HCC) cells can be reversed by inhibiting miR-199a expression (Zhang et al. 2013b ). According to the analysis of target gene prediction website, PAK4 was a target gene of miR-199a. Songwang Cai et al. also reported that PAK4 was up-regulated in NSCLC and PAK4 can mediate phosphorylation of LIMK1 to regulate NSCLC cell migration and invasion (Cai et al. 2015 ). Given the above, we speculated that propofol can mediate miR-199a and its target gene PAK4 to regulate the NSCLC growth. This study investigated if propofol can inhibit proliferation, invasion and migration of NSCLC cells via mediating miR-199a/PAK4 axis and provided some theoretical basis for the potential treatment methods of NSCLC. Materials And Methods Ethics statement This study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals (Carbone 2012 ), and all anmal experiments were under the supervision of the Medical Ethics Committee of Laboratory Animals in our hospital. Culture and propofol treatment of NSCLC cells Human NSCLC cells, H1299 and A549, were purchased from American type culture collection (ATCC, USA). All NSCLC cells were cultured in DMEM medium which was supplemented with 10% fetal bovine serum (FBS) and the culture conditions included 37°C, relative humidity of 95%, 5% CO 2 and 95% O 2 . Culture medium was renewed every two days. Cell passaging was performed when cells covered 80% of the plate. Cells in logarithmic growth phase (approximately the 3rd-5th generation) were used for subsequent experiments. H1299 and A549 cells were treated with 0, 5 and 10 µg/mL propofol (Sigma-Aldrich Chemical Co.) which was dissolved in dimethyl sulfoxide (DMSO, Solarbio) (Gao et al. 2021 ), and miR-199a expression was determined at 12, 24, and 48 h, respectively. Cell grouping and transfection Human NSCLC cells, A549 and H1299, were divided into four groups: Control group, Propofol group, Propofol + miR-NC group, and Propofol + miR-199a inhibitor group. Cells in Control group did not receive any treatment, but cells in Propofol group were treated with 10 µg/mL propofol for 48 h. Cells in Propofol + miR-NC group and Propofol + miR-199a inhibitor group were transfected with miRNA negative control and miR-199a inhibitor respectively before treatment with 10 µg/mL Propofol for 48 h. The miRNA negative control and miR-199a inhibitor were provided by Shanghai Genechem Co., LTD. Cells collected in the logarithmic growth phase were seeded to 6-well plates and cultured. When cell confluence reached 70%-80%, culture medium was replaced with fresh one and cell transfection performed based on the instructions of Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA). After transfection, cells were incubated routinely for 4–6 h and culture medium was replaced for another 48 h of incubation. Cells in each group were collected for later experiments. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) Total RNA of cells was extracted with TRIZOL reagent (Invirtrogen, USA) and quantified for RNA concentration using ultraviolet spectrophotometry (Beckman, Germany). RNA was reversely transcribed into cDNA using Taq Man MicroRNA Reverse Trallscription Kit (Applied Biosystems, USA). The appropriate volume of cDNA was used as templates for PCR reaction. Primer sequences were designed using the software Primer 5.0 and then synthesized by Sangon Biotech (Shanghai) Co., Ltd. For miR-199a: upstream primer: 5’-GCCACAGTAGTCTGCACAT-3’, downstream primer: 5’-CAGTGCGTGTCGTGGAGT-3’. For U6: upstream primer: 5’-CTCGCTTCGGCAGCACA-3’, downstream prime: 5’-AACGCTTCACGAATTTGCGT-3’. ABI Step One quantitative PCR instrument was used for qRT-PCR. Reaction conditions included initialization at 95°C for 10 min, and 40 cycles of denaturation at 95°C for 15 s and annealing at 60°C for 60 s. Relative expression of target genes was normalized with U6 as internal reference and calculated using 2 −△△Ct method, with ΔCt = Ct target gene - Ct U6 ; ΔΔCt = ΔCt Experimental group - ΔCt Control group - Relative transcription level of target mRNA equals 2 −△△Ct . Cell proliferation assay detected by CCK-8 Cells (1 × 10 4 cells/mL) were seeded to 96-well plates and cultured until they were adherent to the plate wall. The supernatant was discarded and 10 µL of CCK8 reagent (DOJINDO, Japan) was added to each well for 4 h of incubation at 37°C. After disposal of the supernatant, cells were washed with PBS and reacted with 100 µL dimethylsulfoxide (DMSO) (Sigma, USA) in each well before 10 min of oscillation. Microplate reader (MK3, Thermo, Pittsburgh, PA, USA) was used to examine the optical density (OD) at wavelength of 450 nm. Cell viability was calculated according to the formula: (OD value Experimental group - OD value Blank group )/(OD value Control group - OD value Blank group ) × 100%. The experiment was performed three times independently to obtain mean value. Cell apoptosis measured by flow cytometry Cells were digested by 0.25% trypsin (without EDTA), washed with PBS, suspended and adjusted to density of 1 × 10 6 cells/mL. Based on the instructions of apoptosis reagent kit, cells were suspended in 400 µL of binding buffer and reacted with 5 µL Annexin V-FITC and 5 µL PI for 15 min in dark environment. Cell apoptosis was evaluated using flow cytometer. In the scatter plot of the flow cytometer, the right-lower quadrant indicates early-apoptotic cells, right-upper quadrant necrotic and apoptotic cells, left-upper quadrant mechanically injured or necrotic cells, and left-lower quadrant viable cells. Cell apoptosis rate (%) = Early apoptotic cell percentage + Late apoptotic cell percentage. Wound healing assay Cells were collected at logarithmic growth phase and inoculated to 96-well plates with cell density of 4 × 10 5 cells/mL. When the plate was covered by cells, the sterile pipette tip was used to scratch lines, with the pipette perpendicular to the plate. Detached cells were washed away by PBS and photos of cells were taken. Serum-free medium was added to culture cells for 24 h, which were observed and photographed under the microplate. Image Pro Plus 6.0 was used to test the width of scratch lines. Migration rate = (Scratch width 0 h - Scratch width 24 h )/Scratch width 0 h × 100%. The experiment was repeated three times. Transwell invasion assay in vitro Matrigel was diluted to 50 mg/L with serum-free medium and 80 µL Matrigel was added to cover the upper surface of the Transwell chamber. At 48 h after transfection, cells were fasted for 24 h in serum-free medium. After disposal of the culture medium, cells were washed with PBS, suspended in serum-free medium, and adjusted to cell density of 2 × 10 5 cells/mL. And 200 µL cell suspension was added to the upper Transwell chamber, while 500 µL serum-containing medium added to the lower chamber, for 24 h of culture at 37°C with 5% CO 2 . Next, Matrigel and detached cells in the upper chamber were wiped away with cotton swabs, and cells were fixed in 500 µL methanol for 10 min. Then, cells were stained in 500 µL 0.1% crystal violet for 15 min. Cells were observed and photographed under an inverted microscope and five visual fields were randomly selected for invasive cell counting. The experiment was repeated three times. Dual-luciferase reporter gene assay A549 and H1299 cells were collected at logarithmic growth phase and inoculated to 24-well plates with cell density of 2 × 10 5 /well. When cell confluence reached 80%-90%, transfection was performed with Lipofectamine TM 2000 (Invitrogen, USA). Wild-type PAK4 3’-UTR-WT or mutant-type PAK4 3’UTR-MUT plasmid were constructed respectively and used for co-transfection of cells with miR-199a-mimic/miR-NC, with Renilla luciferase (100 ng/well) as the control. At 48 h after co-transfection, the luciferase activity was tested with the dual-luciferase report analysis system (Promega, USA). The ratio of firefly luciferase activity to renilla luciferase activity was regarded as the relative luciferase activity. Besides, A549 and H1299 cells were transfected with miR-NC/miR-199a-mimic/miR-199a inhibitor to evaluate PAK4 expression by western blotting. Western blotting Total proteins in cells were extracted with radioimmunoprecipitation assay (RIPA) lysate and quantified for protein concentration with a bicinchoninic acid (BCA) kit (Sigma, USA). During electrophoresis with 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS- PAGE), proteins were transferred to Polyvinylidene Fluoride (PVDF) membrane using a semi-dry transfer system (Bio-Rad, USA). PVDF membrane was blocked in 5% defatted milk powder for 2 h. Next, primary antibody PAK4 (CST, USA) was added for overnight incubation at 4°C. Then, PVDF membrane was washed with PBS (3 times × 5 min) and reacted with Horseradish Peroxidase (HRP)-labeled secondary antibody (CST, USA) for 1 h at 37°C. The membrane was washed again with PBS (3 times × 5 min), prior to the development and visualization by enhanced chemiluminescence (ECL) solution. Image Pro Plus 6.0 was used for gray value analysis. With GAPDH as the internal reference, the relative expression of target proteins was expressed as the gray value ratio of target protein to GAPDH. Establishment of the nude mouse xenograft model In this study, 20 BALB/c nude mice (4–5 weeks old and weighing 17–21 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. A549 cells were transfected with miR-NC and miR-199a inhibitor and suspended in PBS to adjust cell density to 1 × 10 7 mL. Cell suspension (4 × 10 6 cells) of miR-NC group and miR-199a inhibitor group was injected subcutaneously into the back of nude mice, with 6 mice in each group. The tumor length (L) and width (W) were measured every three days to calculate tumor volume, with the formula: V = L × W 2 /2. After 8 d of tumor establishment, nude mice were injected with DMSO or propofol (45 mg/kg) every three days, and tumor volume was monitored. After 23 d, the mice were sacrificed and xenograft tumors weighed. Statistical methods All data were analyzed with the statistical software package SPSS 21.0 (SPSS, Inc, Chicago, IL, USA). Measurement data were presented by mean ± standard deviation (SD) and tested by Student’s t -test. Comparison among multiple groups was analyzed by one-way ANOVA with Tukey’s post-hoc test. The value of P < 0.05 indicated the statistical significance of differences. Results Propofol reduces NSCLC proliferation and enhances miR-199a expression As illustrated in Fig. 1 , the viability of NSCLC cells H1299 and A549 declined after treatment with 5 and 10 µg/mL propofol for 24 h (all P < 0.05). And the expression of miR-199a was up-regulated in H1299 and A549 cells after treatment with 5 and 10 µg/mL propofol for 12 h (all P < 0.05). Thus, we decided to use 10 µg/mL propofol for 48 h to conduct subsequent experiments, since it could effectively reduce cell viability to 50%. Propofol increases miR-199a expression to limit proliferation and promote apoptosis of NSCLC cells CCK-8 method and flow cytometry were used to evaluate the proliferation and apoptosis of NSCLC cells, respectively (Fig. 2 ). Compared with Control group, H1299 and A549 cells in Propofol group had decreased cell viability and increased cell apoptosis rate (all P < 0.05). By contrast, these cells in the Propofol + miR-199a inhibitor group had increased cell viability and declined cell apoptosis relative to the Propofol group (all P < 0.05). Propofol increases miR-199a expression to restrict invasion and migration of NSCLC cells Transwell assay and wound healing assay were used to evaluate migration and invasion of NSCLC cells (A549 and H1299) respectively, as shown in Fig. 3 . Both Propofol group and Propofol + miR-NC group had declined invasive cell number and cell migration rate when compared to Control group (all P < 0.05). However, Propofol + miR-199a inhibitor group was significantly higher than Propofol group regarding the invasive cell number and migration rate (all P < 0.05). PAK4 can be directly targeted by miR-199a Online database TargetScan supported the hypothesis that miR-199a can specifically bind to PAK4 mRNA 3’UTR (Fig. 4 A). According to the dual-luciferase reporter assay (Fig. 4 B), compared with miR-NC group, co-transfection with miR-199a mimic and wild-type plasmid PAK4 3’-UTR-WT led to the decreased luciferase activity of A549 and H1299 cells (all P 0.05). Besides, compared with miR-NC in A549 and H1299 cells, miR-199a mimic can effectively reduce PAK4 protein level, while miR-199a inhibitor can significantly improve PAK4 protein expression (Fig. 4 C-D, all P < 0.05), which suggested that miR-199a can regulate PAK4 expression in NSCLC. These results proved that PAK4 was a target gene of miR-199a. Expression of miR-199a and PAK4 in NSCLC cells The expression of miR-199a and PAK4 in NSCLC A549 and H1299 cells was detected by qRT-PCR and western blotting, respectively (Fig. 5 ). Both Propofol group and Propofol + miR-NC group were significantly up-regulated in miR-199a expression and down-regulated in PAK4 protein expression when compared to Control group (all P < 0.05). The miR-199a expression was decreased while PAK4 protein expression was increased in Propofol + miR-199a inhibitor group, as compared with Propofol group (all P < 0.05). Propofol regulates miR-199a to affect the growth of xenograft tumor in nude mice As presented by Fig. 6 , compared with miR-NC group, nude mice in miR-199a inhibitor group were faster in xenograft tumor growth and bigger in tumor weight, while those in miR-NC + Propofol group were slower in tumor growth and lower in tumor weight (all P < 0.05). Besides, miR-199a inhibitor + Propofol group had much faster xenograft tumor growth and much bigger tumor weight than miR-NC + Propofol group (all P < 0.05). Discussion In the current study, propofol effectively suppressed the cell viability of NSCLC cell lines. Similarly, S-G Xing et al. also reported propofol could inhibit the survival and induce the apoptosis of NSCLC A549 cells by up-regulating ERK1/2-dependent PUMA expression (Xing et al. 2018 ). In the study by Qian Zhang et al. , the proliferation, invasion and migration of NSCLC was remarkably inhibited by propofol in a dose-dependent manner and miR-326/FOXM1 axis was mediated by propofol via the hinderance of circ-RHOT1 expression, consequently the growth of NSCLC cells was successfully blocked by propofol (Zhang et al. 2021 ). These findings supported the effective effect of propofol on the inhibition of NSCLC cell growth. Besides, we also observed the up-regulation of miR-199a induced by propofol. In fact, miR-199a was reported to be abnormally expressed in many types of tumor cells, which was down-regulated in hepatocellular carcinoma (Kim et al. 2016 ), breast cancer (Li et al. 2016 ), bladder cancer (Ecke et al. 2017 ), and prostate cancer (Qu et al. 2017 ), but up-regulated in osteosarcoma (Tian et al. 2014 ), gastric cancer (He et al. 2014 ), pancreatic cancer (Kuninty et al. 2016 ), suggesting that miR-199a