Anti-proliferation And Inducing Apoptosis Effects Of LUNX-siRNA On A549 Lung Cancer Cell Lines

As a lung tumor surgeon,we must master the basic knowledge of biological tumor markers and molecules,genomics,in order to grasp the latest progress in the field of lung cancer research。Lung cancer is one cancer worldwide morbidity and mortality highest,a total of about seven million people die from lung cancer and cancer-related complications each year.With the intensification of industrial pollution and air pollution,lung cancer has become China’s population morbidity and mortality fastest rising cancer,male cancer patients have occupied the cause of death of the first,the second leading cause of death of women cancer patients,the number of dead more than six hundred thousand each year,the overall number of people died of lung cancer accounts for overall cancer deaths22.7%.From the point of view of thoracic surgeons,surgery is the best treatment for lung cancer,but most patients with lung cancer has entered our first visit in late,lost chance of surgery,even without lymph node metastasis IA IB Patients period,five years after receiving potentially curative resection,recurrence rate is still as high as 25%.Studies have shown that most patients with lung cancer occult micro-metastases.But the existing CT,Ultrasound,and even PET-CT andother conventional clinical examination cannot find it for early detection methods.Lung cancer treatment model has entered the era of accurate treatment,early micro-metastasis is currently checking for lung cancer in the blood concentration of tumor markers direction,but there are still many shortcomings,while the rapid development of liquid biopsy techniques,including blood circulating tumor cells,plasma free DNA and exosomes,which is proteomics,clinical transformation progress of genomics,but also the inevitable trend of the times into the lung cancer gene therapy.By differential display protein technique,Japan oncologist Kyoko Iwao Koizumi,isolated a protein which expressed in the human respiratory system organ lung specific X protein RNA(LUNX mRNA).In recent years,studies have found,LUNX mRNA in lung cancer,metastatic lymph nodes,sentinel lymph node,skip lymph node metastases,lung metastases were highly expressed,therefore,prove LUNX mRNA in lung cancer occurrence,development and metastasis plays an important role,LUNX m RNA is an effective means of detecting subclinical lung micro-metastases early detection,LUNX mRNA is also a potential target for gene therapy of lung cancer clinical.AS a very popular method,it can down-regulate gene expression specially.RNA interference technique(RNA interference,RNA i)by small interfering RNA(small interference RNA,siRNA)cause specific target mRNA degradation,so that a phenomenon of post-transcriptional silencing.This phenomenon is widespread in nature life body.In biological evolution can effectively resist foreign gene change the original structure of the role of genes.The mechanism,play the important role in maintaining biological genome stability.RNA i technology can be used as a high effective knockout tool.Widely used in functional genomics study field,and gradually entered the era of gene therapy clinical lung cancer.As a carrier,lentivirus expression plasmid can transduction,integration target gene mRNA to acceptor genome DNA and further to achieve a lasting expression of lentivirus-mediated RNA interference.Efficient gene silencing can achieve good results.In this experimental study,lentivirus vector-mediated RNA interference technology to A549 lung cancer cell line.In the study,LUNX mRNA is the purpose of a target gene,transfect LUNX siRNA into the lung cancer cells.And by immunohistochemistry,real-time PCR(RT-PCR)technology,MTT assay,enzyme immunocytochemistry method scratch test method,transmission electron microscopy techniques,the study LUNX mRNA on biological behavior of cells decreased the expression.Further by detectingA549 lung cancer cell line of alkaline phosphates(AKP),Surfactant protein(SPC),with Deoxy-uridine bromide(BrdUrd),further analysis LUNX mRNA regulate A549 signaling pathways and molecular mechanisms of biological functions lung cancer cells.This study was designed to investigate the above experiment LUNX mRNA.Its mechanism of action in the early micro-metastasis lung cancer screening LUNXAs the feasibility of lung cancer targeted gene therapy for the treatment of lung cancer gene accurate,explore generic drugs provide the necessary experimental