| Lung cancer(lung cancer,LC)is the top malignancy with the highest incidence and mortality rate,and non-small-cell lung cancer(NSCLC)accounts for about 85%of the total lung cancer cases.As the main tissue type of NSCLC,lung adenocarcinoma is prone to mutations,common mutations are ALK,KRAS,HER2RET,and EGFR,mutated genes will vary by region,like Asian regions such as China,EGFR mutations in up to 50%of the patients with lung caradoma.Although the significant progress in lung adenocarcinoma screening and personalized treatment,including surgery,chemotherapy,radiotherapy,targeted therapy and immunotherapy comprehensive treatment has improved the condition of lung cancer patients,due to the different types of gene mutations,individual heterogeneity and resistance to chemotherapy drugs,there is still poor prognosis in lung adenocarcinoma,individual differences in treatment effect,poor antibody response,low survival rate.Therefore,it is crucial to explore the pathogenesis of lung adenocarcinoma by newly regulating cell death and to study bio-diagnostic markers and therapeutic targets that regulate lung adenocarcinoma specificity.Tumor cells often escape normal death,and current chemotherapeutic agents mainly induce programmed tumor death.Due to the tolerance and unresponse of tumor cells to apoptotic signals,the effectiveness of anti-tumor drugs in inducing apoptosis in tumors is very limited.This is one of the main reasons for the resistance to some antitumor drugs.To promote growth,cancer cells show a higher iron demand than normal cells,and this iron dependence makes cancer cells more sensitive to the environment-dependent death pathway of ferroptosis.Ferroptosis a new form of irondependent programmed cell death distinct from apoptosis,characterized by massive iron accumulation and glutathione peroxidase 4(GPX4)inactivation and reactive oxygen species(ROS)and lipid peroxides accumulation.At present,the mechanism of ferroptosis as follows:1,abnormal iron metabolism,iron,in normal state,is involved in oxygen transportation and DNA biosynthesis.When other reasons such as transferrin break the iron metabolism pathway,leading to intracellular accumulation of iron ions,resulting in a large production of hydroxyl-radicals,and then intracellular accumulation of reactive oxygen species.2.The redox system is unbalanced,and the normal oxidative stress pathway is involved in the synthesis of intracellular glutathione(GSH)after the cysteine intake.GSH reduces reactive oxygen species and reactive nitrogen under the action of glutathione peroxidase 4(GPX4),plays an antioxidant role,and maintains redox homeostasis in vivo.If glutathione synthesis is abnormal,glutathione peroxidase 4 is inactivated or reduced,its antioxidant capacity is greatly weakened,and the reactive oxygen species continues to show explosive accumulation,leading to the cell death.Data from several studies have shown that ferroptosis is a new way of regulating the cell death participates in multiple diseases,such as neurodegenerative diseases,cerebrovascular diseases,and tumors,etc.This project conducted bioinformatics-analysis and screening through existing databases combining clinical survival and prognosis,and found the genes involved in the ferroptosispathway and involved in the development of NSCLC,7 members of the solute carrier family,11-SLC7A11.SLC7A11 is the cystine/glutamate hair transporter xc-The light-chain subunit of the system,which can exchange the extracellular cysteine to the intracellular glutamate,promotes glutathione synthesis.S LC7A11 is the executive protein that maintains cellular redox balance and prevents lipid peroxidation-induced induction.Its mediated cysteine uptake plays a key role in inhibiting the oxidative response and maintaining cell viability under oxidative stress conditions.At present,it has been found for SLC7A11 to show high expression in multiple malignancies,and is closely related to the malignant process of multiple tumors,such as breast cancer and liver cancer.High SLC7A11 expression is also costly for cancer cells.First,acting as a reverse transporter,the SLC7A11-mediated cysteine uptake binds to the glutamate export.Alternatively,due to the oxidative extracellular environment,the extracellular cysteine is much more stable than the extracellular cysteine,resulting in much higher concentrations of the extracellular cysteine than the extracellular cysteine concentration.Therefore,in order to obtain the cysteine,the cancer cells mainly rely on the uptake of the extracellular cysteine to the intracellular cell,and then convert it into a cysteine,and this process consumes the glunicotinamide adenine dinucleotide phosphate(NADPH)produced by the conose-pentose pathway.Thus,cancer cells with high SLC7A11 expression are more sensitive to glutamine starvation and glucose starvation.Based on the above protein function characteristics of SLC7A11,the main known anti-tumor strategies for using SLC7A11 are:1,blocking the transcriptional activity of SLC7A11;2,targeting glucose with high SLC7A11 expression,3,and targeting glutaminase with high levels of SLC7A11 expression.Multiple regulatory pathways for SLC7A11 expression and protein activity,and how they fine control the development of normal cells to cancer cells,remain unknown,and have aroused wide