| BackgroundBackground:It is known that lung cancer,with its highest incidence and mortality rates,is one of the greatest threat to human's health and life in the world.Since last 50 years,many countries have reported a significant increase in the incidence and mortality of lung cancer.It is reported that lung cancer accounts for the first place in the incidence and mortality rates between all malignant tumors in men,and in women it accounts for second place in incidence rate,the second in mortality rate.But the pathogenesis of lung cancer is still not entirely clear.When our team compared dendritic cells in the mouse spleen we found PCNP was contained in the high expression genes of the CD8alpha + DC.Over the years,our group has been doing many studies about PCNP,Mainly in relationship with inflammation and cancer.Previous studies have shown that PCNP along with TIPE2,JNKl and other protein molecules plays a key role in leukemia,rheumatoid arthritis and pancreatic cancer and other diseases,mainly impacts the growth and proliferation of cells.It is known that the level of PCNP expression has a certain impact on the ability of antigen presentation in mice DC cells.PCNP(PEST-Containing Nuclear Protein)is a nuclear protein containing PEST motif.It is reported that it reacts with ubiquitin ligase NIRF(Np95/ICBP90-like RING Figer Protein),which may be the substrate of its reaction.NIRF contains four main functional domains that are a ubiquitin-like domain),YDG/SRA region(a YDG/SRA domain),PHD region(a PHD domain)and finger region(a RING domain).It is shown that NIRF expresses high in normal cell proliferation,low in GO/G1 while continued high in a variety of tumor cells,suggesting that NIRF was involved in cell cycle regulation,and in certain aspects of the tumorigenesis.Nucleoprotein PCNP contains the PEST motif,which is rich in proline(P),glutamic acid(E),serine(S)and threonine(T),which is usually involved in the regulation of the proteolytic pathway,in which most proteins are degraded by ubiquitination-mediated pathway.NIRF itself has the characteristics of ubiquitin ligase and the function like automatic ubiquitin.The degradation of ubiquitin-dependent protein can be involved in variety cell processes in many types of cells,which includes cell cycle progression,protein transport,DNA damage and mutation.NIRF ubiquitin ligase binds to PCNP,the specific substrate of the protein,by which the function and efficiency of ubiquitination is in turn affected.For example,nucleoprotein PCNP has a high ubiquitination rate in 293T cells and COS-7 cells,and PCNP can be ubiquitinated by NIRF both in vivo and in vitro,which suggests that NIRF and PCNP pathway may play a role in regulating cell proliferation by regulating cell cycle.At the same time,it is found that NIRF of 9p23-24.1 gene in many tumor cell genes is significantly higher than in normal cells.It is presumed that PCNP and NIRF might be related to tumorigenesis.The study of PCNP is rarely reported besides our team.We consulted amount of literatures on the PEST structure from the PEST motif of the PCNP to design a better technical route.It is found that proteins containing PEST motifs are mostly involved in the regulation of protein translation.They are mainly regulated by their own ubiquitination-proteasome degradation pathways and the phosphorylation of serine and tyrosine in the motifs to regulate protein expression and keep the function of protein.In order to study the relationship between PCNP and lung adenocarcinoma and the molecular mechanism,we conducted a large data mining again,finding that PCNP expresses highly in the malignant cells of endometrial cancer,cervical cancer,rectal cancer,lung cancer and gastric cancer and other tumors.As for the normal tissues,the expression of PCNP is high except the liver tissues,hippocampus and myocardium.It can be inferred that nuclear protein PCNP is involved in the development of a variety of tumors,and stably expresses in most tissues.Being engaged with diagnosis of lung cancer in clinical work for many years,I have a wealth of experience in the diagnosis,treatment and prognosis of lung adenocarcinoma.After being touched deeply by seeing the suffering of lung cancer patients and the burden of their families,I determine to do research on lung cancer,with hoping to find new target drugs,from which lung cancer patients can benefit.The expression of PCNP in lung adenocarcinoma tissue was significantly higher