| BackgroundLung cancer is one of the most common malignant tumors in the world.Lung cancer is the leading cause of cancer death in the United States.The incidence and mortality of lung cancer in China are the first malignant tumor,global cancer data survey shows that Chinese lung cancer patients account for the highest proportion of lung cancer patients in the world,and the incidence is still rising in recent years.Lung cancer is divided into adenocarcinoma(mainly including papillary adenocarcinoma,bronchoalveolar carcinoma and mixed adenocarcinoma),squamous carcinoma,small cell lung cancer,large cell lung cancer,sarcomatoid carcinoma,carcinoid carcinoma,carcinosarcoma,adenosquamous carcinoma and so on.Clinically,lung cancer is mainly divided into two categories:Small cell lung cancer(SCLC)and non-small cell lung cancer(NSCLC),in which Small cell lung cancer accounts for 20%-50%of lung cancer,with extremely poor differentiation and early metastasis.The resection rate was low and the sensitivity of radiotherapy and chemotherapy was high.Non-small cell lung cancer is the most common type of lung cancer,lung cancer accounted for about 80%,compared with small cell lung cancer,non-small cell lung cancer grows slower,transfer relatively late,the degree of differentiation from low to high,are squamous cell carcinomas of the most common(30%-40%),followed by adenocarcinoma(accounts for 20%-40%),and the rest of bronchioloalveolar carcinoma and large cell carcinoma and mixed.Non-small cell lung cancer has a high surgical resection rate and low sensitivity to radiotherapy and chemotherapy.The pathogenesis of this disease has not been fully clarified,which increases the difficulty of clinical treatment.In addition,the diversity of tumor biological behavior,although the clinical diagnosis and treatment of the disease is constantly updated,the survival rate of patients is still very low.At present,surgery and chemotherapy are still the main treatment methods for lung cancer,but most patients are in the middle and late stage after diagnosis,missing the best opportunity for surgical treatment.The median survival period of patients with first-line chemotherapy is short,about 9-10 months.Therefore,actively seeking effective treatment for lung cancer plays a very important role in improving the survival rate of patients.With the continuous development of medical technology and in-depth research on the mechanism of lung cancer,targeted therapy has become a new trend in the treatment of lung cancer in recent years,and its clinical effect is remarkable.In view of the poor prognosis of advanced non-small cell lung cancer(NSCLC),it is important to adopt a reliable diagnostic method for early clinical treatment of the disease.Human Wnt(Wingless-Int1,Wnt)gene family consists of 19 members,encoding conserved glycoproteins with 22 or 24 cysteine residues.Wnt gene was first discovered in 1982 as the preferred integration site of mouse breast tumor virus,which can transmit information of proliferation and differentiation between cells and is an oncogene.It is a homologous gene with drosophila wingless gene,so it was named Wnt gene.With the deepening of research,it was found that the Wnt family is highly conserved,with 83%homology from lower animals to human beings.The proteins encoded by Wnt can initiate intracellular signaling pathways,conduct growth-stimulating signals,participate in development,cell differentiation and proliferation.Wnt signaling consists of three important steps:first,the specific binding of Wnt ligand to cell membrane receptor,and second,the initiation of intracellular cascade reactions to regulate gene expression in the nucleus.Studies have found that Wnt interlocks with other cell functions and signal transduction to form a network regulation mode,the main framework of which is as follows:1.Canonical Wnt/β-Cantenin Pathway(Wnt/β-Cantenin pathway)is a typical Wnt/β-Cantenin signaling pathway that activates transcriptional activity of target genes through nuclear translocation of β-Cantenin.2.The Planar Cell Polarity Pathway(PCP):regulates cytoskeletal rearrangement during embryonic development and is involved in gastrum formation.No tumor events have been reported to date.3.The Wnt/Ca2+ pathway,activated by Wnt5a and Wntl 1,activates Protein kinase C(PKC),Phospholipase C(PLC)and transcription factor NF-AT by increasing