| BackgroundLung cancer is a malignant tumor with the highest mortality and the third highest morbidity in the world.It is the main malignant tumor that seriously threatens the health of residents in China.The latest information shows that lung cancer has become the leading cause of death of malignant tumors.Although the wide use of low-dose computed tomography(CT)has greatly increased the detection rate of early lung cancer,lung cancer is still a malignant disease with poor prognosis.The treatment of lung cancer has changed dramatically,from the three traditional approaches of surgery,chemotherapy and radiotherapy to targeted therapy and immunotherapy.Molecular targeting of lung cancer driving gene mutations has dramatically changed the treatment of lung cancer.Since the driving genes of lung cancer play an important role in the activation of signaling pathways that promote tumor development and progression,targeted therapy is highly effective compared with traditional cytotoxic chemotherapy.Tyrosine kinase inhibitors(TKIs),the driving genes of lung cancer,are used to target patients with mutations in receptor Tyrosine kinase regions(EGFR 18,19,20 and 21 exons).According to relevant statistics,the use of EGFR-TKI has benefited Chinese patients with NSCLC,and the survival time of some patients has been greatly improved.There are many kinds of tumor driving genes in lung cancer,many of which are related to tumor targeted therapy.Important tumor drivers include:EGFR,ALK,Kras/Nras,Braf,ROS1,MET,etc.With further research,more and more lung cancer driving genes have been found.Tyrosine kinase protein 7(PTK7)is a tyrosine kinase receptor protein without tyrosine kinase activity,which has been found to be abnormally expressed in human tumors such as colorectal cancer,prostate cancer,gastric cancer,breast cancer,and cervical cancer in many studies at home and abroad,and is related to tumor proliferation,invasion and metastasis.However,so far,there have been few studies on PTK7 gene in NSCLC,and its role and function in NSCLC remain unclear.In this study,the mRNA and protein expression levels of PTK7 gene in a group of lung cancer cell lines and non-small cell lung cancer(NSCLC)tissue samples were detected to determine its expression level in non-small cell lung cancer.The correlation between its expression and clinicopathological indicators was analyzed.Lung cancer cell lines with high expression of PTK7 were screened out,the expression of the gene was inhibited by transfection of small interference RNA(siRNA)targeting PTK7 gene,and the effect of PTK7 protein on the biological function of lung cancer cells was observed after knock-out of PTK7,and the effect of PTK7 gene on lung cancer cells was preliminarily studied.On the other hand,we examed the correlations of PTK7 expression levels with EGFR mutation and EML4-ALK fusion.Materials and methodsIn this study,human embryonic lung normal cells and lung cancer cells were purchased from Shanghai cell bank,Chinese academy of sciences.A549 non-small cell lung cancer from white lung cancer tissues;NCI-H460 human cell carcinoma originated from the patient's pleural effusion.NCI-H1734 non-small cell lung cancer;MRC-5 human embryonic lung cells derived from normal fetal lung tissue.The mRNA and protein expression levels of PTK7 in human embryo lung cell lines(MRC-5)and lung cancer cell lines(NCI-H292,A549,NCI-H460 and NCI-H1734)were determined by quantitative PCR,Western blot and immunochemical staining.The cells were cultured in a 25mL Corning culture flask.10%fetal bovine serum was added to the RPMI1640 basic culture medium.The culture conditions of the CO2 incubator were 37?,5%CO2 and certain humidity.Cell total RNA was extracted using Trizol,260nm/280nm wavelength to determine the purity and concentration of the total RNA extracted,and the quality of the extracted RNA was checked by using MOPS denaturing gel.The mixed genomic DNA was digested with DNase.2 ?g of total RNA was reversed to cDNA using the Promega kit.The mRNA expression level of PTK7 was detected in the cell line,and the housekeeping gene GAPDH was used as an internal reference standardization results.When the cultured cells reach a fusion rate of more than 90%,discard the medium,wash with PBS buffer three times,and add 1 ml of RIPA lysate containing 10?l of protease inhibitor mixture and 10?l of phosphatase inhibitor mixture pre-chilled on ice.Total protein in lung cancer cells was extracted.This study collected paraffin tissue specimens from 95 patients with non-small cell lung cancer who were hospitalized for surgery between January 2017 and December 2018 in Taixin People's Hospital of Jiangsu Province.None of the patients received chemotherapy,radiation therapy,and other treatments before surgery.The patients included 48 males and 47 females;the average age was 58 years;34 cases of poorly differentiated tumors,35 cases of moderately differentiated tumors,16 cases of highly differentiated tumors;31 cases of lymph node metastasis,and 62 cases of no lymph node metastasis.The surgical tissue specimens were sent to the pathology department within 30 minutes after ex vivo,soaked in a sufficient amount of 