The Mechanism Study On MID1-PP2A Signaling Pathway Functions In The Development Of Lung Adenocarcinoma

Background:One of the major diseases endangering human health continues to be lung adenocarcinoma,which has the highest mortality rate of all malignancies worldwide and attracted the attention of many experts and scholars.The 5-year relative survival of which is only 17% despite innovations in techniques and the application of targeted therapy drugs.However,the molecular mechanisms active in NSCLC are still poorly understood,thus investigating new active pathways in the disease to understand its occurrence and development is imperative,and may provide new insights into pharmacological mechanisms for antitumor therapies.The gene Midline 1(MID1)was found in 1997 in a patient with Opitz G/BBB syndrome(OS),which is a congenital disorder that affects the formation of ventral midline structures as a member of tripartite motif(TRIM)family proteins.Besides OS,MID1 was found in other nervous system diseases,such as Alzheimer's disease and Huntington's disease(HD).In follow-up,MID1 was found in prostate cancer,breast cancer and colorectal cancer.In the same time,we had demonstrated that MID1 played a key role in respiratory system diseases,such as asthma and idiopathic fibrosis,with airway hyper reactivity(AHR)and the release of inflammation factor,inducing the development of disease.MID1 is an E3 ubiquitin ligase(the RING,B-box1 and B-box2 domains have ubiquitin E3 ligase activity)that associates with microtubules.A known MID1 target is protein phosphatase 2A(PP2A),which is targeted by the B-box1 domain,reduce the activity of protein phosphatase 2A(PP2A)holoenzyme.The PP2 A complex is a member of the protein serine/threonine phosphatase(PP)type 2A family,composed of three types of subunits,scaffo lding subunit A,regulatory subunit B and catalytic subunit C.It has been reported that PP2 A levels are decreased in many cancers,including colorectal and breast cancers,PP2 A plays many roles in carcinogenesis including regulating apoptosis,proliferation,cell migration,cytoskeletal rearrangement and regulating cell cycle.As mentioned above,this is related literature on the role and function of MID1 in the pathogenesis and development of lung adenocarcinoma,and the function of MID1 in tumor system is still unknown as a tumor suppressor or oncogene.Thus,to study on mechanism of MID1-PP2 A signal pathway in lung adenocarcinoma,to clarify the expression and function of MID1 and PP2 A,and to explore the regulation of MID1-PP2 A signal pathway in the proliferation,cycle and apoptosis of lung adenocarcinoma cells could provide an important cytological and molecular basis for the further development of antitumor drugs and the advancement of gene targeting therapy technology.Methods:1.Comparing the expression of MID1 and PP2 A in lung adenocarcinoma cells(A549?H1975?H1650)and bronchial epithelial cell(BEAS-2B)Taking the cells which were stable in vitro,total mRNA and protein were extracted from A549?H1975?H1650 and BEAS-2B.Detecting the expressio n of MID1 and PP2 A using quantitative real-time PCR(RT-qPCR),Western Blot and ELISA methods.2.Comparing the expression of MID1 and PP2 A in lung adenocarcinoma and adjacent tissuesThirty-paired human lung adenocarcinoma and adjacent tissues were ob tained from the Department of Thoracic Surgery of the Second Hospital of Jilin University(Changchun,People's Republic of China).Detecting the expression of MID1 and PP2 A using quantitative real-time PCR(RT-qPCR),Western Blot and ELISA methods.And we analyzed the correlation between MID1,PP2 A protein and clinical characteristics of lung adenocarcinoma patients,such as age,sex,TNM stage,smoking history and so on.Immunohistochemistry(IHC)was done to analyze the expression and localization of MID1 and PP2 A in human lung adenocarcinoma and adjacent tissues.3.Comparing the expression of MID1 and PP2 A after MID1-siRN A transfection and activation by FTY-720(10nM)in A549?H1975?H1650 cel sAfter transfection in MID1-siRNA and negative control siRNA(NC-siRNA)in A549,H1975 and H1650 cells.RT-qPCR?Western Blot and ELISA methods was done to detect the MID1 expression and calculate transfection efficiency,besides PP2 A expression.After FTY720 was diluted with culture medium to a final concentration of 10 nM for 24 h as a PP2 A activator,and detect PP2 A expression.4.Comparing the regulation