Autophagic Phosphorylation Modifies PAK1-cofilin Pathway To Mediate Lung Adenocarcinoma Cell Migration Induced By Apelin-13

Objective：Apelin is a newly discovered bioactive peptides which had been proved to bean endogenous ligand of the APJ receptor. Recent evidences had confirmedthat apelin is a novel cancer factor involved in angiogenesis and anti-apoptosisin non-small cell lung cancer and oral squamous cell carcinoma. Our previousresearch has found that apelin promotes lung adenocarcinoma A549cellsautophagy via ERK1/2. Here, we evaluated whether apelin influences themigration of lung adenocarcinoma cells, as well as whether it is mediated byautophagy by increasing PAK-1-cofilin signal pathway. Consequently，thisstudy may provide a target for antimetastatic therapies.Method:1. To detect the effect of apelin-13on cell migration：wound healing,transwell.2. ELISA，To study the effect of CoCl2on apelin generation.3. Western blotting，Detecting the related protein APJ, p-PAK-1, PAK-1,p-cofilin, cofilin, LC3, beclin-1expression.4. Gene transfect Detecting the function of the high expression and lowexpression of APJ on migration by transfection.5. MTT，determine effects of doxorubicin and Razoxane on cell viability.6. AO/EB A549cells were fluorescent immunostained by AO/EB to test theeffect of doxorubicin and Razoxane on cell apoptosis.Results:1. Apelin-13induces lung adenocarcinoma cells migrationTranswell result showed that apelin-13promoted lung adenocarcinomaA549cells migration in a dose-dependent manner, where0.01μM apelin-13significantly promote cell migration, and the effect of apelin-13on migration reached the peak in0.1μM concentration. Wound healing analysis confirmedthat0.1μM apelin-13time-dependent promoted A549cells migration.Transwell and wound healing study further testified that0.1μM apelin-13significantly induced lung adenocarcinoma H460and human high metastaticlung adenocarcinoma95-D cell migration.2. Apelin-13induced lung adenocarcinoma cell migration via APJThe blast proved that the sequence of construction of recombinantplasmid of pcMV-Tag2B-APLNR and APJ interfering RNA miR-APLNR-494,miR-APLNR-1712, miR-APLNR-1572are exact without mutation.Fluorescence results suggest that the transfection is successfully andefficient. Western blotting showed that transfected with empty vectorpcMT-Tag2B did not affect the expression of APJ in A549cells, andpcMV-Tag2B-APLNR transfection significantly increased APJ expressionmiR-APLNR-494significantly inhibited the expression of APJ whilemiR-APLNR-1712, miR-APLNR-1572has no influence on APJ expression.Wound healing showed that compared with empty vector,pcMV-Tag2B-APLNR dramatically promoted while miR-APLNR-494significantly inhibited A549cell migration treatment with or without apelin-13.Transwell result further confirmed that the different is significant.3. Apelin-13induced phosphorylation of PAK1-cofilin to promoted lungadenocarcinoma A549cells migrationString server was applied to predict the protein-protein interaction ofapelin and PAK1. The result indicated that apelin may combine with PAK1.PAK1inhibitor IPA-3incubation significantly decreased A549cellsmigration induced by apelin-13. The Western blot showed that apelin-13increased PAK1and cofilin phosphorylation in a time-dependent mannerwhich was abolished by IPA-3. What’s more, Western blot further confirmedthat apelin-13significantly induced PAK1protein expression, but had noeffect on the expression of cofilin.4. Apelin-13promotes A549cells migration mediates by autophagyApelin-13increased the expression of LC3total protein and LC3Ⅱ inA549cells. Autophagy inhibitor3-MA inhibited LC3Ⅱ generation inducedby apelin-13. Fluorescence results implied that apelin-13induced LC3 membrane localization in A549cells. Transwell results confirmed thatautophagy inhibitor3-MA can inhibit A549cell migration, and inhibits theapelin-13-induced migration, rapamycin significantly promote the migrationof A549cells, rapamycin in combination with apelin strongest effect on cellmigration promoting.5. Autophagy induced phosphrylation of PAK1-cofilin to mediate migration ofA549cells induced by apelin-13Autophagy inhibitor3-MA time-dependent inhibited PAK1phosphorylationin A549cells, and3-MA abolished the effect of apelin-13on PAK1phosphorylation. And autophagy inhibitor3-MA equally time-dependentinhibition cofilin phosphorylation in A549cells and inhibited the cofilinphosphrylation induced by apelin-13.6. Apelin-APJ for the treatment of lung adenocarcinoma potentially effectivemigration targetThe effects of doxorubicin on A549cell viability were determined in anMTT assay. And the IC50of doxorubicin is1.332μM. AO/EB stainingresults suggested that low concentrations of doxorubicin didn’t significantlyinduced apoptosis. The wound healing results indicated that1μMdoxorubicin inhibited A549cell migration. Transwell result proved that1μMdoxorubicin significantly inhibited A549cell migration which was abolishedby apelin-13. In transfected pcMV-Tag2B-APLNR cells, effect of doxorubicinis reversed. In miR-APLNR-494transfection cells doxorubicin significantlyinhibited A549cell migration even treatment with apelin-13. The IC50ofrazoxane on A549cells is892.164μM. Wound healing displayed that10μMrazoxane inhibited A549cell migration. Moreover, AO/EB staining showedthat50μM razoxane can not cause apoptosis. Transwell resultsdemonstrated that10μM razoxane significantly inhibited A549cell migration,and apelin-13abolished the effect of doxorubicin on A549cells. TransfectionpcMV-Tag2B-APLNR significantly increased A549cell migration compariedwith non-transfected cells and transfection miR-APLNR-494significantlyinhibited A549cell migration.7. Hypoxia induces APJ expression and apelin secretion in A549cells.Cobalt chloride (CoCl2) was used to induced hypoxia. Western blot confirmed that CoCl2time-and concentration-dependent inducedexpression of APJ in A549cells, and ELISA results confirmed that CoCl2time-and dose-dependent increased the release of apelin.Conclusion:Autophagic phosphorylation modifies PAK1-cofilin pathway to mediate lungadenocarcinoma cell migration promoted by apelin-13...