Role And Mechanism Of TMOD3 In Regulating Proliferation,migration,invasion And Iron Death Of Non-small Cell Lung Cancer Ferroptosis Of Non-small Cell Lung Cancer Cells By

Objective:Both the incidence and mortality of lung cancer are among the highest in the world.Although the diagnosis and treatment conditions and treatment level of lung cancer in modern society have made great progress,most patients are still can not be controlled timely and effectively,which has brought a heavy burden to global medical care.According to the histological type,lung cancer can be divided into two categories:small cell lung cancer(SCLC)and non-small cell lung cancer(NSCLC),in which non-small cell lung cancer(NSCLC)accounts for 80%-85% of patients.In recent years,great progress has been made in the research of surgery,chemotherapy,radiotherapy and targeted therapy for NSCLC.However,there are still many serious problems to be solved,mainly including differences in drug sensitivity caused by tumor cell heterogeneity,inevitable drug resistance and metastasis in patients,serious drug side effects and limited therapeutic targets,which seriously affect the clinical therapeutic effect of NSCLC.Therefore,it is of great theoretical significance and clinical value to thoroughly reveal the pathogenesis of NSCLC,identify new targets of NSCLC and explore new therapeutic strategies.Tropomyosin regulatory protein 3(TMOD3)belongs to the tip cap protein family and participates in the regulation of mechanical signals of tip actin filaments.Actin filaments are an important part of the cytoskeleton in all types of cells.F-actin has two structures:barb and tip.Polymerization and depolymerization occur at both ends.However,the faster polymerization speed is at the barb end.Actin filaments continue to polymerize at the barb end and depolymerize from the tip end.TMOD3 can block the elongation and depolymerization of actin filaments at the tip,and promote various physiological processes such as cell morphology determination,migration and muscle contraction by regulating the dynamic changes of actin.At the same time,TMOD3 can also seal actin monomers or polymerize actin into nuclei by binding to actin.TMOD3 related diseases include amyotrophic lateral sclerosis type 5 and aromatase excess syndrome.Studies have shown that TMOD3 plays a key role in cancer cell migration and mitosis.It has also been reported that the activation of TMOD3 is related to the occurrence and development of liver cancer and esophageal cancer.TMOD3 can control the balance between protrusion structure and contraction structure by stabilizing actin myosin filaments.However,in general,there are few studies on TMOD3 in tumors,and its potential regulatory functions and mechanisms need to be further studied and clarified.Ferroptosis is a kind of programmed cell death caused by lipid peroxidation induced by iron ions and reactive oxygen species.Its essence is that the inducer reduces the activity of glutathione peroxidase(Glutathione peroxidase,GPXs),which leads to the accumulation of membrane lipid ROS and cell oxidative death.Studies have shown that activation of the AKT signaling pathway inhibits iron death in tumor cells through SREBP-mediated adipogenesis.It was found that GPX4,x CT and FACL4,three key genes regulated by iron death signal,play key roles in the process of iron death in tumor cells.Induction of iron death can inhibit the growth of tumor cells,significantly improve the therapeutic effect of tumor,making it an important target for cancer treatment,and may become an effective cancer treatment strategy in the future.Adenylate succinate lyase(ADSL)is an essential enzyme for the de novo synthesis of purine.It catalyzes the synthesis of purine in two steps:(1)the conversion of succinylamimidazole carbamide riboside to Amimidazole carbamide riboside;(2)adenylate succinate is converted to adenosine monophosphate;The human ADSL gene is located on chromosome 22q13.1-q13.2,and the original study found that defects in human ADSL have been shown to predisposes to neurodevelopmental syndroms characterized by autism.ADSL deficiency is a recessive genetic disorder that can cause a variety of but most common severe mental retardation,often accompanied by epilepsy and/or autism.This study reported that high-throughput urine screening can be used as an early diagnosis of ADSL deficiency patients,and then find that the mutant ADSL enzyme function is severely affected,resulting in abnormal urine metabolism in patients.Recent studies have found that ADSL is involved in the development of a variety of tumors.It was found that the topological characteristics of anticancer drug targets(ADTs)in biological pathways showed that ADSL could be used as a target for anticancer drug development.ADSL is a carcinogen driver of triple negative breast cancer(TNBC),promoting the malignant progression of breast cancer by driving the expression of oncogene C-MYC.ADSL plays a carcinogenic role in the development and progression of prostate cancer through the cell cycle pathway.Another study reported that ADSL promotes the malignant progression of colorectal cancer by affecting mitochondrial function.However,up to now,no studies on ADSL and NSCLC have been reported.Previous studies have found that iron death plays a critical role in the