YME1L affects the biological function of non-small cell lung cancer by promoting Gαi1 expression and Akt activation

YME1L affects the biological function of non-small cell lung cancer by promoting Gαi1 expression and Akt activation | 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 YME1L affects the biological function of non-small cell lung cancer by promoting Gαi1 expression and Akt activation MinDan Wu, Feng Gao, MeiJie Xu, JieRu Zhang, Chen Ni, WenXia Qian, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3972823/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 Background : As a key regulatory enzyme in mitochondria, YME1L is crucial for maintaining mitochondrial morphology, function and plasticity, and plays a catalytic role in PDAC. The research team has found that the overexpression of YME1L can promote the expression of Gαi1 and the activation of Akt, and promote the progression of glioma. Taking NSCLC as the research material, this project aims to reveal the effect of YME1L regulating mitochondrial remodeling on the progress of NSCLC and its molecular mechanism, and provide new ideas for the treatment of NSCLC patients. Results : YME1L is highly expressed in NSCLC tissues Overexpression of yme1l can promote Gαi1 expression and Akt activation, which in turn regulate cell proliferation, growth, migration and survival, and promote the progression of NSCLC. Conclusions : In this study, we found that YME1L have a novel oncogenic role in promoting NSCLC tumorigenesis and progression via the Gαi1-AS-pAKT axis. The implementation of this project may provide a new entry point for the treatment of lung cancer. NSCLC Mitochondrial Remodeling YME1L Targeted therapy Gαi1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Lung cancer, the most frequent malignant tumor in both men and women, is the leading cause of cancer-related deaths worldwide [ 1 ] . Approximately 15% of lung cancers have the histologic subtype of SCLC and the remaining 85% are classified as non–small cell lung cancer NSCLC [ 2 ] . Lung cancer is a heterogeneous disease with an abundance of genetic and epigenetic alterations. These genetic changes result in activation of oncogenic pathways and/or inhibition of tumor suppressor genes that are critical for malignant transformation of tumor precursor cells [ 3 ] . In recent years, the identification of tumor subgroups with distinct genetic alterations has changed the landscape of lung cancer treatment and pushed thoracic oncology to the forefront of personalized oncology.However, due to the late diagnosis and the existence of treatment resistance, although the combined application of targeted therapy and comprehensive drugs has improved the prognosis of some patients, the 5-year survival rate of patients with stage IV lung cancer is extremely low [ 4 , 5 ] . Recent research has demonstrated that oxidative stress changes are associated with the reprogramming of cellular metabolism during different cancer progression stages, exhibiting a role in maintaining redox homeostasis and supporting the metabolic demand imposed at each stage. Mitochondrial redox metabolism is pivotal in this process, promoting the progression of cancer cells through tumor development, metastasis, and colonization in distant organs [ 6 ] . Therefore, comprehensively understanding the changes in mitochondrial redox metabolism during lung cancer and identifying the mechanisms involved in drug resistance development will help design new treatment strategies and facilitate the treatment plan to continuously adapt as the disease progresses [ 7 , 8 ] . The YME1L gene on chromosome 10p is a member of the AAA superfamily of metalloproteases and is localized to the inner mitochondrial membrane [ 9 ] . Morever YME1L encodes a predicted protein of 716 amino acids highly similar to the mitochondrial AAA proteases (particularly yeast Yme1p), with conserved roles in mitochondrial assembly, integrity, and possibly DNA metabolism [ 10 ] . Previous studies have revealed that YME1L regulates the processing and stability of OPA1, where the exogenous expression of OPA1 isoform 1 or L-OPA1 accelerates the mitochondrial fragmentation induced by sh-YME1L [ 11 ] . Additionally, deleting YME1L has been shown to decrease cell proliferation and apoptosis resistance, alter mitochondrial ultrastructure, and increased susceptibility to oxidative damage, emphasizing its critical role in the homeostatic maintenance of the inner mitochondrial membrane protein and the proteolytic regulation of respiratory chain biogenesis [ 12 ] . Multiple studies have suggested that the human YME1L may be related to cancer progression. For example, YME1L-mediated mitochondrial reshaping has been reported to support the growth of pancreatic ductal adenocarcinoma cells as spheroids or xenografts, indicating that YME1L is a promising therapeutic target for cancer [ 13 ] . Enzymes such as YME1L and other mitochondrial proteases may be explored as potential druggable targets in cancer treatment; however, identifying their substrates or regulatory mechanisms is essential for targeting the cancer-specific metabolic pathways [ 14 ] . Heterotrimeric G-proteins, comprising three subunits (α, β, and γ), are crucial components in varied cellular events. Hence, dysregulation in these proteins can severely affect the signaling pathways and overall cellular state. The G-protein inhibitory α-subunit (Gαi) family has the highest number of individual members, (including Gαi1, Gαi2, Gαi3, Gαo, Gαt, Gαg, and Gαz), and constitutes the majority of G-protein α-subunits expressed in tissuess or cells [ 15 ] . Furthermore, the Gαi proteins bind to G protein-coupled receptors to inhibit adenylate cyclase and decrease cyclic AMP levels [ 16 ] . Our previous research has identified that Gαi1 is a pivotal signaling protein required for Akt-mTOR activation via multiple receptor tyrosine kinases. In this context, YME1L overexpression may promote Gαi1 expression and Akt activation in human glioma, exerting a pro-tumorigenic activity in this tumor type [ 17 ] . In light of these findings, our study aimed to investigate the potential role of YME1L in Gαi1-induced signaling in NSCLC. Results YME1L is overexpressed in human NSCLC tissues. In the qRT-PCR analysis, YME1L mRNA expression was examined in human NSCLC tissues (“tumor [T]”) and paired normal lung tissues (“normal [N]”) obtained from 16 patients with primary NSCLC. As shown in Fig. 1 A, YME1L mRNA expression in the T tissues was significantly higher than that in N tissues (P < 0.05). Moreover, YME1L protein expression was elevated in the T tissues from eight patients (i.e., patient 1 to patient 8, which represented the first eight patients enrolled in the study; Fig. 1 B). In all 16 sets of the human tissues, we found a significant upregulation of the YME1L protein (P < 0.05 versus N tissues; Fig. 1 C). Furthermore, we determined YME1L expression in the human tissues via immunohistochemistry (IHC). TMA showed that YME1L expression was significantly upregulated in the T tissues compared to the N tissues (P < 0.05; Fig. 1 D). Figure 1 YME1L is overexpressed in human NSCLC tissues. YME1L mRNA and protein expressions in 16 different human NSCLC tissues (“Tumor”) and paired surrounding normal lung tissues (“Normal”) were tested by qRT-PCR (A), Western blotting (B, C), and IHC staining (D) assays. Scale bar = 100 µm. Construction of NSCLC cell lines with interference and overexpression of YME1L We transduced NSCLC cells with a lentiviral construct encoding YME1L cDNA (“OE-YME1L”) to analyze the function of YME1L in NSCLC cells (Fig. 2 A, B, C). Additionally, we silenced YME1L using targeted shRNAs. For this purpose, three lentiviral shRNAs targeting the non-overlapping sequences of YME1L (sh-YME1L-1, sh-YME1L-2, and sh-YME1L-3) were individually transduced into H1299 and A549 cells to establish stable cell lines with a 90% reduction in YME1L mRNA (Fig. 2 D) and protein (Fig. 2 E, F). Figure 2 Construction of NSCLC cell lines with interference and overexpression of YME1L NSCLC cell lines(H1299 and A549) with lentiviral construct encoding the YME1L cDNA(“OE-YME1L”) or the control empty vector (“Control”) and YME1L shRNA(“sh-YME1L-1/2/3”) or control nonsense shRNA(“sh-NC”) were established and tested by qRT-PCR and Western blotting assays (A-F). YME1L promotes NSCLC cell proliferation in vitro. Subsequent examination of cell proliferation using the CCK8 assay revealed that the OD significantly decreased in the YME1L-shRNA-expressing H1299 and A549 cells (Fig. 3 G). Thus, YME1L knockdown robustly decreased the number of H1299 and A549 cell colonies (Fig. 3 H), indicating inhibited proliferation. Moreover, analyzing cell cycle progression via a PI-FACS assay demonstrated that YME1L overexpression increased the proportion of NSCLC cells in the S-phase and reduced the number of G0/G1 cells. Conversely, YME1L-shRNA transduction decreased the number of S-phase cells and elevated the number of G0/G1 cells (Fig. 3 I), implying G1-S arrest. All these results indicate that YME1L upregulation is crucial in the proliferation of NSCLC cells. Figure 3 YME1L promotes NSCLC cell proliferation in vitro. Cells were further cultured for applied time periods, cell viability (A), colony formation (B, C) and cell cycle distribution (PI-FACS, D, E) were tested by the listed assays, with results quantified. YME1L promotes NSCLC cell migration and invasion in vitro. Next, we explored the potential mechanism of elevated YME1L levels in NSCLC. In this examination, stable NSCLC cell lines (H1299 and A549) overexpressing YME1L (“OE-YME1L”) or expressing YME1L shRNAs (“sh-YME1L-1”) were cultured for specified