basis.1.Purpose1.1 To study the anti-proliferation and inducing apoptosis effects of Lung-specific X protein(LUNX)on A549 lung cancer cell lines.In accordance with LUNX Gene coding region sequence,it was designed and synthesized LUNX siRNA of DNA to Lentivirus with(pGCL and pHelper 1.0)vector to construct and transfect cells.1.2 Research LUNX transfection and expression of cell morphology on A549 lung cancer cell lines,proliferation and biological function of apoptosis,and metastasis.2.Materials and methods2.1 Use of lentiviral gene transfer method of detecting expression of the LUNX anti proliferative effect on A549 cells and biological function.2.2 MTT test and cell scratch test was observed changes in A549 lung cancer cell proliferation and migration.2.3 RT-PCR to detect A549 lung cancer cell lines LUNX-siRNA of expression.2.4 The anti-proliferation and the cytotoxicity of LUNX on A549 lung cancer cell line were detected by MTT assay.To observe LUNX expression in A549 lung cancer cell lines,and the mRNA level were evaluated with Real-time PCR.The morphological changes of the apoptosis cell were observed by invert microscope,HE stain,transmission electron microscope and AO/EB double fluorescent staining.Expression levels in A549 cells by immunohistochemistry to detect LUNX-siRNA.2.5 RT-PCR assay proliferative index BrdUrd A549 lung cancer cells after transfection,expression of m RNA levels AKP and SPC.2.6 And morphological changes observed apoptosis of A549 cells by a microscope,HE staining and transmission electron microscopy.2.7 Statistical analysis: SPSS 14.0 statistical analysis software,data were expressed as mean ± standard deviation(`x±s),and using analysis of variance and pair wise comparison(SNK)method,P <0.05 was considered statistically significant.3 Results3.1 Determining which sequence-specific genes using LUNX,LUNX-si RNA were designed and synthesized in the DNA,annealing,digested connection pGCL-GFP vector into the expression plasmid.And mixing pHelperl.0 pGCL-GFP vector was successfully constructed LUNX-si RNA lentivirus vector.3.2 A549 cells were divided into 3 groups: Group A: LUNX-siRNA and pGCL-GFPco-transfection group,Group B: untransfected group,Group C: pGCL and co-transfection pHelper 1.0,A group of experimental group B and C group was the control group.Success on the transfection rear,join 400μg PmlG419 screening.After three weeks the group formed positive clones,expansion cultures.3.3 Strain A549 cells migration LUNX transfected A549 cells than normal(P =0.026).3.4 The LUNX expression in A549 cells transfected with LUNX-siRNA was confirmed by Real-time PCR and immunocytochemical method,respectively.RT-PCR and immunocytochemistry in A549 lung cancer cells have demonstrated stable expression after LUNX transfection.3.5 Compared with that in the control group,AKP,SPC and BrdUrd mRNA level decreased significantly in PEFLUNX transfected A549 cells(P<0.05).RT-PCR confirmed the A549 cells after transfection of AKP,SPC BrdUrd and other proliferation index was significantly decreased(P <0.05).3.6 MTT assay showed that the inhibitory rate enhanced obviously with the addition of effect/target rate and extension of time(P<0.05).MTT assay showed that A549 cells and effectors to target ratio is increased and prolonged duration of action,inhibition was significantly enhanced(P <0.05).3.7 Lung cancer cell apoptosis or necrosis were morphologically observed after lung cancer cells were induced by LUNX for 48 hours(P<0.05).LUNX-si RNA effect apoptosis or necrosis after 48 h,cell morphology lung cancer cells,compared with the control group,the difference was statistically significant(P <0.05).4 Conclusions4.1 Targeted gene was successfully constructed with specific small interfering RNA lentivirus,which infected lung cancer cells,can effectively reduce the target protein-lung-specific X protein expression.4.2 LUNX is highly effective protein which a stronger anti-proliferation and inducing apoptosis affect on lung cancer cells.LUNX-siRNA prepared in vitro proliferation and apoptosis of lung cancer A549 cells.4.3 LUNX protein might inhibit the proliferation of A549 cells by decreasing the expression of AKP,SPC and BrdUrd regulate pathway negatively.LUNX mRNA low expression of protein molecule resulting in reduced expression of the downstream portion,mechanism by reducing the negative regulatory pathway the AKP,expression of the SPC and BrdUrd.4.4 LUNX as precise treatment of lung cancer,a new target for targeted gene therapy is a certain feasible.