may play as the tumor suppressor gene or oncogene in tumor cells. Of note, Gang Ding et al. exhibited the down-regulation of miR-199a in NSCLC, while miR-199a could inhibit NSCLC cell proliferation induced by hypoxia via the target regulation of HIF1a (Ding et al. 2013 ). By contrast, in the study of Jian Zhang et al. , propofol up-regulated miR-199a expression to inhibit the invasion and migration of HCC cells (Zhang et al. 2013b ). Given the above, miR-199a plays an important role in the inhibition of NSCLC growth by propofol. In view of the finding by experiment in vitro , propofol apparently inhibited the proliferation, invasion and migration of NSCLC cells, which, however, was reversed by miR-199a inhibitor. Yanli Li et al. reported that miR-199a-5p can directly target MAP3K11 to play its anti-tumor effect in NSCLC, thus inhibiting cell proliferation and arresting cell cycle at G1 phase (Li et al. 2019 ). L-M Wang et al. found that over-expressed miR-199a can down-regulate the expression of its target gene HIF-1α, thereby inhibiting NSCLC cell proliferation (Wang et al. 2019a ). As proven by G Mudduluru et al. , miR-199a was poorly expressed in lung cancer cells and its expression level was associated with the proliferation, migration and invasion of lung cancer cells via the regulation of its target gene Axl (Mudduluru et al. 2011 ). Importantly, propofol could also inhibit the growth and promote the apoptosis of HCC cells by up-regulating miR-199a expression (Zhang et al. 2013a ). Taken together, propofol can elevate miR-199a expression to play its anti-tumor role in NSCLC. Dual-luciferase reporter assay confirmed that PAK4 could be directly targeted and regulated by miR-199a. Another important finding in this study was that propofol can up-regulate miR-199a and down-regulate PAK4 expression, whereas miR-199a inhibitor can significantly reduce miR-199a and elevate PAK4 expression. Similarly, inhibiting PAK4, as indicated by Byung Jun Ryu et al. , can down-regulate MMP-2/MMP-9 to suppress invasion and migration of lung cancer cells (Ryu et al. 2014 ). It has been well-established that PAK4 is an important proto-oncogene up-regulated in many cancer tissues, which could promote the proliferation and migration of tumor cells but inhibit tumor cell apoptosis (Zhang et al. 2011 ), (Li et al. 2010b ). In general, PAK4 can regulate the motility of tumor cells through intracellular signal transduction pathways (Wang et al. 2019b ), and meanwhile, it can induce the phosphorylation of SSH-1L (Slingshot-1L) and LIMK, inactivate the actin-binding protein Cofilin and reduce the actin-binding activity of phosphorylated Cofilin, eventually leading to actin polymerization and stabilization (Li et al. 2020 ), (Li et al. 2010a ). LIMK1/Cofilin pathway can regulate the expression of cytoskeleton protein and the formation of microfilament actin stress fibers and macula adherens, which means it can regulate microfilament skeleton system to affect migrating and invasive abilities of cancer cells (Jang et al. 2012 ; Spratley et al. 2011 ). Xincheng Liu et al. found that miR-193a-3p can directly target PAK4 to inhibit the expression of downstream p-Slug and L1CAM and hinder the migration of invasion of NSCLC cells (Liu et al. 2019 ). As reported by Bin Zeng et al. , miR-199a/b-3p was decreased in gastric cancer and over-expressed miR-199a/b-3p reduced PAK4 expression to inhibit gastric cancer proliferation (Zeng et al. 2018 ). The above evidence suggested that propofol can elevate miR-199a expression to reduce PAK4 expression, thus affecting the activity of downstream pathways and inhibiting proliferation, invasion and migration of NSCLC. Furthermore, nude mouse xenograft model was used to verify that propofol can inhibit NSCLC growth in vivo , which was consistent with the finding of a previous study (Gong et al. 2019 ). However, such effect of propofol can be reversed by miR-199a inhibitor, which added further weight to the hypothesis that propofol can increase miR-199a expression to inhibit NSCLC growth in vivo . To sum up, we found propofol can up-regulate miR-199a expression to decrease PAK4 level, thereby inhibiting the proliferation, invasion and migration of NSCLC. 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(2014) miR-199a-3p negatively regulates the progression of osteosarcoma through targeting AXL American journal of cancer research 4:738-750 Wang LM, Zhang LL, Wang LW, Zhu L, Ma XX (2019a) Influence of miR-199a on rats with non-small cell lung cancer via regulating the HIF-1alpha/VEGF signaling pathway European review for medical and pharmacological sciences 23:10363-10369 doi:10.26355/eurrev_201912_19675 Wang M, Gao Q, Chen Y, Li Z, Yue L, Cao Y (2019b) PAK4, a target of miR-9-5p, promotes cell proliferation and inhibits apoptosis in colorectal cancer Cellular & molecular biology letters 24:58 doi:10.1186/s11658-019-0182-9 Wu X, Li X, Xu G (2020) Propofol suppresses the progression of non‑small cell lung cancer via downregulation of the miR‑21‑5p/MAPK10 axis Oncology reports 44:487-498 doi:10.3892/or.2020.7619 Xing SG, Zhang KJ, Qu JH, Ren YD, Luan Q (2018) Propofol induces apoptosis of non-small cell lung cancer cells via ERK1/2-dependent upregulation of PUMA European review for medical and pharmacological sciences 22:4341-4349 doi:10.26355/eurrev_201807_15431 Yu H, Ma M, Wang X, Zhou Z, Li R, Guo Q (2019) Propofol suppresses proliferation, invasion, and migration of human melanoma cells via regulating microRNA-137 and fibroblast growth factor 9 Journal of cellular physiology 234:23279-23288 doi:10.1002/jcp.28896 Zeng B, Shi W, Tan G (2018) MiR-199a/b-3p inhibits gastric cancer cell proliferation via down-regulating PAK4/MEK/ERK signaling pathway BMC cancer 18:34 doi:10.1186/s12885-017-3949-2 Zhang HJ et al. (2011) Overexpressed PAK4 promotes proliferation, migration and invasion of choriocarcinoma Carcinogenesis 32:765-771 doi:10.1093/carcin/bgr033 Zhang J, Wu GQ, Zhang Y, Feng ZY, Zhu SM (2013a) Propofol induces apoptosis of hepatocellular carcinoma cells by upregulation of microRNA-199a expression Cell biology international 37:227-232 doi:10.1002/cbin.10034 Zhang J, Zhang D, Wu GQ, Feng ZY, Zhu SM (2013b) Propofol inhibits the adhesion of hepatocellular carcinoma cells by upregulating microRNA-199a and downregulating MMP-9 expression Hepatobiliary & pancreatic diseases international : HBPD INT 12:305-309 doi:10.1016/s1499-3872(13)60048-x Zhang Q, Cheng F, Zhang Z, Wang B, Zhang X (2021) Propofol suppresses non-small cell lung cancer tumorigenesis by regulation of circ-RHOT1/miR-326/FOXM1 axis Life sciences:119042 doi:10.1016/j.lfs.2021.119042 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2340235","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":157445875,"identity":"d53510e1-3d26-464d-b65f-2785f5de01be","order_by":0,"name":"Rui-Long Yu","email":"","orcid":"","institution":"Shouguang Dongcheng Hospital","correspondingAuthor":false,"prefix":"","firstName":"Rui-Long","middleName":"","lastName":"Yu","suffix":""},{"id":157445876,"identity":"3f781d89-e784-4c22-b270-4719f9ee3dd1","order_by":1,"name":"Kai Li","email":"","orcid":"","institution":"Shouguang People’ s