periods. Furthermore, cell migration and invasion tests were performed using cell scratch, transwell, and Matrigel transwell assays. These assay findings showed that YME1L overexpression enhanced NSCLC cell migration (Fig. 3 A-D) and invasion (Fig. 3 E, F), whereas these processes were inhibited in the NSCLC cells transduced with YME1L shRNAs. All these results further suggest that YME1L overexpression may promote NSCLC cell malignant behaviors, including migration and invasion, in vitro. Figure 4 YME1L promotes NSCLC cell migration and invasion in vitro. Cell Scratch (A, B) as well as cell migration and invasion (“Transwell” assays, C-F) were tested by the listed assays, and results were quantified. Scale bar = 100 µm. YME1L is critical for Gαi1 expression and Akt activation. We further investigated the role of YME1L in Gαi1 expression within NSCLC cells. As depicted in Fig. 5 , shRNA-induced silencing of YME1L led to a marked downregulation in Gαi1 protein expression in NSCLC cells. Moreover, Akt activation, as assessed by p-Akt levels, was inhibited after YME1L silencing (Fig. 5 C, D). Conversely, cells overexpressing YME1L (“OE-YME1L”; Fig. 5 A, B) exhibited a significant increase in Gαi1 protein levels and enhanced Akt activation. All these findings indicate that YME1L is vital for Gαi1 expression and Akt activation in NSCLC cells. Figure 5 YME1L is important for Gαi1 expression and Akt activation. Cervical cancer cell lines(H1299 and A549) with lentiviral construct encoding the YME1L cDNA(“OE-YME1L”)or the control empty vector (“Control”) and YME1L shRNA(“sh-YME1L”) or control nonsense shRNA(“sh-NC”) were established and cultured for applied time periods;Expression of listed proteins were tested by Western blotting assays(A-D). YME1L is crucial for NSCLC xenograft growth in nude mice. Finally, we investigated the potential role of YME1L in NSCLC growth in vivo. Stable NSCLC cells with YME1L-expressing vectors or empty vectors were injected (1 × 10 7 cells per mouse) into the flanks of nude mice (Fig. 6 A). The results of the tumor growth curve (Fig. 6 B, C) demonstrated that the NSCLC cells with YME1L-expressing vectors grew significantly faster than the control tumors. In contrast, the growth of the NSCLC xenografts expressing YME1L shRNAs was much slower than that of the control xenografts (Fig. 6 B, C). Thus, YME1L is essential for NSCLC xenograft growth in nude mice. Furthermore, the YME1L-Gαi1 axis genes were assessed in fresh tumor tissues using qRT-PCR and Western blotting analyses. Compared to the control xenografts, YME1L-expressing NSCLC xenografts showed significantly higher Gαi1 levels (Fig. 6 D-F). Conversely, NSCLC xenograft tissues expressing YME1L shRNA demonstrated an accumulation of Gαi1 and a decrease in YME1L mRNA (Fig. 6 D-F). Western blotting assay results exhibited increased expression levels of p-Akt in the xenograft tissues with YME1L overexpression (Fig. 6 D-F), whereas Akt signaling was decreased in xenografts with YME1L knockdown (Fig. 6 D-F). Additionally, IHC indicated that Gαi1 and p-AKT levels were downregulated in sh-YME1L-AS-injected A549 xenografts (Fig. 6 G). This finding suggests that the intratumoral injection of sh-YME1L-AS lentivirus inhibited NSCLC xenograft growth and vice versa. Figure 6 YME1L is required for NSCLC xenograft growth in nude mice. Stable A549 cells transfected with YME1L-overexpressed (“OE-YME1L”) vector, control nonsense empty vector (“control”), YME1L shRNA (“sh-YME1L-1”), or nonsense control shRNA (“sh-NC”) were injected into the flanks of nude mice (four mice per group), and xenograft tumors established (A). Tumor volumes were recorded every 5 days (B). At 35 days after the experiment (“day 35”), all tumors were isolated (A) and weighed individually (C). In fresh tumor tissues, YME1L mRNA expression was determined via qRT-PCR assays (D), with relevant proteins detected by Western blotting assays (E, F). Representative HE and YME1L IHC images are presented (G). Scale bar = 100 µm. Discussion Lung cancer is a prominent public health challenge due to its high morbidity and mortality rates. Moreover, the metabolic changes observed in lung cancer can affect prognosis and treatment response [ 18 ] . These tumors can also involve mitochondrial dysfunction, resulting from mitochondrial DNA mutations, TCA cycle enzyme dysfunction, electronic respiratory chain defects, and subsequent oxidative stress, and/or abnormal oncogenic and tumor suppressive signaling. These alterations can in turn modify the cellular metabolic pathways, disrupt redox balance, and lead to apoptosis and resistance, playing a critical role in sustaining the growth and survival of tumor cells [ 19 , 20 ] . YME1L is the human orthologue of the Yme1 subunit of the yeast mitochondrial i-AAA complex. This complex represents a so-called i-AAA protease (where AAA denotes ATPases associated with various cellular activities), which exerts peptidase, chaperone-like, and translocase activities on the intermembrane space side of the inner mitochondrial membrane [ 12 ] . Therefore, YME1L couples mitochondrial morphology and function by maintaining mitochondrial fusion and fission via OPA1 turnover and processing as well as by regulating metabolic output via the degradation of a broad range of substrates in response to nutrient availability and cellular stress [ 14 ] . Our previous research revealed that YME1L overexpression could induce Gαi1 expression and Akt activation, influencing cell proliferation, growth, migration, and survival and consequently promoting glioma progression [ 21 ] . Moreover, limited information exists on the YME1L expression status among Chinese patients with NSCLC. Therefore, this study investigated the role of YME1L in NSCLC occurrence and development and evaluated the signaling pathways regulated by YME1L. Our study demonstrated that YME1L overexpression promotes tumor cell growth and migration by modulating the Gαi1-Akt signaling pathway, suggesting that targeting this process may contribute to devising new treatment strategies for lung cancer. In the current study, IHC was used to detect the expression levels of YME1L in surgically excised tissues consisting of 16 pairs of paraffin-embedded specimens from patients with NSCLC. The IHC results showed that almost 90% of the lung cancer tissues had higher YME1L expression levels than the adjacent lung tissues. Furthermore, we conducted functional studies and confirmed that YME1L overexpression enhances cell proliferation in vitro and in vivo, where silencing YME1L expression inhibits tumor cell proliferation and migration. Therefore, our data confirms that YME1L has a pro-oncogenic role in NSCLC. Heterotrimeric G-proteins, comprising Gα, Gβ, and Gγ subunits, are crucial signaling components. These G-proteins act as molecular switches, controlling the transmission of information from various extracellular cues to the numerous intracellular effectors regulating cell behavior. Gαi, a member of this G-protein family, was initially identified for its inhibitory function on adenylyl cyclase activity that led to decreased cyclic AMP levels [ 22 ] . Our research group previously highlighted that Gαi1 and Gαi3 are pivotal signaling proteins required for Akt-mTOR activation via multiple receptor tyrosine kinases [ 16 ] . In this study, we employed lentiviral interference and YME1L overexpression and estimated the protein expression levels of Gαi1 and Akt via Western blotting analysis to verify whether this signaling pathway plays a similar role in NSCLC. Our results showed that YME1L overexpression might promote Gαi1 expression and Akt activation in NSCLC. Additionally, qRT-PCR, Western blot, and IHC analyses in this study demonstrated that the mRNA and protein expression of YME1L was significantly higher in NSCLC tissue than in control tissues. Moreover, this study revealed for the first time that YME1L might promote NSCLC cell proliferation and migration by targeting the Gαi1-Akt signaling pathway. All these research findings suggest that developing inhibitors targeting this signaling pathway may be a promising strategy for treating NSCLC. Conclusions In this study, we found that YME1L have a novel oncogenic role in promoting NSCLC tumorigenesis and progression via the Gαi1-AS-pAKT axis. Methods Reagents. Puromycin were purchased from Sigma-Aldrich (St. Louis, MO). The antibodies were provided by Cell Signaling Technology (Beverly, MA) and Abcam (Cambridge, UK). Fetal bovine serum (FBS, Gibco), Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park Memorial Institute (RPMI)-1640 medium and antibiotics were provided by Gibco-BRL (Suzhou, China). TRIzol and other RNA assay reagents were purchased from Thermo-Fisher Invitrogen (Shanghai, China). Cell culture. NSCLC cell lines (H1299, and A549) were acquired commercially from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). Cell culture was respectively performed in RPMI-1640 medium and DMEM, which were supplemented with 10% FBS and antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin; Invitrogen, Carlsbad, CA) in a humidified air atmosphere at 37℃ with 5% CO 2 . Human tissues. Fresh NSCLC tissues (“T”) and matched adjacent normal lung tissues (“N”) were obtained from 16 patients with primary lung cancer who were admitted to the Affiliated Zhangjiagang First People’s Hospital of Soochow University. The tissue samples were stored in liquid nitrogen and homogenized in a tissue lysis buffer with proteasome inhibitors before further analyses. In the immunofluorescence studies, the human tissue sections (4-µm thick) were incubated with anti-YME1L antibodies at 4°C overnight, followed by incubation with