The Role Ofamyloid Precursor Protein-binding Protein 2(APPBP2) And Its Potential Mechanism In The Occurrence And Development Of Non-small Lung Cancer

Lung cancer is one of the most common malignant tumors and it's the leading cause of cancer related morbidity and mortality among malignant tumors in China and around the world.The current treatment of lung cancer is surgical resection,chemotherapy,radiotherapy,targeted therapy and immunotherapy.From the perspective of clinical treatment and biological characteristics,lung cancer can be divided into two categories:non-small cell lung cancer(NSCLC)and small cell lung cancer(SCLC).In recent years,the spectrum of lung cancer has shifted to adenocarcinoma,which has gradually taken the place of highest incidence from squamous.Adenocarcinoma is characterized by easy distant metastasis and recurrence and contributes 50%of incidence of NSCLC,which accounts for about 85%of all lung cancer.The majority of NSCLC cases are diagnosed at later stages with local invasion or distal metastases,consequently leading to poor effectiveness of surgical orother interventions.The 5 year survival rate was less than 20%.Early diagnosis and treatment can effectively prevent the recurrence and metastasis of lung cancer and improve survival rate and life quality.Currently,a mount of efforts are engage in the early diagnosis and early treatment of lung cancer,but the occurrence of lung cancer is the results of a complex biological process that involves many factors and the pathogenesis is still unclear.Therefore,exploring the abnormal genes in the process of lung cancer and identifying key molecules that regulate cell proliferation and apoptosis,have become the core connection in the study of lung cancer.The amyloid precursor protein-binding protein 2(APPBP2)gene is located in the 17q21-q24 region of chromosome and the encoded protein can specifically bind to the amyloid precursor protein(APP),which participate in the intracellular transport and secretion of APP.APP is a membrane protein which expressed in many tissues,among which concentrated in the synapse of neurons.In recent years,studies have found that APP is closely related to the occurrence and development of malignant tumors.Meanwhile,researchers detected sAPP,a fragment of APP,in the medium with cultured pancreatic cancer cells.The blocking of sAPP can inhibit the proliferation of pancreatic cancer cells,suggesting that APP is involving the biological process of pancreatic cancer.Studies also revealed that APP is closely related to the pathogenesis of oral and colon cancer.Above all,APP is involved in the pathophysiological processes of various malignant tumors.APPBP2 is a hydrophilic tubulin,which not only regulates the transport and secretion of APP,but also specifically binds to microtubules,affecting the activity and function of microtubules.Microtubules are the main components of the cytoskeleton and are present in all eukaryotic cells,which are involved in the assembly of spindles,centromeres,flagella,and neural tubes.The microtubules play critical role in the maintance of cell morphology,cell migration,cell mitosis,and intracellular transportation and signal transduction.The microtubules polymerize into a spindle in the early stage of cell division,which pulls the chromosome into the two daughter cells during the mitosis,thereby completing the cell proliferation process.Therefore,microtubules play a very important role in the proliferation of tumor cells.APP and microtubules play an important role in the pathogenesis of malignant tumors,and their activities and functions are affected by APPBP2.It is reasonable to hypothesize that APPBP2 participate in the process of malignant tumors.Studies have confirmed that APPBP2 is closely related to the malignant phenotype and prognosis of ovarian clear cell carcinoma.In breast cancer,APPBP2 was significantly up-regulated and involved in the invasion process of breast cancer.Moreover,the copy number of APPBP2 in neuroblastoma is significantly higher than that in adjacent tissues.The amplification of APPBP2 was also found in medulloblastoma.Thus,APPBP2 plays a role in the pathogenesis of various malignant tumors,but its role in the pathogenesis of lung cancer is still unclear.Therefore,this project focuses on the effects of APPBP2 in the biological process of lung cancer and underlying mechanisms.[Objective]1)To clarify the clinical correlation between APPBP2 gene and non-small cell lung cancer;2)To elucidate the role of APPBP2 gene in the process of non-small cell lung cancer;3)To reveal the molecular mechanism of APPBP2 in promoting the proliferation and invasion of non-small cell lung cancer.[Methods]1 The examination of the clinical correlation between APPBP2 and NSCLC(1)The expression of APPBP in NSCLC was analyzed based on GEO-LAUD data array.(2)Collection ofadenocarcinoma tissues and adjacent normal tissues.(3)RT-PCR assay for the measurement of mRNA level of APPBP2 between adenocarcinoma tissues and adjacent normal tissues.(4)Western blotting(WB)and immunostaing assays for the measurement of protein level of APPBP2 between adenocarcinoma tissues and adjacent normal tissues.2 The functional examination of APPBP2 in NSCLC2.1 The construction of APPBP2 silencing vector(RNAi,lentivirus backbone)(1)Design several RNAi sequence that targeting APPBP2 gene.