| Objective:Lung cancer is the second most frequently diagnosed cancer and the leading cause of all cancer-related deaths worldwide,with a steadily increasing incidence.Approximately 85% to 90% of lung cancer cases are diagnosed as non-small-cell lung cancer(NSCLC).When possible,surgery is the best treatment strategy for NSCLC;however,even patients treated with curative intent may have local or systemic recurrence and ultimately succumb to the disease.The prognosis of patients with NSCLC principally correlates with tumor metastasis,which is associated with transcriptional regulation of certain critical genes;therefore,it is necessary to reveal these pro-metastatic genes and investigate their effects in detail.Notch signaling has been shown to participate in development and cell proliferation,differentiation,and apoptosis,and several studies have demonstrated that deregulation of the Notch pathway is involved in such malignancies as breast cancer and T cell leukemia.The role of Notch in lung cancer was experimentally demonstrated in a transgenic mouse model,where activated Notch overexpressed in the alveolar epithelium cooperated with the Myc pathway in promoting NSCLC development.Another study indicated that Notch1 suppressed p53-mediated apoptosis through the regulation of p53 stability and was required for the growth of lung adenocarcinoma.Notch signaling is activated by ligand-receptor binding involving five ligands(Delta-like 1,3,and 4,and Jagged-1 and-2)and four receptors(Notch1–4).Ligand binding induces a conformational change in Notch,leading to the exposure of the S2 site for the sequential cleavage by a protease of the disintegrin and metalloproteinase(ADAM)family and the gamma-secretase complex.As a result,the Notch intracellular domain(NICD)is liberated and translocated to the nucleus where it induces the expression of target oncogenes coding for cyclin D1,c-Myc,p21,p27,nuclear factor-kappa B(NF-?B),survivin,slug,and Nanog,which promote carcinogenesis.KIAA0247 is a novel gene encoding a 303-amino acid single-pass type I membrane protein,which is highly conserved among species.Although KIAA0247 is not mutated in cancers,it has been suggested to play a role in oncogenesis because of a p53-responsive element found in its promoter region,indicating that KIAA0247 is a prospective target for the tumor suppressor p53.KIAA0247 has also been named DRAGO(drug-activated gene overexpressed)because treatment with certain drugs(cisplatin,alkylating agents,antimetabolites,topoisomerase II inhibitors,taxanes,and nutlin-3)induces its expression in HCT116 p53+/+ cells but not in HCT116 p53-/-cells.KIAA0247 overexpression is associated with the therapeutic benefits of 5-fluorouracil,and the presence of KIAA0247 m RNA in fecal samples of colon cancer patients correlates with a more favorable prognosis.In ovarian cancer,advanced stage tumors express approximately 30% less KIAA0247 m RNA compared to levels in early stage tumors.Another study showed that KIAA0247 m RNA was downregulated in glioma compared to normal brain tissue,whereas KIAA0247 overexpression suppressed the proliferation and angiogenesis of glioma cell lines and promoted apoptosis through inactivation of the AKT and Stat3 signaling pathways.The KIAA0247 gene is located on human chromosome 14q24.1,which also contains the SERPINA1 gene responsible for ?1-antitrypsin deficiency that leads to lung tissue damage,pulmonary emphysema,and lung cancer.However,the biological function of KIAA0247 in lung cancer is currently unclear,and there are no data regarding the KIAA0247 expression pattern or its clinical significance in NSCLC.In this study,we investigated the role of KIAA0247 in NSCLC by examining KIAA0247 m RNA and protein expression in cancer tissues by real-time PCR and immunohistochemistry.We also analyzed the effects of KIAA0247 levels on the proliferation,migration,and invasion of lung cancer cell lines and explored the underlying molecular mechanisms.Methods:1.immunohistochemical staining experiments were conducted in 197 cases of non-small cell lung cancer from 2013 to 2015 in the First Affiliated Hospital of China Medical University.All 197 patients who participated in this pathological analysis were not treated with chemotherapy or radiotherapy before the operation.Male patients(N=127),female patients(N=70),aged between 30 and 90 years old.According to the International Union against cancer(Union for International Cancer Control,UICC)Eighth Edition TNM staging system,according to the pathological information of patients with stage I(N=83),phase II and III patients(N=114 cases).According to the WHO classification system,histological diagnosis and histological differentiation were seen in squamous cell carcinoma(N=101 cases),adenocarcinoma(N=96 cases),moderately differentiated to highly differentiated(N=126 cases),and poorly differentiated(N=71 cases).Tumor size classification,less than 3cm cases(N=108 cases),more than 3cm cases(N=89 cases).Cases of lymph node metastases(N=84 cases)and non metastasis of lymph nodes(N=113).Through immunohistochemical experiments,staining detection,staining intensity and staining cells of all sections of the percentage,semi quantitative scoring evaluation,analysis,clinical data were double blind,which,according to the staining intensity,the expression level of KIAA0247 is set to the score:0 points(not expressed),1 points(weak expression,light yellow particles),2 points(moderate expression,yellow particles),3 points(strong expression,brown granules particles);staining cell percentage evaluation score: 0 points(0%),1(1%-30%),2(31%-70%),3(71%-100%).In a single sample pathological section,the scores of staining intensity and the percentage of staining cells were multiplied.The final score was between 0 and 9 points,and the final product score was used for statistical analysis.Tumor samples with pathological score larger than three were identified as KIAA0247 positive,and those whose tumors were less than or equal to three were identified as KIAA0247 low expression.The SPSS 16.0 software was used to analyze the data.The single sample paired t test was used to analyze the statistical relationship between KIAA0247 expression level and clinicopathological factors.The P value is less than 0.05 as