a green fluorescence secondary antibody at 37°C for 60 min. None of the patients had received chemotherapy or radiotherapy before surgery. Written informed consent was obtained from each included patient. The protocols for using human tissues were in accordance with the Declaration of Helsinki, and their use in this study was approved by the Ethics Board of Soochow University. Immunohistochemical staining. Tissue microarrays (TMAs) from the patients and mice were constructed, and core tissue samples of 2-mm diameter were acquired from the tissue sections, which were embedded with paraffin and stored in paraffin-recipient blocks. Next, the slides were heated for 10 min in a sodium citrate buffer for antigen retrieval. The slides were then blocked with bovine serum albumin, followed by incubation with primary antibodies for anti-YME1L (dilution, 1:50) at 4°C overnight. Further, the slides were washed three times with PBS and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody at the indoor temperature for 60 min. Finally, the slides were stained using a DAB solution. Two pathologists evaluated the slides for YME1L expression based on the staining intensity of the positive immunoreactive cells. The staining intensity was graded as follows: 0 (no staining), 1 (weak staining), 2 (moderate staining), and 3 (strong staining). Silencing or overexpression of YEM1L. A set of three different YEM1L shRNA sequences was designed and synthesized by Genechem (Shanghai, China) and individually sub-cloned into lentiviral vectors. The virus was enriched (at MOI = 30) and added to the cells cultivated in a complete medium with HitransG P (Genechem) to generate YEM1L shRNA-/YEM1L-expressing NSCLC cells. Subsequently, the cells were separately cultured in a complete medium (with 2.0 µg/mL of puromycin) for 96 h. YEM1L knockdown or overexpression in the stable cells was confirmed by Western blotting and qRT-PCR assays. Additionally, control cells were infected with scrambled shRNA lentivirus (“sh-NC”; Genechem) or empty vectors (“control”; Genechem). In the in vivo studies, the YME1L shRNA (-seq1), sh-NC, empty vector, and YEM1L-expressing lentivirus were sub-cloned into lentiviral vectors. The resulting constructs were then separately transfected into A549 cells to generate shRNA- and YEM1L-expressing cells. Ultimately, the transfected A549 cells were enriched and injected into xenograft tumors. Transwell migration and invasion assays. Cell migration and invasion tests were conducted in transwell chambers. In the invasion experiment, Matrigel was pre-coated on the inserts in the upper surface. After transfection, cells (5 × 10 4 ) were temporarily maintained in 200 µL of serum-free medium and later seeded in the upper compartment. Subsequently, 600 µL of complete medium containing 10% FBS was added to the lower compartment, serving as a chemoattractant. After 48 h of incubation at 37°C, the inserts were washed. Next, the cells on the lower surface were fixed with 4% paraformaldehyde, washed, and stained with 0.1% crystal violet. Finally, the migrated cells were counted. In all transwell assays, five random views of each condition were included to obtain the average number of migrated/invaded cells. Quantitative real-time reverse-transcription PCR. Total RNA was extracted from tissues and cells using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. For each treatment, total RNA (500 ng) was reverse transcribed to cDNA using a SuperScript III kit (Invitrogen). Subsequently, qRT-PCR assays were performed utilizing the SYBR Green Supermix with an ABI 7500 FAST Prism system (Applied Biosystems, Foster City, CA, USA). Melting curve analysis was conducted for all assays. Targeted mRNA was quantified via the ΔΔCt method (Schmittgen and Zakrajsek, 2000), with GAPDH as the internal control (Liu et al., 2018). The mRNA primers for YEM1L have been described in a previous study (MacVicar et al., 2019). Western blotting. Cells and tissues were homogenized in a RIPA lysis buffer with a protease inhibitor cocktail (Biyuntian, Wuxi, China). A 10% SDS-PAGE was then run to separate equal amounts of proteins (30–40 µg per treatment), and the proteins were transferred to a PVDF membrane. Subsequently, the membrane was blocked and incubated overnight at 4°C with the appropriate primary antibody. After this period, the membrane was incubated with an HRP-conjugated secondary antibody. The antibody-antigen binding was visualized using an enhanced chemiluminescence detection kit (Biyuntian, Wuxi, China). In this method, the same sets of lysates were run in parallel gels to assess different proteins. The protein bands were quantified utilizing ImageJ software (NIH). Cell viability. Cell viability was examined via a cell counting kit-8 assay (CCK-8; Dojindo Molecular Technologies, Inc.). Briefly, cells were seeded in poly-L-lysine-coated 96-well microplates (5 × 10 3 cells/well). After incubation for 96 h, CCK-8 solution was added to each well, and the microplates were incubated for 2 h at 37°C. The absorbance of each well was measured at 450 nm using a microplate reader. Nude mice xenograft assay. In this assay, 5- to 6-week-old female BALB/c nude mice (18–19 g) purchased from the Animal Center of Soochow University were raised indoors at standard conditions. A549 cells (1 × 10 6 cells per mouse in 0.2 mL of 10% FBS DMEM/Matrigel solution) with indicated genetic modifications were subcutaneously injected into the left armpit of the nude mice. The mice tumor volumes, calculated via a previously described formula (Liu et al., 2018), were measured every 5 days with digital calipers. At 35 days after the experiment, the mice in all groups were sacrificed, and the tumors were surgically isolated. All animal experiments in this study were approved by the Institutional Animal Care and Use Committee and Ethics Committee of Soochow University. Statistical analyses. The investigators were blinded to the group allocation for the in vitro experiments, which were replicated at least three times. For all the functional assays, the exact same number of viable cells of different genetic modifications were initially seeded into each well/dish (at 0h/day-0). Data with normal distribution were expressed as means ± standard deviation (SD). To examine statistical differences among multiple groups, one-way ANOVA followed by a Scheffe’s f-test (SPSS 23.0, SPSS Co., Chicago, CA) was utilized. A two-tailed unpaired t test (Excel 2007) was applied to examine significance between two treatment groups. P values < 0.05 were considered statistically significant. Abbreviations PDAC pancreatic ductal adenocarcinoma NSCLC non-small cell lung cancer SCLC small cell lung cancer IHC immunohistochemistry TMAs tissue microarrays OPA1 optic atrophy 1. Declarations Ethics approval and consent to participate Not applicable Consent for publication All authors consent for publication. Availability of data and materials Not applicable Competing interests All authors declare that they have no competing interests Funding Not applicable Authors' contributions WMD wrote and edited the manuscript. Both authors have read and approved the final manuscript Acknowledgements Not applicable Authors' information 1 Department of Respiratory Diseases, The First People’s Hospital of Zhangjiagang City, Soochow University, Zhangjiagang, China Availability of data and material. All data generated or analyzed during this study are included in this published article (and its supplementary information files). References Li C, Zhang L, Meng G, Wang Q, Lv X, Zhang J, Li J. Circular RNAs: pivotal molecular regulators and novel diagnostic and prognostic biomarkers in non-small cell lung cancer[J]. J Cancer Res Clin Oncol. 2019;145(12):2875–89. Nooreldeen R, Bach H. Current and Future Development in Lung Cancer Diagnosis[J]. Int J Mol Sci, 2021, 22(16). Salehi-Rad R, Li R, Paul MK, Dubinett SM, Liu B. The Biology of Lung Cancer: Development of More Effective Methods for Prevention, Diagnosis, and Treatment[J]. Clin Chest Med. 2020;41(1):25–38. Yuan M, Huang LL, Chen JH, Wu J, Xu Q. The emerging treatment landscape of targeted therapy in non-small-cell lung cancer[J]. Signal Transduct Target Ther. 2019;4:61. Lu S, Yu Y, Yang Y. Retrospect and Prospect for Lung Cancer in China: Clinical Advances of Immune Checkpoint Inhibitors[J]. Oncologist. 2019;24(Suppl 1):21–S30. Choudhury FK. Mitochondrial Redox Metabolism: The Epicenter of Metabolism during Cancer Progression[J]. Antioxid (Basel), 2021, 10(11). Mendes C, Serpa J. Metabolic Remodelling: An Accomplice for New Therapeutic Strategies to Fight Lung Cancer[J]. Antioxid (Basel), 2019, 8(12). Lennon FE, Salgia R. Mitochondrial dynamics: biology and therapy in lung cancer[J]. Expert Opin Investig Drugs. 2014;23(5):675–92. Shah ZH, Hakkaart GA, Arku B, de Jong L, van der Spek H, Grivell LA, Jacobs HT. The human homologue of the yeast mitochondrial AAA metalloprotease Yme1p complements a yeast yme1 disruptant[J]. FEBS Lett. 2000;478(3):267–70. Coppola M, Pizzigoni A, Banfi S, Bassi MT, Casari G, Incerti B. Identification and characterization of YME1L1, a novel paraplegin-related gene[J]. Genomics. 2000;66(1):48–54. Ruan Y, Li H, Zhang K, Jian F, Tang J, Song Z. Loss of Yme1L perturbates mitochondrial dynamics[J]. Cell Death Dis. 2013;4:e896. Stiburek L, Cesnekova J, Kostkova O, Fornuskova D, Vinsova K, Wenchich L, Houstek J, Zeman J. YME1L controls the accumulation of respiratory chain subunits and is required for apoptotic resistance, cristae morphogenesis, and cell proliferation[J]. Mol Biol Cell. 2012;23(6):1010–23. MacVicar T, Ohba Y, Nolte H, Mayer FC, Tatsuta T, Sprenger HG, Lindner B, Zhao Y, Li J, Bruns C, Kruger M, Habich M, Riemer J, Schwarzer R, Pasparakis M, Henschke S, Bruning JC. Zamboni N,Langer T. Lipid signalling drives proteolytic rewiring of mitochondria by YME1L[J]. Nature. 