intracellular calcium content.The Wnt/Ca2+ pathway and Wnt/β-Cantenin signaling pathway interact with each other and are associated with tumorgenesis,but the specific process and mechanism remain unclear.Wnt signaling is a complex process composed of many factors,involving many links and regulation.Normal cell growth and development have certain rules,usually the Wnt signaling pathway is closed,when activated,cell differentiation,apoptosis and proliferation will appear multiple changes,which is also one of the reasons for tumor formation.The Wnt signal enters the cell and transfers the signal to Dishevelled protein(Dvl/Dsh),which inhibits Axin,adenomatous polyposis coli,APC)and glycogen synthase kinase 3β(GSK-3β)complex,which does not phosphorylate β-Cantenin.As non-phosphorylated β-cantenin cannot be degraded by intracellular protease,β-Cantenin accumulates in the cell and travels to the nucleus,where it interacts with T cell factor/lymphoid enhancer factor(T cell factor/lymphoid enhancer factor,TCF/LEF),TCF/LEF binding activates TCF transcription and regulates the expression of target genes.Therefore,whether β-Cantenin is phosphorylated is the key to whether Wnt signaling pathway can play a role.β-Cantenin-TCF/LEF complex is a key link in Wnt signaling.β-Cantenin is transferred into the nucleus and binds to TCF/LEF to activate Wnt signaling,resulting in massive accumulation of β-Cantenin-TCF/LEF.Abnormal accumulation ofβ-Cantenin activates TCF/LEF,resulting in transcription of downstream target genes and promoting cell cycle development to produce abnormal proteins,which ultimately lead to cell carcinogenesis.A large number of studies have found thatβ-Cantenin gene mutations exist in a variety of tumors,among which the mutation rate of thyroid cancer,colon cancer and ovarian cancer can be as high as more than 50%.The activity status of β-Cantenin is also affected by upstream Wnt molecules,mainly APC,GSK-3β and Axin.Similarly,the upregulation of Wnt3 in NSCLC is also closely associated with the survival rate of NSCLC patients,but the tumorigenic mechanism of Wnt3 remains unclear.PurposeBy studying the expression of Wnt3 in non-small cell lung cancer tissues,the relationship between Wnt3 and non-small cell lung cancer was clarified,and the expression of Wnt3 in non-small cell lung cancer was inhibited by RNAi technology,so as to further study the role of Wnt3 in non-small cell lung cancer cell differentiation,cell cycle and apoptosis.In this paper,the research results of new drug targets for non-small cell lung cancer to provide basic experiment results,the experimental results can be directly applied to the new drug development and clinical treatment of non-small cell lung cancer,to improve the therapeutic efficacy of non-small cell lung cancer chemotherapy drugs,reduce the pain of patients and families,reduce the burden of social economy is of great importance.MethodsThe first part1.1 Collection and fixation of pathological tissues and adjacent tissues of NSCLCAll experimental lung cancer tissues were reviewed by the ethics Committee of Southern Medical University and the ethics Committee of PLA Eastern Theater Command General Hospital(the former Nanjing Military Command General Hospital).All subjects are patients signed the informed consent form,from February 2014 to June 2015 were 30 cases of non-small cell lung cancer tissue adjacent to carcinoma of pathological tissue and its matching(tissue adjacent to carcinoma is 1 cm based on the edge of the tumor tissue),pathological tissue based selection without chemotherapy,radiation therapy,and other drug treatment of patients with lung cancer tissues and tissue adjacent to carcinoma.After sampling,all specimens were quickly frozen with 1%formaldehyde solution or liquid nitrogen and stored in a-80℃ refrigerator.All experimental lung cancer tissues were clinically and pathologically classified.1.2 Immunohistochemical staining of Wnt3 in pathological tissuesNon-small cell lung cancer tissues were fixed with 4%formaldehyde,after fixation,paraffin was fixed in thin sections,and stained with Wnt3 immunohistochemical reagent.The basic procedure of immunohistochemical experiment was as follows:Tissue cell samples were repaired by antigen to block endogenous peroxidase,then sealed with serum,followed by dropping specific primary antibody,then dropping secondary antibody,dropping