10%neutral formalin fixative,and pathological samples were taken after 8-12 hours of fixation.Tissue specimens are fixed with formalin,and then dehydrated,transparent,and wax-impregnated,and then embedded into wax blocks with embedding agent.All tissue wax blocks used were pathologically diagnosed as non-small cell lung cancer.The diagnostic criteria refer to the World Health Organization Tumor Classification and Diagnostic Standards,pathology and genetics of lung,pleura,thymus and heart tumors.Immunohistochemical staining was used to detect the expression level of PTK7 protein in tissue samples from patients with non-small cell lung cancer,and the correlation between PTK7 protein expression in lung cancer tissues and clinicopathological indicators was studied through statistical analysis.Judgment criteria for immunohistochemical results:The determination of PTK7 protein expression results depends on the comprehensive score of staining intensity of stained cells and the ratio of positive cells.Five fields of view were randomly selected under the × 400 microscope for observation.Staining intensity scoring criteria:The tissue has almost no yellow color as 0 points;light yellow coloration as 1 point;more pronounced yellow coloration as 2 points;strong brownish coloration as 3 points.Positive cell ratio scoring criteria:positive cell number<5%is 0 points;positive cell number<25%is 1 point;positive cell numbers?25%and<50%and 2 points;positive cell numbers? 50%and<75%Is 3 points;the number of positive cells?75%is 4 points.The staining intensity is multiplied by the score of the positive cells,and the score is positive if? 2,otherwise it is negative.The selected lung cancer cell line A549 with high expression of PTK7 was transfected with PTK7-specific siRNA,and its proliferation,apoptosis and migration ability were observed.One day before transfection,the cells were passaged in a 24-well plastic culture plate to achieve a cell fusion rate of 90-95%.The transfection medium did not contain antibiotics.Lipofectamine 2000 was diluted with OPti-MEMI special medium.Mix the diluted PTK7 siRNA and Lipofectamine 2000 gently,add the cells and continue to culture for 24-48 hours.Calculate the number of cells after the cultured cells was digested.Add 1×104 cells and 100?l of culture medium to each well in a 96-well plate.Set up three parallel wells of each cell at 37 ?,5%CO2 and incubate overnight.Add 10?l CCK-8 test liquid to avoid air bubbles.Incubate for another 2 hours.After mixing,use a microplate reader to detect the absorbance of each well at 450 nm.Cells were digested with 2.5%trypsin without EDTA,and the reaction was stopped by adding 1640 medium.The cells were washed with PBS buffer,centrifuged at 2000 rpm for 5 minutes,repeated once,and cells 2×105 cells were collected.Add 500?l of Binding Buffer to pipette the suspended cells,add 5?l of AnnexiV-FITC and mix,then add 5?l of PI(Propidium Iodide)and mix,and react at room temperature for 10 minutes in the dark.Detection was carried out by flow cytometry.The excitation light wavelength was selected to be 488 nm,and the emission light wavelength was selected to be 530 nm.The green fluorescence of AnnexiV-FITC passed the FITC channel(FL1),and the PI red fluorescence was detected through the FL3 channel.The transfected cells and control cells were subcultured for 24 hours,and when the cell fusion reached 60%or more,a 20?1 pipette tip was used to uniformly draw a "-" line at the bottom of the culture dish.After rinsing twice with PBS buffer,the serum-free 1640 medium was replaced and the culture was continued for 24 hours.Observe and take photos.Image J software was used to measure the distance between cell scratches to evaluate the migration ability of transfected and control cells.The epidermal growth factor receptor(EGFR)gene mutations in lung cancer tissues were detected using paraffin tissue sample DNA produced by TianGen Company and EGFR gene exon 18-21 mutation kit produced by Beijing Yakangbo Biotechnology Co.,Ltd.Ventana Medical Systems automated immunohistochemical analyzer,Ventana anti-ALK(D5F3)antibody was used to detect ALK protein,and ALK two-color separation probe produced by Beijing Jinpujia was used for fluorescence in situ hybridization to detect anaplastic lymphoma kinase gene(ALK)Integration situation.The correlation between PTK7 protein expression and EGFR gene mutation and EML4-ALK gene fusion mutation was analyzed.SPSS 13.0 software was used for statistical analysis,and the correlation analysis was analyzed by chi-square test.P<0.05 was considered statistically significant.ResultsThe expression levels of PTK7 mRNA in non-small cell lung cancer cell lines NCI-H292,A549,NCI-H460,NCI-H1734 and human embryonic normal lung cell line MRC-5 were determined,and the expression level was normalized with the reference gene GAPDH.PTK7 is not expressed in the normal lung cell line MRC-5,and is expressed to varying degrees in NCI-H292,A549,NCI-H460,and NCI-H1734 lung cancer cells.The expression of PTK7 mRNA in lung cancer cells decreased in order:A549>NCI-H292>NCI-H1734>NCI-H460>MRC-5.NCI-H292 and A549 were highly expressed,and NCI-H460 and NCI-H1734 were low