2019;575(7782):361–5. Ohba Y, MacVicar T, Langer T. Regulation of mitochondrial plasticity by the i-AAA protease YME1L[J]. Biol Chem. 2020;401(6–7):877–90. Villaseca S, Romero G, Ruiz MJ, Perez C, Leal JI, Tovar LM, Torrejon M. Galphai protein subunit: A step toward understanding its non-canonical mechanisms[J]. Front Cell Dev Biol. 2022;10:941870. Xu G, Qi L-n, Zhang M-q, Li X-y, Chai J-l, Zhang Z-q, Chen X, Wang Q, Li K. -r,Cao C. Gαi1/3 mediation of Akt-mTOR activation is important for RSPO3-induced angiogenesis[J]. Protein & Cell; 2022. Liu F, Chen G, Zhou L-N, Wang Y, Zhang Z-q. Qin X,Cao C. YME1L overexpression exerts pro-tumorigenic activity in glioma by promoting Gαi1 expression and Akt activation[J]. Protein & Cell; 2022. Ye Z, Zhang H, Kong F, Lan J, Yi S, Jia W, Zheng S, Guo Y, Zhan X. Comprehensive Analysis of Alteration Landscape and Its Clinical Significance of Mitochondrial Energy Metabolism Pathway-Related Genes in Lung Cancers[J]. Oxid Med Cell Longev, 2021, 2021: 9259297. Luo Y, Ma J, Lu W. The Significance of Mitochondrial Dysfunction in Cancer[J]. Int J Mol Sci, 2020, 21(16). Han M, Bushong EA, Segawa M, Tiard A, Wong A, Brady MR, Momcilovic M, Wolf DM, Zhang R, Petcherski A, Madany M, Xu S, Lee JT, Poyurovsky MV, Olszewski K, Holloway T, Gomez A, John MS, Dubinett SM, Koehler CM, Shirihai OS, Stiles L, Lisberg A, Soatto S, Sadeghi S, Ellisman M. H,Shackelford D B. Spatial mapping of mitochondrial networks and bioenergetics in lung cancer[J]. Nature. 2023;615(7953):712–9. Yin L, Zhang Y, Yin L, Ou Y, Lewis MS, Wang R, Zhau HE, Zhou Q. Chung L W K. Novel Mitochondria-Based Targeting Restores Responsiveness in Therapeutically Resistant Human Lung Cancer Cells[J]. Mol Cancer Ther. 2021;20(12):2527–38. Villaseca S, Romero G, Ruiz MJ, Pérez C, Leal JI, Tovar LM. ,Torrejón M. Gαi protein subunit: A step toward understanding its non-canonical mechanisms[J]. Front Cell Dev Biology, 2022, 10. 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-3972823","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":275148268,"identity":"6cb00170-36d9-4200-a6df-7aa6a24027c0","order_by":0,"name":"MinDan Wu","email":"","orcid":"","institution":"The First People’s Hospital of Zhangjiagang City","correspondingAuthor":false,"prefix":"","firstName":"MinDan","middleName":"","lastName":"Wu","suffix":""},{"id":275148269,"identity":"a06bc327-b07c-4198-b66f-807eb54c6189","order_by":1,"name":"Feng Gao","email":"","orcid":"","institution":"The First People’s Hospital of Zhangjiagang City","correspondingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"Gao","suffix":""},{"id":275148270,"identity":"a29f134b-934d-497a-b22c-8565dd72374e","order_by":2,"name":"MeiJie Xu","email":"","orcid":"","institution":"The First People’s Hospital of Zhangjiagang City","correspondingAuthor":false,"prefix":"","firstName":"MeiJie","middleName":"","lastName":"Xu","suffix":""},{"id":275148271,"identity":"7124dad7-7a58-482d-86db-ea4507cd0c95","order_by":3,"name":"JieRu Zhang","email":"","orcid":"","institution":"The First People’s Hospital of Zhangjiagang City","correspondingAuthor":false,"prefix":"","firstName":"JieRu","middleName":"","lastName":"Zhang","suffix":""},{"id":275148272,"identity":"4dee8d54-4e3e-49e6-b65a-34aef6cacadc","order_by":4,"name":"Chen Ni","email":"","orcid":"","institution":"The First People’s Hospital of Zhangjiagang City","correspondingAuthor":false,"prefix":"","firstName":"Chen","middleName":"","lastName":"Ni","suffix":""},{"id":275148273,"identity":"6b69257d-47d4-4a47-a325-3ff995363653","order_by":5,"name":"WenXia Qian","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAApklEQVRIiWNgGAWjYBACPghlw8PP30CkFjYQcYAhTUZyxgHStBy2MWhIIFaL9OGDjz/8Oc9jwHCA8cPHHGK08KUlGxzguc1jztzALDlzGzFaeHjMJA5I3OaxbDjAxsxLpBbzHwcMzvEYHEggXosZw4GEAyRpYUuWOHMgmUdyxsFm4vzCz8N88EPFHzt7fv7mgx8+EqMFCTA2kKZ+FIyCUTAKRgFuAAD3SjAZxsgOggAAAABJRU5ErkJggg==","orcid":"","institution":"The First People’s Hospital of Zhangjiagang City","correspondingAuthor":true,"prefix":"","firstName":"WenXia","middleName":"","lastName":"Qian","suffix":""},{"id":275148274,"identity":"b755b0b0-307f-42ab-bb98-2c5e6b6fc8b4","order_by":6,"name":"LiXiu Chen","email":"","orcid":"","institution":"The First People’s Hospital of Zhangjiagang City","correspondingAuthor":false,"prefix":"","firstName":"LiXiu","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2024-02-20 12:59:44","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3972823/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3972823/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51823533,"identity":"58615101-6b96-4fa0-90fd-7515583380fa","added_by":"auto","created_at":"2024-02-29 16:29:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":14104354,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eYME1L is overexpressed in human NSCLC tissues.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYME1L mRNA and protein expressions in 16 different human NSCLC tissues (“Tumor”) and paired surrounding normal lung tissues (“Normal”) were tested by qRT-PCR (A), Western blotting (B, C), and IHC staining (D) assays. Scale bar = 100 μm.\u003c/p\u003e","description":"","filename":"Figure116.png","url":"https://assets-eu.researchsquare.com/files/rs-3972823/v1/b4381f67891a7ace4199b8af.png"},{"id":51823531,"identity":"e3845d6c-7a0c-45e8-a793-18227cb2cf74","added_by":"auto","created_at":"2024-02-29 16:29:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":858821,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConstruction of NSCLC cell lines with interference and overexpression of YME1L\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNSCLC cell lines(H1299 and A549) with lentiviral construct encoding the YME1L cDNA(“OE-YME1L”) or the control empty vector (“Control”) and YME1L shRNA(“sh-YME1L-1/2/3”) or control nonsense shRNA(“sh-NC”) were established and tested by qRT-PCR and Western blotting assays (A-F).\u003c/p\u003e","description":"","filename":"Figure213.png","url":"https://assets-eu.researchsquare.com/files/rs-3972823/v1/b150169b813ee85b28538109.png"},{"id":51824522,"identity":"ad69134a-5ce5-4376-904a-5f2108b4b0a5","added_by":"auto","created_at":"2024-02-29 16:37:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2249419,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eYME1L promotes NSCLC cell proliferation in vitro.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were further cultured for applied time periods, cell viability (A), colony formation (B, C) and cell cycle distribution (PI-FACS, D, E) were tested by the listed assays, with results quantified.\u003c/p\u003e","description":"","filename":"Figure311.png","url":"https://assets-eu.researchsquare.com/files/rs-3972823/v1/bb4aaa8232a05d228732ef2a.png"},{"id":51823535,"identity":"d879bad8-ebfd-4bd6-a342-712915b2c918","added_by":"auto","created_at":"2024-02-29 16:29:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5785187,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eYME1L promotes NSCLC cell migration and invasion in vitro.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell Scratch (A, B) as well as cell migration and invasion (“Transwell” assays, C-F) were tested by the listed assays, and results were quantified. Scale bar = 100 μm.\u003c/p\u003e","description":"","filename":"Figure46.png","url":"https://assets-eu.researchsquare.com/files/rs-3972823/v1/3b987d5e1406638d821a0aa8.png"},{"id":51824523,"identity":"a1e61496-34d6-40b7-9300-61e4c52f769b","added_by":"auto","created_at":"2024-02-29 16:37:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":947647,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eYME1L is important for Gαi1 expression and Akt activation.\u003c/strong\u003e Cervical cancer cell lines(H1299 and A549) with lentiviral construct encoding the YME1L cDNA(“OE-YME1L”)or the control empty vector (“Control”) and YME1L shRNA(“sh-YME1L”) or control nonsense shRNA(“sh-NC”) were established and cultured for applied time periods;Expression of listed proteins were tested by Western blotting assays(A-D).\u003c/p\u003e","description":"","filename":"Figure54.png","url":"https://assets-eu.researchsquare.com/files/rs-3972823/v1/0625c9d832253dcea81d2e64.png"},{"id":51823536,"identity":"d6e9f34e-7b84-4f89-8c6b-190e96ff4a70","added_by":"auto","created_at":"2024-02-29 16:29:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":9034325,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eYME1L is required for NSCLC xenograft growth in nude mice.\u003c/strong\u003e Stable A549 cells transfected with YME1L-overexpressed (“OE-YME1L”) vector, control nonsense empty vector (“control”), YME1L shRNA (“sh-YME1L-1”), or nonsense control shRNA (“sh-NC”) were injected into the flanks of nude mice (four mice per group), and xenograft tumors established (A). Tumor volumes were recorded every 5 days (B). At 35 days after the experiment (“day 35”), all tumors were isolated (A) and weighed individually (C). In fresh tumor tissues, YME1L mRNA expression was determined via qRT-PCR assays (D), with relevant proteins detected by Western blotting assays (E, F). Representative HE and YME1L IHC images are presented (G). Scale bar = 100 μm.\u003c/p\u003e","description":"","filename":"Figure63.png","url":"https://assets-eu.researchsquare.com/files/rs-3972823/v1/a0fdfe53ad772a5900f090dc.png"},{"id":57000246,"identity":"5391839a-6a92-4122-bfe8-c2211fdbe2f8","added_by":"auto","created_at":"2024-05-23 08:32:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":35596154,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3972823/v1/d4ca11c9-719d-4fe9-a990-685957b7d33a.pdf"},{"id":51823538,"identity":"32bbef82-0130-4a28-8a45-6c941a7b133e","added_by":"auto","created_at":"2024-02-29 16:29:03","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9148091,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfile.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3972823/v1/80272519de53e1f2efcb502e.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"YME1L affects the biological function of non-small cell lung cancer by promoting Gαi1 expression and Akt activation","fulltext":[{"header":"Background","content":"\u003cp\u003eLung cancer, the most frequent malignant tumor in both men and women, is the leading cause of cancer-related deaths worldwide\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Approximately 15% of lung cancers have the histologic subtype of SCLC and the remaining 85% are classified as non\u0026ndash;small cell lung cancer NSCLC\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Lung cancer is a heterogeneous disease with an abundance of genetic and epigenetic alterations. These genetic changes result in activation of oncogenic pathways and/or inhibition of tumor suppressor genes that are critical for malignant transformation of tumor precursor cells\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. In recent years, the identification of tumor subgroups with distinct genetic alterations has changed the landscape of lung cancer treatment and pushed thoracic oncology to the forefront of personalized oncology.However, due to the late diagnosis and the existence of treatment resistance, although the combined application of targeted therapy and comprehensive drugs has improved the prognosis of some patients, the 5-year survival rate of patients with stage IV lung cancer is extremely low \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRecent research has demonstrated that oxidative stress changes are associated with the reprogramming of cellular metabolism during different cancer progression stages, exhibiting a role in maintaining redox homeostasis and supporting the metabolic demand imposed at each stage. Mitochondrial redox metabolism is pivotal in this process, promoting the progression of cancer cells through tumor development, metastasis, and colonization in distant organs\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Therefore, comprehensively understanding the changes in mitochondrial redox metabolism during lung cancer and identifying the mechanisms involved in drug resistance development will help design new treatment strategies and facilitate the treatment plan to continuously adapt as the disease progresses \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe YME1L gene on chromosome 10p is a member of the AAA superfamily of metalloproteases and is localized to the inner mitochondrial membrane \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Morever YME1L encodes a predicted protein of 716 amino acids highly similar to the mitochondrial AAA proteases (particularly yeast Yme1p), with conserved roles in mitochondrial assembly, integrity, and possibly DNA metabolism\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Previous studies have revealed that YME1L regulates the processing and stability of OPA1, where the exogenous expression of OPA1 isoform 1 or L-OPA1 accelerates the mitochondrial fragmentation induced by sh-YME1L \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Additionally, deleting YME1L has been shown to decrease cell proliferation and apoptosis resistance, alter mitochondrial ultrastructure, and increased susceptibility to oxidative damage, emphasizing its critical role in the homeostatic maintenance of the inner mitochondrial membrane protein and the proteolytic regulation of respiratory chain biogenesis\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Multiple studies have suggested that the human YME1L may be related to cancer progression. For example, YME1L-mediated mitochondrial reshaping has been reported to support the growth of pancreatic ductal adenocarcinoma cells as spheroids or xenografts, indicating that YME1L is a promising therapeutic target for cancer \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Enzymes such as YME1L and other mitochondrial proteases may be explored as potential druggable targets in cancer treatment; however, identifying their substrates or regulatory mechanisms is essential for targeting the cancer-specific metabolic pathways\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHeterotrimeric G-proteins, comprising three subunits (α, β, and γ), are crucial components in varied cellular events. Hence, dysregulation in these proteins can severely affect the signaling pathways and overall cellular state. The G-protein inhibitory α-subunit (Gαi) family has the highest number of individual members, (including Gαi1, Gαi2, Gαi3, Gαo, Gαt, Gαg, and Gαz), and constitutes the majority of G-protein α-subunits expressed in tissuess or cells\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Furthermore, the Gαi proteins bind to G protein-coupled receptors to inhibit adenylate cyclase and decrease cyclic AMP levels\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Our previous research has identified that Gαi1 is a pivotal signaling protein required for Akt-mTOR activation via multiple receptor tyrosine kinases. In this context, YME1L overexpression may promote Gαi1 expression and Akt activation in human glioma, exerting a pro-tumorigenic activity in this tumor type\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. In light of these findings, our study aimed to investigate the potential role of YME1L in Gαi1-induced signaling in NSCLC.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eYME1L is overexpressed in human NSCLC tissues.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn the qRT-PCR analysis, YME1L mRNA expression was examined in human NSCLC tissues (\u0026ldquo;tumor [T]\u0026rdquo;) and paired normal lung tissues (\u0026ldquo;normal [N]\u0026rdquo;) obtained from 16 patients with primary NSCLC. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, YME1L mRNA expression in the T tissues was significantly higher than that in N tissues (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Moreover, YME1L protein expression was elevated in the T tissues from eight patients (i.e., patient 1 to patient 8, which represented the first eight patients enrolled in the study; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In all 16 sets of the human tissues, we found a significant upregulation of the YME1L protein (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 versus N tissues; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Furthermore, we determined YME1L expression in the human tissues via immunohistochemistry (IHC). TMA showed that YME1L expression was significantly upregulated in the T tissues compared to the N tissues (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e \u003cb\u003eYME1L is overexpressed in human NSCLC tissues.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eYME1L mRNA and protein expressions in 16 different human NSCLC tissues (\u0026ldquo;Tumor\u0026rdquo;) and paired surrounding normal lung tissues (\u0026ldquo;Normal\u0026rdquo;) were tested by qRT-PCR (A), Western blotting (B, C), and IHC staining (D) assays. Scale bar =\u0026thinsp;100 \u0026micro;m.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of NSCLC cell lines with interference and overexpression of YME1L\u003c/h2\u003e \u003cp\u003eWe transduced NSCLC cells with a lentiviral construct encoding YME1L cDNA (\u0026ldquo;OE-YME1L\u0026rdquo;) to analyze the function of YME1L in NSCLC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B, C). Additionally, we silenced YME1L using targeted shRNAs. For this purpose, three lentiviral shRNAs targeting the non-overlapping sequences of YME1L (sh-YME1L-1, sh-YME1L-2, and sh-YME1L-3) were individually transduced into H1299 and A549 cells to establish stable cell lines with a 90% reduction in YME1L mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) and protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e \u003cb\u003eConstruction of NSCLC cell lines with interference and overexpression of YME1L\u003c/b\u003e\u003c/p\u003e \u003cp\u003eNSCLC cell lines(H1299 and A549) with lentiviral construct encoding the YME1L cDNA(\u0026ldquo;OE-YME1L\u0026rdquo;) or the control empty vector (\u0026ldquo;Control\u0026rdquo;) and YME1L shRNA(\u0026ldquo;sh-YME1L-1/2/3\u0026rdquo;) or control nonsense shRNA(\u0026ldquo;sh-NC\u0026rdquo;) were established and tested by qRT-PCR and Western blotting assays (A-F).\u003c/p\u003e \u003cp\u003e \u003cb\u003eYME1L promotes NSCLC cell proliferation in vitro.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSubsequent examination of cell proliferation using the CCK8 assay revealed that the OD significantly decreased in the YME1L-shRNA-expressing H1299 and A549 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Thus, YME1L knockdown robustly decreased the number of H1299 and A549 cell colonies (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH), indicating inhibited proliferation. Moreover, analyzing cell cycle progression via a PI-FACS assay demonstrated that YME1L overexpression increased the proportion of NSCLC cells in the S-phase and reduced the number of G0/G1 cells. Conversely, YME1L-shRNA transduction decreased the number of S-phase cells and elevated the number of G0/G1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI), implying G1-S arrest. All these results indicate that YME1L upregulation is crucial in the proliferation of NSCLC cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e \u003cb\u003eYME1L promotes NSCLC cell proliferation in vitro.