MKRN2 Inhibits Migration And Invasion Of Non-small-cell Lung Cancer By Negatively Regulating The PI3K/Akt Pathway

Objective:Lung cancer which is always regarded as one of the main causes of all kinds of deaths that is closely connected with lung disease around the world,thus its incidence continues to rise.There are roughly 85% to 90% cases of lung cancer are diagnosed as non-small-cell lung cancer(NSCLC),which is the most common type of lung cancer.Overall,the 5-year survival rate has kept at 15%,with the prognosis of patients with NSCLC principally relating to tumor metastasis and the transcriptional regulation of certain key genes.Therefore,it is necessary to understand the mechanisms involved in NSCLC progression.The phosphatidylinositol 3-kinase(PI3K)/Akt pathway is recognized as a key pathway in carcinogenesis and commonly identified in diverse human tumors.The central role of PI3K/Akt signaling in this complex network of cellular processes makes this pathway highly important in cancer cells.Accordingly,phosphorylated(p-)Akt is overexpressed in a multitude of human cancers and related to poor overall survival in some cancer types.Akt activation can be determined through the use of phospho-specific antibodies against either p-Akt(Ser473)or p-Akt(Thr308).Patterns of p-Akt staining vary between follicular and papillary carcinomas,but more prominent nuclear p-Akt(Ser473)has been reported in regions of invasion by both types.Similarly,although differentiated thyroid cancers demonstrate cytoplasmic staining,contiguous anaplastic thyroid cancers display both nuclear and cytoplasmic p-Akt(Ser473).These findings might suggest a role for nuclear p-Akt in aggressive or invasive disease.Additionally,significant evidence suggests that the PI3K/Akt pathway plays critical roles in multiple tumors of endocrinetissues.Makorin RING zinc finger-2(MKRN2)belongs to the makorin RING zinc finger family,and the MKRN2 gene partially overlaps the RAF1 proto-oncogene in antisense transcriptional orientation.This family encodes putative ribonucleoproteins with a distinctive array of zinc finger domains.To date,nine MKRN-family loci at various sites in the human genome have been identified.Makorins are zinc finger proteins with a typical C3HC4 motif(the RING domain)associated with arrays of one to four C3 H domains and representing a type of zinc finger found in a variety of ribonucleoproteins.MKRN2 harbors four C3 H zinc fingers and a signature C3HC4 RING zinc finger domain.MKRN proteins also contain a protein-protein interaction motif rich in Cys and His residues,but that exhibits a currently unknown function specific to MKRNs.This motif is found in most E3 ubiquitin ligases,a category of enzymes mediating the transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to target protein substrates.The RING domain is responsible for ubiquitin ligase activity,leading to monoubiquitination and/or to synthesis of polyubiquitin chains on lysine residues.Accordingly,some MKRN proteins work as E3 ubiquitin ligases,with a previous study reporting that MKRN2 is a novel ubiquitin E3 ligase targeting the p65 subunit of NF-?B to negatively regulate inflammatory responses.In this study,we demonstrated that MKRN2 inhibited cell migration and invasion of NSCLC cells by reducing the p-Akt(Ser473)levels.Additionally,we showed that MKRN2 was involved in ubiquitin-dependent degradation of the p85? subunit of PI3K(PI3Kp85?).Moreover,we examined the expression of MKRN2 in NSCLC tissues andcell lines by immunohistochemistry and western blot and altered MKRN2 expression in these cells to evaluate changes in cancer-related phenotypes in order to determine its role in NSCLC.Methods : 1.Commonly the specimens of foundamental lung cancer were sampled randomly from 261 different patients(156 males and 105 females).From 2013 to 2017,all of the patients went through complete surgical resection in the First Affiliated Hospital of China Medical University,and analyzed the correlation between clinical and pathological factors.All the 261 patients who participated in the pathologic correlation analysis did not receive preoperative chemotherapy or radiotherapy.According to the International Union of Cancer(the Union for International Cancer Control,UICC)the eighth edition of lung Cancer TNM staging system,statistics,in ? stage patients(N =143),stage ? and ? patients(N = 118).According to the WHO classification system,histological diagnosis and degree of tissue differentiation were squamous cell carcinoma(N=102 cases),adenocarcinoma(N= 159 cases),moderately to highly differentiated(N=143 cases),and poorly differentiated(N= 118 cases).Tumor size was classified as less than or equal to 3 cm(N=168 cases)and greater than 3 cm(N=93 cases).Cases with lymph node metastasis(N=150)and cases without lymph node metastasis(N=111).By immunohistochemical staining the