Hospital","correspondingAuthor":false,"prefix":"","firstName":"Kai","middleName":"","lastName":"Li","suffix":""},{"id":157445877,"identity":"31691330-afc6-4b3e-8ca1-b55fe18ca0a2","order_by":2,"name":"Jia-Jun Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+klEQVRIiWNgGAWjYBACxmYGBgMgLcPGwH7wwYcKIFOCSC08bAw8yYYzzsC0JBC2jQeIzYQ524jQwtzO/KCYdwcDD59EQhoz47zDcvyzG9gefPyBz2FsBsa8Z4AOk0g89rhw22FjiTsH2A1n4LEF6BegljaQloR045nbDic23Ehgk+bBq4X9A0yLmTTvnMP180Fa/uDVwmOApKXhcIIBSAs+7wO1FBjOBWnheQMM5GPphhtvJLZJ9qTh1mLYf3ybwds2Bjn59nRgVNZYy8vdSD4m8cMGj5YGBjZgVP6H8ZtBNjfgVg8E8sCoeYDEr8OrehSMglEwCkYmAACgqUoGXwCkHQAAAABJRU5ErkJggg==","orcid":"","institution":"Shiyan Hospital of Traditional Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Jia-Jun","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2022-12-03 11:14:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2340235/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2340235/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":30017403,"identity":"c46173e6-c221-48b4-a2da-cf9c15eff5ac","added_by":"auto","created_at":"2022-12-07 15:53:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":666924,"visible":true,"origin":"","legend":"\u003cp\u003ePropofol inhibits NSCLC proliferation and up-regulates miR-199a expression\u003c/p\u003e\n\u003cp\u003eNote: A-B, The cell viability of NSCLC H1299 (A) and A549 (B) cells after treatment with propofol detected by CCK-8 assay; C-D, The miR-199a expression in NSCLC H1299 (A) and A549 (B) cells after treatment with propofol evaluated by qRT-PCR; *, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05 compared with 0 μg/mL treatment group.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-2340235/v1/624fe7a0befd286294895ce4.png"},{"id":30018356,"identity":"4c60f5b1-233d-497a-a645-02ff9e099f48","added_by":"auto","created_at":"2022-12-07 16:01:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1044220,"visible":true,"origin":"","legend":"\u003cp\u003ePropofol increases miR-199a expression to limit proliferation and promote apoptosis of NSCLC cells\u003c/p\u003e\n\u003cp\u003eNote: A-B, The cell viability of NSCLC A549 (A) and H1299 (B) cells determined by CCK-8 method; C, The apoptosis of NSCLC cells examined by flow cytometry; D-E, The apoptosis rate of A549 (D) and H1299 (E) cells; *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 compared with Control group; #, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05 compared with Propofol group.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-2340235/v1/2703105ecee785fee90652b3.png"},{"id":30017398,"identity":"ac2fa1b2-311b-4428-88da-041cd0bbfa51","added_by":"auto","created_at":"2022-12-07 15:53:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6417114,"visible":true,"origin":"","legend":"\u003cp\u003ePropofol increases miR-199a expression to restrict invasion and migration of NSCLC cells\u003c/p\u003e\n\u003cp\u003eNote: A-B, Transwell assay and wound healing assay were used to evaluate migration and invasion of NSCLC cells A549 (A) and H1299 (B); C-D, Invasive cell number and cell migration rate of NSCLC A549 (C) and H1299 (D) cells; *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 compared with Control group; #, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 compared with Propofol group.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-2340235/v1/90c7340869e0f48c89313ed6.png"},{"id":30017402,"identity":"15546934-6fb3-4570-927c-1cede55a4181","added_by":"auto","created_at":"2022-12-07 15:53:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1478885,"visible":true,"origin":"","legend":"\u003cp\u003ePAK4 was a target gene of miR-199a\u003c/p\u003e\n\u003cp\u003eNote: A, TargetScan analysis revealed a site on PAK4 3’-UTR that can bind to miR-199a; B, Dual-luciferase reporter assay results; C-D,\u003cstrong\u003e \u003c/strong\u003eThe alteration of PAK4 protein expression in A549 and H1299 cells after transfected with miR-199a mimic and miR-199a inhibitor was measured by western blotting; *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 compared with miR-NC group; #, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 compared with miR-199a mimic group.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-2340235/v1/021bb141f503f3ba6653b4aa.png"},{"id":30017399,"identity":"5d1a3ff8-6a27-448c-b057-1ecef68b80a5","added_by":"auto","created_at":"2022-12-07 15:53:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1286728,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression of miR-199a and PAK4 in NSCLC A549 and H1299 cells\u003c/p\u003e\n\u003cp\u003eNote: A-B, The expression of miR-199a in NSCLC A549 (A) and H1299 (B) cells determined by qRT-PCR; C-D, The protein expression of PAK4 in NSCLC A549 (C) and H1299 (D) cells tested by western blotting; E-F, Comparison of PAK4 protein expression in A549 (E) and H1299 (F) cells; *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 compared with Control group; #, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 compared with Propofol group.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-2340235/v1/d4e1a9c45b32627a2b074a28.png"},{"id":30017401,"identity":"9f9439a2-ab45-41ec-8995-51d313f444af","added_by":"auto","created_at":"2022-12-07 15:53:22","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":878670,"visible":true,"origin":"","legend":"\u003cp\u003ePropofol mediates miR-199a to regulate the growth of xenograft tumor in nude mice\u003c/p\u003e\n\u003cp\u003eNote: A, The tumor growth of nude mice in each group; B, Xenograft tumor specimen of nude mice in each group; C, The tumor weight of nude mice in each group; *, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05 compared with miR-NC group; #, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 compared with miR-199a inhibitor group; \u0026amp;, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 compared with miR-NC + Propofol group.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-2340235/v1/bd1144eff6ba7d6f53d86003.png"},{"id":31211460,"identity":"b3694301-3948-45aa-8c13-e65de5632282","added_by":"auto","created_at":"2023-01-06 10:59:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2818986,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2340235/v1/0ce3db9d-d902-470b-a8e1-37d2a9396fa9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Propofol mediates miR-199a/PAK4 axis to regulate the proliferation, invasion and migration of non-small cell lung carcinoma cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLung cancer is one of the most common malignant tumors with the highest incidence and mortality, becoming a serious risk to human health and quality of life (Bray et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Histologically, it can divide into small-cell lung cancer and non-small cell lung carcinoma (NSLCC), with the latter accounting for more than 80% of all lung cancer patients (Osmani et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Currently, the major treatment of NSCLC includes surgery, chemotherapy and radiotherapy, but the therapeutic outcome is far from satisfactory (Chen et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Most patients already have distal metastasis at the time of diagnosis and cannot undertake surgical resection, and therapeutic methods like radiotherapy and chemotherapy used in middle- or late-stage usually have side effects, leading to a poor prognosis with the five-year survival rate of as low as only 15% (Chen et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Hirsch et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Therefore, it is urgent to develop new therapeutic strategies for NSCLC.