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCells were further cultured for applied time periods, cell viability (A), colony formation (B, C) and cell cycle distribution (PI-FACS, D, E) were tested by the listed assays, with results quantified.\u003c/p\u003e \u003cp\u003e \u003cb\u003eYME1L promotes NSCLC cell migration and invasion in vitro.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eNext, we explored the potential mechanism of elevated YME1L levels in NSCLC. In this examination, stable NSCLC cell lines (H1299 and A549) overexpressing YME1L (\u0026ldquo;OE-YME1L\u0026rdquo;) or expressing YME1L shRNAs (\u0026ldquo;sh-YME1L-1\u0026rdquo;) were cultured for specified periods. Furthermore, cell migration and invasion tests were performed using cell scratch, transwell, and Matrigel transwell assays. These assay findings showed that YME1L overexpression enhanced NSCLC cell migration (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-D) and invasion (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, F), whereas these processes were inhibited in the NSCLC cells transduced with YME1L shRNAs. All these results further suggest that YME1L overexpression may promote NSCLC cell malignant behaviors, including migration and invasion, in vitro.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e \u003cb\u003eYME1L promotes NSCLC cell migration and invasion in vitro.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCell Scratch (A, B) as well as cell migration and invasion (\u0026ldquo;Transwell\u0026rdquo; assays, C-F) were tested by the listed assays, and results were quantified. Scale bar =\u0026thinsp;100 \u0026micro;m.\u003c/p\u003e \u003cp\u003e \u003cb\u003eYME1L is critical for Gαi1 expression and Akt activation.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe further investigated the role of YME1L in Gαi1 expression within NSCLC cells. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, shRNA-induced silencing of YME1L led to a marked downregulation in Gαi1 protein expression in NSCLC cells. Moreover, Akt activation, as assessed by p-Akt levels, was inhibited after YME1L silencing (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D). Conversely, cells overexpressing YME1L (\u0026ldquo;OE-YME1L\u0026rdquo;; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B) exhibited a significant increase in Gαi1 protein levels and enhanced Akt activation. All these findings indicate that YME1L is vital for Gαi1 expression and Akt activation in NSCLC cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e \u003cb\u003eYME1L is important for Gαi1 expression and Akt activation.\u003c/b\u003e Cervical cancer cell lines(H1299 and A549) with lentiviral construct encoding the YME1L cDNA(\u0026ldquo;OE-YME1L\u0026rdquo;)or the control empty vector (\u0026ldquo;Control\u0026rdquo;) and YME1L shRNA(\u0026ldquo;sh-YME1L\u0026rdquo;) or control nonsense shRNA(\u0026ldquo;sh-NC\u0026rdquo;) were established and cultured for applied time periods;Expression of listed proteins were tested by Western blotting assays(A-D).\u003c/p\u003e \u003cp\u003e \u003cb\u003eYME1L is crucial for NSCLC xenograft growth in nude mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFinally, we investigated the potential role of YME1L in NSCLC growth in vivo. Stable NSCLC cells with YME1L-expressing vectors or empty vectors were injected (1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e cells per mouse) into the flanks of nude mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The results of the tumor growth curve (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, C) demonstrated that the NSCLC cells with YME1L-expressing vectors grew significantly faster than the control tumors. In contrast, the growth of the NSCLC xenografts expressing YME1L shRNAs was much slower than that of the control xenografts (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, C). Thus, YME1L is essential for NSCLC xenograft growth in nude mice.\u003c/p\u003e \u003cp\u003eFurthermore, the YME1L-Gαi1 axis genes were assessed in fresh tumor tissues using qRT-PCR and Western blotting analyses. Compared to the control xenografts, YME1L-expressing NSCLC xenografts showed significantly higher Gαi1 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-F). Conversely, NSCLC xenograft tissues expressing YME1L shRNA demonstrated an accumulation of Gαi1 and a decrease in YME1L mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-F). Western blotting assay results exhibited increased expression levels of p-Akt in the xenograft tissues with YME1L overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-F), whereas Akt signaling was decreased in xenografts with YME1L knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-F). Additionally, IHC indicated that Gαi1 and p-AKT levels were downregulated in sh-YME1L-AS-injected A549 xenografts (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). This finding suggests that the intratumoral injection of sh-YME1L-AS lentivirus inhibited NSCLC xenograft growth and vice versa.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e \u003cb\u003eYME1L is required for NSCLC xenograft growth in nude mice.\u003c/b\u003e Stable A549 cells transfected with YME1L-overexpressed (\u0026ldquo;OE-YME1L\u0026rdquo;) vector, control nonsense empty vector (\u0026ldquo;control\u0026rdquo;), YME1L shRNA (\u0026ldquo;sh-YME1L-1\u0026rdquo;), or nonsense control shRNA (\u0026ldquo;sh-NC\u0026rdquo;) were injected into the flanks of nude mice (four mice per group), and xenograft tumors established (A). Tumor volumes were recorded every 5 days (B). At 35 days after the experiment (\u0026ldquo;day 35\u0026rdquo;), all tumors were isolated (A) and weighed individually (C). In fresh tumor tissues, YME1L mRNA expression was determined via qRT-PCR assays (D), with relevant proteins detected by Western blotting assays (E, F). Representative HE and YME1L IHC images are presented (G). Scale bar =\u0026thinsp;100 \u0026micro;m.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eLung cancer is a prominent public health challenge due to its high morbidity and mortality rates. Moreover, the metabolic changes observed in lung cancer can affect prognosis and treatment response\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. These tumors can also involve mitochondrial dysfunction, resulting from mitochondrial DNA mutations, TCA cycle enzyme dysfunction, electronic respiratory chain defects, and subsequent oxidative stress, and/or abnormal oncogenic and tumor suppressive signaling. These alterations can in turn modify the cellular metabolic pathways, disrupt redox balance, and lead to apoptosis and resistance, playing a critical role in sustaining the growth and survival of tumor cells\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. YME1L is the human orthologue of the Yme1 subunit of the yeast mitochondrial i-AAA complex. This complex represents a so-called i-AAA protease (where AAA denotes ATPases associated with various cellular activities), which exerts peptidase, chaperone-like, and translocase activities on the intermembrane space side of the inner mitochondrial membrane\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Therefore, YME1L couples mitochondrial morphology and function by maintaining mitochondrial fusion and fission via OPA1 turnover and processing as well as by regulating metabolic output via the degradation of a broad range of substrates in response to nutrient availability and cellular stress\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Our previous research revealed that YME1L overexpression could induce Gαi1 expression and Akt activation, influencing cell proliferation, growth, migration, and survival and consequently promoting glioma progression\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Moreover, limited information exists on the YME1L expression status among Chinese patients with NSCLC. Therefore, this study investigated the role of YME1L in NSCLC occurrence and development and evaluated the signaling pathways regulated by YME1L. Our study demonstrated that YME1L overexpression promotes tumor cell growth and migration by modulating the Gαi1-Akt signaling pathway, suggesting that targeting this process may contribute to devising new treatment strategies for lung cancer.\u003c/p\u003e \u003cp\u003eIn the current study, IHC was used to detect the expression levels of YME1L in surgically excised tissues consisting of 16 pairs of paraffin-embedded specimens from patients with NSCLC. The IHC results showed that almost 90% of the lung cancer tissues had higher YME1L expression levels than the adjacent lung tissues. Furthermore, we conducted functional studies and confirmed that YME1L overexpression enhances cell proliferation in vitro and in vivo, where silencing YME1L expression inhibits tumor cell proliferation and migration. Therefore, our data confirms that YME1L has a pro-oncogenic role in NSCLC.\u003c/p\u003e \u003cp\u003eHeterotrimeric G-proteins, comprising Gα, Gβ, and Gγ subunits, are crucial signaling components. These G-proteins act as molecular switches, controlling the transmission of information from various extracellular cues to the numerous intracellular effectors regulating cell behavior. Gαi, a member of this G-protein family, was initially identified for its inhibitory function on adenylyl cyclase activity that led to decreased cyclic AMP levels\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Our research group previously highlighted that Gαi1 and Gαi3 are pivotal signaling proteins required for Akt-mTOR activation via multiple receptor tyrosine kinases \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. In this study, we employed lentiviral interference and YME1L overexpression and estimated the protein expression levels of Gαi1 and Akt via Western blotting analysis to verify whether this signaling pathway plays a similar role in NSCLC. Our results showed that YME1L overexpression might promote Gαi1 expression and Akt activation in NSCLC.