MiR-618 suppresses the proliferation, invasion, and migration of non-small lung cancer via the JAK2/STAT3 axis

MiR-618 suppresses the proliferation, invasion, and migration of non-small lung cancer via the JAK2/STAT3 axis | 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 MiR-618 suppresses the proliferation, invasion, and migration of non-small lung cancer via the JAK2/STAT3 axis Ziyuan Chen, Wei Chen, Zhiqi Hong, Xianqiao Wu, Tianzheng Fang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4695743/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Dec, 2024 Read the published version in Journal of Cardiothoracic Surgery → Version 1 posted 7 You are reading this latest preprint version Abstract Objective The regulatory role of the miR-618/JAK2/STAT3 axis in non-small cell lung cancer (NSCLC) cells was investigated with the objective of identifying a target for the precise treatment of patients with NSCLC. Methods Differentially expressed genes were identified in the GEO database and were analyzed bioinformatically. The tissue and cell levels of miR-618 were assessed using qRT-PCR, while the protein levels of JAK2 and STAT3 were determined through western blotting analysis.The association between miR-618 and JAK2 was scrutinized through bioinformatics analysis and dual-luciferase experiments. To evaluate cell proliferation, migration, and invasion, MTT, wound-healing, and Transwell assays were employed. RESULTS MiR-618 expression decreased in NSCLC, leading to the inhibition of growth, invasiveness, and migratory properties of non-small cell lung cancer. This is achieved by MiR-618 modulating the JAK2/STAT3 signaling pathway. In addition, miR-618 inhibited cell proliferation, migration, and invasion by targeting JAK2/STAT3. Conclusion Our study demonstrates that a novel miR-618/JAK2/STAT3 signaling axis is involved in suppressing malignancy in NSCLC and provides a promising target for NSCLC therapy. Non-small cell lung cancer miRNA JAK2 miR-618 STAT3 Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Lung carcinoma is one of the leading causes of global cancer mortality.Based on the information from GLOBOCAN 2018 data, approximately 2.09 million new cases and 1.76 million deaths are reported each year[ 1 ]. Lung cancer ranks among the most prevalent malignant neoplasms in China. The 2015 data published by the National Cancer Center reveals that the 5-year prevalence rate of lung cancer in China during the period of 2006 to 2011 stood at 130.2 cases per 100,000 individuals.Of these cases, the prevalence rate for men was 84.6 (1/100 000), ranking second for malignant tumors, while the rate for women was 45.6 (1/100 000), ranking fourth for malignant tumors[ 2 ]. Histopathologically, lung malignancy can be categorized as small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC), with NSCLC accounting for approximately 80% of all cases based on histological analysis.Early-stage metastatic lung cancer can be treated by tumor resection but advanced or metastatic lung cancer still requires radiotherapy alone or in combination with chemotherapy[ 3 ]. Despite the recent development of innovative treatments, the survival rate of lung cancer patients is still only about 15% with late-stage disease manifestations, histological heterogeneity of the tumor subtypes, and resistance to anti-tumor drugs being the key reasons for poor prognosis[ 4 ]. Understanding the underlying mechanisms of lung cancer tumorigenesis is essential in enhancing patient diagnosis, treatment, and prognosis. The body of research indicates that non-coding RNAs (ncRNAs) are significantly involved in the development and advancement of non-small cell lung cancer (NSCLC). [ 5 – 7 ]. MicroRNAs (miRNAs), comprising small ncRNAs with a length of 20–24 nucleotides, have emerged as key players in this context[ 8 , 9 ]. They were first discovered in 1993 in Caenorhabditis elegans [ 10 , 11 ]. Since then, miRNAs have been shown to have significant importance in the regulatory pathways of both unicellular and multicellular eukaryotes. Small RNA molecules have the ability to selectively identify and attach to matching regions found in the 3'-untranslated portions (UTRs) of target messenger RNAs. This interaction can impede translation or induce degradation of the mRNA, ultimately causing post-transcriptional gene silencing[ 12 ]. In addition, miRNAs can also activate gene expression under certain conditions either directly or indirectly. The miRBase database currently contains over 2500 mature miRNAs derived from 1188 miRNA precursors[ 13 ]. Thousands of miRNAs have been shown to be associated with various human diseases, including malignant tumors. In 2002, a study by Calin et al. showed a relationship between miRNA dysregulation and cancer[ 14 ]. In 2004, Takamizawa et al. demonstrated a relationship between miRNA expression and lung cancer[ 15 ]. miR-519d-3p has been found to inhibit the expression of Bcl-w and hypoxia-inducing factor (HIF)-1α, reducing hypoxia-induced tumorigenesis[ 16 ], while miR-487a-3p down-regulation inhibits the progression of NSCLC by targeting Smad7[ 17 ]. Janus kinase 2 (JAK2) serves as a non-receptor tyrosine kinase signaling molecule responsible for transducing the effects of a range of hormones and cytokines such as interferon, erythropoietin, leptin, and growth hormone.[ 18 , 19 ]. JAK2 plays a critical role in the regulation of cellular volume, safeguarding cells during energy utilization and proliferation, and facilitating the survival of tumor cells. The JAK/STAT pathway is an evolutionarily conserved signaling pathway, involving many basic cell functions, such as cell growth and metastasis, which can lead to the development and progression of cancer[ 20 ]. In this study, we identified several miRNAs that are differentially expressed in lung cancer through data mining and the sorting of lung cancer gene chips (Accession No. GSE24709)[ 21 ]. Through tissue sample verification, we found that miR-618 showed low expression in NSCLC tissue cells. Our findings indicate that miR-618 specifically targets JAK2 within tumor cells, resulting in the suppression of migration and invasion in NSCLC cells by modulating the JAK2/STAT3 pathway, thus providing new insights into the pathogenesis of NSCLC. MATERIALS AND METHODS Human tissue specimens Tissue samples (tumor and normal adjacent tissue) were obtained from lung cancer patients at the First Affiliated Hospital of Ningbo University. The specimens were procured from surgical excision of neoplasms, with the individuals having had no prior exposure to radiotherapy, chemotherapy, or targeted therapy prior to the operative procedure.Clinical information and case data were obtained from the patients at the time of surgery. All the patients provided written informed consent, and the research methodology was sanctioned by the Medical