Effect Of Nuclear Protein PCNP On Proliferation?Migration And Invasion Of Human Lung Adenocarcinoma Cells

BackgroundIn recent years,the incidence and mortality of lung cancer have continued to rise globally,and lung cancer has become one of the most common malignant tumors.The mechanism of lung cancer is not completely clear.A large number of research data indicate that the occurrence of lung cancer is related to smoking,occupational factors,air pollution,ionizing radiation,genetic and genetic changes.The early stage of lung cancer is similar to benign lung disease,with no special symptoms.Most of the patients are in the late stage and lose the best time for treatment.Non-small cell lung cancer(NSCLC)is the most common type of lung cancer.Surgery is often used for the early treatment of NSCLC.Patients who cannot undergo surgery are often treated with radiotherapy,chemotherapy,interventional therapy,biological immunization,etc.,and their 5-year survival rate is low.Therefore,in-depth study of the molecular mechanisms of NSCLC pathogenesis,and to find more new targets for lung cancer treatment,can provide more opportunities and possibilities for the treatment of lung cancer.In 2004,Japanese scientists first reported PCNP as a substrate for NIRF.PCNP is a protein that contains PEST in the nucleus.After reviewing a large number of literatures,it was found that these proteins are mainly involved in regulating cell cycle and cell proliferation.Therefore,we suspect that PCNP plays a role in the development of tumors.After reviewing the big data,we found that PCNP is highly expressed in a series of malignant tumors such as cervical cancer,rectal cancer and lung cancer.In order to understand the relationship between PCNP and tumor,our group first studied the relationship between PCNP and neuroblastoma.The results showed that PCNP can inhibit the proliferation of human neuroblastoma cells,attenuate the ability of cell invasion and migration,inhibit allogeneic tumor formation,and enhance the ability of apoptosis,but the specific mechanism needs further study.Therefore,we decided to continue to study the relationship between PCNP and lung adenocarcinoma.Before the experiment,we analyzed the lung adenocarcinoma tissue chip provided by Shanghai Core Superchip Company and found that PCNP was highly expressed in lung adenocarcinoma tissues and low in adjacent tissues.Next,we selected A549 cells from human non-small cell lung cancer and performed some preliminary experiments,including MTS,EDU,Plate Cloning,Cell Scratching,Transwell and Western blot to detect Bax protein expression,and found that PCNP can promote the process of cell proliferation and migration.In order to know the specific role of PCNP in lung adenocarcinoma,we selected two kinds of cells,A549 and H1299 cells as the research object,and re-examined the previous experiments in order to deeply study the relationship between PCNP and lung adenocarcinoma cells,and also attempt to provide more feasible theoretical basis for the prevention,diagnosis,treatment and prognosis of lung adenocarcinoma.ObjectiveTo explore the relationship between PCNP and proliferation,migration,invasion and tumorigenicity of human lung adenocarcinoma cells,and to explore possible molecular mechanisms.MethodsThe database was examined;PCNP expression levels were detected by IHC in 63 human lung adenocarcinoma specimens and corresponding non-tumor lung tissues(National Human Genetic Resources Sharing Service Platform,Shanghai,China).Four surgical specimens were collected to detect protein levels of PCNP.The relationship between the expression of PCNP in lung adenocarcinoma and adjacent tissues,the relationship between PCNP expression and gender,tumor size,pathological grade,clinical stage andother factors and the relationship between survival and survival were analyzed.After drug sensitivity experiments with G418 and puromycin,PCNP over-expression and knockdown plasmids were transfected into A549 and H1299 cells,respectively.Western blot and RT-PCR were used to verify and confirm the successful construction of each stable strain,and the apoptosis ability of the cells was detected by TUNEL assay.Cell scratch test,Transwell test,Invasion test and soft agar test were used to detect the migration and invasion of lung cancer cells.In addition,Western blot was used to detect the expression of apoptotic protein and STAT3/STAT5 signaling pathway,and the PI3K/AKT/mTOR signaling pathway and autophagy protein P62/Becline-1/Lc3 were detected to explore the possible relationship between them.The lung cancer cells were injected into nude mice to detect the effect of PCNP on the tumorigenicity of lung adenocarcinoma in nude mice,and the mouse tumor tissues were subjected to HE staining and immunohistochemical analysis.Results1.PCNP is mainly expressed in the nucleus.PCNP levels are high in lung adenocarcinoma tissues,but low in adjacent non-tumor tissues.PCNP levels are associated with T classification of lung adenocarcinoma.2.The expression of PCNP in lung adenocarcinoma cells A549 and H1299 was detected by Western blot and RT-PCR.The over-expressing cell group and the silencing cell group were successfully constructed.3.EDU experiment,MTS experiment and plate cloning experiment to detect the proliferation of A549 and H1299 cells showed that PCNP mediates the proliferation and viability of human lung adenocarcinoma cells.4.Cell scratch assay and Transwell assay were used to detect the migration ability of cells.Invasion assay and soft agar assay were used to detect the invasive ability of cells.The results showed that PCNP increased the migration and invasion of human lung adenocarcinoma cells.5.The TUNEL assay was used to detect the apoptotic ability of cells.The results showed that PCNP over-expression inhibited cell apoptosis and the silencing group promoted apoptosis.The expression of apoptotic protein was detected by Western blot.The results were the same as those of TUNEL.The expression levels of Cleaved Caspase3 and Cleaved PARP in PCNP over-expression group were decreased,and the expression of cells in silencing group was increased.The expression of p-STAT3 and p-STAT5 was increased in the over-expression group.Therefore,it can be seen from the above results that PCNP inhibits apoptosis of human lung adenocarcinoma cells and may mediate apoptosis of lung adenocarcinoma cells through STAT3 signaling pathway.6.Western blot was used to detect the expression of PI3K/AKT/mTOR signaling pathway and the expression of Becline1,LC3 and p62.The results showed that P-PI3 K,P-AKT and P-mTOR in PCNP over-expression group compared with the control group expression increased,and the expression level of the silent group decreased.The protein expression levels of Becline1 and P62 were increased in the PCNP over-expression group,and the expression level in the silencing group was decreased.In addition,the protein level of LC3 A / B showed an opposite trend.Therefore,PCNP increased the level of autophagy,and PCNP knockdown showed the opposite effect.Taken together,these results indicate that PCNP may regulate autophagy through the PI3 K / Akt / mTOR pathway in human lung adenocarcinoma cells.7.Transplantation tumor model experiments in nude mice indicate that PCNP promotes growth and angiogenesis in human lung adenocarcinoma tumors.ConclusionNuclear protein PCNP enhances the proliferation,migration and invasion of human lungadenocarcinoma cells,and may regulate cell apoptosis and autophagy through STAT3/5 and PI3K/Akt/mTOR signaling pathways.In turn,it promotes the occurrence and development of human lung adenocarcinoma.