\u003c/p\u003e \u003cp\u003eAdditionally, qRT-PCR, Western blot, and IHC analyses in this study demonstrated that the mRNA and protein expression of YME1L was significantly higher in NSCLC tissue than in control tissues. Moreover, this study revealed for the first time that YME1L might promote NSCLC cell proliferation and migration by targeting the Gαi1-Akt signaling pathway. All these research findings suggest that developing inhibitors targeting this signaling pathway may be a promising strategy for treating NSCLC.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, we found that YME1L have a novel oncogenic role in promoting NSCLC tumorigenesis and progression via the Gαi1-AS-pAKT axis.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003eReagents.\u003c/b\u003e Puromycin were purchased from Sigma-Aldrich (St. Louis, MO). The antibodies were provided by Cell Signaling Technology (Beverly, MA) and Abcam (Cambridge, UK). Fetal bovine serum (FBS, Gibco), Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM), Roswell Park Memorial Institute (RPMI)-1640 medium and antibiotics were provided by Gibco-BRL (Suzhou, China). TRIzol and other RNA assay reagents were purchased from Thermo-Fisher Invitrogen (Shanghai, China).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell culture.\u003c/b\u003e NSCLC cell lines (H1299, and A549) were acquired commercially from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). Cell culture was respectively performed in RPMI-1640 medium and DMEM, which were supplemented with 10% FBS and antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin; Invitrogen, Carlsbad, CA) in a humidified air atmosphere at 37℃ with 5% CO\u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHuman tissues.\u003c/b\u003e Fresh NSCLC tissues (\u0026ldquo;T\u0026rdquo;) and matched adjacent normal lung tissues (\u0026ldquo;N\u0026rdquo;) were obtained from 16 patients with primary lung cancer who were admitted to the Affiliated Zhangjiagang First People\u0026rsquo;s Hospital of Soochow University. The tissue samples were stored in liquid nitrogen and homogenized in a tissue lysis buffer with proteasome inhibitors before further analyses. In the immunofluorescence studies, the human tissue sections (4-\u0026micro;m thick) were incubated with anti-YME1L antibodies at 4\u0026deg;C overnight, followed by incubation with a green fluorescence secondary antibody at 37\u0026deg;C for 60 min. None of the patients had received chemotherapy or radiotherapy before surgery. Written informed consent was obtained from each included patient. The protocols for using human tissues were in accordance with the Declaration of Helsinki, and their use in this study was approved by the Ethics Board of Soochow University.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunohistochemical staining.\u003c/b\u003e Tissue microarrays (TMAs) from the patients and mice were constructed, and core tissue samples of 2-mm diameter were acquired from the tissue sections, which were embedded with paraffin and stored in paraffin-recipient blocks. Next, the slides were heated for 10 min in a sodium citrate buffer for antigen retrieval. The slides were then blocked with bovine serum albumin, followed by incubation with primary antibodies for anti-YME1L (dilution, 1:50) at 4\u0026deg;C overnight. Further, the slides were washed three times with PBS and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody at the indoor temperature for 60 min. Finally, the slides were stained using a DAB solution. Two pathologists evaluated the slides for YME1L expression based on the staining intensity of the positive immunoreactive cells. The staining intensity was graded as follows: 0 (no staining), 1 (weak staining), 2 (moderate staining), and 3 (strong staining).\u003c/p\u003e \u003cp\u003e \u003cb\u003eSilencing or overexpression of YEM1L.\u003c/b\u003e A set of three different YEM1L shRNA sequences was designed and synthesized by Genechem (Shanghai, China) and individually sub-cloned into lentiviral vectors. The virus was enriched (at MOI\u0026thinsp;=\u0026thinsp;30) and added to the cells cultivated in a complete medium with HitransG P (Genechem) to generate YEM1L shRNA-/YEM1L-expressing NSCLC cells. Subsequently, the cells were separately cultured in a complete medium (with 2.0 \u0026micro;g/mL of puromycin) for 96 h. YEM1L knockdown or overexpression in the stable cells was confirmed by Western blotting and qRT-PCR assays. Additionally, control cells were infected with scrambled shRNA lentivirus (\u0026ldquo;sh-NC\u0026rdquo;; Genechem) or empty vectors (\u0026ldquo;control\u0026rdquo;; Genechem). In the in vivo studies, the YME1L shRNA (-seq1), sh-NC, empty vector, and YEM1L-expressing lentivirus were sub-cloned into lentiviral vectors. The resulting constructs were then separately transfected into A549 cells to generate shRNA- and YEM1L-expressing cells. Ultimately, the transfected A549 cells were enriched and injected into xenograft tumors.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTranswell migration and invasion assays.\u003c/b\u003e Cell migration and invasion tests were conducted in transwell chambers. In the invasion experiment, Matrigel was pre-coated on the inserts in the upper surface. After transfection, cells (5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e) were temporarily maintained in 200 \u0026micro;L of serum-free medium and later seeded in the upper compartment. Subsequently, 600 \u0026micro;L of complete medium containing 10% FBS was added to the lower compartment, serving as a chemoattractant. After 48 h of incubation at 37\u0026deg;C, the inserts were washed. Next, the cells on the lower surface were fixed with 4% paraformaldehyde, washed, and stained with 0.1% crystal violet. Finally, the migrated cells were counted. In all transwell assays, five random views of each condition were included to obtain the average number of migrated/invaded cells.\u003c/p\u003e \u003cp\u003e \u003cb\u003eQuantitative real-time reverse-transcription PCR.\u003c/b\u003e Total RNA was extracted from tissues and cells using TRIzol reagent (Invitrogen) according to the manufacturer\u0026rsquo;s instructions. For each treatment, total RNA (500 ng) was reverse transcribed to cDNA using a SuperScript III kit (Invitrogen). Subsequently, qRT-PCR assays were performed utilizing the SYBR Green Supermix with an ABI 7500 FAST Prism system (Applied Biosystems, Foster City, CA, USA). Melting curve analysis was conducted for all assays. Targeted mRNA was quantified via the ΔΔCt method (Schmittgen and Zakrajsek, 2000), with GAPDH as the internal control (Liu et al., 2018). The mRNA primers for YEM1L have been described in a previous study (MacVicar et al., 2019).\u003c/p\u003e \u003cp\u003e \u003cb\u003eWestern blotting.\u003c/b\u003e Cells and tissues were homogenized in a RIPA lysis buffer with a protease inhibitor cocktail (Biyuntian, Wuxi, China). A 10% SDS-PAGE was then run to separate equal amounts of proteins (30\u0026ndash;40 \u0026micro;g per treatment), and the proteins were transferred to a PVDF membrane. Subsequently, the membrane was blocked and incubated overnight at 4\u0026deg;C with the appropriate primary antibody. After this period, the membrane was incubated with an HRP-conjugated secondary antibody. The antibody-antigen binding was visualized using an enhanced chemiluminescence detection kit (Biyuntian, Wuxi, China). In this method, the same sets of lysates were run in parallel gels to assess different proteins. The protein bands were quantified utilizing ImageJ software (NIH).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell viability.\u003c/b\u003e Cell viability was examined via a cell counting kit-8 assay (CCK-8; Dojindo Molecular Technologies, Inc.). Briefly, cells were seeded in poly-L-lysine-coated 96-well microplates (5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells/well). After incubation for 96 h, CCK-8 solution was added to each well, and the microplates were incubated for 2 h at 37\u0026deg;C. The absorbance of each well was measured at 450 nm using a microplate reader.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNude mice xenograft assay.\u003c/b\u003e In this assay, 5- to 6-week-old female BALB/c nude mice (18\u0026ndash;19 g) purchased from the Animal Center of Soochow University were raised indoors at standard conditions. A549 cells (1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells per mouse in 0.2 mL of 10% FBS DMEM/Matrigel solution) with indicated genetic modifications were subcutaneously injected into the left armpit of the nude mice. The mice tumor volumes, calculated via a previously described formula (Liu et al., 2018), were measured every 5 days with digital calipers. At 35 days after the experiment, the mice in all groups were sacrificed, and the tumors were surgically isolated. All animal experiments in this study were approved by the Institutional Animal Care and Use Committee and Ethics Committee of Soochow University.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analyses.