three antibodies and chromogenic agent,and finally sealed after restaining.The sections were immersed in citrate buffer for 15 min to fully combine the antigen.The antibody was incubated with mouse anti-human Wnt3 antibody at 37℃ for 1h,and then incubated with horseradish peroxidase-anti-mouse antibody at room temperature for 30 min.Then the Wnt3 antigen antibody conjugate was stained with hematoxylin.1.3 Protein isolation and western blot reactionFrom non-small cell lung cancer cells using cell dissolved buffer and protease inhibitors,extraction of protein in the first place with 10%SDS-PAGE gel electrophoresis separation,membrane to nitrocellulose membrane,then with 2%bovine serum albumin sealing membrane 4℃ for the night,with mouse anti human Wnt3 antibody incubation glyceraldehyde antibody(phosphoric acid as the control group),37℃ incubation for 1 hour,The bands were then incubated with horseradish peroxidase complex anti-mouse antibody for 40 min at room temperature,and washed with washing solution after each incubation.Finally,the bands were detected by electrochemiluminescence system.The second part2.1 cell culture and differentiationHuman non-small cell lung cancer A549 cells were cultured in DMEM medium.Culture with 10%calf serum in 37℃ humid 5%C02 incubator.When the cell concentration was 85%,the cells were isolated and cultured in a new culture flask.The cells were interfered with cisplatin,and 2uM cisplatin was directly added to the medium.2.2 real-time fluorescence quantitative PCR analysis of Wnt3 mRNA levelsTRIzol reagent mRNA,extracted from lung cancer A549 cells in the process of extraction using RNase inhibitor,Wnt3 mRNA was analyzed by fluorescence quantitative PCR,Wnt3 mRNA level and internal quality control glyceraldehyde 3-phosphate were detected,and ΔΔCt relative quantity was calculated.2.3 Protein isolation and western blot reactionProteins were extracted from A549 cells using cytosolic buffer and protease inhibitors.The proteins were separated by 10%SDS-PAGE gel electrophoresis and then transferred to nitrocellulose membrane.The membrane was then sealed with 2%bovine serum protein at 4℃ overnight and incubated with mouse anti-human Wnt3 antibody(phosphoglyceraldehyde antibody as the control group)at 37℃ for 1 hour.Then,horseradish peroxidase combined anti-mouse antibody was incubated for 40 min at room temperature,and the bands were washed with washing solution after each incubation.Finally,the bands were detected by electrochemiluminescence system.The third part3.1 Cell culture and differentiationHuman non-small cell lung cancer A549 cells were cultured in DMEM m edium with 10%calf serum in a humid 5%CO2 incubator at 37℃.When th e cell concentration was 85%,the cells were isolated and cultured in a new c ulture flask.The cells were interfered with cisplatin,and 2uM cisplatin was di rectly added to the medium.3.2 Cell proliferation and clonal formation experimentsCell proliferation was detected by CCK:Cell proliferation of A549 cells w as detected by CCK-8.Cells were transfected with Wnt3-specific siRNA(siRN Al/2-Wnt3)and control siRNA(siRNA-Ctrl)at 0,24,48 and 72 hours,then 10μL CCK-8 reagent was added.The absorbance at 450nm was measured by s pectrophotometry(A)after incubation at 37℃ with 5%CO2 for 2 hours.Cell proliferation rate was calculated.Cell proliferation rate=(transfected progenitor c ell A/control cell A)× 100%.Plate clone formation experiment:A549 cells were planted on 12-well cell plates and transfected with 50 nM siRNA1/2-Wnt3 and siRNA-Ctrl.The cells were incubated for 72 hours,and the cell clones were stained with 0.007%c rystal violet for 10 min.The clone formation rate was detected by UVP biosp ectroscopic 500 imaging system.3.2 Cell cycle and apoptosis were detected by flow cytometryMain steps of cell cycle experiment:A549 cells were digested with 0.25%EDTA trypsin,incubated with 700 μL buffer and 7-amino-actinomycin at 37℃ for 10 min,then 300 μL buffer was added with 2ul endocin-V-phycored protein,and A549 cells were fixed with 70%alcohol at 4℃ for 2 h.The cells were incubated with 0.5 mg/mL RNase A37℃ for 10 min,then 5μL propidium iodide was added and incubated at room temperature for 30 min.The cell cycle was detected by flow cytometry according to the practical instructions.Apoptosis analysis procedures:A549 cells were added 500 μL buffer and 1μL 7AAD and incubated