The Expression Of Thrombospondin 1 In Lung Adenocarcinoma And The Effect Of Its Regulation On The Biological Behavior Of Lung Adenocarcinoma Cells

Lung cancer is the leading cause of cancer mortality worldwide,statistics in 2012 show that about 1.8 million new cases of lung cancer each year,accounting for nearly 13% of the total number of new tumors globally.It is the most common cancer among men and the second most common cancer among women.Lung cancer is a highly aggressive and challenging disease,it is the most common cause of cancer-related death worldwide.The major histologic subtypes of lung cancer are small cell lung cancer(SCLC)and non-small cell lung cancer(NSCLC).Non-small cell lung cancer(NSCLC)reaches about the 80% of lung cancer cases.It contains adenocarcinoma,squama carcinoma,large cell carcinoma and other cancer types,and adenocarcinoma is the major subtype.Surgical resection is the current optimal therapy for NSCLC,but <50% of NSCLC patients can receive surgical treatment due to late diagnosis and advanced stage of disease.Therefore,with the exception of surgery,chemotherapy is also an essential strategy in the treatment of NSCLC,particularly for molecular targeted therapy.The majority of patients will have to rely on chemotherapy,radiotherapy and chemotherapy,molecular targeted therapy and other comprehensive treatment.Especially the application of molecular targeted therapy has become the medical profession at this stage,a great deal research focused on molecular alteration of lung cancer.In the past 10 years,the overall survival and quality of life of lung cancer patients has greatly improved due to the introduction of new drugs and individualized therapy based on different histological subtypes and driver mutations that determine the biology of lung cancers and can be used to predict drug efficacy.Epidermal growth factor receptor(EGFR)and anaplastic lymphoma kinase(ALK),have changed the way these cancers are diagnosed and treated.However,some patients do not exist in the gene-related changes,despite efforts and active treatment,the prognosis is still poor.As a result,more molecular-driven genes remain to be studied.Thrombospondin1(THBS1)is a glycoprotein initially found in platelets,it is also synthesized and secreted by many normal and transformed cells.THBS1 was the first endogenous angiogenesis inhibitor to be identified,and it involved in the regulation of tumor progression and metastases.THBS1 suppresses endothelial cell proliferation,migration,blood vessel-formation and induces endothelial apoptosis.Thus,THBS1 is an endogenous inhibitor of angiogenesis under physiological and pathological conditions,including in the context of malignancy.However,THBS1 can also stimulate angiogenesis,the angiogenesis-related function of THBS1 is very complex.Furthermore,the expression and function of THBS1 in tumors were also complicated.The expression of THBS1 in solid tumors such as breast cancer,cervical cancer,pancreatic ductal adenocarcinoma,prostate cancer and bladder cancer was decreased,and the expression level of THBS1 was significantly correlated with thyroid carcinoma,carcinoma of the prostate,adenocarcinoma of the prostate,and malignant degree of bladder cancer.However,THBS1 expression in breast cancer and colorectal cancer is increased and promotes the migration of cancer cells.In addition,the contradictory results regarding the pathological significance and effects of THBS1 in the progression of malignant tumors have also been described in some animal experiments,human pathology studies and comprehensive reviews.The study of THBS1 in human lung adenocarcinoma is limited,a study has shown that its expression has no correlation with lung adenocarcinoma,the other studies confirmed that it may be associated with lung adenocarcinoma prognosis.The relationship between THBS1 and adenocarcinoma of the lung has not been clarified.In order to further explore the relationship between THBS1 and the development of lung adenocarcinoma,to find a new target gene for the treatment of lung adenocarcinoma,the expression of THBS1 protein in 53 cases of lung adenocarcinoma tissues and 53 cases of normal tissues was detected by