\u003c/b\u003e The investigators were blinded to the group allocation for the in vitro experiments, which were replicated at least three times. For all the functional assays, the exact same number of viable cells of different genetic modifications were initially seeded into each well/dish (at 0h/day-0). Data with normal distribution were expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). To examine statistical differences among multiple groups, one-way ANOVA followed by a Scheffe\u0026rsquo;s f-test (SPSS 23.0, SPSS Co., Chicago, CA) was utilized. A two-tailed unpaired t test (Excel 2007) was applied to examine significance between two treatment groups. P values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePDAC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epancreatic ductal adenocarcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNSCLC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enon-small cell lung cancer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSCLC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esmall cell lung cancer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIHC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eimmunohistochemistry\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTMAs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etissue microarrays\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOPA1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eoptic atrophy 1.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors consent for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWMD wrote and edited the manuscript. Both authors have read and approved the final manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eDepartment of Respiratory Diseases, The First People\u0026rsquo;s Hospital of Zhangjiagang City, Soochow University, Zhangjiagang, China\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material.\u003c/strong\u003e All data generated or analyzed during this study are included in this published article (and its supplementary information files).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLi C, Zhang L, Meng G, Wang Q, Lv X, Zhang J, Li J. Circular RNAs: pivotal molecular regulators and novel diagnostic and prognostic biomarkers in non-small cell lung cancer[J]. J Cancer Res Clin Oncol. 2019;145(12):2875\u0026ndash;89.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNooreldeen R, Bach H. Current and Future Development in Lung Cancer Diagnosis[J]. Int J Mol Sci, 2021, 22(16).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalehi-Rad R, Li R, Paul MK, Dubinett SM, Liu B. The Biology of Lung Cancer: Development of More Effective Methods for Prevention, Diagnosis, and Treatment[J]. Clin Chest Med. 2020;41(1):25\u0026ndash;38.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYuan M, Huang LL, Chen JH, Wu J, Xu Q. The emerging treatment landscape of targeted therapy in non-small-cell lung cancer[J]. Signal Transduct Target Ther. 2019;4:61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu S, Yu Y, Yang Y. Retrospect and Prospect for Lung Cancer in China: Clinical Advances of Immune Checkpoint Inhibitors[J]. Oncologist. 2019;24(Suppl 1):21\u0026ndash;S30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChoudhury FK. Mitochondrial Redox Metabolism: The Epicenter of Metabolism during Cancer Progression[J]. Antioxid (Basel), 2021, 10(11).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMendes C, Serpa J. Metabolic Remodelling: An Accomplice for New Therapeutic Strategies to Fight Lung Cancer[J]. Antioxid (Basel), 2019, 8(12).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLennon FE, Salgia R. Mitochondrial dynamics: biology and therapy in lung cancer[J]. Expert Opin Investig Drugs. 2014;23(5):675\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShah ZH, Hakkaart GA, Arku B, de Jong L, van der Spek H, Grivell LA, Jacobs HT. The human homologue of the yeast mitochondrial AAA metalloprotease Yme1p complements a yeast yme1 disruptant[J]. FEBS Lett. 2000;478(3):267\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoppola M, Pizzigoni A, Banfi S, Bassi MT, Casari G, Incerti B. Identification and characterization of YME1L1, a novel paraplegin-related gene[J]. Genomics. 2000;66(1):48\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRuan Y, Li H, Zhang K, Jian F, Tang J, Song Z. Loss of Yme1L perturbates mitochondrial dynamics[J]. Cell Death Dis. 2013;4:e896.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStiburek L, Cesnekova J, Kostkova O, Fornuskova D, Vinsova K, Wenchich L, Houstek J, Zeman J. YME1L controls the accumulation of respiratory chain subunits and is required for apoptotic resistance, cristae morphogenesis, and cell proliferation[J]. Mol Biol Cell. 2012;23(6):1010\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMacVicar T, Ohba Y, Nolte H, Mayer FC, Tatsuta T, Sprenger HG, Lindner B, Zhao Y, Li J, Bruns C, Kruger M, Habich M, Riemer J, Schwarzer R, Pasparakis M, Henschke S, Bruning JC. Zamboni N,Langer T. Lipid signalling drives proteolytic rewiring of mitochondria by YME1L[J]. Nature. 2019;575(7782):361\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOhba Y, MacVicar T, Langer T. Regulation of mitochondrial plasticity by the i-AAA protease YME1L[J]. Biol Chem. 2020;401(6\u0026ndash;7):877\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVillaseca S, Romero G, Ruiz MJ, Perez C, Leal JI, Tovar LM, Torrejon M. Galphai protein subunit: A step toward understanding its non-canonical mechanisms[J]. Front Cell Dev Biol. 2022;10:941870.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu G, Qi L-n, Zhang M-q, Li X-y, Chai J-l, Zhang Z-q, Chen X, Wang Q, Li K. -r,Cao C. Gαi1/3 mediation of Akt-mTOR activation is important for RSPO3-induced angiogenesis[J]. Protein \u0026amp; Cell; 2022.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu F, Chen G, Zhou L-N, Wang Y, Zhang Z-q. Qin X,Cao C. YME1L overexpression exerts pro-tumorigenic activity in glioma by promoting Gαi1 expression and Akt activation[J]. Protein \u0026amp; Cell; 2022.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYe Z, Zhang H, Kong F, Lan J, Yi S, Jia W, Zheng S, Guo Y, Zhan X. Comprehensive Analysis of Alteration Landscape and Its Clinical Significance of Mitochondrial Energy Metabolism Pathway-Related Genes in Lung Cancers[J]. Oxid Med Cell Longev, 2021, 2021: 9259297.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuo Y, Ma J, Lu W. The Significance of Mitochondrial Dysfunction in Cancer[J]. Int J Mol Sci, 2020, 21(16).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan M, Bushong EA, Segawa M, Tiard A, Wong A, Brady MR, Momcilovic M, Wolf DM, Zhang R, Petcherski A, Madany M, Xu S, Lee JT, Poyurovsky MV, Olszewski K, Holloway T, Gomez A, John MS, Dubinett SM, Koehler CM, Shirihai OS, Stiles L, Lisberg A, Soatto S, Sadeghi S, Ellisman M. H,Shackelford D B. Spatial mapping of mitochondrial networks and bioenergetics in lung cancer[J]. Nature. 2023;615(7953):712\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYin L, Zhang Y, Yin L, Ou Y, Lewis MS, Wang R, Zhau HE, Zhou Q. Chung L W K. Novel Mitochondria-Based Targeting Restores Responsiveness in Therapeutically Resistant Human Lung Cancer Cells[J]. Mol Cancer Ther. 2021;20(12):2527\u0026ndash;38.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVillaseca S, Romero G, Ruiz MJ, P\u0026eacute;rez C, Leal JI, Tovar LM. ,Torrej\u0026oacute;n M. Gαi protein subunit: A step toward understanding its non-canonical mechanisms[J]. Front Cell Dev Biology, 2022, 10.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"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},"keywords":"NSCLC, Mitochondrial Remodeling, YME1L, Targeted therapy, Gαi1","lastPublishedDoi":"10.21203/rs.3.rs-3972823/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3972823/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: As a key regulatory enzyme in mitochondria, YME1L is crucial for maintaining mitochondrial morphology, function and plasticity, and plays a\u003c/p\u003e\n\u003cp\u003ecatalytic role in PDAC. The research team has found that the overexpression of YME1L can promote the expression of Gαi1 and the activation of Akt, and promote the progression of glioma. Taking NSCLC as the research material, this project aims to reveal the effect of YME1L regulating mitochondrial remodeling on the progress of NSCLC and its molecular mechanism, and provide new ideas for the treatment of NSCLC patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: YME1L is highly expressed in NSCLC tissues Overexpression of yme1l can promote Gαi1 expression and Akt activation, which in turn regulate cell proliferation, growth, migration and survival, and promote the progression of NSCLC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: In this study, we found that YME1L have a novel oncogenic role in promoting NSCLC tumorigenesis and progression via the Gαi1-AS-pAKT axis. The implementation of this project may provide a new entry point for the treatment of lung cancer.\u003c/p\u003e","manuscriptTitle":"YME1L affects the biological function of non-small cell lung cancer by promoting Gαi1 expression and Akt activation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-29 16:28:57","doi":"10.21203/rs.3.rs-3972823/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":"aa575f14-8693-411d-a390-7bb5f2090d0d","owner":[],"postedDate":"February 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-23T08:24:29+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-29 16:28:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3972823","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3972823","identity":"rs-3972823","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}