at room temperature for 15 min,450 μL buffer and 7-amino-actinomycin were added and incubated at 37℃ for 15 min.Apoptosis was detected by flow cytometry,and the data were analyzed by FlowJo software.3.3 Protein isolation and western blotting reactionThe proteins extracted from A549 cells with cytosolic buffer and protease inhibitors were separated by 10%SDS-PAGE gel electrophoresis,then transferr ed to nitrocellulose membrane,and then sealed with 2%bovine serum protein at 4℃ overnight.Mouse anti-human Caspase 9 and Caspase 3 antibodies(phos phoglyceraldehyde antibody as the control group)were incubated at 37℃ for 1 hours,and then at room temperature for 40min with horseradish peroxidase co mplex anti-mouse antibodies.After each incubation,the bands were washed wit h washing solution.Finally,the bands were detected by electrochemiluminescen ce system.3.4 Tumor implantation in nude micePerform aseptic operation on ultra-clean worktable,disinfect with peracetic acid or negeramine,and wear protective clothing.Sterile disinfection was performed according to operating room operating procedures,and a number of non-small cell lung cancer cells(8 normal A549 cell group,16 transfected with Wnt3-specific siRNA(siRNA 1/2-Wnt3)A549 cell group)were injected into the right hind limb of nude mice with an injection dose of 0.4 mL.1 × 108 cells were contained.When the tumor size reached 15-20mm,the successfully modeled mice were randomly divided into 3 groups(normal A549 cell group;Transfection group;Transfection+ cisplatin chemotherapy group,in which the first two groups were injected with the same volume of normal saline),8 mice in each group,including cisplatin(cischloroamplatin,1.0mg/kg,subcutaneously administered once a day),were sacrificed at the same time on the 9th day,and the tumor tissues were separated to compare the tumor volume between different groups.ResultsThe first part1.1 ImmunohistochemistryNon small cell lung cancer,according to the results of immunohistochemical Wnt3 positive expression,compared with the tissue adjacent to carcinoma,Wnt3 in non small cell lung cancer tissues expression,by comparison,the difference is statistically significant(p=0.0037).1.2 Expression of Wnt3mRNA and protein in non-small cell lung cancer tissuesTo study Wnt3 signaling pathways in the role of non small cell lung cancer microenvironment,we tested the non small cell lung cancer tissues Wnt3 mRNA level and protein expression,Wnt3 at mRNA level in non small cell lung cancer tissues were higher than tissue adjacent to carcinoma,by comparison,the difference is statistically significant(p=0.0018),western blot,according to the results of Wnt3 protein expression levels were higher than tissue adjacent to carcinoma,by comparison,the difference is statistically significant.The second part2.1 Expression of Wnt3 protein in A549 cells of NSCLCIn order to study the Wnt3 signaling pathways in the role of non small cell lung cancer A549 cells,we tested the non small cell lung cancer A549 cells Wnt3 protein expression,western blot,according to the results of Wnt3 protein expression levels were higher than the control group,by comparison,the difference is statistically significant.2.2 Expression of Wnt3 protein in A549 cells of NSCLCIn order to study the Wnt3 signaling pathways in the role of non small cell lung cancer A549 cells,we further tested the non small cell lung cancer A549 cells Wnt3 mRNA level,Wnt3 in non small cell lung cancer A549 cells mRNA levels were higher than control group,by comparison,the difference has statistical significance(p=0.0018).The third part3.1 Wnt3-siRNA transfection of non-small cell lung cancer A549 cells inhibited cell differentiationTo study Wnt3 in the role of non small cell lung cancer,we synthesized specificity Wnt3-siRNA plasmid,transfection into non small cell lung cancer A549 cells,30 and 60 nM transfection Wnt3-specific siRNA to non-small cell lung cancer A549 cells,compared with control group siRNA-Ctrl,cell transfection group Wnt3 significantly lower,including 60 nM Wnt3-specific siRNA transfection cells Wnt lowest,1/2-Wnt3-and at the same time,we measured the siRNA siRNA Ctrltransfection after A549 cell growth curve,we found that the siRNA 2-1-Wnt3-and siRNA Wnt3-transfection A549 cell,its growth rate was lower than those of siRNACtrl-carrying A549 cells,by comparison,the difference is statistically