RUNX1 Promotes Proliferation,Migration Of Non-small Cell Lung Cancer Through The MTOR Pathway

Objective:Lung cancer is one of the most ordinary malignant tumors in the world,and its main characteristics are the large number of patients and the short survival period.Talking about the etiology,the occurrence of lung cancer is related to personal bad living habits,external environment,genetics and other factors.In the 1990s,there were no effective options for cytotoxic treatment of lung cancer.In the 21st century,the emergence of the first generation of EGFR tyrosine kinase inhibitors(TKIs)brought a major breakthrough,followed by MET inhibition,targeting gene mutation,molecular targeted therapy and other related drugs.Although the current selection of drugs is relatively mature,the evolution of tumors is the outcome of various factors,and it is particularly significant to make sure that how lung cancer takes place for early detection and early treatment.RUNX,belonging to the metazoan transcription factor family,has transcription factor activity,participates in the regulation of proliferation,apoptosis,differentiation and other processes in growth and development.They are also involved as important members in the occurrence and development of tumors.The RUNX family is composed of three members,RUNX1,RUNX2,and RUNX3.They have the same runt domain(RD),which can combine with the promoter of the target gene to regulate the transcriptional activation or repression of the target gene.RUNX1 was the first gene identified as a t(8;21)translocation breakpoint in approximately 10%of AML patients,causally implicated in the development of AML.In hematopoietic cells,RUNX1 can affect cell cycle progression by regulating the expression of cyclin D2/D3,CDK inhibitor P21,apoptosis-related gene Bcl-2,hematopoietic growth factors and/or their receptors.RUNX1 induces the expression of MMPs and VEGFA through the p38-MAPK signaling pathway to promote cell metastasis and invasion and angiogenesis in glioblastoma blast cells.However,it is not clear whether RUNX1 participates in the regulation of the onset and development of non-small cell lung cancer,so we searched online databases and found that RUNX1 was highly expressed in non-small cell lung cancer tissues than that in normal tissues.Therefore,this survey was intent on excavating the function of RUNX1 in non-small cell lung cancer,and related molecular mechanisms.Methods:1.Database information retrieval and analysis:we evaluate the expression of RUNX1 in non-small cell lung cancer tissue and normal lung tissue,and its correlation with cancer stage,lymph node metastasis through GEPIA and UALCAN online databases.The relationship between RUNX1 and disease prognosis was forecasted by Kaplan-Meier database.The correlation between RUNX1 and the pathway was explored by searching KEGG and Bio Carta databases.2.Immunohistochemical staining:RUNX1 staining was performed on lung cancer tissue chips,including 58 pairs of lung adenocarcinoma tissue and adjacent normal tissue,52pairs of lung squamous cell carcinoma tissue and adjacent normal tissue.Sodium citrate high temperature and high pressure antigen retrieval and DAB color development were mainly selected for staining.Intensity of staining and percentage of stained area were assessed by two researchers on a double-blind basis.The staining intensity was divided into four grades:0(not expressed),1(weakly expressed),2(moderately expressed),3(strongly expressed).The percentage of stained area is divided into five grades:0(0%),1(1%-25%),2(26%-50%),3(51%-75%),4(76%-100%)).The multiplication results of staining intensity and stained area ranged from 0 to 12 points,with a score of≥6 indicating high expression of RUNX1,and a score of<6 indicating low or no expression of RUNX1.3.Cell culture:DMEM medium is used to culture normal lung bronchial epithelial cell line HBE;RPMI1640 medium is used to culture human non-small cell lung cancer cell lines A549,H1299,H1975 and H292;MEM medium is used to culture human non-small cell lung cancer cell line SK-MES-1.The concentration of fetal bovine serum in the medium was 10%.They are all cultured in a 37°C incubator containing 5%carbon dioxide.4.Cell immunofluorescence experiment:A certain number of cells were seeded on24-well plate slides one day in advance.In the next day,cleaned with filtered PBS three times for 5 minutes each time,fixed with tissue fix solution for 15 minutes,and punched through the membrane with 0.5%Triton X-100 for 15 minutes,blocked with 3%BSA for1.5 hours,and incubated with the primary antibody overnight at last.On the third day,firstly washed with filtered PBS,the fluorescent secondary antibody was incubated at room temperature without light for 2 hours,and the nucleus was stained with DAPI for10 minutes,use anti-quenching medium to cover the slides,observe and take pictures with a microscope after covering.5.Cell transfection,interference,pathway inhibitor and construction of stable transfection cell line:Lipofectamine 3000 was used to transfect and interfere with non-small cell lung cancer cell lines according to the instructions.In this experiment,A549 and H1299 cell lines were selected for transfection;SK-MES-1 and H1299 cell lines were selected for interference,and the transfection and interference efficiencies of cells were clear by western blotting after 48 hours and 72 hours,respectively.After 24hours of transfection,G418 was added for stable screening in H1299 cells.After 4 weeks of stable screening and culture,the transfection efficiency was detected by western blotting.The stable transfected cell line was successfully constructed.Rapamycin is an inhibitor of mTOR pathway.After 24 hours of transfection of A549 and H1299 cells,100n M of rapamycin was added to the cells and cultured for 24 hours for subsequent experiments.6.Western blotting experiment:After the cells were washed and air-dried,and then added cell lysate on ice to lyse protein,prepared the protein sample of equal mass(40μg).Firstly,proteins were separated by 10%SDS-PAGE gel,transferred to PVDF membrane,blocked in 5%skimmed milk powder at room temperature for 1 hour on a shaker,then cut the membrane,finally cultured with the corresponding antibodies at 4°C