\u003c/p\u003e \u003cp\u003eAs a common intravenous anesthetic, propofol can not only induce or maintain general anesthesia, but also have potential anti-tumor effects (Martin and Jiang \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Accumulating evidence has revealed that the anti-tumor effect of propofol is associated with the regulation of microRNAs (miRNAs) (Du et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Yu et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Coincidentally, Xiaoyu Zheng \u003cem\u003eet al.\u003c/em\u003e also reported that propofol can inhibit the growth and accelerate the apoptosis of NSCLC by mediating miR-21/PTEN/AKT pathway (Martin and Jiang \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In the study by Xinhua Wu \u003cem\u003eet al.\u003c/em\u003e, propofol down-regulated the miR-21-5p/MAPK10 axis, thus hindering the survival and promoting the apoptosis of NSCLC cells (Wu et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, it is still unclear if propofol can mediate other miRNAs to play its regulatory role in NSCLC.\u003c/p\u003e \u003cp\u003eMiR-199a is a class of miRNA widely distributed in human tissues (Liu et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). As reported by many studies, miR-199a was abnormally expressed in many tumor cells and involved in cell proliferation, metabolism, angiogenesis and apoptosis (Fornari et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Gu and Chan \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Of note, the study by Jian Zhang \u003cem\u003eet al.\u003c/em\u003e proved that propofol can up-regulate miR-199a expression substantially and its effect on invasion and migration of hepatocellular carcinoma (HCC) cells can be reversed by inhibiting miR-199a expression (Zhang et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013b\u003c/span\u003e). According to the analysis of target gene prediction website, PAK4 was a target gene of miR-199a. Songwang Cai \u003cem\u003eet al.\u003c/em\u003e also reported that PAK4 was up-regulated in NSCLC and PAK4 can mediate phosphorylation of LIMK1 to regulate NSCLC cell migration and invasion (Cai et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Given the above, we speculated that propofol can mediate miR-199a and its target gene PAK4 to regulate the NSCLC growth.\u003c/p\u003e \u003cp\u003eThis study investigated if propofol can inhibit proliferation, invasion and migration of NSCLC cells via mediating miR-199a/PAK4 axis and provided some theoretical basis for the potential treatment methods of NSCLC.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEthics statement\u003c/h2\u003e \u003cp\u003eThis study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals (Carbone \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), and all anmal experiments were under the supervision of the Medical Ethics Committee of Laboratory Animals in our hospital.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCulture and propofol treatment of NSCLC cells\u003c/h2\u003e \u003cp\u003eHuman NSCLC cells, H1299 and A549, were purchased from American type culture collection (ATCC, USA). All NSCLC cells were cultured in DMEM medium which was supplemented with 10% fetal bovine serum (FBS) and the culture conditions included 37\u0026deg;C, relative humidity of 95%, 5% CO\u003csub\u003e2\u003c/sub\u003e and 95% O\u003csub\u003e2\u003c/sub\u003e. Culture medium was renewed every two days. Cell passaging was performed when cells covered 80% of the plate. Cells in logarithmic growth phase (approximately the 3rd-5th generation) were used for subsequent experiments. H1299 and A549 cells were treated with 0, 5 and 10 \u0026micro;g/mL propofol (Sigma-Aldrich Chemical Co.) which was dissolved in dimethyl sulfoxide (DMSO, Solarbio) (Gao et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and miR-199a expression was determined at 12, 24, and 48 h, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCell grouping and transfection\u003c/h2\u003e \u003cp\u003eHuman NSCLC cells, A549 and H1299, were divided into four groups: Control group, Propofol group, Propofol\u0026thinsp;+\u0026thinsp;miR-NC group, and Propofol\u0026thinsp;+\u0026thinsp;miR-199a inhibitor group. Cells in Control group did not receive any treatment, but cells in Propofol group were treated with 10 \u0026micro;g/mL propofol for 48 h. Cells in Propofol\u0026thinsp;+\u0026thinsp;miR-NC group and Propofol\u0026thinsp;+\u0026thinsp;miR-199a inhibitor group were transfected with miRNA negative control and miR-199a inhibitor respectively before treatment with 10 \u0026micro;g/mL Propofol for 48 h. The miRNA negative control and miR-199a inhibitor were provided by Shanghai Genechem Co., LTD. Cells collected in the logarithmic growth phase were seeded to 6-well plates and cultured. When cell confluence reached 70%-80%, culture medium was replaced with fresh one and cell transfection performed based on the instructions of Lipofectamine\u0026trade; 2000 (Invitrogen, Carlsbad, CA, USA). After transfection, cells were incubated routinely for 4\u0026ndash;6 h and culture medium was replaced for another 48 h of incubation. Cells in each group were collected for later experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative reverse transcriptase polymerase chain reaction (qRT-PCR)\u003c/h2\u003e \u003cp\u003eTotal RNA of cells was extracted with TRIZOL reagent (Invirtrogen, USA) and quantified for RNA concentration using ultraviolet spectrophotometry (Beckman, Germany). RNA was reversely transcribed into cDNA using Taq Man MicroRNA Reverse Trallscription Kit (Applied Biosystems, USA). The appropriate volume of cDNA was used as templates for PCR reaction. Primer sequences were designed using the software Primer 5.0 and then synthesized by Sangon Biotech (Shanghai) Co., Ltd. For miR-199a: upstream primer: 5\u0026rsquo;-GCCACAGTAGTCTGCACAT-3\u0026rsquo;, downstream primer: 5\u0026rsquo;-CAGTGCGTGTCGTGGAGT-3\u0026rsquo;. For U6: upstream primer: 5\u0026rsquo;-CTCGCTTCGGCAGCACA-3\u0026rsquo;, downstream prime: 5\u0026rsquo;-AACGCTTCACGAATTTGCGT-3\u0026rsquo;. ABI Step One quantitative PCR instrument was used for qRT-PCR. Reaction conditions included initialization at 95\u0026deg;C for 10 min, and 40 cycles of denaturation at 95\u0026deg;C for 15 s and annealing at 60\u0026deg;C for 60 s. Relative expression of target genes was normalized with U6 as internal reference and calculated using 2\u003csup\u003e\u0026minus;△△Ct\u003c/sup\u003e method, with ΔCt\u0026thinsp;=\u0026thinsp;Ct\u003csub\u003etarget gene\u003c/sub\u003e - Ct\u003csub\u003eU6\u003c/sub\u003e; ΔΔCt\u0026thinsp;=\u0026thinsp;ΔCt\u003csub\u003eExperimental group\u003c/sub\u003e - ΔCt\u003csub\u003eControl group\u003c/sub\u003e - Relative transcription level of target mRNA equals 2\u003csup\u003e\u0026minus;△△Ct\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCell proliferation assay detected by CCK-8\u003c/h2\u003e \u003cp\u003eCells (1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/mL) were seeded to 96-well plates and cultured until they were adherent to the plate wall. The supernatant was discarded and 10 \u0026micro;L of CCK8 reagent (DOJINDO, Japan) was added to each well for 4 h of incubation at 37\u0026deg;C. After disposal of the supernatant, cells were washed with PBS and reacted with 100 \u0026micro;L dimethylsulfoxide (DMSO) (Sigma, USA) in each well before 10 min of oscillation. Microplate reader (MK3, Thermo, Pittsburgh, PA, USA) was used to examine the optical density (OD) at wavelength of 450 nm. Cell viability was calculated according to the formula: (OD value \u003csub\u003eExperimental group\u003c/sub\u003e - OD value \u003csub\u003eBlank group\u003c/sub\u003e)/(OD value \u003csub\u003eControl group\u003c/sub\u003e - OD value \u003csub\u003eBlank group\u003c/sub\u003e) \u0026times; 100%. The experiment was performed three times independently to obtain mean value.