the standard to define whether there is statistical significance.2.We randomly selected from 35 cases of surgical operation in fresh non-small cell lung cancer specimens,including tumor tissues and adjacent normal lung tissue,real-time PCR detection of KIAA0247 m RNA expression by using SPSS 16.0 software,selection of t test method of single sample pairing,whether analysis of KIAA0247 m RNA level has clinical pathological correlation.The Kaplan-Meier database was used to analyze the correlation between KIAA0247 and the prognosis of lung cancer patients.3.The experimental group purchase of non small cell lung cancer cell lines commonly used from Shanghai Institute of cell library,according to the official website,RPMI1640 culture medium from GIBICO containing 10% fetal bovine serum,anhydrous glucose,sodium bicarbonate,sodium pyruvate,cultured H292,H1299,A549,H460,H661 cell lines,using medium GIBICO MEM,containing 10% fetal bovine serum,anhydrous glucose,sodium bicarbonate,sodium pyruvate,cultured SK-MES-1 cells,normal human bronchial epithelial cell line HBE cell culture medium using Dulbecco modified Eagle(DMEM)high blood glucose.All cells were cultured in flasks with filter holes in the sterile,were cultured in 6-well plates in aseptic processing.4.Cell climbing pieces into the 24 cell culture plate,cultured with different kinds of cell lines,fixed with 4% paraformaldehyde for 10 to 15 minutes,with 0.1% Triton X-100 membrane perforated 10-15 minutes,blocked for two hours with non immune fetal bovine serum albumin,KIAA0247 antibody overnight at 4 degrees on fertility,TRITC treated for two hours,finally under dark conditions DAPI staining.Under the fluorescence microscope,the target light was excited and the location and relative expression of KIAA0247 in various cell lines were observed.5.By using Lipofectamine 3000 to transfer intracellular KIAA0247 si-RNA and KIAA0247 plasmids to down-regulate or up-regulate the expression of KIAA0247 target protein,and provide a basis for subsequent cell function experiments.6.In the sterile cell cell cycle synchronization 6-well culture plate,down-regulated or up-regulated expression of KIAA0247 protein cells were collected without mutual adhered cells,fixation,staining,changes of cell cycle by flow cytometry,analysis on the effect of KIAA0247 on cell cycle.7.In the sterile cell cell cycle synchronization 6-well culture plate,down-regulated or up-regulated expression of KIAA0247 protein cells was determined by cell counting,each with the same number,repeat the same hole,was inoculated into 96 well culture plates,five consecutive days,fixed time everyday with MTT,cell proliferation ability continuously.8.In the sterile cell cell cycle synchronization 6-well culture plate,down-regulated or up-regulated expression of KIAA0247 protein cells count among groups at the same number,repeat three times holes and inoculated into sterile six culture plate,cultured for10-15 days,until the cell colony formation,fixed and stained,count number and size of colony formation,analysis of cell colony forming ability in the role of KIAA0247.9.In the sterile cell cell cycle synchronization 6-well culture plate,down-regulated or up-regulated expression of KIAA0247 protein cells,cell counting,according to the group the same number counted into the Transwell chamber,the same time remove the Transwell chamber,fixation,staining,count the cells in down chambers.The effect of KIAA0247 on cell invasiveness was detected when the matrix was applied to the upper chamber.The effect of KIAA0247 on cell migration was not detected when no matrix adhesive was applied.10.After cut or increase the KIAA0247 protein expression cells,cells were collected by using NP40 lysis protein lysate,and collect the total protein of the cells were detected and analyzed by protein immunoblotting experiments related to cell proliferation,migration and invasion,looking for possible pathway.11.Using Western blot analysis,KIAA0247 may influence the biological behavior of lung cancer cell lines through Notch signal pathway.By using Notch signal pathway inhibitor DAPT,a specific inhibitor of the Notch signaling pathway,determined that KIAA0247 inhibited the verification of non-small cell lung cancer biological behavior through the Notch signaling pathway.Results: 1.KIAA0247 located in cytoplasm and nucleus.In clinical cases of lung cancer,the expression of KIAA0247 in lung cancer tissues of m RNA was significantly lower than that in adjacent normal lung tissue,analysis of clinical pathological correlation,the expression level of KIAA0247 protein and the degree of differentiation,lymph node metastasis and P-TNM staging have negative correlation,but with age,gender,tumor type,tumor size had no correlation.Kaplan-Meier analysis of KIAA0247 was associated with the prognosis of patients with lung adenocarcinoma.2.In non-small cell lung cancer cell lines,up-regulated and down-regulated the expression level of KIAA0247 protein.It was found that KIAA0247 could inhibit the proliferation of non-small cell lung cancer cell lines and inhibit the transformation of G1 to S phase.3.Up-regulation and down-regulation of KIAA0247 protein expression in non-small cell lung cancer cell lines showed that KIAA0247 could inhibit the migration and invasion of non-small cell lung cancer cell lines.4.Western blotting showed that KIAA0247 had the same effect on cell proliferation and migration and invasion function.It was found that it could inhibit the activation of Notch signaling pathway.5.After the action of Notch signaling pathway inhibitor DAPT,combined with functional tests and Western blotting analysis,we determined that KIAA0247 plays a role in inhibiting the biological behavior of non-small cell lung cancer cell lines through Notch signaling pathway.Conclusion: 1.Downregulation of KIAA0247 expression has clinical significance2.KIAA0247 inhibits migration and invasion of lung cancer cells3.KIAA0247 inhibits the growth of lung cancer cells.4.KIAA0247 negatively regulates the expression of proteins related to epithelial-mesenchymal transition(EMT)5.KIAA0247 inhibits tumorigenic behavior and downregulates expression of EMT proteins in NSCLC cells through Notch signaling... |
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