overnight.The next day,the membrane was cleaned with TTBS for 20 minutes and incubated with the secondary antibody for 1 hour at room temperature on a shaker.Then ECL ultra-sensitive luminescent solution was added to detect protein expression,and finally Image J software was used to quantitatively evaluate the gray value of each band.7.CCK8 proliferation experiment:seeded in a 96-well cell culture plate at a concentration of 2500 cells per well,spread into 5 plates,and incubated for 5 consecutive days.Add 10μl of CCK8 reagent to each well of one plate every day,and after incubation in the dark for 2 hours,use a microplate reader to measure the OD value of each well,and set the wavelength of the instrument to 450 nm.8.Colony formation experiment:seeded in a 6-well cell culture plate at a concentration of 800 cells per well,incubated at 37°C with 5%carbon dioxide incubator for 10-15 days.Washed three times with PBS,after discarding PBS,fixed cells with 4%paraformaldehyde,and then stained with crystal violet.Finally,take pictures and count with a high-sensitivity chemiluminescence imaging system.9.Transwell experiment:the bottom layer was 20%FBS,then the chamber was placed in a 24-well cell culture plate.At last,the cell suspension containing 2%fetal bovine serum was added to the upper chamber with 40000 cells.Incubated at 37°C with 5%carbon dioxide incubator for about 22 hours.Cells were cleaned three times with PBS,after discarding PBS,fixed with tissue fix solution,and stained with crystal violet.Finally,observe with a microscope and take pictures for counting.10.Wound healing assay:when the cell density grows to about 90%,discard the medium,replace it with serum-free medium containing 1%mitomycin,incubate in a 37°C incubator for 2 hours,then use a 200μl pipette tip to draw a“well”shape at the bottom of the 6 wells plate,and the cells were slowly washed with PBS,to make sure that floating cells were thoroughly washed away.10%FBS was added to culture,and took pictures with a microscope at the same time.11.RT-q PCR experiment:Use Trizol method to extract total RNA of cells,and then carry out follow-up experiments according to the instructions of reverse transcription and q PCR kit.The experimental results used GAPDH as the endogenous reference,and the relative expression of the target gene was obtained by calculating the numerical value of2-ΔΔCt.12.In vivo tumorigenesis experiment in nude mice:The stably transfected cells were made into a cell suspension with serum-free medium,and a 200μl volume of cell suspension containing 10~7 cells was subcutaneously injected into the right armpit of each nude mouse,and cultured aseptically.Twenty-eight days later,the nude mice were killed by cervical dislocation,then the nude mice were dissected to take the tumors,the sizes of the tumors were measured,and the tumors were photographed.13.Ch IP experiment,agarose gel electrophoresis:After the cells in the 10cm cell culture dish are full,follow the steps of the kit step by step,and finally get the purified DNA.Agarose gel electrophoresis:use 2%agarose gel to separate the DNA samples obtained after Ch IP,conduct gel electrophoresis at a constant voltage field,and use a gel imaging system to image the images after electrophoresis.14.Statistical analysis:Statistical analysis of data was performed using Graph Pad prism version 8.0 software,the results of each group were represented by mean±standard deviation(SD),and t-test or one-way ANOVA was used for comparison between two groups.All experiments were independently repeated three times under the same conditions.The relationship between RUNX1 expression and clinicopathological features was calculated by chi-square test for analysis.The P value<0.05 was considered statistically significant.Results:1.Searching the GEPIA and UALCAN databases,we found that RUNX1 was highly expressed in non-small cell lung cancer than that in normal tissues,and high expression of RUNX1 was positively correlated to lung cancer stage and lymph node metastasis;Searching the Kaplan-Meier database found that RUNX1 was associated with poor prognosis of patients.Next,the tissue chip immunohistochemistry experiment was improved,and the results suggested that high expression of RUNX1 was positively correlated to the lymph node metastasis and TNM stage of patients,which was consistent with the database results.2.Overexpression of RUNX1 expression promotes the proliferation and migration of non-small cell lung cancer,while down-regulation of RUNX1 expression inhibits the proliferation and migration of non-small cell lung cancer.Western blotting confirmed that up-regulation of RUNX1 expression promotes the expression of related functional proteins,and vice versa.3.The database predicts that RUNX1 is related to the mTOR pathway.Western blotting and RT-q PCR experiments confirmed that up-regulation of RUNX1 expression promotes the phosphorylation of mTOR,and the m RNA level of mTOR;Ch IP experiments suggest that RUNX1 can directly regulate mTOR transcription.4.After adding the mTOR pathway inhibitor rapamycin,compared with the control group,overexpression of RUNX1 weakened the promoting effect of cell proliferation and migration,and the promotion of related functional protein expression was attenuated.5.The protein and m RNA levels of PD-L1 decreased after RUNX1 knockdown;RUNX1overexpression could increase the protein and m RNA levels of PD-L1;after adding mTOR pathway inhibitor rapamycin,compared with the control group,the promoting effect of PD-L1 expression by the overexpression of RUNX1 was attenuated.6.In the tumorigenesis experiment of nude mice,the tumor volume and growth rate of the RUNX1 stable transfection group were higher than those of the control group.Western blotting experiments in the tumor confirmed that the overexpression of RUNX1promoted the phosphorylation of mTOR.Conclusion:1.RUNX1 is highly expressed in non-small cell lung cancer and is related to tumor TNM stage,lymph node metastasis and poor prognosis;2.RUNX1 promotes the proliferation and migration of non-small cell lung cancer by activating the mTOR pathway;3.RUNX1 regulates the expression of mTOR/PD-L1 axis;4.RUNX1 plays a role in promoting cancer in non-small cell lung cancer.