experimental results showed that all of the slice staining intensity and dyeing cell percentage,semi-quantitative score evaluation,double-blind clinical data analysis result,among them,according to the intensity of staining,MKRN2 expression level evaluation scores as: 0(no),1(low expression,light yellow granular),2(moderate intensity expression,yellow particles),3(high expression,brown granules);The percentage of staining cells was assessed as 0(0%),1(1%-30%),2(31%-70%)and 3(71%-100%).The final score between 0 and 9 is obtained by means of multiplying the different grades of every tumor sample.The final product score was used for statistical analysis.If the pathological section score of the tumor sample is more than three points,it will be considered as high expression of MKRN2;if the pathological section score is less than or equal to three points,it will be considered as low expression of MKRN2.SPSS 17.0 software was used for analysis,and single sample paired t test was used to analyze the relationship between MKRN2 expression level and clinicopathological factors.P < 0.05 was used as the criterion to determine whether it was statistically significant.Kaplan–Meier analyses was analyzed and compared by logarithmic rank test to study the correlation between MKRN2 and prognosis of lung cancer patients.2.Cell culture: Cell lines used in the experiment were purchased from Shanghai cell bank of Chinese academy of sciences of the commonly used non-small cell lung cancer cell lines,according to the cultivation of the cell bank official website open formula,use Gibco 1640 medium,adding 10% fetal bovine serum,anhydrous glucose,sodium bicarbonate,sodium pyruvate,to cultivate A549,H292,H1299,H460,H226 cell lines,use Gibco MEM medium,adding 10% fetal bovine serum,anhydrous glucose,sodium bicarbonate,sodium pyruvate,to cultivate SK-MES-1 cell lines,Human normal bronchial epithelial HBE cell lines were cultured using DMEM high glucose medium.All cells are in the sterile,the cultivation of the filter with a hole in the condition of 5%CO2,37 ? apply box for culture,in the six orifice of sterile interference transfection anda series of processing.3.Cellular immunofluorescence: The cells to climb in the cell culture 24 orifice,the cells with different kinds of cell lines,shop in 24 hole plate,in culture box,after cells stick on the wall in cells to climb,remove the 24 hole plate,wash three times with PBS,and then with 4% paraformaldehyde fixed 10 to 15 minutes,then use 0.1% of Triton X-100 membrane punch 10-15 minutes,then non immune fetal bovine serum albumin closed two hours,add MKRN2 antibody at 4 ? overnight,the second day,fluorescent two resistance to avoid light under normal temperature conditions apply for two hours,Finally,the nuclei were stained with DAPI dye for 8-10 minutes under dark conditions.The localization and expression of MKRN2 in various cell lines were observed by stimulated target light under a fluorescence microscope.4.According to manufacturer's instructions,Lipofectamine 3000 reagent was largely used for cell transfection.We used si-RNA targeting MKRN2 to knockdown the expression of MKRN2,For upregulating the expression of MKRN2,we use MKRN2-expression plasmid.5.Transwell: Cell migration and invasion assays were performed in 24-well Transwell chambers containing inserts with a pore size of 8 ?m.For the invasion assay,inserts were coated with 100 ?L Matrigel.Cells were trypsinized 24 h after transfection,and 5 × 104 cells in 100 ?L medium supplemented with 2% fetal bovine serum(FBS)were transferred to the upper Transwell chamber,whereas 600 ?L medium supplemented with10% FBS was added to the lower chamber.After incubation for 24 h,cells on the upper membrane surface were removed with a cotton tip,and those passed through themembrane were fixed with paraformaldehyde and stained with hematoxylin.The number of migrated/invaded cells was counted in 10 randomly selected fields under a microscope at high magnification.When the upper chamber was coated with matrix gel,was used to detect the effect of MKRN2 on cell invasion ability,while to detect the effect of MKRN2 on cell migration ability when the upper chamber was not coated with matrix gel.6.Western blot: knockdown or overexpress MKRN2 expression,total cellular protein was extracted using lysis buffer containing a protease-inhibitor cocktail and in the presence or absence of a phosphatase-inhibitor cocktail according to manufacturer instructions and quantified using the Bradford method.Proteins(80 ?g/lane)were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis,transferred to polyvinylidene fluoride membranes,and the membranes were blocked in 5% skim milk in Tris-buffered saline with Tween-20 at room temperature,followed by incubation overnight at 4°C with the appropriate primary antibodies.Membranes were washed and then incubated with horseradish