Ethics Committee of the First Affiliated Hospital of Ningbo University.Helsinki Declaration has been followed for involving human subjects in this study. Cell culture The cell lines utilized in the study were procured from the Cell Bank of the Chinese Academy of Sciences(CASCB,China),including one human normal bronchial epithelial cell line BEAS-2B and four human lung adenocarcinoma cell lines namely NCI-H1299,LTEP-A-2,SPC-A-1 and A549. BEAS-2B cells were cultured in DMEM medium (Hyclone, USA), while all human lung cancer cell lines were cultured in RPMI-1640 medium (Hyclone, USA). The cells mentioned above were all cultured in an environment containing 10% fetal bovine serum, and all cell lines were placed in a 5% CO2, 37°C incubator. Total RNA extraction and RT-qPCR Total RNA was isolated from lung cancer tissues and cell lines employing the TRIzol reagent (Invitrogen, Waltham. MA, USA).mRNA was obtained by reverse-transcription of the total RNA using a reverse-transcription kit (Toyobo, Japan), and cDNA was synthesized from miRNA using a miRNA reverse-transcription kit (GenePharma, China). RT-qPCR was performed using SYBR Green 1 (Takara, Japan). β-actin and U6 were employed as internal reference standards, with the specific primers utilized detailed in Table S1 (additional file 1). siRNAs, miRNA mimic, miRNA inhibitor, and transfection experiments JAK2 siRNA, the miR-618 mimic and inhibitor, and a non-targeting negative control were purchased from GenePharma (Shanghai, China). Lipofectamine 2000 (Invitrogen, Germany) was used for transfection.After a span of forty-eight hours subsequent to transfection, the cellular entities were employed for the purpose of RNA extraction and identification through RT-qPCR. The transfected sequences of the miR-618 mimics and siRNA oligonucleotides are shown in Table S2, Additional file 1. MTT assay The transfected cells were seeded and cultured in 96-well plates for 12 h. Twenty microliters of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) solution were added to each well, after which the plate was incubated at 37˚C for 4 h. After removing the medium and MTT solution, 150µl of DMSO was introduced, and the absorbance was measured using a microplate reader. (Bio-Rad, Hercules, CA, USA) every hour for 0, 24, 48, 72, and 96 hours. Colony formation assay The transfected cells were evenly allocated into individual wells of 6-well plates, with each well containing a concentration of 500 cells. The plates underwent a 12-day incubation in a cell culture incubator, and then were subjected to three washes using phosphate-buffered saline (PBS). The colonies were then fixed with 4% paraformaldehyde for a duration of 30 minutes, followed by staining with 150µl of 0.1% crystal violet per well for a period of 15 minutes. Transwell assay A Transwell chamber with its upper and lower chambers separated by a polycarbonate microporous membrane (pore size 8 µm) and coated with artificial base glue was used. Cells were harvested and resuspended to a density of 1×10 6 cells/mL. One hundred microliters of this cell suspension were added to the upper chamber, and 500 µL of DMEM complete culture medium was added to the lower chamber. The Transwell chamber was then placed in a 37°C, 5% CO 2 incubator and cultured for 8 h. The polycarbonate microporous membrane was then removed from the chamber and the base glue and cells on its upper surface were gently wiped with a cotton swab. The cells were fixed in neutral formaldehyde for 20 minutes, followed by staining with hematoxylin and eosin. Finally, the cells were examined under a microscope and the cells in five randomly selected fields of view were counted. Wound-healing experiment The transfected cells were distributed into a 6-well plate at a density of 2x10 5 cells per well, followed by an overnight incubation at 37°C in a 5% CO2 environment. After ensuring that the cells were adherent, 2,4-diamino-4,6-dihydroxypyrimidine (DDP) was added for treatment, after which the cells were cultured until fully confluent. A linear scratch was made on the cell monolayer with a 200 µL sterile pipette tip and photomicrographs were taken immediately. After culturing for a further 48 h, the cells were again photographed and recorded. Image J software was used to measure the migration distance of each group of cells. Western blotting Total protein was extracted from the cells using RIPA lysis buffer (Solarbio, Beijing, China) and quantified using a BCA protein assay kit (Beyotime, China). The Western blotting procedure was executed following established protocols.The primary antibodies were anti-JAK2 (Bioss Antibodies, Woburn, MA, USA), anti-STAT3 (Bioss), anti-p-STAT3 (Bioss), and anti-β-actin (Santa Cruz Biotechnology, Dallas, TX, USA). Statistical analysis Statistical analysis of the data was conducted using GraphPad Prism 8 software (GraphPad Software, La Jolla, CA, USA). Data were presented in the form of means ± standard deviations.The study was conducted with a minimum of three replications, with statistical significance determined at a p-value of less than 0.05. Photoshop CS6 software (Adobe Photoshop CS, Berkeley, CA, USA) was used for the statistical analysis of cell clone numbers, and quantitative analysis of the gray values of the Western blot bands was performed using Image J software. RESULTS Expression of miR-618 in NSCLC First, we confirmed the presence of miR-618 in NSCLC tissues. We employed qRT-PCR to assess the expression of miR-618 in 25 sets of NSCLC tumors along with their neighboring non-cancerous tissues. The levels of miR-618 were notably reduced in the cancerous tissues in contrast to the neighboring non-cancerous tissues. (Fig. 1 A). The starbase database[ 22 ] showed that miR-618 was significantly underexpressed in 512 lung cancer tissues compared with 20 controls (Fig. 1 B).Subsequent studies showed that the expression of miR-618 in NSCLC cells (LTEP-A-2, SPC-A-1, NCI-H1299, and A549) was significantly reduced compared with BEAS-2B cells (Fig. 1 C).NSCLC cells were assessed to investigate the impact of miR-618 on their proliferation, migration, and invasion through gain and loss of function analyses. First, we designed and synthesized a miR-618 inhibitor, and observed that it could downregulate the expression of miR-618 by 48–63%. (Fig. 1 D). Furthermore, we employed miR-618 mimics for inducing ectopic expression of miR-618, observing a substantial up-regulation of miR-618 by 247-879-fold. (Fig. 1 E). MiR-618 inhibits lung cancer cell proliferation,migration, and invasion. The results obtained from MTT and colony formation assays demonstrated that the downregulation of miR-618 significantly enhanced the growth and proliferation of LTEP-A-2 and NCI-H1299 cells.Conversely, upregulating miR-618 had the opposite impact on cell proliferation, suggesting that miR-618 suppressed the proliferation