than that in adjacent tissues by the immunohistochemistry,which may reveal that the expression of PCNP was involved in the process of lung adenocarcinoma through an unclear mechanism.Finding the relationship between PCNP and lung adenocarcinoma and determining the molecular mechanism is conducive to the diagnosis of lung adenocarcinoma,treatment and assessment of prognosis.we analyzed the expression of nuclear protein PCNP in lung adenocarcinoma and the correlation of clinical symptoms,staging and prognosis of adenocarcinoma by detecting the expression of nuclear protein PCNP in human lung adenocarcinoma by analyzing the expression of nuclear protein PCNP in Shanghai lung superchip company.At the cellular level,we selected human lung adenocarcinoma cell line A549 as the object of study.We explore its cell proliferation,migration,apoptosis and tumorigenicity to provide a theoretical basis for the lung adenocarcinoma prevention,clinical diagnosis,treatment and prognosis by changing the expression of nuclear protein PCNP in A549 cells.Objective1.To confirm the expression and clinical correlation of nuclear protein PCNP in human lung adenocarcinoma by tissue chips and clinical data correlation analysis.2.To investigate the effect of PCNP expression on the proliferation,migration,apoptosis and tumorigenicity of A549 cells by overexpress and knockdown the level of PCNP in A549 cells.MethodsThe expression of PCNP in lung adenocarcinoma tissues and cells was analyzed by collecting 78 cases of clinical lung adenocarcinoma cases,histological tissue wax and prognosis clinical data and tissue microarray analysis followed.The data were analyzed by multivariate correlation analysis to confirm the correlation between nuclear protein PCNP and clinical symptoms,pathological grade,clinical stage and prognosis of lung adenocarcinoma.We constructed human PCNP eukaryotic expression vector GV-230-PCNP.Then the recombinant plasmid GV-230-PCNP and GV-240-RNAi-PCNP were transfected into A549 cells separately and filtered with G418 neomycin and puromycin.The cells with G418 resistance were PCNP overexpressing A549 cells,while the cells with of puromycin resistance were PCNP knock-down A549 cells.The two stable cells had their own empty vector(control).The expression of PCNP protein in stable strains was confirmed by Western Blot to confirm the level of PCNP.The proliferation of stable strains was confirmed by MTS and EDU.The migration ability of each stable strain was detected by scratch test and Transwell assay.The invasive ability of each stable strain was detected by Invision assay.The expression of Bax was detected by Western Blot and the apoptosis of the cells was detected by flow cytometry.The clone formation ability of stable strains was detected by plate cloning assay.result:1.The expression of PCNP in lung adenocarcinoma cells was significantly higher than that in adjacent tissues(P<0.01).The expression of PCNP in the nucleus of lung adenocarcinoma was significantly higher than that in the cytoplasm(P<0.01).2.The expression of PCNP was not correlated with clinical correlation analysis(P>0.05).The survival time of patients was significantly correlated with clinical stages(P<0.05)3.Overexpress and knockdown of PCNP expression in A549 cells(1)It is found that proliferation of PCNP overexpressing A549 cell is fast,while knock-down PCNP A549 cell slow by MTS and EDU(P<0.05).(2)Scratch test and Transwell test showed that the migration of PCNP overexpression A549 cells was fast(P<0.01),while knock-down PCNP A549 cell slow(P<0.01).(4)The expression of Bax and the result of staining were detected by flow cytometry and Western Blot.The apoptosis of A549 cells decreased when PCNP overexpressed,and increased when PCNP knocked down(P<0.01).(5).The clonogenic ability of A549 cells was higher when PCNP overexpressed(P<0.01),and lower when PCNP knocked down(P<0.01).Conclusion(1)The expression of the nuclear protein PCNP in the nucleus and cytoplasm of lung adenocarcinoma was higher than that in the paracancerous tissue.(2)The survival time of the patients with human lung adenocarcinoma was negatively correlated with the size of the tumor and the clinical TNM staging,and had no correlation with the expression of PCNP in the cancer cells.(3)The nuclear protein PCNP promotes the proliferation and migration of A549 cells,invasiveness and tumorigenicity,and reduces apoptosis... |
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