peroxidase-conjugated anti-mouse/rabbit Ig G at 37°C for2 h.Immune reactivity was detected by enhanced chemiluminescence using a Bio Imaging system.Relative protein expression was calculated after normalization to glyceraldehyde 3-phosphate dehydrogenase(GAPDH),which was used as a loading control.Proteins related to cell migration and invasion were detected and analyzed by western blot assay to find possible pathways and molecular mechanisms.7.Co-immunoprecipitation assays: collect cells,add NP40 protein cracking cracking liquid,centrifugal removing cell debris,cracking liquid file,join the magnetic beads closed two hours,centrifugal remove magnetic beads,cracking liquid,add MKRN2 andPI3Kp85? antibody,4 ? shaking bed overnight,then join the magnetic beads,4 ?shaking bed six to eight hours,removed the protein lysate,the magnetic beads were left,co-immunoprecipitation assays testing whether there is interaction between MKRN2 and PI3Kp85?.8.Quantitative real-time PCR: Real-time PCR was performed in a 7900 HT fast real-time PCR system using SYBR Green PCR master mix in a total volume of 20 ?L under the following cycling conditions: 95°C for 30 s and 45 cycles of 95°C for 5 s and 60°C for30 s.The dissociation step was used to generate a melting curve and confirm amplification specificity.Relative gene expression was calculated using the 2-??Ct method,with ?-actin used as a reference.9.Cycloheximide experiments: the cells spread in a germ-free 6 hole plate,add cycloheximide,respectively at 0,3,6,9 hours to collect cells,knockdown MKRN2,after48 hours joined cycloheximide,respectively after joining cycloheximide 0,3,6,9 hours collecting cells,western blot was performed to analyze the effect of MKRN2 expression on PI3Kp85? protein synthesis.10.Ubiquitination assays: Cells were transfected with the indicated plasmids,and at 48-h post-transfection,the 26 S proteasome inhibitor MG132 was added at a final concentration of 10 ?M for 5 h,after which samples were collected.Cells were lysed in lysis buffer,and cell debris was pelleted by centrifugation at 12,000 rpm for 10 min at4°C.Supernatants were collected for IP,which was performed using whole-cell lysates(~200 ?g protein),4 ?g to 10 ?g antibody,and 20 ?L protein A/G agarose.Cell lysates were precleared with 20 ?L agarose A/G beads by rocking for 1 h at 4°C,after whichbeads were removed,and appropriate antibodies were added.Samples were then incubated with 20 ?L agarose A/G beads by rocking for 4 h to 6 h at 4°C,followed by the addition of lysates and incubation with rocking overnight at 4°C.Immune complexes were collected by centrifugation,followed by washing in cell lysis buffer before analysis.Levels of PI3Kp85? ubiquitination were evaluated by IP of PI3Kp85? using anti-PI3Kp85? antibodies,followed by anti-hemagglutinin(HA)immunoblotting.Results: 1.Immunohistochemistry and cellular immunofluorescence showed that MKRN2 was localized in cytoplasm and nucleus.In clinical cases of lung cancer and non-small cell lung cancer cell lines,MKRN2 showed lower expression,in the normal bronchial and alveolar and HBE cell line,MKRN2 was high expression.In the correlation analysis of clinicopathological factors,the expression level of MKRN2 protein was correlated with the degree of differentiation,lymph node metastasis and p-TNM stage,but was not correlated with patient age,gender,tumor typing and tumor size.Kaplan-meier database analysis showed that MKRN2 was correlated with the prognosis of lung cancer patients.2.In non-small cell lung cancer cell lines A549 and H1299,MKRN2 inhibit the migration and invasion ability of non-small cell lung cancer cell lines.3.Western blot revealed that MKRN2 can regulate the expression of Rho A,ROCK1 and MMP9,which are related to migration and invasion,and screen related pathways founded that MKRN2 inhibit the activation of PI3K/Akt signaling pathway.4.After the addition of PI3K/Akt signaling pathway inhibitor LY294002,combined withfunctional experiments and western blot analysis,it was determined that MKRN2 exerts an inhibitory effect on the migration and invasion of non-small cell lung cancer cell lines through the PI3K/Akt signaling pathway.5.Immunoprecipitation confirmed the interaction between MKRN2 and PI3Kp85.6.MKRN2 inhibited PI3Kp85? protein synthesis.The ubiquitination experiment confirmed that MKRN2 affected the protein expression of PI3Kp85? through the ubiquitination degradation pathway.Conclusion: 1.MKRN2 is low expressed in non-small cell lung cancer cell lines,and it was associated with clinicopathological factors and poor prognosis2.In vitro experiments showed that MKRN2 inhibited the migration and invasion of non-small cell lung cancer cells through Rho A,ROCK1,MMP9 proteins.3.MKRN2 can degrade PI3Kp85? by ubiquitination pathway,and inhibit the activation of Akt pathway and affect the migration and invasion ability of NSCLC.