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell apoptosis measured by flow cytometry\u003c/h2\u003e \u003cp\u003eCells were digested by 0.25% trypsin (without EDTA), washed with PBS, suspended and adjusted to density of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/mL. Based on the instructions of apoptosis reagent kit, cells were suspended in 400 \u0026micro;L of binding buffer and reacted with 5 \u0026micro;L Annexin V-FITC and 5 \u0026micro;L PI for 15 min in dark environment. Cell apoptosis was evaluated using flow cytometer. In the scatter plot of the flow cytometer, the right-lower quadrant indicates early-apoptotic cells, right-upper quadrant necrotic and apoptotic cells, left-upper quadrant mechanically injured or necrotic cells, and left-lower quadrant viable cells. Cell apoptosis rate (%)\u0026thinsp;=\u0026thinsp;Early apoptotic cell percentage\u0026thinsp;+\u0026thinsp;Late apoptotic cell percentage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eWound healing assay\u003c/h2\u003e \u003cp\u003eCells were collected at logarithmic growth phase and inoculated to 96-well plates with cell density of 4 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/mL. When the plate was covered by cells, the sterile pipette tip was used to scratch lines, with the pipette perpendicular to the plate. Detached cells were washed away by PBS and photos of cells were taken. Serum-free medium was added to culture cells for 24 h, which were observed and photographed under the microplate. Image Pro Plus 6.0 was used to test the width of scratch lines. Migration rate = (Scratch width\u003csub\u003e0 h\u003c/sub\u003e - Scratch width\u003csub\u003e24 h\u003c/sub\u003e)/Scratch width\u003csub\u003e0 h\u003c/sub\u003e \u0026times; 100%. The experiment was repeated three times.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTranswell invasion assay\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ein vitro\u003c/span\u003e\u003c/p\u003e \u003cp\u003eMatrigel was diluted to 50 mg/L with serum-free medium and 80 \u0026micro;L Matrigel was added to cover the upper surface of the Transwell chamber. At 48 h after transfection, cells were fasted for 24 h in serum-free medium. After disposal of the culture medium, cells were washed with PBS, suspended in serum-free medium, and adjusted to cell density of 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/mL. And 200 \u0026micro;L cell suspension was added to the upper Transwell chamber, while 500 \u0026micro;L serum-containing medium added to the lower chamber, for 24 h of culture at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. Next, Matrigel and detached cells in the upper chamber were wiped away with cotton swabs, and cells were fixed in 500 \u0026micro;L methanol for 10 min. Then, cells were stained in 500 \u0026micro;L 0.1% crystal violet for 15 min. Cells were observed and photographed under an inverted microscope and five visual fields were randomly selected for invasive cell counting. The experiment was repeated three times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eDual-luciferase reporter gene assay\u003c/h2\u003e \u003cp\u003eA549 and H1299 cells were collected at logarithmic growth phase and inoculated to 24-well plates with cell density of 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e/well. When cell confluence reached 80%-90%, transfection was performed with Lipofectamine\u003csup\u003eTM\u003c/sup\u003e2000 (Invitrogen, USA). Wild-type PAK4 3\u0026rsquo;-UTR-WT or mutant-type PAK4 3\u0026rsquo;UTR-MUT plasmid were constructed respectively and used for co-transfection of cells with miR-199a-mimic/miR-NC, with Renilla luciferase (100 ng/well) as the control. At 48 h after co-transfection, the luciferase activity was tested with the dual-luciferase report analysis system (Promega, USA). The ratio of firefly luciferase activity to renilla luciferase activity was regarded as the relative luciferase activity. Besides, A549 and H1299 cells were transfected with miR-NC/miR-199a-mimic/miR-199a inhibitor to evaluate PAK4 expression by western blotting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eTotal proteins in cells were extracted with radioimmunoprecipitation assay (RIPA) lysate and quantified for protein concentration with a bicinchoninic acid (BCA) kit (Sigma, USA). During electrophoresis with 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS- PAGE), proteins were transferred to Polyvinylidene Fluoride (PVDF) membrane using a semi-dry transfer system (Bio-Rad, USA). PVDF membrane was blocked in 5% defatted milk powder for 2 h. Next, primary antibody PAK4 (CST, USA) was added for overnight incubation at 4\u0026deg;C. Then, PVDF membrane was washed with PBS (3 times \u0026times; 5 min) and reacted with Horseradish Peroxidase (HRP)-labeled secondary antibody (CST, USA) for 1 h at 37\u0026deg;C. The membrane was washed again with PBS (3 times \u0026times; 5 min), prior to the development and visualization by enhanced chemiluminescence (ECL) solution. Image Pro Plus 6.0 was used for gray value analysis. With GAPDH as the internal reference, the relative expression of target proteins was expressed as the gray value ratio of target protein to GAPDH.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEstablishment of the nude mouse xenograft model\u003c/h2\u003e \u003cp\u003eIn this study, 20 BALB/c nude mice (4\u0026ndash;5 weeks old and weighing 17\u0026ndash;21 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. A549 cells were transfected with miR-NC and miR-199a inhibitor and suspended in PBS to adjust cell density to 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e mL. Cell suspension (4 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells) of miR-NC group and miR-199a inhibitor group was injected subcutaneously into the back of nude mice, with 6 mice in each group. The tumor length (L) and width (W) were measured every three days to calculate tumor volume, with the formula: V\u0026thinsp;=\u0026thinsp;L \u0026times; W\u003csup\u003e2\u003c/sup\u003e/2. After 8 d of tumor establishment, nude mice were injected with DMSO or propofol (45 mg/kg) every three days, and tumor volume was monitored. After 23 d, the mice were sacrificed and xenograft tumors weighed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical methods\u003c/h2\u003e \u003cp\u003eAll data were analyzed with the statistical software package SPSS 21.0 (SPSS, Inc, Chicago, IL, USA). Measurement data were presented by mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) and tested by Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test. Comparison among multiple groups was analyzed by one-way ANOVA with Tukey\u0026rsquo;s post-hoc test. The value of \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicated the statistical significance of differences.