of NSCLC cells. (Fig. 2 A-B). Cellular scratching and invasion assays elucidated that suppression of miR-618 facilitated the migratory and invasive capacities of LTEP-A-2 and NCI-H1299 cells, whereas upregulation hindered cell migration and invasion. (Fig. 2 C-D). MiR-618 targets the JAK2 3' UTR. The experimental investigations concerning cellular functions have indicated that miR-618 possesses the capability to impede the proliferation, invasion, and migration of NSCLC cells.We suspected that miR-618 might influence the progression of NSCLC via a distinct molecular mechanism. Research indicates that miRNAs have the ability to selectively attach to and control the activity of target genes in order to influence the advancement of NSCLC.TargetScan software predicted that miR-618 binds to JAK2 (Fig. 3 A). To verify this prediction, we knocked down or overexpressed miR-618 in LTEP-A-2 and NCI-H1299 cells, confirming that JAK2 was regulated by miR-618 (Fig. 3 B). For further exploration of this interaction, we conducted a dual-luciferase reporter assay. The findings indicated that after co-transfection of miR-618 mimics with JAK2 wild-type, the luciferase activity was significantly reduced, while co-transformation with the JAK2 mutant sequence had no effect (Fig. 3 C). Western blotting showed that JAK2 knockdown significantly reduced the expression of both JAK2 and p-STAT3 protein, while the simultaneous knockdown of JAK2 and mir-618 restored the JAK2 and p-STAT3 protein levels (Fig. 3 F). These findings suggested that mir-618 can interact with JAK2. Knockdown of JAK2 restores cell proliferation, migration, and invasion inhibited by miR-618 in NCI-H1299 and LTEP-A-2 cells. To confirm if miR-618 enhances lung cancer advancement through JAK2 regulation, we co-transfected the miR-618 inhibitor and JAK2 siRNA into LTEP-A-2 and NCI-H1299 cells.We designed MTT and clone formation assays to examine the effects of miR-618 and JAK2 on cells. The results showed that inhibiting JAK2 could inhibit cell proliferation, while inhibiting miR-618 could alleviate the inhibitory effect of JAK2. (Fig. 4 A-B). Consistent with the above findings, it was found by wound-healing and transwell experiments that downregulation of miR-618 promoted cell migration and invasion that were inhibited by JAK2 knockdown (Fig. 4 C-D). DISCUSSION MiRNAs, a subtype of small non-coding RNAs, have been extensively supported in the research literature regarding their involvement in tumor progression. Specifically, miR-618 has been identified to exert regulatory influences in various types of tumors.For example, miR-618 inhibits migration and invasion in gastric and prostate cancer cells by targeting FOXP2[ 23 ], and miR-618 targets the cell growth-promoting PI3K/Akt pathway in human thyroid cancer[ 24 ]. In this study, we demonstrated an association between miR-618 and lung cancer. MiR-618 expression was found to be notably decreased in lung cancer tissues, indicating a potential association between this decreased expression and the onset and progression of lung cancer.We further analyzed the specific molecular action of miR-618 in lung cancer. The findings from our experimental investigation indicated a decreased expression of miR-618 in cell lines associated with lung cancer.The findings from the MTT, clone formation, wound-healing, and Transwell assays collectively indicate that miR-618 exerts inhibitory effects on the proliferation, migration, and invasion capabilities of lung cancer cells.The findings align with the documented function of miR-618 in various malignancies.Overall, it can be inferred that miR-618 functions as a tumor suppressor in lung cancer.Dual-luciferase experiments showed that miR-618 targets the JAK2 3'-UTR directly. JAK2, being part of the JAK family of protein tyrosine kinases, fulfills various functional roles in carcinogenesis[ 25 ]. Prior research has demonstrated that JAK2 functions as an oncogene through the modulation of STAT3 phosphorylation in human hepatocellular carcinoma, as well as in lung, colorectal, pancreatic, and gastric cancer[ 26 – 28 ]. The JAK/STAT pathway, a signaling pathway conserved throughout evolution, plays a role in cell growth and metastasis, contributing to its association with the initiation and progression of cancer[ 29 ]. JAK is autophosphorylated and activated and, in turn, phosphorylates STAT3. STAT3 then forms a homodimer and translocates to the nucleus, facilitating the transcription of multiple oncogenes such as c-MYC, Bcl-1, cyclin D1/D2, and Bcl-xL[ 30 ]. The present study found that miR-618 has a tumor-suppressor effect in NSCLC through the regulation of JAK/STAT activation. Although our findings showed that miR-618/JAK2/STAT3 signaling axis is involved in suppressing malignancy in NSCLC, the specific mechanism was analyzed in detail. Currently, a variety of JAK2 inhibitors have entered clinical trials. Therefore, it is necessary to carry out related knockout and interference studies on lung cancer cell lines, which can further elucidate the regulatory role of the JAK/STAT pathway in NSCLC. Secondly, while we have explored the role of miR-618 and the JAK/STAT pathway in lung cancer in a variety of lung cancer cell lines, animal data are lacking. The construction of animal models would allow further verification and investigation of the miR-618 regulatory mechanism, and would also indicate the potential clinical benefit of JAK2 inhibitors in mouse lung cancer models. CONCLUSIONS In our research, we found that miR-618 significantly inhibits the growth, mobility, and invasion of lung cancer cells by targeting the JAK/STAT signaling pathway.These findings provide fresh insights into non-small cell lung cancer (NSCLC) development and may present pioneering strategies for NSCLC treatment. Declarations Funding information 2021 Ningbo Health Youth Backbone Talent Training Project(2021SWSONGG-ZCW),2022 Medical New Talent Program,(YTXXZCW-2023),Ningbo Public Welfare Technology Plan Project(2022S042),Zhejiang Medical and Health Program(2023KY1112) and Wu Jieping Medical Foundation Project(320.6750.2022-22-43). CONSENT FOR PUBLICATION Not applicable. CONFLICT OF INTEREST The authors declare no conflict of interest, financial or otherwise. ACKNOWLEDGEMENTS The authors thank their respective laboratory members and collaborators for critical review of this article. This work is supported by 2021 Ningbo Health Youth Backbone Talent Training Project(2021SWSONGG-ZCW),2022 Medical New Talent Program.(YTXXZCW-2023),Ningbo Public Welfare Technology Plan Project(2022S042), Zhejiang Medical and Health Program (2023KY1112) and Wu Jieping Medical Foundation Project(320.6750.2022-22-43). The authors are thankful to patients who took part in this study. 