expressed.Western blot was used to detect the expression of PTK7 protein in non-small cell lung cancer cell lines NCI-H292,A549,NCI-H460,NCI-H1734 and human embryonic normal lung cell line MRC-5.PTK7 protein is not expressed in normal lung cells and is expressed to varying degrees in non-small cell lung cancer cells.Using cellular immunochemical methods,the expression of PTK7 protein in non-small cell lung cancer cell lines NCI-H292,A549,NCI-H460,NCI-H1734,and human embryonic normal lung cell line MRC-5 was further verified.Two methods verified that PTK7 mRNA expression level and protein expression level were consistent.The expression of PTK7 protein in 95 lung cancer tissue samples containing normal alveolar epithelial cells and tumor cells was detected by polyclonal antibody specific to PTK7.PTK7 protein was negatively expressed in all normal alveolar epithelial cells,while its expression in tumor cells varied from negative,weakly positive,moderately positive to strongly positive.PTK7 protein was mainly expressed in tumor cytoplasm,and PTK7 protein expression was negative in 50 cases(52.6%)and positive in 45 cases(47.4%)of lung cancer.Statistical analysis showed that PTK7 protein expression was associated with gender(P=0.024)and lymph node metastasis(P<0.001),regardless of age,tumor differentiation,primary tumor and tumor cell type.The lung cancer cell line A549 is a cell line that highly expresses PTK7.It was selected as a cell-level PTK7 gene knockout research object.The CCK8 kit was used to detect the proliferation ability of PTK7 siRNA transfected and control cells.The results showed that the proliferation activity of PTK7 knockdown in lung cancer cell line A549 cells significantly decreased(P<0.05).The cells were tested after 48 hours of culture.The results of cell proliferation experiments showed that the average cell viability of the A549 control group was 91%,and the average viability of A549 cells transfected with siRNA was 63%.Annexin V-FITC/PI apoptosis detection kit was used to detect the apoptosis between PTK7 cells transfected with PTK7 siRNA and control cells.The results showed that the apoptosis rate of PTK7 increased in lung cancer cell line A549 cells.The cells were tested after 48 hours of culture.The results of apoptosis experiments showed that the average apoptosis rate of the A549 control group was 21%,the average apoptosis rate of A549 cells transfected with siRNA and the PTK7 gene was knocked out was 38%.The apoptosis rate of A549 was significantly higher than that of control cells(P<0.05).The cell migration ability between the transfected PTK7 siRNA and the control cells was tested using a scratch test on cultured cells.The results showed that the migration ability of A549 that knocked out of PTK7 gene was significantly reduced.The blank control group of lung cancer cell line A549 with high expression of PTK7 gene and A549 cells knocked out of PTK7 gene by siRNA were cultured for 24 hours.Image J software was used to analyze the cell migration and repair ability of the two groups.The relative scratch distance of the A549 blank control group was:103.32±12.54;the relative distance of the scratch of A549 cells knocked out by the PKT7 gene was:1357.32±23.65.The migration ability of A549 knocked out PTK7 gene was significantly lower than that of the control group,and there was a significant difference between the two groups(P<0.05).95 cases of non-small cell lung cancer tissue samples were tested for EGFR mutation and EML4-ALK fusion gene.47 cases of exon 18-21 mutations were detected by EGFR and 48 cases of wild type.The positive rate of EGFR mutation was 49.5%.EGFR mutations mostly occurred in patients with negative PTK7 protein expression(P=0.014).Of the 95 cases of non-small cell lung cancer detected,7 cases(7.4%)of EML4-ALK fusion were detected.Positive samples were further verified by FISH.This study found that EML4-ALK gene fusion was more likely to occur in patients with positive PTK7 protein expression(P=0.050).ConclusionsPTK7 gene is not expressed in human embryonic lung cell line and normal alveolar epithelial cells,but in non-small cell lung cancer cells.mRNA and protein expression of PKT7 gene was increased in NSCLC compared with normal lung tissue cells.The expression level of PTK7 protein in NSCLC was correlated with gender and lymph node metastasis,but not with other pathological indicators.PTK7 protein expression is also associated with EGFR mutations and EML4-ALK fusion.After the PTK7 gene was knocked out at the cell level,the proliferation force of the cells decreased,the apoptosis rate increased and the migration force decreased.The results of this study preliminarily suggest the role of PTK7 gene in non-small cell lung cancer.This study is a preliminary study on the function of PTK7 gene in non-small cell lung cancer,and its results provide important clues for further study on its role in the pathogenesis,development,metastasis and prognosis of lung cancer and its molecular mechanism in the future.It may also provide an important basis for further research on targeted therapy for high expression of PTK7. |
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