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePropofol reduces NSCLC proliferation and enhances miR-199a expression\u003c/h2\u003e \u003cp\u003eAs illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the viability of NSCLC cells H1299 and A549 declined after treatment with 5 and 10 \u0026micro;g/mL propofol for 24 h (all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). And the expression of miR-199a was up-regulated in H1299 and A549 cells after treatment with 5 and 10 \u0026micro;g/mL propofol for 12 h (all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Thus, we decided to use 10 \u0026micro;g/mL propofol for 48 h to conduct subsequent experiments, since it could effectively reduce cell viability to 50%.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003ePropofol increases miR-199a expression to limit proliferation and promote apoptosis of NSCLC cells\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eCCK-8 method and flow cytometry were used to evaluate the proliferation and apoptosis of NSCLC cells, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Compared with Control group, H1299 and A549 cells in Propofol group had decreased cell viability and increased cell apoptosis rate (all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). By contrast, these cells in the Propofol\u0026thinsp;+\u0026thinsp;miR-199a inhibitor group had increased cell viability and declined cell apoptosis relative to the Propofol group (all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ePropofol increases miR-199a expression to restrict invasion and migration of NSCLC cells\u003c/h2\u003e \u003cp\u003eTranswell assay and wound healing assay were used to evaluate migration and invasion of NSCLC cells (A549 and H1299) respectively, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Both Propofol group and Propofol\u0026thinsp;+\u0026thinsp;miR-NC group had declined invasive cell number and cell migration rate when compared to Control group (all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, Propofol\u0026thinsp;+\u0026thinsp;miR-199a inhibitor group was significantly higher than Propofol group regarding the invasive cell number and migration rate (all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003ePAK4 can be directly targeted by miR-199a\u003c/h2\u003e \u003cp\u003eOnline database TargetScan supported the hypothesis that miR-199a can specifically bind to PAK4 mRNA 3\u0026rsquo;UTR (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). According to the dual-luciferase reporter assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), compared with miR-NC group, co-transfection with miR-199a mimic and wild-type plasmid PAK4 3\u0026rsquo;-UTR-WT led to the decreased luciferase activity of A549 and H1299 cells (all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while co-transfection with miR-199a mimic and mutant-type plasmid PAK4 3\u0026rsquo;UTR-MUT didn\u0026rsquo;t bring obvious change to luciferase activity (all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Besides, compared with miR-NC in A549 and H1299 cells, miR-199a mimic can effectively reduce PAK4 protein level, while miR-199a inhibitor can significantly improve PAK4 protein expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-D, all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), which suggested that miR-199a can regulate PAK4 expression in NSCLC. These results proved that PAK4 was a target gene of miR-199a.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eExpression of miR-199a and PAK4 in NSCLC cells\u003c/h2\u003e \u003cp\u003eThe expression of miR-199a and PAK4 in NSCLC A549 and H1299 cells was detected by qRT-PCR and western blotting, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Both Propofol group and Propofol\u0026thinsp;+\u0026thinsp;miR-NC group were significantly up-regulated in miR-199a expression and down-regulated in PAK4 protein expression when compared to Control group (all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The miR-199a expression was decreased while PAK4 protein expression was increased in Propofol\u0026thinsp;+\u0026thinsp;miR-199a inhibitor group, as compared with Propofol group (all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003ePropofol regulates miR-199a to affect the growth of xenograft tumor in nude mice\u003c/h2\u003e \u003cp\u003eAs presented by Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, compared with miR-NC group, nude mice in miR-199a inhibitor group were faster in xenograft tumor growth and bigger in tumor weight, while those in miR-NC\u0026thinsp;+\u0026thinsp;Propofol group were slower in tumor growth and lower in tumor weight (all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Besides, miR-199a inhibitor\u0026thinsp;+\u0026thinsp;Propofol group had much faster xenograft tumor growth and much bigger tumor weight than miR-NC\u0026thinsp;+\u0026thinsp;Propofol group (all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the current study, propofol effectively suppressed the cell viability of NSCLC cell lines. Similarly, S-G Xing \u003cem\u003eet al.\u003c/em\u003e also reported propofol could inhibit the survival and induce the apoptosis of NSCLC A549 cells by up-regulating ERK1/2-dependent PUMA expression (Xing et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In the study by Qian Zhang \u003cem\u003eet al.\u003c/em\u003e, the proliferation, invasion and migration of NSCLC was remarkably inhibited by propofol in a dose-dependent manner and miR-326/FOXM1 axis was mediated by propofol via the hinderance of circ-RHOT1 expression, consequently the growth of NSCLC cells was successfully blocked by propofol (Zhang et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These findings supported the effective effect of propofol on the inhibition of NSCLC cell growth. Besides, we also observed the up-regulation of miR-199a induced by propofol. In fact, miR-199a was reported to be abnormally expressed in many types of tumor cells, which was down-regulated in hepatocellular carcinoma (Kim et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), breast cancer (Li et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), bladder cancer (Ecke et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), and prostate cancer (Qu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), but up-regulated in osteosarcoma (Tian et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), gastric cancer (He et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), pancreatic cancer (Kuninty et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), suggesting that miR-199a may play as the tumor suppressor gene or oncogene in tumor cells. Of note, Gang Ding \u003cem\u003eet al.\u003c/em\u003e exhibited the down-regulation of miR-199a in NSCLC, while miR-199a could inhibit NSCLC cell proliferation induced by hypoxia via the target regulation of HIF1a (Ding et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). By contrast, in the study of Jian Zhang \u003cem\u003eet al.\u003c/em\u003e, propofol up-regulated miR-199a expression to inhibit the invasion and migration of HCC cells (Zhang et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013b\u003c/span\u003e). Given the above, miR-199a plays an important role in the inhibition of NSCLC growth by propofol.\u003c/p\u003e \u003cp\u003eIn view of the finding by experiment \u003cem\u003ein vitro\u003c/em\u003e, propofol apparently inhibited the proliferation, invasion and migration of NSCLC cells, which, however, was reversed by miR-199a inhibitor. Yanli Li \u003cem\u003eet al.