The Studies Of Protein Z's Effect On The Growth And Metastasis In Lung Adenocarcinoma

Background Lung carcinoma is a malignancy characterized by uncontrolled growth of cancer cells in lung tissue.Lung cancer cells metastasize rapidly to other organs and tissue without appropriate treatment.Morbidity of lung cancer ranks second in all cancers and mortality ranks first.In recent years,with the further understanding of genetic characteristics and bio-molecular mechanism of lung cancer,new molecular target drug(EGFR-TKIs: Osmertinib)and immunochemical drugs(CRLX101,TAS-102)were developed that led patients to a more effective treatment and promising prognosis.However,metastasis and invasiveness remaindifficulty problems.Lung adenocarcinoma,a pathological type that early metastasis is prevalent.Adevanced stage at primary diagnosis.At present,treatment of metastasis and recurrence of lung adenocarcinoma is not optimistic leads high mortality that puts a threat to human life.Therefore,it is significant to explore its metastatic and infiltrating mechanism in more details.Protein Z is a serum glycoprotein synthesized by the liver that acts in anticoagulation.Physiologically,protein Z binds with protein Z dependent protease inhibitor in circulation.Protein Z plays a significant effect by enhancing the anticoagulation by 1000 times with ZPI,which inhibits Factor Xa in the course of anticoagulation.Inhibition of Factor Xa through ZPI is reduced when protein Z deficiency that makes coagulant activity increases and so does the thrombotic risk.Recent reports discover expressions of PZ in malignancies like gastric,colon and lung cancers.Our early study points out that PZ level decreases along with the progression of cancer,which in some ways implies that PZ may be a factor of poor prognosis.Furthermore,PZ is expressed in both cells and tissues of lung cancer in our result that is inconsistent with previous studies.Above all,PZ must have other relation with cancer.By now,the effect of PZ on cancer proliferation,metastasis,and other biological aspects remains unclear.Based on our previous findings,our study explores the effect of PZ on lung adenocarcinoma's proliferation and metastasis,which may contribute a theoretical basis for later clinical practice.Objectives: 1.To investigate the expression of protein Z in lung adenocarcinoma.2.To investigate the effects of PZ on migration,invasion,proliferation and apoptosis of lung adenocarcinoma cells.3.Preliminary study on the role of protein Z in lung adenocarcinoma metastasis mechanism.Methods: Immunohistochemistry and Western blot assay were separately used to detect lung adenocarcinoma tissue and lung adenocarcinoma cells expression of Protein Z.1.Immunohistochemistry was used to detect the expression of Protein Z in lung adenocarcinoma and pericarcinous: the specimens of lung adenocarcinoma from patients who had not been treated with radiotherapy and chemotherapy were collected.Immunohistochemical staining was used to detect the expression of PZ in 30 cases of lung adenocarcinoma and 16 cases of pericarcinous.Data were collected and statistical analysis was performed to compare the expression of protein Z between lung adenocarcinoma and pericarcinus.2.Western blot method was used to detect the expression of protein Z in lung adenocarcinoma cells: there were three humen lung adenocarcinoma cell lines: ACC212102,A549 and SPCA-1.Logarithmic growth phase cells were collected.PBS was used to gently wash the cell metabolites,then the RIPA lysis solution was added to the cells.The cells were placed on ice and were fully cracked.BCA method was used to detect the protein concentration.Data was collected and statistical analysis was performed to investigate the expression of Protein Z in different lung adenocarcinoma cell lines and the cell line high expressed PZ was screened.Synthesized the si RNA-PZ and transfected into A549 lung adenocarcinoma cell line,the effect of si RNA in A549 cells was detected by Western blot assay.According to the results of protein Z expression in lung adenocarcinoma cells in the first part,the A549 cell line was screened.Logarithmic growth phase cells were collected and divided into 5 groups: blank control group-Mock,invalid sequence negative control groupsi RNA-NC,experimental group-si RNA-PZ001,002,003.Completed the experiment in strict accordance with the si RNA kit.The si RNA targeted lipid vector was transfected into A549 lung adenocarcinoma cells,and put the cells into the 37?,5%CO2 incubator for 24-96 hours,collected the target protein.BCA assay was used to detect the protein concentration.Western blot assay detected the effect of transfection.Collected data and statistical analysis.The most effective target sequence was screened and prepared for the next experiment: healing assay,transwell migration assay,invasion assay,cell proliferation and apoptosis.Migration and invasion capability of A549 cells before and after silencing PZ gene by si RNA were detected by healing assay and transwell asssys.1.Healing assay was used to detecte the invasion and metastasis capability of lung adenocarcinoma: we divided the cells into 3 groups: blank control group Mock,negative control group si RNA-NC,and experimental group si RNA-PZ group.Maing marks at the buttom of the 6 holes plate and the 3 groups of cells were inoculated into the hole with right amount,vertical scratching,PBS washing and adding serumfree medium,at 37? and 5%CO2 incubator,choose 0 hours,24 hours,48 hours and taking pictures.Data collection and statistical analysis were performed to compare the effects of protein Z on metastasis in lung adenocarcinoma cells.2.Transwell assay was used to detect the migration and invasion ability of A549 cells.Logarithmic growth phase cells were collected and divided into 3 groups: Mock group,si RNA-NC group and si RNA-PZ group.The 0.8um size Transwell was