| Background and PurposePrimary lung cancer is the most common malignant tumor of the respiratory tract and its pulmonary glands,which seriously endangers human life and health due to its high morbidity and mortality as well as insidious symptoms and rapid development.In China,the incidence rate of lung cancer is 17.9% and the mortality rate is 23.8%,which is the highest incidence and mortality rate of malignant tumors in China and the highest number of incidence and death in the world.In recent years,the causes of lung cancer have been diversified.In addition to smoking,lung cancer is triggered by factors such as secondhand smoke,air pollution,occupational exposure and genetic factors.Lung cancer is a complex multi-genetic and multi-factorial disease induced by the combination of environmental pathogenic factors and genetic factors.The prognosis of lung cancer treatment is closely related to the pathogenesis cycle,and early diagnosis and early treatment are still the best treatment principles,however,the current means of lung cancer diagnosis and treatment have not yet reached the ideal effect.Therefore,an in-depth study of the abnormal genes and oncogenic signaling pathways associated with the development of lung cancer can help us to better prevent and treat the disease.Fibrillin 2(FBN2)is an important component of microfibrils and is involved in the formation of elastic fibers in human connective tissues.abnormal expression of FBN2 gene is associated with various genetic connective tissue diseases,including congenital contracture subarachnoid effusion(CCA),macular degeneration(MD)and myopathy.With the development of bioinformatics research techniques,FBN2 is thought to be closely related to cancer development,with abnormal expression in a variety of cancer tissues including esophageal,colon,and ovarian cancers,and closely associated with their survival prognosis.On this basis,we hypothesized that the FBN2 gene may play a broad pro-cancer role in the development and progression of lung cancer.In this project,we analyzed the relationship between lung cancer and FBN2 expression and its relationship with prognosis by gene sequencing combined with bioinformatics database in the early stage,to clarify the potential of FBN2 as a diagnostic marker and therapeutic target for lung cancer,and to further analyze the mechanism of FBN2’s role in lung cancer development and progression.Part I.Abnormal expression of FBN2 gene in lung cancer patients Objective:To explore gene expression differences in lung cancer clinical samples,search and study abnormal genes associated with the development and malignant progression of lung cancer,and provide diagnostic markers and therapeutic targets for the diagnosis and treatment of lung cancer.Methods:In this study,multiple gene mutation profiling was performed by whole exome sequencing on five clinically collected lung tumor samples and corresponding paraneoplastic tissues to explore their relationship with clinicopathological features and to search for mutated genes that could be used as diagnostic and therapeutic targets for lung cancer;the m RNA transcript levels of FBN2 gene in 97 lung cancer tissues and corresponding paraneoplastic tissues were detected by fluorescence quantitative PCR;lung cancer tissues embedded in paraffin The correlation between FBN2 protein expression levels and patients’ basic conditions(age,gender,tumor size,TNM stage,lymph node metastasis,histological type,differentiation degree and smoking history)was analyzed through paraffin-embedded lung cancer tissue sections and immunohistochemistry experiments;the TCGA database and GTEx The association of FBN2 gene expression differences and prognostic survival between lung adenocarcinoma and lung squamous carcinoma tumor tissues and normal tissues were compared and analyzed by TCGA database and GTEx database.Meanwhile,the actual survival of 97 lung cancer patients was recorded and analyzed to screen for independent risk factors affecting lung cancer progression by COX method.ROC curves of FBN 2 and common lung cancer serum markers(NSE,SCC,CA50,CEA,VE GF)for the lung cancer diagnosis.Results: The results of whole-exome sequencing analysis showed that missense mutations with C-T substitution dominated in early lung cancer,and the genes with the highest mutation frequency were TTN,OBSCN,SRPX,PIEZO1,PCNXL2,NEB,MDN1,MACF1,FBN2 and BRCA2;based on the analysis results and literature research,this study concluded that FBN2 was associated with tumorigenesis and development were closely correlated.The