\u003c/em\u003e reported that miR-199a-5p can directly target MAP3K11 to play its anti-tumor effect in NSCLC, thus inhibiting cell proliferation and arresting cell cycle at G1 phase (Li et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). L-M Wang \u003cem\u003eet al.\u003c/em\u003e found that over-expressed miR-199a can down-regulate the expression of its target gene HIF-1α, thereby inhibiting NSCLC cell proliferation (Wang et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e). As proven by G Mudduluru \u003cem\u003eet al.\u003c/em\u003e, miR-199a was poorly expressed in lung cancer cells and its expression level was associated with the proliferation, migration and invasion of lung cancer cells via the regulation of its target gene Axl (Mudduluru et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Importantly, propofol could also inhibit the growth and promote the apoptosis of HCC cells by up-regulating miR-199a expression (Zhang et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2013a\u003c/span\u003e). Taken together, propofol can elevate miR-199a expression to play its anti-tumor role in NSCLC.\u003c/p\u003e \u003cp\u003eDual-luciferase reporter assay confirmed that PAK4 could be directly targeted and regulated by miR-199a. Another important finding in this study was that propofol can up-regulate miR-199a and down-regulate PAK4 expression, whereas miR-199a inhibitor can significantly reduce miR-199a and elevate PAK4 expression. Similarly, inhibiting PAK4, as indicated by Byung Jun Ryu \u003cem\u003eet al.\u003c/em\u003e, can down-regulate MMP-2/MMP-9 to suppress invasion and migration of lung cancer cells (Ryu et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). It has been well-established that PAK4 is an important proto-oncogene up-regulated in many cancer tissues, which could promote the proliferation and migration of tumor cells but inhibit tumor cell apoptosis (Zhang et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), (Li et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2010b\u003c/span\u003e). In general, PAK4 can regulate the motility of tumor cells through intracellular signal transduction pathways (Wang et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e), and meanwhile, it can induce the phosphorylation of SSH-1L (Slingshot-1L) and LIMK, inactivate the actin-binding protein Cofilin and reduce the actin-binding activity of phosphorylated Cofilin, eventually leading to actin polymerization and stabilization (Li et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), (Li et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010a\u003c/span\u003e). LIMK1/Cofilin pathway can regulate the expression of cytoskeleton protein and the formation of microfilament actin stress fibers and macula adherens, which means it can regulate microfilament skeleton system to affect migrating and invasive abilities of cancer cells (Jang et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Spratley et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Xincheng Liu \u003cem\u003eet al.\u003c/em\u003e found that miR-193a-3p can directly target PAK4 to inhibit the expression of downstream p-Slug and L1CAM and hinder the migration of invasion of NSCLC cells (Liu et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). As reported by Bin Zeng \u003cem\u003eet al.\u003c/em\u003e, miR-199a/b-3p was decreased in gastric cancer and over-expressed miR-199a/b-3p reduced PAK4 expression to inhibit gastric cancer proliferation (Zeng et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The above evidence suggested that propofol can elevate miR-199a expression to reduce PAK4 expression, thus affecting the activity of downstream pathways and inhibiting proliferation, invasion and migration of NSCLC. Furthermore, nude mouse xenograft model was used to verify that propofol can inhibit NSCLC growth \u003cem\u003ein vivo\u003c/em\u003e, which was consistent with the finding of a previous study (Gong et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, such effect of propofol can be reversed by miR-199a inhibitor, which added further weight to the hypothesis that propofol can increase miR-199a expression to inhibit NSCLC growth \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eTo sum up, we found propofol can up-regulate miR-199a expression to decrease PAK4 level, thereby inhibiting the proliferation, invasion and migration of NSCLC. This study provides the new clue for the treatment of NSCLC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u0026nbsp;\u003c/strong\u003eThe data that support the findings of this study are available on request form the corresponding author. The data are not publicly available due to privacy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThere was no funding in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003eThe authors appreciate the reviewers for their useful comments in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A (2018) Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries CA: a cancer journal for clinicians 68:394-424 doi:10.3322/caac.21492\u003c/li\u003e\n \u003cli\u003eCai S et al. 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Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed to evaluate miR-199a expression. A549 and H1299 cells were divided into Control, Propofol, Propofol\u0026thinsp;+\u0026thinsp;miR-NC and Propofol\u0026thinsp;+\u0026thinsp;miR-199a inhibitor groups. The proliferation, apoptosis, migration, and invasion were examined by CCK-8, flow cytometry, wound healing, and Transwell, respectively. Western blotting was used to measure the protein expression of PAK4. Xenograft model was established in nude mice to observe if propofol can mediate miR-199a expression to regulate the growth of subcutaneous xenograft tumor.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003ePropofol can effectively enhance the expression of miR-199a in NSCLC. Compared with Control group, H1299 and A549 cells in Propofol group decreased in viability, invasion and migration, and increased in apoptosis. The inhibitory effect of propofol on NSCLC growth was reversed by miR-199a. In comparison with Propofol group, Propofol\u0026thinsp;+\u0026thinsp;miR-199a inhibitor group was declined in miR-199a expression and increased in PAK4 protein expression. According to dual-luciferase reporter assay, PAK4 was a target gene of miR-199a. Experiment \u003cem\u003ein vivo\u003c/em\u003e revealed propofol can inhibit the growth and reduce the weight of xenograft tumor, which can be reversed by miR-199a inhibitor.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003ePropofol can suppress PAK4 expression by inducing miR-199a up-regulation, thereby inhibiting the proliferative, invasive and migrating abilities of NSCLC.\u003c/p\u003e","manuscriptTitle":"Propofol mediates miR-199a/PAK4 axis to regulate the proliferation, invasion and migration of non-small cell lung carcinoma cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-12-07 15:53:17","doi":"10.21203/rs.3.rs-2340235/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4ae473f1-66b9-489c-821c-aeb2276c1c86","owner":[],"postedDate":"December 7th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-08-21T06:44:30+00:00","versionOfRecord":[],"versionCreatedAt":"2022-12-07 15:53:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2340235","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2340235","identity":"rs-2340235","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}