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Supplementary Files ZiyuanChensupplementaryInformation.docx Cite Share Download PDF Status: Published Journal Publication published 23 Dec, 2024 Read the published version in Journal of Cardiothoracic Surgery → Version 1 posted Editorial decision: Revision requested 23 Sep, 2024 Reviews received at journal 19 Sep, 2024 Reviewers agreed at journal 30 Aug, 2024 Reviewers invited by journal 09 Aug, 2024 Editor assigned by journal 09 Jul, 2024 Submission checks completed at journal 09 Jul, 2024 First submitted to journal 06 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-4695743","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":333783803,"identity":"1186b65e-6952-4672-a3d0-3d73b36ae681","order_by":0,"name":"Ziyuan Chen","email":"","orcid":"","institution":"the First Affiliated Hospital Of Ningbo University","correspondingAuthor":false,"prefix":"","firstName":"Ziyuan","middleName":"","lastName":"Chen","suffix":""},{"id":333783805,"identity":"11d90452-3cdd-4ca4-851c-9792c0204e59","order_by":1,"name":"Wei Chen","email":"","orcid":"","institution":"the First Affiliated Hospital Of Ningbo University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Chen","suffix":""},{"id":333783806,"identity":"8c83a358-4a57-4c2a-8d29-f58d3b68e1c3","order_by":2,"name":"Zhiqi Hong","email":"","orcid":"","institution":"the First Affiliated Hospital Of Ningbo University","correspondingAuthor":false,"prefix":"","firstName":"Zhiqi","middleName":"","lastName":"Hong","suffix":""},{"id":333783807,"identity":"ebea3835-9d4d-4f54-a6af-77655b6db1dc","order_by":3,"name":"Xianqiao Wu","email":"","orcid":"","institution":"the First Affiliated Hospital Of Ningbo University","correspondingAuthor":false,"prefix":"","firstName":"Xianqiao","middleName":"","lastName":"Wu","suffix":""},{"id":333783808,"identity":"2c02baad-d2a2-44d2-a274-321d093369f1","order_by":4,"name":"Tianzheng Fang","email":"","orcid":"","institution":"the First Affiliated Hospital Of Ningbo University","correspondingAuthor":false,"prefix":"","firstName":"Tianzheng","middleName":"","lastName":"Fang","suffix":""},{"id":333783810,"identity":"29f715e3-456a-4314-944e-ca2be6e4d055","order_by":5,"name":"Yufei Sheng","email":"","orcid":"","institution":"Ningbo University","correspondingAuthor":false,"prefix":"","firstName":"Yufei","middleName":"","lastName":"Sheng","suffix":""},{"id":333783812,"identity":"4aceb225-d340-4f66-b94d-a6dd3919b006","order_by":6,"name":"Shuai Fang","email":"","orcid":"","institution":"the First Affiliated Hospital Of Ningbo University","correspondingAuthor":false,"prefix":"","firstName":"Shuai","middleName":"","lastName":"Fang","suffix":""},{"id":333783816,"identity":"622b0b99-da15-4e44-8336-a2b3be02ee3a","order_by":7,"name":"Chengwei Zhou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIiWNgGAWjYDACCQY2EJUA4RnY8PDzN5CmJU1GcsYBkrQwHLYxaEjAqRgMzKXbnz342GaXxy/dfk3iQ8F5HgOGA4wfPubg1mI550C64cy25GLJOWfKJGcY3OYxZ25glpy5DbcWgxsJx6R52w4kbriRk3abB6jFsuEAGzMvXi2JbWAt+0Fa/hic4zE4kEBISzIbxBaJ9GO3GQwOEKHlzjE2yRnnkhNn3Mhh/9ljkMwjOeNgM36/3G5/JvGhzC6xf0b6Y4Mff+zs+fmbD374iEcLEgAGLwQwNhClHgjYHxCrchSMglEwCkYYAAAMwVZMMLMd8wAAAABJRU5ErkJggg==","orcid":"","institution":"the First Affiliated Hospital Of Ningbo University","correspondingAuthor":true,"prefix":"","firstName":"Chengwei","middleName":"","lastName":"Zhou","suffix":""}],"badges":[],"createdAt":"2024-07-06 08:18:40","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4695743/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4695743/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13019-024-03160-5","type":"published","date":"2024-12-23T15:57:36+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":62150923,"identity":"3ab5f9a9-5b42-4a6e-8a69-c2d19b47ee17","added_by":"auto","created_at":"2024-08-09 20:35:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":265149,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of miR-618 in NSCLC. (A) The differential expression of miR-618 was analyzed in 25 pairs of non-small cell lung cancer tissues and corresponding noncancerous tissues.(B) The starbase database predicts the expression level of miR-618 in 512 lung cancer tissue samples and 20 normal samples. (C) qRT-PCR analysis of miR-618 expression in NSCLC cell lines. (D) The miR-618 expression levels were examined following the transfection of miR-NC and an miR-618 inhibitor in NCI-H1299 and LTEP-A-2 cells.(E) MiR-618 expression levels were analyzed post transfection of miR-NC and miR-618 mimics in NCI-H1299 and LTEP-A-2 cells. * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003e P\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"ZiyuanChenfig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4695743/v1/2b3bd0edfce034fe617df6bc.png"},{"id":62150926,"identity":"0dfe3efe-7d56-43d1-a034-d8734e84398d","added_by":"auto","created_at":"2024-08-09 20:35:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1904395,"visible":true,"origin":"","legend":"\u003cp\u003eMiR-618 inhibits lung cancer cell proliferation,migration, and invasion. (A) Proliferation of NCI-H1299 and LTEP-A-2 cells after overexpression or knockdown of miRNA-618, shown by MTT assays and (B) colony-forming assays; (C) Migration of miRNA-618-overexpressing or knockdown NCI-H1299 and LTEP-A-2 cells shown by wound-healing assays; (D) Invasion of miRNA-618-overexpressing or knockdown NCI-H1299 and LTEP-A-2 cells shown by Transwell assays. * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, *** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 .\u003c/p\u003e","description":"","filename":"ZiyuanChenfig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4695743/v1/742641e3ac88ded3408e36ba.png"},{"id":62150922,"identity":"f1749503-0460-4a36-998e-3c7b88e37775","added_by":"auto","created_at":"2024-08-09 20:35:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":334136,"visible":true,"origin":"","legend":"\u003cp\u003eMiR-618 targets the JAK2 3' UTR. (A) Prediction of interaction between miR-618 and the JAK2 3' UTR the TargetScan database; (B) After overexpressing or knocking down miR-618, qPCR was used to detect expression of JAK2 in NCI-H1299 and LTEP-A-2 cells; (C) After transfection of NCI-H1299 and LTEP-A-2 cells with miR-618, the relative luciferase activity of the JAK2 3' UTR wild-type or mutant luciferase vector was measured; (D) After overexpression or knockdown of miR-618, western blotting was used to detect expression of JAK2, STAT3, and p-STAT3 in NCI-H1299 and LTEP-A-2 cells. * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003e P\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"ZiyuanChenfig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4695743/v1/64724d2b384ce7474e0957a6.png"},{"id":62151459,"identity":"86942149-413b-401f-bfb1-3ee3ddcaae37","added_by":"auto","created_at":"2024-08-09 20:43:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2126320,"visible":true,"origin":"","legend":"\u003cp\u003eKnockdown of JAK2 