(2)Synthesize double strand oligo for RNA-interfere,which flanked with restriction endonuclease.The oligo was cloned into the vector of RNAi.(3)Screen the most efficient RNAi vector for APPBP2 degradation.(4)Package sh-APPBP2 lentivirus and sh-NC lentivirus with the most efficient RNAi vector and control vector,respectively.The virus were qualified with titer determination.2.2 The infection of H1299 cells with RNAi virus(1)The H1299 cells with logarithmic phase are digested with trypsin and are suspended with culture medium.(2)The cells are cultured into 6-well.(3)Infection the cells with sh-NC or sh-APPBP2 according to the MOI while cell attach to the wall.(4)Evaluated the knockdown efficiency of APPBP2 with RT-PCR and Western blotting.2.3 The effects of APPBP2 to H1299 cell proliferation,migration,apoptosis and tumorigenesis(1)Employ sh-APPBP2 virus to silence the expression of APPBP2 in H1299 cells,and set sh-NC as control group.(2)Employ BrdU assay and MTT assay to detect the proliferation between experimental and control group.(3)Employ clone formation assay to detect the ability of tumorigenesis between experimental and control group.(4)Employ PI staining flow cytometry assay to value cell cycle between experimental and control group.(5)Employ Annexin Vstaining flow cytometry assay to value cell apoptosis between experimental and control group.(6)Employ transwell assay and wound healing assay to examine the abilities of migration and invasiveness between experimental and control group.2.4 Repeat the experimental schemes from 2.2 to 2.3 on A549 cells and further study the biological functions of APPBP2.2.5 Tumor formation in nude mice(1)Culture sh-APPBP2 or sh-NC infected NSCLC cells and resuspend cells.(2)Transplant cells into the underarm of the right forelimb of nude mice..(3)Value the tumor burden at 5?7 days post injection.Measure the tumor size after tumor formation.(4)The mice were sacrificed at 28?30 days post injection and the tumor was isolated for measurement of body weight.(5)Tumor volume and tumor weight were compared between the experimental group and control group.3.Explore APPBP2 closely correlated signal pathway3.1 Screen genes associated with APPBP2 in NSCLC(1)Screen APPBP2 associated genes from TCGA-LAUD array.(2)Confirm the clinical relation between APPBP2 and candidates in NSCLC sample.(3)Employ sh-APPBP2 virus to silence the expression of APPBP2 in H1299 cells,and set sh-NC as control group.(4)Employ RT-PCR and western blotting assay to examine the regulation of APPBP2 to candidate genes.3.2 Study the binding between APPBP2 and associated genes by co-immunoprecipitation(co-IP)3.3 Study the mediation of associated genes on the effects of APPBP2 biological functions by gain of function strategy(1)Overexpress associated genes in sh-APPBP2 infected H1299 cells and sh-NC infected H1299 cells.(2)Employ clone formation assay to detect the function of associated genes in regulating tumorigenesis of APPBP2 silenced H1299 cells.(3)Employ MTT assay to detect the function of associated genes in regulating the proliferation of APPBP2 silenced H1299 cells.(4)Employ Annexin Vstaining/flow cytometry assay to detect the function of associated genes in regulating the apoptosis of APPBP2 silenced H1299 cells.(5)Employ transwell assay to detect the function of associated genes in regulating the migration and invasiveness of APPBP2 silenced H1299 cells.3.4 Repeat the experimental schemes from 3.1(3)to 3.3 on sh-APPBP2 infected A549 cells and sh-NC infected A549 cells.To further study the mediation of associated genes on the effects of APPBP2 biological functions.[Results]We employed immunostaining,RT-PCR,and Western blotting to determine the expression level of APPBP2,PPM1D,and SPOP in NSCLC tissues and adjacent normal tissues.The results indicated that the expression of APPBP2,PPM1D,and SPOP are significant upregulated in NSCLC tissues compare with normal tissues.The expression level of APPBP2 is positive correlated with the expression of PPM1D and SPOP in NSCLC.The functional results of cell biology indicated that ectopic expression of APPBP2 significantly promoted NSCLC cell proliferation,migration,and invasiveness,but inhibit the apoptosis of NSCLC cells.The knockdown of APPBP2 decreased the expression of PPM1D and SPOP,resulting the inhibiting of NSCLC cell proliferation and the inducing of cell apoptosis in vitro.Meanwhile,the inhibition of APPBP2 could slow the migration and invasion of NSCLC cells and reduce the proliferation of NSCLC cells in a natural environment.Moreover,overexpression of PPM1D and SPOP attenuated APPBP2-knockdown inhibition of cells proliferation,migration and invasiveness in NSCLC.[Conclusion]This study shows that the expression level of APPBP2 is positive correlated with NSCLC cell proliferation,migration,and invasiveness.APPBP2 promotes the progression of NSCLC through regulating PPM1D and SPOP signal pathway.This study not only provides an novel molecular mechanism underlying NSCLC,but also supports that APPBP2 can act as a potential diagnostic biomarker and target for intervention in NSCLC.