results of fluorescence quantitative PCR experiments showed that the m RNA level of FBN2 gene was significantly elevated in lung tumor tissues(P<0.05);The results of immunohistochemical analysis showed that FBN 2 was highly expressed in lung tumor tissue,mainly distributed at the edge of cancer cells,and had significant correlation with TNM stage,lymph node metastasis and histological type,SCC and VE GF,suggesting that the expression of the gene may be correlated with the growth and proliferation of tumor cells(P <0.05).The results of TCGA database and GTEx database analysis showed that the expression of FBN2 was significantly higher in lung squamous carcinoma tissues,while there was no significant difference in lung adenocarcinoma tissues,and furthermore,the high expression of FBN2 gene was positively correlated with the prognostic survival of lung cancer patients.Analythe actual survival of 97 patients,the results showed that different TNM stages,VE GF expression level,FBN 2 expression level,and distal metastasis were significantly associated with progression-free survival(Progression-free survival,PFS),with FBN 2(-)having PFS than FBN 2(+)[(34.57 ± 0.65)vs(24.71 ± 0.97),P <0.05].COX analysis showed FBN 2 as an independent risk factor for the prognosis of lung cancer(P <0.05).The ROC curve of FBN 2 and common lung cancer serum markers(NSE,SCC,CA50,CEA,VE GF)for diagnostic lung cancer showed that the diagnostic value of NSE and SCC was high(AUC =0.81,P <0.01),followed by the diagnostic value of CA50 and FBN 2(AUC=0.80,P <0.01).Part II.Study on the effect and mechanism of FBN2 gene on the malignant biological function of lung cancer cells Objective:The expression of FBN2 was found to be significantly correlated with the prognosis of lung cancer.To further investigate the mechanism of FBN2 in lung cancer and to clarify the potential of FBN2 as a tumor marker,the following experiments were conducted in this study.Methods: The m RNA and protein expression levels of FBN2 gene in lung cancer cell lines(PC-9,H1975,H1640,H441,A549)and human normal lung epithelial cells BEAS-2B were detected by PCR assay and Western blot assay;FBN2 knockdown PC-9 cell line and H1640 cell line were constructed and quantified by real-time fluorescence PCR and Western blot assays were performed to verify the knockdown results;the effects of FBN2 gene on lung cancer cell growth,cell proliferation and cell metastasis and invasion were determined by Tissue blue staining,clone formation and Transwell assays;the knockdown of FBN2 gene in PC-9 and H1640 cell lines were investigated by quantitative real-time fluorescence PCR and Western blot assays.The effects of knockdown of FBN2 on the FAK/ERK signaling pathway activity in PC-9 cells were investigated by real-time fluorescence PCR and Western blot;the effects of knockdown of FBN2 on PC-9 cells were investigated by nude mice subcutaneous transplantation tumor experiment.The effect of FBN2 knockdown on the proliferation ability of PC-9 cells in vivo was investigated by subcutaneous transplantation of nude mice.Results: The results of real-time fluorescence quantitative PCR and Western blot assays showed that the expression levels of FBN2 gene in lung cancer cell lines were significantly higher than those in human normal lung epithelial BEAS-2B,with the highest expression in H160 and PC-9 cells.The results of Taiban blue staining assay,clone formation assay and Transwell assay showed that knockdown of FBN2 gene inhibited the growth,proliferation,metastasis and invasion of lung cancer cells,and the specific mechanism may be through the inhibition of EMT-related molecule expression and FAK/ERK signaling pathway activity in cancer cells.In addition,the results of subcutaneous transplantation tumor experiment in nude mice showed that knockdown of FBN2 gene inhibited the proliferation of lung cancer cells in vivo.Conclusion: FBN2 regulates the EMT of lung cancer cells through the FAK/ERK signaling pathway and is thus involved in their proliferation and metastasis.Summary:The results of this study showed that knockdown of FBN2 significantly inhibited the phosphorylation of FAK protein,which in turn affected the activation of the downstream molecule MEK/EKR.In addition,in vivo nude mouse experiments further confirmed that knockdown of FBN2 significantly inhibited the growth rate of tumor cells in vivo.Thus,our data suggest that FBN2 could be a predictive indicator or therapeutic target for lung carcinogenesis and development. |
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