immunohistochemistry En Vision method in this study;The expressions of THBS1 protein and m RNA were detected by immunohistochemistry,Western blotting and real-time quantitative polymerase chain reaction(q RT-PCR)in 4 fresh lung adenocarcinoma tissues and 4 normal tissues;And then the relationship between THBS1 expression and clinicopathologic parameters in patients with lung adenocarcinoma was analyzed,the role of THBS1 in the pathogenesis of lung adenocarcinoma was elucidated.Then,the expression of THBS1 in various lung adenocarcinoma cell lines was analyzed,the THBS1 eukaryotic expression vector was successfully constructed and then transfected into lung adenocarcinoma cells,the expression of THBS1 protein and m RNA in lung adenocarcinoma cells before and after transfection were detected by Western blotting and q RT-PCR respectively;THBS1 si RNA was transfected into THBS1 cells with high expression of THBS1.Western blotting and q RT-PCR were used to detect the expression of THBS1 protein and m RNA in lung adenocarcinoma cells before and after transfection.The apoptosis and growth cycle of adenocarcinoma cells were detected by flow cytometry before and after transfection.The effects of THBS1 on caspase-3,MMP-2(matrix metalloprotein 2)and MMP-9(matrix metalloprotein 9)were analyzed.The changes of cell migration ability were observed before and after transfection.Finally,through nude mice transplanted tumor experiment,immunohistochemistry,the tumor size of the nude mice was compared,and western blotting and q RT-PCR were used to detect the expression of THBS1 in nude mice xenografts.In order to elucidate the significance of THBS1 expression in the development and progression of lung adenocarcinoma,to further clarify the role of THBS1 gene in the development of lung adenocarcinoma and its mechanismand to provide theoretical basis and experimental evidence for THBS1 as a new molecular therapeutic target of lung adenocarcinoma patients,the role of down-regulation of THBS1 expression and activity in the development and progression of lung adenocarcinoma was explored from the aspects of gene,protein,cell localization and tumor cell biological behavior,in vivo nude mice tumorigenesis experiment.This study is divided into the following three parts.The first part: The clinicopathologleal signincance of the expression of THBS1 in lung adenocarcinomaMethods 1.Detected the THBS1 protein expression in 53 cases lung adenocarcinoma tissue,and 53 cases normal lung tissue by immunohistochemistry.2.Detected the THBS1 protein expression of THBS1 protein in 4 cases lung adenocarcinoma tissues and corresponding normal tissues by western blotting.3.Detected the expression of THBS1 m RNA in 4 cases lung adenocarcinoma tissues and corresponding 4 cases normal tissues by q RT-PCR.4.Statistical analysis: All the data were analyzed by SPSS 17.0 statistical software.The statistical data were expressed as mean ± standard deviation(SD).Immuno-histochemical results were analyzed by chi-squared analysis.The mean of multiple samples was compared using one-way ANOVA analysis(One-way ANOVA),P<0.05 was significant.Results 1.Immunohistochemistry showed that the expression of THBS1 protein was mainly located in the cytoplasm of tumor cells,but not expression or low expression in normal alveolar epithelial cells.There was significant difference between the two groups(P<0.05).2.The expression of THBS1 protein in lung adenocarcinoma tissues was higher than that in normal lung tissues by Western blotting.3.The level of THBS1 m RNA in lung adenocarcinoma tissues was higher than that in normal lung tissues by q RT-PCR.4.The expression of THBS1 protein in lung adenocarcinoma of patients with lymph node metastasis was significantly higher than that of patients without lymph node metastasis(P<0.05).5.The expression of THBS1 protein was not related to age,gender,tumor diameter and histological grade in patients with lung adenocarcinoma(P>0.05).The second part: Molecular cloning of THBS1 gene and construction of eukaryotic expression vector and its effect on the biological behavior of human lung adenocarcinoma cell lines A549 and H1975Methods 1.The total RNA was extracted from lung cancer tissue and