restores cell proliferation, migration, and invasion inhibited by miR-618 in NCI-H1299 and LTEP-A-2 cells. After co-transfection of the miR-618 inhibitor and si-JAK2, (A) MTT measurement of proliferation in cells; (B) Colony formation assay measurement of proliferation in cells; (C) Wound-healing assay measurement of migration in cells; (D) Transwell assay measurement of cell invasion. * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003e P\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"ZiyuanChenfig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4695743/v1/23a138749e6d0f4c9fbf0fa7.png"},{"id":72640653,"identity":"35480937-3440-43ea-9a37-5bfc47d59c98","added_by":"auto","created_at":"2024-12-30 16:08:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4954588,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4695743/v1/d2e6b08d-e611-4208-889e-4ddf0eee87d0.pdf"},{"id":62150925,"identity":"b85bc58e-7a56-4306-a3b8-e04771069504","added_by":"auto","created_at":"2024-08-09 20:35:21","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":12314,"visible":true,"origin":"","legend":"","description":"","filename":"ZiyuanChensupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-4695743/v1/ba22751ab1ef9dc761878e3c.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"MiR-618 suppresses the proliferation, invasion, and migration of non-small lung cancer via the JAK2/STAT3 axis","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eLung carcinoma is one of the leading causes of global cancer mortality.Based on the information from GLOBOCAN 2018 data, approximately 2.09\u0026nbsp;million new cases and 1.76\u0026nbsp;million deaths are reported each year[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Lung cancer ranks among the most prevalent malignant neoplasms in China. The 2015 data published by the National Cancer Center reveals that the 5-year prevalence rate of lung cancer in China during the period of 2006 to 2011 stood at 130.2 cases per 100,000 individuals.Of these cases, the prevalence rate for men was 84.6 (1/100 000), ranking second for malignant tumors, while the rate for women was 45.6 (1/100 000), ranking fourth for malignant tumors[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Histopathologically, lung malignancy can be categorized as small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC), with NSCLC accounting for approximately 80% of all cases based on histological analysis.Early-stage metastatic lung cancer can be treated by tumor resection but advanced or metastatic lung cancer still requires radiotherapy alone or in combination with chemotherapy[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Despite the recent development of innovative treatments, the survival rate of lung cancer patients is still only about 15% with late-stage disease manifestations, histological heterogeneity of the tumor subtypes, and resistance to anti-tumor drugs being the key reasons for poor prognosis[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Understanding the underlying mechanisms of lung cancer tumorigenesis is essential in enhancing patient diagnosis, treatment, and prognosis.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eThe body of research indicates that non-coding RNAs (ncRNAs) are significantly involved in the development and advancement of non-small cell lung cancer (NSCLC).\u003c/span\u003e[\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. MicroRNAs (miRNAs), comprising small ncRNAs with a length of 20\u0026ndash;24 nucleotides, have emerged as key players in this context[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. They were first discovered in 1993 in \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Since then, miRNAs have been shown to have significant importance in the regulatory pathways of both unicellular and multicellular eukaryotes. Small RNA molecules have the ability to selectively identify and attach to matching regions found in the 3'-untranslated portions (UTRs) of target messenger RNAs. This interaction can impede translation or induce degradation of the mRNA, ultimately causing post-transcriptional gene silencing[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In addition, miRNAs can also activate gene expression under certain conditions either directly or indirectly. The miRBase database currently contains over 2500 mature miRNAs derived from 1188 miRNA precursors[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThousands of miRNAs have been shown to be associated with various human diseases, including malignant tumors. In 2002, a study by Calin et al. showed a relationship between miRNA dysregulation and cancer[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In 2004, Takamizawa et al. demonstrated a relationship between miRNA expression and lung cancer[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. miR-519d-3p has been found to inhibit the expression of Bcl-w and hypoxia-inducing factor (HIF)-1α, reducing hypoxia-induced tumorigenesis[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], while miR-487a-3p down-regulation inhibits the progression of NSCLC by targeting Smad7[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eJanus kinase 2 (JAK2) serves as a non-receptor tyrosine kinase signaling molecule responsible for transducing the effects of a range of hormones and cytokines such as interferon, erythropoietin, leptin, and growth hormone.[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. JAK2 plays a critical role in the regulation of cellular volume, safeguarding cells during energy utilization and proliferation, and facilitating the survival of tumor cells. The JAK/STAT pathway is an evolutionarily conserved signaling pathway, involving many basic cell functions, such as cell growth and metastasis, which can lead to the development and progression of cancer[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we identified several miRNAs that are differentially expressed in lung cancer through data mining and the sorting of lung cancer gene chips (Accession No. GSE24709)[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Through tissue sample verification, we found that miR-618 showed low expression in NSCLC tissue cells. Our findings indicate that miR-618 specifically targets JAK2 within tumor cells, resulting in the suppression of migration and invasion in NSCLC cells by modulating the JAK2/STAT3 pathway, thus providing new insights into the pathogenesis of NSCLC.