progression and treatment of non-small cell lung cancer.Therefore,in the malignant progression of non-small cell lung cancer,exploring the regulatory mechanism of iron death is of great significance to elucidate the molecular regulatory network of the occurrence and development of non-small cell lung cancer and to explore the therapeutic targets of NSCLC.This study found that TMOD3 is highly expressed in NSCLC,and established the evidence that TMOD3 may be used as a new oncogene.At the same time,it was found that TMOD3 could regulate the iron death level of non-small cell lung cancer cells,and the targeted regulation relationship between TMOD3 and ADSL was established by protein interaction technology and protein mass spectrometry sequencing technology;In conclusion,the team made scientific hypotheses: TMOD3 regulates the iron death process of NSCLC cells by inhibiting the expression of ADSL and mediating the activation of AKT signaling kinase.The association between TMOD3 and iron death in NSCLC was established,and the evidence chain between TMOD3 and ADSL was found in the regulation of iron death in NSCLC.Methods : 1.In this study,95 patients with non-small cell lung cancer(NSCLC)confirmed by pathology after surgical resection were selected from the First Affiliated Hospital of China Medical University from 2014 to 2016.None of the 95 patients who participated in this pathological correlation analysis had received preoperative radiotherapy or chemotherapy.Immunohistochemical analysis showed high expression of TMOD3 in non-small cell lung cancer with clinicopathological correlation.2.GEPIA database was used to analyze the relationship between TMOD3 gene and prognosis of NSCLC patients online.3.NSCLC cell lines A549,SK-MES-1,H1299,H460 and HBE2(normal lung epithelial cell control)were selected(Caco2 cell was used as irrelevant control),and Western Blot was used to screen out cell lines A549 and SK-MES-1 with high expression of TMOD3.4.si RNA-TMOD3 knockdown: The si RNA sequence targeting TMOD3 was synthesized and transfected into A549 and SK-MES-1 cells.The cells were collected to extract total protein,and the optimal knockdown sequence was confirmed by Western Blot.5.Cell proliferation activity test: 96 Well cells were laid with A549 cells(si RNA-NC /Control,si RNA-TMOD3)and SK-MES-1 cells(si RNA-NC/Control,si RNA-TMOD3),4 compound wells were laid for each group,the reagents were prepared according to the instructions of CCK8 reagent every 24 h,and added into the cell wells.The absorbance was measured by OD450,and CCK8 reagent was added to the control group for 4h after cell placement.The absorbance at each time point was statistically analyzed to draw the proliferation curve.6.Cell cloning: A549 cells(si RNA-NC /Control,si RNA-TMOD3)and SK-MES-1 cells(si RNA-NC /Control,si RNA-TMOD3)were placed in the 6 well plate.Cell proliferation continued for 7-14 days.Cell proliferation was compared.7.Cell scratch test: A549 cells(si RNA-NC/Control,si RNA-TMOD3)and SK-MES-1 cells(si RNA-NC /Control,si RNA-TMOD3)were placed in 6 well plates,and the cells proliferated to 50%-60% density on the next day.Sterile yellow head was used.The cells at the bottom of the orifice plate were scraped vertically from top to bottom along the middle of the orifice plate,and the medium was removed.The cells were cleaned once with PBS,and the fresh complete medium was added.The cells were cultured for 96 h,and the photos of the scratches were taken under the microscope every 24 h.8.Cell invasion experiment: A549 cells(si RNA-NC /Control,si RNA-TMOD3)and SK-MES-1 cells(si RNA-NC /Control,si RNA-TMOD3)were laid in 8 um,24 well cells.Transwell cells were collected for 48 h after cell invasion and stained.Microscopic counting of invasive cells and statistical analysis.9.Western Blot detection of proliferation and cycle signaling proteins: A549cells(si RNA-NC /Control,si RNA-TMOD3)and SK-MES-1 cells(si RNA-NC /Control,si RNA-TMOD3)were placed in the 6 well plate.The cell density reached over90%.SDS-PAGE was used to detect the expression of related indexes.10.Western Blot detection of tumor EMT protein: A549 cells(si RNA-NC /Control,si RNA-TMOD3)and SK-MES-1 cells(si RNA-NC /Control,si RNA-TMOD3)were placed in the 6 well plate.The cell density reached over 90%.SDS-PAGE was used to detect the expression of EMT related indexes.11.Fe ion detection: A549 cells(si RNA-NC /Control,si RNA-TMOD3)and SK-MES-1 cells(si RNA-NC /Control,si RNA-TMOD3)were placed in 6 well plates,and Ferroptosis activator Erastin was added to stimulate the cells for a period of time.When the cell density reached over 90%,the cell supernatant was collected and operated according to Fe ion detection kit to detect the content difference of Fe ions between different groups.12.ROS-lipid reactive oxygen enzyme detection: A549 cells(si RNA-NC,si RNA-TMOD3-1,si RNA-TMOD3-2)and SK-MES-1 cells(si RNA-NC,si RNA-TMOD3-1,si RNA-TMOD3-2)were placed in the6 well plate.The cell density increased to more than 90%.Cell ROS was labeled and the difference of intracellular lipid reactive oxygen species was detected by flow cytometry.13.Construction of TMOD3-sh RNA stable strains: The TMOD3-sh RNA plasmid was synthesized and packaged with lentivirus,and A549 and SK-MES-1 cells were infected.The stable strains were screened on 2 