of MID1-PP2 A signaling pathway in cell cycle progression,proliferation and apoptosis in vitroAfter the treatment of MID1-siRNA transfection and activation by FTY-720,using MTS methods to detect cell proliferation and activity.And also detecting the expression of cell cycle and apoptosis related gene such as Bax?Bcl-2?p53?p21?Cleave-caspase9?CDK4?CDK6?CyclinD1?ERK1/2?Phopho-ERK1/2 by using Western Blot methods.Result:1.lung adenocarcinoma cells showed significantly higher MID1 expression compared with the nonmalignant BEAS-2B cell line.These results showed that adenocarcinoma cell lines had higher MID1 levels than BEAS-2B cells(P<0.05).And the expression of PP2 A had an opposite result(P<0.05).2.lung adenocarcinoma tissues showed significantly higher MID1 expression compared with the adjcent tissues.We then studied the correlation between MID1 expression and clinical characteristics of lung adenocarcinoma patients.The older patients had more MID1 expression than young patients(P<0.001).However,no significant differences were observed with respect to sex,TNM stage or smoking history in lung adenocarcinoma.These results showed that MID1 was significantly upregulated in lung adenocarcinoma compared with normal tissues and cell lines,and that MID1 expression may have a relationship with patient age.The 30 lung adenocarcinoma tissues had lower PP2 A expression than adjacent normal tissues(P<0.05).Consistent with MID1 expression,we also studied correlations between PP2 A expression and clinical characteristics of lung adenocarcinoma patients.Again,only age had a significant correlation,older patients had lower PP2 A expression than younger ones(P<0.001).No significant differences were observed with respect to sex,TNM stage or smoking history in lung adenocarcinoma.According to these results,we suggest that PP2 A is a tumor suppressor that is downre gulated in lung adenocarcinoma,patient age may influence PP2 A expression.3.After transfection with MID1-siRNA,MID1 expression was significantly reduced at the mRNA and protein levels compared with NC-siRNA(P<0.05),PP2 A protein levels were found to have increased compared with cells transfected with NC-siRNA(P<0.05).The protein expression of PP2 A was higher upregulated by the treatment of FTY-720(P<0.05).4.After the three lung adenocarcinoma cell lines(A549,H1975,H1650)were treated with MID1-siRN A and NC-siRNA,MID1-siRNA significantly increased levels of C leave-Caspase9 and Bax,but decreased levels of Bcl-2,ERK1/2 and phospho-ERK1/2,the phospho-ERK/ERK ratio also decreased.We also analyzed the relative levels of cell cycle regulator expression,which indicated that treatment of MID1-siRNA reduced the relative levels of C DK4,CDK6,Cyclin D1,but increased the relative levels of p53 and p21.After using FTY-720 to active PP2 A,the expression of PP2 A was significantly increased.Furthermore,the expression of cleave-Caspase9,Bax,Bcl-2,ERK1/2,phopho-ERK1/2,p53,p21,C DK4,CDK6,and cyclin D1 were the same as treatment with MID-siRNA in A549,H1975 and H1650 cells.We also did cell proliferation assays,after treated with transfection and activation of FTY-720,the viability of cells was downregulated(P<0.05).Conclusion:1.We demonstrated the function of MID1 in lung adenocarcinoma for the first time.We examined MID1 and PP2 A expression in lung adenocarcinoma tissues and cell lines compared with nonmalignant samples ones,finding that MID1 was highly expressed in lung cancer.This suggested that MID1 could act as an oncogene in lung adenocarcinoma,opposite of PP2 A.Moreover,our studies also showed that both MID1 upregulation and PP2 A downregulation were significantly associated with patients age;the older the patient,the higher their MID1 expression and the lower their PP2 A expression.2.We silenced MID1 in vitro,we found that PP2 A expression was higher than in control cells.Thus,we conclude that increased MID1 e xpression leads to a reduction in PP2 A,making PP2 A a tumor suppressor in this signaling pathway.3.We also demonstrated that the MID1-PP2 A signaling pathway could induce apoptosis,proliferation and cell cycle arrest.O verall,our results revealed that the MID1-PP2 A signaling pathway plays a role in lung adenocarcinoma.O ur findings are novel,improve our understanding of the molecular mechanisms underlying lung tumorigenesis,and may provide new insights into pharmacological mechanisms for antitumor therapies.