the human THBS1 gene was amplified by reverse transcription-polymerase chain reaction(RT-PCR).The recombinant product was cut with Hind III and Xba I and pc DNA3.1 empty vector,T4 DNA ligase was used to ligate the pc DNA3.1 empty vector fragment and the target fragment to transform the competent E.coli DH5? cells.PCR was used to identify the correct clone.The recombinant plasmid pc DNA-THBS1 or empty vector pc DNA3.1(+)was constructed to construct the eukaryotic expression vector pc DNA3-THBS1 of THBS1 gene and identified by restriction enzyme digestion.2.Control si RNA(100 n M)and THBS1 si RNA(100 n M)were transfected into lung adenocarcinoma A549 cells with the highest expression of THBS1 by Liposome 2000.Liposome 2000 introduced eukaryotic expression vector pc DNA3-THBS1 and empty vector pc DNA3.1 into H1975 cells with the lowest expression of THBS1.The expression of THBS1 protein in lung adenocarcinoma A549 cells and H1975 cells was detected by Western blotting and RT-PCR.3.The CCK-8 kit was used to analyze the proliferation of lung adenocarcinoma A549 cells and H1975 cells in different groups before and after transfection.4.Flow cytometry was used to analyze the cell cycle and apoptosis of lung adenocarcinoma A549 cells and H1975 cells in different groups.And the activity of caspase-3 in A549 cells and H1975 cells was detected.Further analysis of the possible molecular mechanisms of THBS1-induced changes in apoptosis.5.The effect of THBS1 expression on the migration of A549 and H1975 cells was detected by cell scratches.6.Western blotting was used to analyze the expression of MMP-2 and MMP-9,which were closely related to migration and invasion in lung adenocarcinoma cells of different treatment groups.7.Statistical analysis: All the data were statistically analyzed by SPSS 17.0 statistical software.Statistical data were expressed as mean ± standard deviation(SD).Immunohistochemical results were analyzed by chi-square analysis.Multiple samples were compared with single factor variance Analysis(One-way ANOVA),P<0.05 was significant.Results 1.Lung cancer cell lines A549,PC9 and Calu3 showed relatively high levels of THBS1 m RNA expression,while H1299,H358,H1975 and PG49 cells showed a relatively low expression level of THBS1 m RNA.THBS1 protein was the most expressed in A549 cell line and had the lowest expression level in H1975 cell line.THBS1 protein expression level in other cell lines was between A549 and H1975.2.PCR amplification resulted in a specific size of approximately 3500 bp gene fragment.PCR was used to identify pc DNA-THBS1 recombinant vector,which could successfully amplify about 500 bp fragment from the recombinant plasmid,suggesting that eukaryotic expression vector pc DNA-THBS1 was successfully constructed.After transfection of pc DNA-THBS1 eukaryotic expression vector,H1975 cells could stably express THBS1 m RNA and protein.3.The expression of THBS1 m RNA and protein in A549 cells was significantly lower in THBS1 si RNA group than that in untreated and control si RNA group(P<0.05),but there was no significant difference in THBS1 m RNA and protein expression between the latter two groups(P>0.05).The expression of THBS1 m RNA and protein in H1975 cells was significantly up-regulated in pc DNA3.1-THBS1 group compared with untreated and empty vector pc DNA3.1 transfected H1975 cells,the difference was statistically significant(P<0.05).There was no significant difference in the expression of THBS1 m RNA and protein between untreated and transfected with empty vector pc DNA3.1(P>0.05).4.The proliferation of A549 cells treated with THBS1 si RNA was significantly inhibited(P<0.05)compared with untreated A549 cells and control si RNA transfected A549 cells,but there was no significant difference between untreated A549 cells and control si RNA transfected A549 cells(P>0.05).The proliferation ability of pc DNA-THBS1-treated H1975 cells was significantly higher than that of untreated H1975 cells and empty vector pc DNA3.1 transfected H1975 cells(P<0.05),while untreated H1975 cells and empty vector pc DNA3.1 Compared with H1975 cells,the proliferation ability of H1975 cells was not different(P>0.05).5.The ratio of G0 / G1 phase