ug/ml purinomycin complete medium,and TMOD3 knockdown expression was detected by Western Blot.14.Intracellular glutathione detection: A549 cells(sh RNA-NC/Control,sh RNA-TMOD3)and SK-MES-1 cells(sh RNA-NC /Control,sh RNA-TMOD3)were placed in 6 well plates,and the cell density reached over 90%.GSH reagent was added to lysis cells,and the difference of GSH content in cells between different groups was detected according to the kit operation.15.Western Blot detection of iron death signal AKT and iron death transcription factor expression: A549 cells(sh RNA-NC/Control,sh RNA-TMOD3)and SK-MES-1 cells(sh RNA-NC/Control,sh RNA-TMOD3)were placed in 6 well plates,and the cell density was over 90%.M2 type protein lysate was added to collect protein and SDS-PAGE running glue was added to detect the expression of related iron death regulation signal related indicators.16.Co-IP&MS Immunoprecipitation combined with mass spectrometry: Human TMOD3-Flag fusion expression plasmid was constructed and transfected into A549 cell line.72 h after transfection,the cells were collected.IP lysate was added to collect proteins.The TMOD3-Flag protein and its interacting protein complex were fenced,and the samples were sent for protein spectrum analysis to screen the TMOD3-interacting protein.17.Co-IP protein-protein interaction verification: The interaction between TMOD3 and ADSL was verified by Co-IP immunoprecipitation under the transfection system of A549 overexpressing TMOD3-Flag and the interaction system of A549 endogenous protein.18.GEPIA database was used to analyze the relationship between TMOD3 gene and ADSL gene in NSCLC online.19.TMOD3 regulates iron death signals in NSCLC by inhibiting ADSL.si RNA knockdown ADSL sequence was synthesized(si RNA-NC/Control)and transfected into A549-sh RNA-TMOD3 cell line.The cells were collected and the total protein was extracted.Western Blot was used to detect key transcription factors regulating iron death and their effects on AKT signaling.Results: 1.In lung adenocarcinoma and lung squamous cell carcinoma,TMOD3 is located in the nucleus and cytoplasm,and statistical scores were performed for all expressed TMOD3.The expression level of TMOD3 in normal bronchi and alveoli was lower than that in lung cancer tissue,and the expression level of TMOD3 increased with the decrease of differentiation degree.2.Two TMOD3-expressing cell lines A549 and SK-MES-1 were screened to synthesize si RNA-TMOD3 knockdown sequence and determine the optimal TMOD3 knockdown sequence.3.Tumor functional phenotype studies showed that TMOD3 knockdown in lung adenocarcinoma cell line A549 and lung squamous cell carcinoma cell line SK-MES-1 inhibited cell proliferation activity,cell migration,cell invasion and epithelial mesenchymal transformation of EMT.4.Western Blot analysis of A549 and SK-MES-1 cell lines showed that TMOD3 inhibited the expression of Cyclin B1,Cyclin D1,CDK2 and other proliferation and cycle regulating proteins.5.Western Blot analysis of A549 and SK-MES-1 cell lines showed that the expression of epithelial marker E-cadherin was increased and the expression of stromal marker N-cadherin was down-regulated.6.In A549 and SK-MES-1 cell lines,TMOD3 inhibited the iron death process and iron ion levels in NSCLC cells,and the effect was more significant with the addition of iron death activator Erastin.7.In A549 and SK-MES-1 cell lines,TMOD3 inhibited iron death and promoted glutathione expression in NSCLC cells.8.In A549 and SK-MES-1 cell lines,TMOD3 inhibited the iron death process of NSCLC cells,and the expression level of ROS lipid reactive oxygen species increased.9.Western Blot analysis of A549 and SK-MES-1 cell lines showed that TMOD3 promoted the phosphorylation of AKT and ERK,the classical signaling kinases of iron death;Meanwhile,TMOD3 promoted the expression of iron death suppressor genes x CT and GPX4,and inhibited the expression of iron death promoter gene FACL4.10.Co-IP&MS(Immunoprecipitation combined with Mass spectrometry)was used to screen the TMOD3-interacting protein ADSL,and positive and negative interaction experiments proved that both exogenous expression of TMOD3-Flag and endogenous expression of TMOD3 could interact with ADSL.Western Blot showed that TMOD3 inhibited the expression of ADSL.11.After transfection of si RNA-ADSL in A549-si RNA-TMOD3 cell line,Western Blot analysis showed that phosphorylation levels of p-AKT and p-ERK were significantly up-regulated compared with those of si RNA-NC/Control group.The expression of inhibitory genes x CT and GPX4 were significantly up-regulated.The expression of iron death promoting gene FACL4 was significantly down-regulated.These results indicate that TMOD3 mediates the regulation of iron AKT death signal in NSCLC cells by inhibiting the expression of ADSL,thus promoting the malignant progression of NSCLC.Conclusion: 1.The high expression of TMOD3 is associated with adverse pathological features such as tumor lymph node metastasis,TNM stage and prognosis,and is a potential new oncogenic gene in NSCLC.2.TMOD3 promotes cell proliferation,migration,invasion and epithelial-mesenchymal transformation(EMT)of NSCLC cell lines A549 and SK-MES-1;3.TMOD3 interacts with ADSL protein to negatively regulate the iron death process of NSCLC cells by activating AKT/ERK signaling pathway.