choosed.The 3 group cells were inoculated into the upper chamber with the free serum culture medium,culture medium containing 20% fetal bovine serum was added into lower container(Matrigel should be paced into the upper container before added cells in invasion assay),cells were cultivated in the incubator.After 24 hours,PBS wash,methanol fixed,0.1% crystal violet staining,gently wiped the upper unmigrated cells with a cotton swab,lower cells under the membrance were observed under the microscope.Data collection and statistical analysis were performed to compare the effects of protein Z on the migration,invasion and metastasis of lung adenocarcinoma cells.The role of protein Z in epithelial-mesenchymal transition(EMT)tumor metastasis mechanism was detected by western blot.Logarithmic growth phase cells were collected and divided into 3 groups: Mock group,si RNA-NC group and si RNA-PZ group.Slug,Vimentin,N-cadherin proteins were detected on the 3 groups A549 cells.According to three groups different exprtssion of Slug,Vimentin,N-cadherin protein,we can know the role of protein Z in EMT metastasis pathway.Proliferation of A549 cells before and after silencing PZ gene detected by CCK-8 assay.Logarithmic growth phase cells were collected and divided into 3 groups: Mock group,si RNA-NC group and si RNA-PZ group.The 3 group cells were inoculated into the 96 holes plate and cultivated into incubator for 24 hours,added 10 ul CCK liquor in every hole,detected OD seperately after 12 hours,24 hours,48 hours,72 hours.Data collection and statistical analysis were performed to compare the effects of protein Z onproliferation of lung adenocarcinoma cells.Apoptosis of A549 cells before and after silencing PZ gene were detected by flow cytometry assay.Logarithmic growth phase cells were collected and divided into 2 groups: control group and si RNA-PZ group.Collected 5×105 cells and mixed with 500 ul 1×Binding Buffer,then added 5ul Annexin V-FITC ?10ul PI,keep in dark place for 5 min.Data collection and statistical analysis were performed to compare the effects of protein Z on apoptosis of lung adenocarcinoma cells.Results: Strong expression of PZ in lung adenocarcinoma tissues and lung adenocarcinoma cells 1.The immunohistochemistry result: The expression of Protein Z in lung adenocarcinoma was significantly higher than that in precarcinus,and the difference was statistically significant(P<0.05).2.Western blot results: PZ was expressed in three lung adenocarcinoma cells(ACC212102?A549?SPCA-1).The expression of PZ was particularly prominent in A549 cell line,compared with SPCA-1 and ACC212102 cells.There was statistically significant difference(P<0.05). The efficiency of si RNA knocking out the PZ gene and screened the most effective sequence After si RNA were transfered into A549 cells,compared with Mock group and si RNA-NC group,the expression level of protein Z in A549 group was significantly decreased in si RNA-PZ001 group.There was statistically significant diference(P<0.001).It is suggested that si RNA liposome can effectively silence the PZ gene,which can be used for the next experiments Between Mock group and si RNA-NC group,the difference was not statistically significant(P>0.05).The capacity of migration and invasion in si RNA-PZ cells were obviously decreased 1.Healing assay result: si RNA-PZ group cells were the slowest migrational group to the midline compare with Mock group and si RNA-NC group no matter 24 hours later or 48 hours later.There was statistically significant diference(P<0.05).Inhibiting the expression of PZ can inhibit the ability of migration in A549 cells.That suggests PZ may participate or help lung cancer cells to metastasis.Between Mock group and si RNA group,the difference was not statistically significant(P>0.05).2.Migration and invasion assasy results: No matter in migration assay or invasion assay,the least quantity passed through the membrace was the si RNA-PZ group,compared with the Mock group and si RNA-NC group.There was statistically significant diference(P<0.05).Inhibiting the expression of PZ can inhibit the ability of migration and invasion in A549 cells.It is suggest that PZ can redound lung adenocarcinoma to metastasis and strengthen the ability of migration and invasion of lung adenocarcinoma.Between Mock group and si RNA group,the difference was not statistically significant(P>0.05).PZ may affect lung adenocarcinoma metastasis through EMT pathway EMT pathway related proteins Slug,Vimentin and N-cadherin were detected by western blot: The expression of these three proteins were all decreased in si RNA-PZ group,compared with the Mock group and si RNA-NC group.There was statistically significant diference(P<0.05).It means that PZ may affect Slug,Vimentin and N-cadherin proteins and that PZ may participated in lung adenocarcinoma metastssis through EMT pathway.PZ may not be involved in A549 cell proliferation CCK-8 result: There were no statisically significant diference the three groups(P>0.05).It is implied that PZ may not participate in lung adenocarcinoma cells proliferation.PZ may not participate in A549 cell apotosis Flow cytometry results: There were no statisically significant diference the three groups(P>0.05).It is implied that PZ may not participate in lung adenocarcinoma cells apoptosis.Conclusions: 1.PZ was strongly expressed both in lung adenocarcinoma tissues and cells,and the immunohistochemistry results imply that protein Z may be relatived to tumor genesis and development.2.The cell biology experiment and molecular biology experiment results showed that expression of PZ was positively correlated with the migration and invasion of lung adenocarcinoma.3.PZ may affect lung adenocarcinoma metastasis through EMT mechanism.4.Protein Z does not take part in lung adenocarcinoma cells proliferation.5.Protein Z does not participate in lung adenocarcinoma cells apoptosis.