attention from the scientific community.microRNAs(miRNAs)is a recent class of non-coding-small-molecule RNA with 18-24 nucleotides in length.It is mainly involved in the regulation of life activities like ontogeny,apoptosis,proliferation and differentiation by either full or incomplete pairing with the target gene 3 ’ UTR,the degradation of the target gene mRNA,or inhibiting its translation.Multiple studies have shown that miRNA can act as a tumor suppressor gene or as an oncogene,which is closely linked to tumor formation.miRNA can be used as diagnostic biomarker molecules and prognostic biomarker,and is also related to the development,survival,relapse probability,drug resistance mechanism,and the corresponding rate of chemotherapy drugs in non-small cell lung cancer.Data from previous studies showed that miRNA(e.g.,miR-34/183/200/148a)are involved in lung cancer cell metastasis,where miR-212-3p,miR-27a-3p,and miR-132-5p is significantly upregulated in lung adenocarcinoma,while miRNA can serve as a cancerspecific characterization marker as a judgment criterion for cancer classification.The miR-205-5p is specifically expressed in lung SCC and can be used to distinguish between SCLC and NSCLC.Overexpression of miR let-7 in the lung cancer cell line A549 caused the stopped growth of the cells in the G0 phase and inhibited the expression of proto-oncogenes such as MYC.The miR-34s is involved in the regulation of p53 expression,which can induce apoptosis in lung cancer cells.Tumor suppressor genes such as miR-93-5p,miR-378a-3p and miR-98-5p promote tumor suppressor genes such as FUS1/TUSC2,associated with reduced or even no FUS1 expression in the vast majority of clinical NSCLC and SCLC cancer patients.Deep understanding and rational use of miRNA regulation of driver genes,exploring the intrinsic mechanisms of individual tumor differences and not response to patients is possible to better treat lung adenocarcinoma.With the intensive investigation of ferroptosisand non-coding RNA,mi R NA and long non-coding RNA(lncRNA)is increasingly recognized as key mediators of regulating ferroptosis.There are a few studies confirming the function of miRNA in ferroptosis in cancer cells.For example,in radiation-resistant cells,miR-7-5p suppresses ferroptosis by downregulating mitochondrial ferroptosis and thereby reducing iron levels.Both miR-9 and miR-137 enhance ferroptosis by reducing intracellular GSH levels,miR-9 inhibits GSH synthesis,and miR-137 inhibits solute carrier family 1 member 5(SLC1A5),a component of systemXc-31.Moreover,miR-6852,regulated by lncRNA Linc00336,inhibits the growth of lung cancer cells by promoting ferroptosis.By discovering the important role of non-coding RNA in tumor-regulating gene expression to affect the biological process of cancer cells and the mechanistic pathways of novel programmed cell death mode,therefore,we can combine non-coding RNA and ferroptosisto jointly explore the pathogenesis of lung adenocarcinoma,in turn to provide new direction and hope for the treatment and drug resistance of lung adenocarcinoma.Although the miRNA involved in the development or regulation of ferroptosishas been reported,the current study on membrane protein SLC7A11 promotes the tumor growth by inhibiting iron death-dependent and independent death function,the small molecules regulated in vivo in lung adenocarcinomais not clear,given that no miRNA regulation of ferroptosisin lung cancer.The study will be divided into three parts:excavate the correlation between SLC7A11 and non-small cell lung cancer through the database analysis,and verify the analysis with clinical samples,cell samples,confirm the biological phenotype on different lung adenocarcinoma cell lines,and part 3,after bioinformatics analysis,find the potential small molecule miR-27a-3p that regulates SLC7A11.Meanwhile,the interaction of miR-27a-3p with SLC7A11 and the biological effects after the interaction were verified both in vitro and abroad.Purpose:This project aims to study the involvement of the miR-27a-3p/SLC7A11 signaling axis in theferroptosis pathway and the mechanism of action in lung adenocarcinoma,and provides some theoretical basis for the pathogenesis of lung cancer,diagnosis and treatment and prognostic analysis.Method:(1)Cell-level validation of the presence of ferroptosis in lung adenocarcinoma cells After treatment of different lung adenocarcinoma cell lines with ferroptosis inducer and inhibitors,cell mortality and cell viability were measured by flow cytometry,oxidative stress,lipid peroxidation and iron ion levels were detected by kits,and the protein expression of SLC7A11 and GPX4 in theferroptosis pathway was determined by western blot.Lung adenocarcinoma cells were associated withferroptosis signals,and cell membrane morphology and mitochondrialferroptosis morphology were examined by electron microscopy.