MZT2A Promotes NSCLC Biological Function By Regulating Akt Phosphorylation Via The MOZART2 Domain

Background:Lung cancer is the most common cancer in the world.In the classification of its histology,85% are non-small cell lung cancers,and the rest are small cell lung cancers.In most parts of the world,the 5-year survival rate for patients with NSCLC is only 10-20%.Although the current targeted therapy for lung cancer has improved the survival of patients to a certain extent,the drug resistance of TKI(Tyrosine kinase inhibitor)has always been a problem in clinical treatment.Therefore,further research on the molecular mechanism of NSCLC will contribute to the early diagnosis of lung cancer,as well as the prediction of treatment effect and prognosis evaluation of patients.Microtubules are dynamic changes in the cell,they exist in the form of hollow tubes.They can not only participate in the formation of cytoskeleton,but also play different functions in the localization and movement of intracellular organelles.Under normal circumstances,microtubules in cells are assembled and decomposed with the cell cycle to perform their functions.In normal cells,microtubules can function in combination with many proteins,and drugs targeting microtubulins and their activity have been proven to be an effective method for the treatment of various human cancers,but drug resistance problems caused by the high expression of multidrug resistant genes also restrict the effectiveness of cancer treatment.Mitotic spindle tissue protein 2A(MZT2A),also known as GCP8A(Gamma-tubulin complex component 8A)or Fam128A(Family with sequence similarity 128A),as a member of microtubulin,could affect the balance of microtubule polymerization and depolymerization by regulating cell division signals during cell growth.MZT2 A locates in the centrosome and is involved in spindle formation during cell mitosis.The lack of MZT2 A expression in cells can weaken the nucleation activity of microtubules and slow down the proliferation rate of cells.Therefore,exploring the possible biological functions and molecular mechanism of MZT2 A in NSCLC could provide the possibility to develop new therapeutic targets.Methods:We searched The Cancer Genome Atlas(TCGA)and Gene Expression Profiling Interactive Analysis(GEPIA)databases and analyzed Kaplan-Meier survival data.We then assessed the differential MZT2 A expression to determine the association with the prognosis for lung cancer.Tissue specimens collected from 224 NSCLC patients who underwent curative surgical resection without prior chemotherapy or radiation therapy for immunohistochemistry assay.The effect of MZT2 A on the proliferation,migration and invasion ability were detected by MTT,colony formation,scratch and Transwell invasion assays;meanwhile,western blot,cellular immunofluorescence and RT-q PCR assays were detected the effect of MZT2 A on NSCLC at molecular level.Akt pathway inhibitor,Co-IP and co-transfection assays were used to explore the specific mechanism of MZT2 A affecting the biological function of NSCLC at the molecular level.MOZART2(mitotic spindle organizing protein associated with a ring tubulin)mutants were designed to explore the molecular mechanism of MZT2 A regulating its interacting proteins in NSCLC.The regulation effect of MOZART2 domain on the proliferation and Akt pathway of NSCLC cells were investigated in vivo by using suncutaneous tumor formation model in nude mice.Result:1.TCGA,GEPIA,Kaplan-Meier databases and immunohistochemical results suggested that the expression level of MZT2 A in NSCLC were higher than in normal lung tissues,and its high expression were positively correlated with poor prognosis of NSCLC.2.Down-regulation of MZT2 A expression in NSCLC cells could inhibit the proliferation,migration and invasion of tumor cells;on the contrary,the up-regulation of MZT2 A expression could promote the above biological functions of tumor cells.Western blot assay also confirmed that regulating the expression of MZT2 A could affect the expression of related functional proteins in NSCLC cells.3.Regulating the expression of MZT2 A in NSCLC cells could affect the phosphorylation level of Akt.The addition of Akt pathway inhibitor could reduce the Akt phosphorylation which induced by MZT2 A high expression.While this change could not change the expression of MZT2 A and total Akt in NSCLC cells.4.The interaction between MZT2 A and LGALS3 BP in NSCLC cells were confirmed by biomedical database,Co-IP assay and immunohistochemical assay.Meanwhile,the co-transfection experiment proved that MZT2 A activated Akt signal pathway via LGALS3 BP.5.Western blot assay and cytological assay both confirmed that MZT2 A could regulate LGALS3 BP and promote Akt signal pathway only in the presence of the MOZART2 domain.However,when the domain was absent,the expression level of LGALS3 BP and the phosphorylation of Akt did not change significantly compared with the cells transfected with the empty plasmid.6.Result of tumor-bearing experiment in nude mice showed that the volume and growth rate of tumor in wild-type and over-expression groups were significantly enhanced compared with the negative control and empty groups.Western blot assay of tumor tissue proteins showed that only in the presence of the MOZART2 domain could exert MZT2A's regulatory effect on Akt phosphorylation.Conclusions:1.The up-regulated expression of MZT2 A in NSCLC is associated with poor prognosis.2.MZT2 A regulates malignant behavior of NSCLC cells by promoting Akt phosphorylation via LGALS3 BP in vitro.3.MZT2 A regulates Akt phosphorylation through the MOZART2 domain in vivo and in vitro.