significant.In order to further validate Wnt3 inhibiting effect on A549 cell differentiation,we have three transfection group the siRNA 2-1-Wnt3-and siRNA Wnt3-and siRNA-Ctrl-transfection A549 cell group compare 60 nM siRNA 1-Wnt3 transfection group(p<0.01)and siRNA 2-Wnt3(p<0.05)transfection group cell clone rate decreased significantly,by the comparison of difference is statistically significant.3.2 Effects of Wnt3-siRNA transfection on cell cycle of non-small cell lung cancer A549 cellsIn order to further validate Wnt3 inhibition of non small cell lung cancer cells after the influence on cell cycle,using flow cytometry instrument to detect the cell cycle,and siRNA Ctrl-transfection A549 cell group,siRNA 2-1-Wnt3 group and siRNA Wnt3 group cells S phase proportion is less,comparison between the three groups,statistically significant differences,two groups of G0/G1 cell proportion increase,but there was no statistically significant difference.3.3 Effects of Wnt3-siRNA transfection on apoptosis of non-small cell lung cancer A549 cellsIn order to further validate Wnt3 inhibition of non small cell lung cancer cells after the influence on cell apoptosis,2 microns platinum suitable under the disturbance of chemotherapy drugs,compared with blank control group,siRNA 2-1Wnt3 group and siRNA Wnt3 group induced apoptosis rate higher,then we check the cell related protein expression changes of western blot results show that compared with the blank control group,siRNA2-1-Wnt3 group and siRNA Wnt3 caspase9 and caspase3 groups of cells increased.3.4 Changes in tumor volume of Wnt3-specific siRNAs(siRNA 1/2-Wnt3)transfected cells and cisplatin treated miceAfter comparison,the tumor volume of the three groups increased gradually with the extension of the survival time of mice.Further comparison,Tumor volume transfected cells+cisplatin group<Transfected cells<Normal A549 cell group.Conclusion1.Compared with the adjacent tissues,the immunohistochemistry Wnt3 of non-small cell lung cancer was positively expressed.2.The mRNA levels of Wnt3 in non-small cell lung cancer tissues were higher than those in adjacent tissues.Wnt3 protein expression levels were higher in non-small cell lung cancer tissues than in adjacent tissues.3.High expression of Wnt3 mRNA and protein in A549 cells of NSCLC.4.When transfected with Wnt3-specific siRNA into non-small cell lung cancer A549 cells,the cell Wnt3 was significantly decreased,among which the higher the transfection dose was,the lower the Wnt3 was,indicating a quantitative dependence.5.The growth rate of non-small cell lung cancer A549 cells transfected with siRNA 1-Wnt3 and siRNA 2-Wnt3 was lower than that of A549 cells transfected with siRNA-Ctrl.The higher the transfection amount,the lower the cell clone rate in the transfection group.6.The proportion of cell S phase cells in the siRNA 1-Wnt3 group and the siRNA 2-Wnt3 group was small,and the proportion of cells in the two groups G0/G1 was up-regulated to inhibit cell maturation.7.Wnt3 siRNAl group and siRNA 2-Wnt3 group induced apoptosis rate higher,apoptosis,caspase9 and caspase3 of protein expression Wnt3 inhibit expression of non small cell lung cancer inhibition of cell differentiation,promote cell apoptosis.8.Tumor volume of mice transfected with Wnt3-specific siRNAs(siRNA 1/2-Wnt3)and cisplatin treated after transfection were as follows:the transfected cells+cisplatin group<Transfected cells<Normal A549 cell group.Therefore this article through studies Wnt3 expression in non-small cell lung cancer tissues and explore the expression of Wnt3 with non small cell lung cancer clinical pathological characteristics of relevance,looking for non small cell lung cancer treatment of new drug targets provide effective basic experiment study,at the same time we found Wnt3 protein and mRNA in non small cell lung cancer tissues and cells in the positive expression of inhibiting Wnt3 expression in non-small cell lung cancer cells,can increase as the concentration of transfection,inhibit cancer cell differentiation,proliferation and clone.This study its research results can be directly used non small cell lung cancer drug development and clinical treatment,both for improve the therapeutic effect of non small cell lung cancer,reduce the pain of the patients and family social economic burden all have important meaning and function. |
PDF Full Text Request