(2)The functional role of SLC7A11 in the development offerroptosis pathway and lung adenocarcinoma was screened and validated by database combination with clinical specimens1.Screening and analysis of the expression of iron-death key genes in non-small-cell lung cancer and analysis of prognosis and survival correlation in databases and tissue samples to determine the expression level of iron-death key protein SLC7A11 in lung adenocarcinoma and the correlation between the survival of patients with non-small-cell lung cancer;2.In specimens of paracancerous and lung adenocarcinoma with complete follow-up,SLC7A11 protein expression in lung adenocarcinoma tissues and Q-PCR quantification of mRNA levels of SLC7A11,GPX4 and GSS were measured by immunofluorescence and immunohistochemistry techniques with complete follow-up data;3.The collected patients were divided into four stages according to their degree of cancer to detect the relationship between SLC7A11 expression level and the malignant process of non-small cell patients,focusing on clarifying their expression differences in different lung adenocarcinoma stages,and confirming it as a new molecular target for the diagnosis and treatment of lung adenocarcinoma.(3)The mechanisms by which SLC7A11 involvement in theferroptosis pathway affects the development of lung cancer were examined at the cell and animal levels1.SLC7A11 and RNA levels and RNA levels in different lung adenocarcinoma cell lines and normal lung epithelial cells by Q-PCR,WB and immunohistochemistry;2.After the knockdown of SLC7A11 lung adenocarcinoma cell lines by lentivirus,SLC7A11 expression was detected,respectively,and tumor cell viability,oxidative stress,lipid peroxidations,and iron ions were explored for their important role in the occurrence offerroptosis in lung adenocarcinoma;3 SLC7A11 knockdown lung adenocarcinoma transplant mice and in vivo experiments demonstrated the effect of SLC7A11 on the development of lung adenocarcinoma.(4)It confirms that miR-27a-3p is a potential regulatory molecule of SLC7A11 mediating theferroptosis pathway1.Interaction miRNA targeting SLC7A11 was first predicted using TargetScan,miRanda,microT,PITA,miRmap and PicTar.2.Ferroptosis occurred in lung adenocarcinoma cells treated with the inducer erastian,and miR-27a-3p expression at different time points was determined by Q-PCR;3.The miR-27-a-3p overexpression as well as the knockdown cell lines were constructed by using the lentiviral infection,and the SLC7A11 expression was determined by Q-PCR and Western-blot;4.The binding site of miR-27a-3p and SLC7A11 was determined using Q-PCR and dual luciferase-reporter detection system,which confirmed the molecular mechanism of miR-27a-3p affecting SLC7A11;5.Effect of up-regulation/down-regulation of miR-27a-3p by s h RNA on biological properties related toferroptosis in lung adenocarcinoma cell viability,oxidative stress,lipid peroxidation,and iron ion levels;6.The miR-27a-3p overexpressing lung adenocarcinoma graft mice were constructed and tested for the effect of miR-27a-3p on lung adenocarcinoma in vivo.Results:1.Stimstimulation of ferroptosis,A549 and Calu-3,confirmed the presence offerroptosis in cell viability,cell mortality,cell morphology,membrane valgus properties,oxidative stress,lipid peroxidation,intracellular iron ion levels and significant protein expression of SLC7A11 and GPX4.2.By analysis of raw letter data on non-small cell lung cancer patients,the key membrane protein offerroptosis,SLC7A11,was positively associated with non-small cell lung cancer.The clinical tissue of patients with major tissue subtypes of lung adenocarcinoma from the non-small cell lung cancer collected confirmed that the upregulation of SLC7A11 in tissues with lung adenocarcinoma and is associated with patient survival and clinicopathological characteristics.3.SLC7A11 expression was significantly upregulated in lung adenocarcinoma cell lines as compared to normal alveolar epithelial cells.After interfering with SLC7A11 in two lung adenocarcinoma cell lines A549 and Calu-3,the increased cell viability and the degree of lipid peroxidation and intracellular iron ion water were decreased on average,confirming that SLC7A11 is a characteristic protein in the development offerroptosis in lung adenocarcinoma cells.4.The miRNA targeting SLC7A11 was predicted using different databases such as TargetScan,focusing on the novel microRNA-miR-27a-3p in lung adenocarcinoma,confirming the involvement of the upregulated miR-27a-3p upregulated in lung adenocarcinoma cells and tissues.Luciferase-binding site mutation was used to demonstrate that miR-27a-3p acts as a regulator for directly targeting SLC7A11.The cell viability of miR-27a-3p overexpressing cells was found on knockdown and miR-27a-3p,significantly decreased in miR-27a-3p,and increased ROS and Fe2+after knockdown of miR-27a-3p,confirming that miR-27a-3p is involved in lung adenocarcinoma cellferroptosis by regulating SLC7A11 expression.5.SLC7A11 interference and miR-27a-3p overexpressed lung adenocarcinoma cell lines,subcutaneous inoculation with nude mice,and a mouse model of lung adenocarcinoma transplanted tumor volume and body weight showed no significant difference compared with the normal group.Conclusion:1.Ferroptosis exists in lung adenocarcinoma cells;2.SLC7A11 is involved in the regulation of intracellular metabolic balance in lung adenocarcinoma and potentially regulatesferroptosis pathways;3.As miR-27a-3p directly targets SLC7A11,negative feedback regulates SLC7A11 expression and involved in the malignant progression of lung adenocarcinoma cells;4.The miR-27a-3p/SLC7A11 is a potential target for the treatment of lung adenocarcinoma. |
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