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eHuman tissue specimens\u003c/h2\u003e \u003cp\u003eTissue samples (tumor and normal adjacent tissue) were obtained from lung cancer patients at the First Affiliated Hospital of Ningbo University. The specimens were procured from surgical excision of neoplasms, with the individuals having had no prior exposure to radiotherapy, chemotherapy, or targeted therapy prior to the operative procedure.Clinical information and case data were obtained from the patients at the time of surgery. All the patients provided written informed consent, and the research methodology was sanctioned by the Medical Ethics Committee of the First Affiliated Hospital of Ningbo University.Helsinki Declaration has been followed for involving human subjects in this study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eThe cell lines utilized in the study were procured from the Cell Bank of the Chinese Academy of Sciences(CASCB,China),including one human normal bronchial epithelial cell line BEAS-2B and four human lung adenocarcinoma cell lines namely NCI-H1299,LTEP-A-2,SPC-A-1 and A549.\u003c/p\u003e \u003cp\u003eBEAS-2B cells were cultured in DMEM medium (Hyclone, USA), while all human lung cancer cell lines were cultured in RPMI-1640 medium (Hyclone, USA). The cells mentioned above were all cultured in an environment containing 10% fetal bovine serum, and all cell lines were placed in a 5% CO2, 37\u0026deg;C incubator.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eTotal RNA extraction and RT-qPCR\u003c/h2\u003e \u003cp\u003eTotal RNA was isolated from lung cancer tissues and cell lines employing the TRIzol reagent (Invitrogen, Waltham. MA, USA).mRNA was obtained by reverse-transcription of the total RNA using a reverse-transcription kit (Toyobo, Japan), and cDNA was synthesized from miRNA using a miRNA reverse-transcription kit (GenePharma, China). RT-qPCR was performed using SYBR Green 1 (Takara, Japan). β-actin and U6 were employed as internal reference standards, with the specific primers utilized detailed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e (additional file 1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003esiRNAs, miRNA mimic, miRNA inhibitor, and transfection experiments\u003c/h2\u003e \u003cp\u003eJAK2 siRNA, the miR-618 mimic and inhibitor, and a non-targeting negative control were purchased from GenePharma (Shanghai, China). Lipofectamine 2000 (Invitrogen, Germany) was used for transfection.After a span of forty-eight hours subsequent to transfection, the cellular entities were employed for the purpose of RNA extraction and identification through RT-qPCR. The transfected sequences of the miR-618 mimics and siRNA oligonucleotides are shown in Table S2, Additional file 1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMTT assay\u003c/h2\u003e \u003cp\u003eThe transfected cells were seeded and cultured in 96-well plates for 12 h. Twenty microliters of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) solution were added to each well, after which the plate was incubated at 37˚C for 4 h. After removing the medium and MTT solution, 150\u0026micro;l of DMSO was introduced, and the absorbance was measured using a microplate reader. (Bio-Rad, Hercules, CA, USA) every hour for 0, 24, 48, 72, and 96 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eColony formation assay\u003c/h2\u003e \u003cp\u003eThe transfected cells were evenly allocated into individual wells of 6-well plates, with each well containing a concentration of 500 cells. The plates underwent a 12-day incubation in a cell culture incubator, and then were subjected to three washes using phosphate-buffered saline (PBS). The colonies were then fixed with 4% paraformaldehyde for a duration of 30 minutes, followed by staining with 150\u0026micro;l of 0.1% crystal violet per well for a period of 15 minutes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eTranswell assay\u003c/h2\u003e \u003cp\u003eA Transwell chamber with its upper and lower chambers separated by a polycarbonate microporous membrane (pore size 8 \u0026micro;m) and coated with artificial base glue was used. Cells were harvested and resuspended to a density of 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/mL. One hundred microliters of this cell suspension were added to the upper chamber, and 500 \u0026micro;L of DMEM complete culture medium was added to the lower chamber. The Transwell chamber was then placed in a 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e incubator and cultured for 8 h. The polycarbonate microporous membrane was then removed from the chamber and the base glue and cells on its upper surface were gently wiped with a cotton swab. The cells were fixed in neutral formaldehyde for 20 minutes, followed by staining with hematoxylin and eosin. Finally, the cells were examined under a microscope and the cells in five randomly selected fields of view were counted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eWound-healing experiment\u003c/h2\u003e \u003cp\u003eThe transfected cells were distributed into a 6-well plate at a density of 2x10\u003csup\u003e5\u003c/sup\u003e cells per well, followed by an overnight incubation at 37\u0026deg;C in a 5% CO2 environment. After ensuring that the cells were adherent, 2,4-diamino-4,6-dihydroxypyrimidine (DDP) was added for treatment, after which the cells were cultured until fully confluent. A linear scratch was made on the cell monolayer with a 200 \u0026micro;L sterile pipette tip and photomicrographs were taken immediately. After culturing for a further 48 h, the cells were again photographed and recorded. Image J software was used to measure the migration distance of each group of cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eTotal protein was extracted from the cells using RIPA lysis buffer (Solarbio, Beijing, China) and quantified using a BCA protein assay kit (Beyotime, China). The Western blotting procedure was executed following established protocols.The primary antibodies were anti-JAK2 (Bioss Antibodies, Woburn, MA, USA), anti-STAT3 (Bioss), anti-p-STAT3 (Bioss), and anti-β-actin (Santa Cruz Biotechnology, Dallas, TX, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis of the data was conducted using GraphPad Prism 8 software (GraphPad Software, La Jolla, CA, USA). Data were presented in the form of means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations.The study was conducted with a minimum of three replications, with statistical significance determined at a p-value of less than 0.05. Photoshop CS6 software (Adobe Photoshop CS, Berkeley, CA, USA) was used for the statistical analysis of cell clone numbers, and quantitative analysis of the gray values of the Western blot bands was performed using Image J software.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003eExpression of miR-618 in NSCLC\u003c/p\u003e \u003cp\u003eFirst, we confirmed the presence of miR-618 in NSCLC tissues. We employed qRT-PCR to assess the expression of miR-618 in 25 sets of NSCLC tumors along with their neighboring non-cancerous tissues. The levels of miR-618 were notably reduced in the cancerous tissues in contrast to the neighboring non-cancerous tissues. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The starbase database[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] showed that miR-618 was significantly underexpressed in 512 lung cancer tissues compared with 20 controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).Subsequent studies showed that the expression of miR-618 in NSCLC cells (LTEP-A-2, SPC-A-1, NCI-H1299, and A549) was significantly reduced compared with BEAS-2B cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).NSCLC cells were assessed to investigate the impact of miR-618 on their proliferation, migration, and invasion through gain and loss of function analyses. First, we designed and synthesized a miR-618 inhibitor, and observed that it could downregulate the expression of miR-618 by 48\u0026ndash;63%. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Furthermore, we employed miR-618 mimics for inducing ectopic expression of miR-618, observing a substantial up-regulation of miR-618 by 247-879-fold. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMiR-618 inhibits lung cancer cell proliferation,migration, and invasion.\u003c/p\u003e \u003cp\u003eThe results obtained from MTT and colony formation assays demonstrated that the downregulation of miR-618 significantly enhanced the growth and proliferation of LTEP-A-2 and NCI-H1299 cells.Conversely, upregulating miR-618 had the opposite impact on cell proliferation, suggesting that miR-618 suppressed the proliferation of NSCLC cells. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). Cellular scratching and invasion assays elucidated that suppression of miR-618 facilitated the migratory and invasive capacities of LTEP-A-2 and NCI-H1299 cells, whereas upregulation hindered cell migration and invasion. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMiR-618 targets the JAK2 3' UTR.\u003c/p\u003e \u003cp\u003eThe experimental investigations concerning cellular functions have indicated that miR-618 possesses the capability to impede the proliferation, invasion, and migration of NSCLC cells.We suspected that miR-618 might influence the progression of NSCLC via a distinct molecular mechanism. Research indicates that miRNAs have the ability to selectively attach to and control the activity of target genes in order to influence the advancement of NSCLC.TargetScan software predicted that miR-618 binds to JAK2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). To verify this prediction, we knocked down or overexpressed miR-618 in LTEP-A-2 and NCI-H1299 cells, confirming that JAK2 was regulated by miR-618 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). For further exploration of this interaction, we conducted a dual-luciferase reporter assay. The findings indicated that after co-transfection of miR-618 mimics with JAK2 wild-type, the luciferase activity was significantly reduced, while co-transformation with the JAK2 mutant sequence had no effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Western blotting showed that JAK2 knockdown significantly reduced the expression of both JAK2 and p-STAT3 protein, while the simultaneous knockdown of JAK2 and mir-618 restored the JAK2 and p-STAT3 protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). These findings suggested that mir-618 can interact with JAK2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eKnockdown of JAK2 restores cell proliferation, migration, and invasion inhibited by miR-618 in NCI-H1299 and LTEP-A-2 cells.\u003c/p\u003e \u003cp\u003eTo confirm if miR-618 enhances lung cancer advancement through JAK2 regulation, we co-transfected the miR-618 inhibitor and JAK2 siRNA into LTEP-A-2 and NCI-H1299 cells.We designed MTT and clone formation assays to examine the effects of miR-618 and JAK2 on cells. The results showed that inhibiting JAK2 could inhibit cell proliferation, while inhibiting miR-618 could alleviate the inhibitory effect of JAK2. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B). Consistent with the above findings, it was found by wound-healing and transwell experiments that downregulation of miR-618 promoted cell migration and invasion that were inhibited by JAK2 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eMiRNAs, a subtype of small non-coding RNAs, have been extensively supported in the research literature regarding their involvement in tumor progression. Specifically, miR-618 has been identified to exert regulatory influences in various types of tumors.For example, miR-618 inhibits migration and invasion in gastric and prostate cancer cells by targeting FOXP2[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], and miR-618 targets the cell growth-promoting PI3K/Akt pathway in human thyroid cancer[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In this study, we demonstrated an association between miR-618 and lung cancer. MiR-618 expression was found to be notably decreased in lung cancer tissues, indicating a potential association between this decreased expression and the onset and progression of lung cancer.We further analyzed the specific molecular action of miR-618 in lung cancer. The findings from our experimental investigation indicated a decreased expression of miR-618 in cell lines associated with lung cancer.The findings from the MTT, clone formation, wound-healing, and Transwell assays collectively indicate that miR-618 exerts inhibitory effects on the proliferation, migration, and invasion capabilities of lung cancer cells.The findings align with the documented function of miR-618 in various malignancies.Overall, it can be inferred that miR-618 functions as a tumor suppressor in lung cancer.Dual-luciferase experiments showed that miR-618 targets the JAK2 3'-UTR directly. JAK2, being part of the JAK family of protein tyrosine kinases, fulfills various functional roles in carcinogenesis[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Prior research has demonstrated that JAK2 functions as an oncogene through the modulation of STAT3 phosphorylation in human hepatocellular carcinoma, as well as in lung, colorectal, pancreatic, and gastric cancer[\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The JAK/STAT pathway, a signaling pathway conserved throughout evolution, plays a role in cell growth and metastasis, contributing to its association with the initiation and progression of cancer[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. JAK is autophosphorylated and activated and, in turn, phosphorylates STAT3. STAT3 then forms a homodimer and translocates to the nucleus, facilitating the transcription of multiple oncogenes such as c-MYC, Bcl-1, cyclin D1/D2, and Bcl-xL[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The present study found that miR-618 has a tumor-suppressor effect in NSCLC through the regulation of JAK/STAT activation.