cells in THBS1 si RNA group was significantly higher than that in untreated group and control group(P =0.000).The ratio of S phase cells in THBS1 si RNA group was significantly lower than that in untreated group(P = 0.004).The ratio of G2 / M cells in THBS1 si RNA group was significantly lower than that in untreated group and control group(P=0.015).The ratio of G0 / G1 phase cells in pc DNA-THBS1 group was significantly lower than that in untreated group and empty vector group(P=0.000).The percentage of cells in S phase of pc DNA-THBS1 group was significantly higher Higher than the untreated group and empty vector group,and the difference was statistically significant(P=0.000).However,there was no significant difference in the number of cells in the G2/M phase between the pc DNA-THBS1 group,the untreated group and the empty vector group(P>0.05).6.The activity of caspase-3 in THBS si RNA group was significantly higher than that in untreated group and control group(P= 0.001).The activity of caspase-3 in pc DNA-THBS1 group was significantly lower than that in untreated group and empty vector group,and the difference was statistically significant(P=0.001).7.The migration of THBS1 si RNA-transfected lung adenocarcinoma A549 cells was significantly inhibited compared with A549 cells transfected with untreated lung adenocarcinoma A549 cells and control si RNA.Compared with untreated lung adenocarcinoma H1975 cells and empty vector pc DNA3.1 transfected H1975 cells,the migration ability of pc DNA-THBS1 transfected H1975 cells was significantly enhanced.8.The expression of MMP-2 and MMP-9 protein in THBS1 si RNA group was significantly lower than that in untreated lung adenocarcinoma A549 cells and A549 cells transfected with control si RNA(P<0.05).The expression of MMP-2 and MMP-9 protein in pc DNA-THBS1 group was significantly higher than that in untreated H1975 and empty vector transfected H1975 cells,and the difference was statistically significant(P<0.05).The third part: THBS1 gene on the growth of nude mice transplanted tumor and its mechanismMethods 1.The lung adenocarcinoma cell line A549 cells of untreated group,control si RNA group and THBS1 si RNA group were injected subcutaneously into nude mice,and then the tumor size of each group was compared.2.The m RNA expression of THBS1 gene was detected by Western blotting and q RT-PCR in nude mice.3.The expression of THBS1,caspase-3,MMP-2 and MMP-9 in tumor tissues of nude mice was detected by En Vision immunohistochemistry.4.Statistical analysis: All data were statistically analyzed by SPSS 17.0 statistical software.Statistical data were expressed as mean ± standard deviation(SD),using chi-square and variance analysis,P<0.05 was significant.Results 1.The volume of THBS1 si RNA group was less than that of untreated group.The tumor volume of control group was significantly different between control group(P<0.05).2.The expression of THBS1 protein in THBS1 si RNA group was significantly lower than that in untreated group and control group(n = 20).The difference was statistically significant(P<0.05).3.The level of THBS1 m RNA in THBS1 si RNA group was significantly lower than that in untreated group and control group(P<0.05).There was significant difference between the two groups(P<0.05).4.The expression of caspase-3 in THBS1 si RNA group was higher than that in untreated group and control group.The expression of MMP-2 and MMP-9 in nude mice was lower than that in untreated group and control group.Conclusion 1.THBS1 gene is overexpressed in lung adenocarcinoma and is associated with invasion and metastasis of lung adenocarcinoma.2.The eukaryotic expression vector pc DNA3-THBS1 was successfully constructed;Lipofectamine 2000 transfected lung adenocarcinoma cells with eukaryotic expression vector pc DNA3-THBS1 or THBS1 si RNA(100 Nm)to lay the foundation for further study of the biological function and targeted therapy of THBS1.3.THBS1 si RNA or pc DNA3-THBS1 can down-regulate or up-regulate the expression of THBS1 m RNA and protein in lung adenocarcinoma cells.4.Down-regulation of THBS1 may promote the apoptosis of lung adenocarcinoma cells and inhibit the invasion and metastasis of lung adenocarcinoma.