\u003c/p\u003e \u003cp\u003eAlthough our findings showed that miR-618/JAK2/STAT3 signaling axis is involved in suppressing malignancy in NSCLC, the specific mechanism was analyzed in detail. Currently, a variety of JAK2 inhibitors have entered clinical trials. Therefore, it is necessary to carry out related knockout and interference studies on lung cancer cell lines, which can further elucidate the regulatory role of the JAK/STAT pathway in NSCLC. Secondly, while we have explored the role of miR-618 and the JAK/STAT pathway in lung cancer in a variety of lung cancer cell lines, animal data are lacking. The construction of animal models would allow further verification and investigation of the miR-618 regulatory mechanism, and would also indicate the potential clinical benefit of JAK2 inhibitors in mouse lung cancer models.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eIn our research, we found that miR-618 significantly inhibits the growth, mobility, and invasion of lung cancer cells by targeting the JAK/STAT signaling pathway.These findings provide fresh insights into non-small cell lung cancer (NSCLC) development and may present pioneering strategies for NSCLC treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e2021 Ningbo Health Youth Backbone Talent Training Project(2021SWSONGG-ZCW),2022\u0026nbsp;Medical\u0026nbsp;New\u0026nbsp;Talent\u0026nbsp;Program,(YTXXZCW-2023),Ningbo Public Welfare Technology Plan Project(2022S042),Zhejiang Medical and Health Program(2023KY1112) and Wu Jieping Medical Foundation Project(320.6750.2022-22-43).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONSENT FOR PUBLICATION\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICT OF INTEREST\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest, financial or otherwise.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank their respective laboratory members and collaborators for critical review of this article. This work is supported by 2021 Ningbo Health Youth Backbone Talent Training \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Project(2021SWSONGG-ZCW),2022 Medical New Talent Program.(YTXXZCW-2023),Ningbo Public Welfare Technology Plan Project(2022S042), Zhejiang Medical and Health Program (2023KY1112) and Wu Jieping Medical Foundation Project(320.6750.2022-22-43). The authors are thankful to patients who took part in this study.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eArbyn M, Weiderpass E, Bruni L, de Sanjos\u0026eacute; S, Saraiya M, Ferlay J, Bray F. Estimates of incidence and mortality of cervical cancer in 2018: a worldwide analysis. Lancet Global health. 2020;8(2):e191\u0026ndash;203.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen W, Zheng R, Baade PD, Zhang S, Zeng H, Bray F, Jemal A, Yu XQ, He J. Cancer statistics in China, 2015. 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J Virol 2018, 92(14).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-cardiothoracic-surgery","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jcts","sideBox":"Learn more about [Journal of Cardiothoracic Surgery](http://cardiothoracicsurgery.biomedcentral.com)","snPcode":"13019","submissionUrl":"https://submission.nature.com/new-submission/13019/3","title":"Journal of Cardiothoracic Surgery","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Non-small cell lung cancer, miRNA, JAK2, miR-618, STAT3","lastPublishedDoi":"10.21203/rs.3.rs-4695743/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4695743/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eThe regulatory role of the miR-618/JAK2/STAT3 axis in non-small cell lung cancer (NSCLC) cells was investigated with the objective of identifying a target for the precise treatment of patients with NSCLC.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eDifferentially expressed genes were identified in the GEO database and were analyzed bioinformatically. The tissue and cell levels of miR-618 were assessed using qRT-PCR, while the protein levels of JAK2 and STAT3 were determined through western blotting analysis.The association between miR-618 and JAK2 was scrutinized through bioinformatics analysis and dual-luciferase experiments. To evaluate cell proliferation, migration, and invasion, MTT, wound-healing, and Transwell assays were employed.\u003c/p\u003e\u003ch2\u003eRESULTS\u003c/h2\u003e \u003cp\u003eMiR-618 expression decreased in NSCLC, leading to the inhibition of growth, invasiveness, and migratory properties of non-small cell lung cancer. This is achieved by MiR-618 modulating the JAK2/STAT3 signaling pathway. In addition, miR-618 inhibited cell proliferation, migration, and invasion by targeting JAK2/STAT3.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur study demonstrates that a novel miR-618/JAK2/STAT3 signaling axis is involved in suppressing malignancy in NSCLC and provides a promising target for NSCLC therapy.\u003c/p\u003e","manuscriptTitle":"MiR-618 suppresses the proliferation, invasion, and migration of non-small lung cancer via the JAK2/STAT3 axis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-09 20:35:16","doi":"10.21203/rs.3.rs-4695743/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-09-23T09:46:47+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-19T05:14:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"56877339995143031367395734227711219242","date":"2024-08-30T05:01:46+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-09T10:21:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-09T07:33:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-09T07:33:02+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Cardiothoracic Surgery","date":"2024-07-06T08:17:24+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-cardiothoracic-surgery","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jcts","sideBox":"Learn more about [Journal of Cardiothoracic Surgery](http://cardiothoracicsurgery.biomedcentral.com)","snPcode":"13019","submissionUrl":"https://submission.nature.com/new-submission/13019/3","title":"Journal of Cardiothoracic Surgery","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"47909729-963c-437f-bb0e-7f57632151b4","owner":[],"postedDate":"August 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-30T16:02:02+00:00","versionOfRecord":{"articleIdentity":"rs-4695743","link":"https://doi.org/10.1186/s13019-024-03160-5","journal":{"identity":"journal-of-cardiothoracic-surgery","isVorOnly":false,"title":"Journal of Cardiothoracic Surgery"},"publishedOn":"2024-12-23 15:57:36","publishedOnDateReadable":"December 23rd, 2024"},"versionCreatedAt":"2024-08-09 20:35:16","video":"","vorDoi":"10.1186/s13019-024-03160-5","vorDoiUrl":"https://doi.org/10.1186/s13019-024-03160-5","workflowStages":[]},"version":"v1","identity":"rs-4695743","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4695743","identity":"rs-4695743","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}