Analysis Of The Mechanism Of Mir-137 Regulating AMPK/Autophagy Pathway On Cisplatin Resistance In Non-small Cell Lung Cancer

Objective:We aim to analyze the relationship between microRNA-137(miR-137)expression variances and clinicopathologic characteristics as well as prognoses in patients with non-small cell lung cancer(NSCLC).We aim to establish the potential role and mechanism of miR-137 in regulating the cisplatin resistance in A549 lung cancer cells.Methods:(1)Quantitative real-time reverse transcription polymerase chain reaction(qRT-PCR)and In Situ Hybridization(ISH)were used to measure miR-137 expression in cancer tissues and paired adjacent normal tissues.The statistical differences between miR-137 expression and classified variables including age,gender,pathological pattern,tumor location,differentiation degree,tumor size,lymph node status and M stage were measured through using chi-square test.Kaplan-Meier curve was used to determine the relationship between miR-137 expression and prognoses in patients with NSCLC.(2)Lentiviruses that could overexpress or inhibit miR-137 expression were used to infect A549 cells.Infected cells were cultured with cisplatin and the chemo-sensitivity of the cells to cisplatin was measured through MTT assay.Autophagic flux indicators such as P62 and LC3 were detected through immunoblotting and immunofluorescence to determine cell's autophagy level.(3)The target of miR-137 was searched through using gene target predicting softwares including Targetscan?Miranda and Diana-MicroT,and further verified through the luciferase reporter gene system.qRT-PCR and immunoblotting were used to determine the regulative relationship between miR-137 and AMPK ?1.(4)Immunohistochemistry was used to measure AMPK al expression in cancer tissues and paired adjacent tissues.Chi-square test was conducted to determine the statistical differences between AMPK al expression and classified variables including age,gender,pathological pattern,tumor location,differentiation degree,tumor size,lymph node status and M stage.Kaplan-Meier curve was used to determine the relationship between AMPK al expression and prognoses in patients with NSCLC.(5)Lentiviruses that could overexpress or inhibit AMPK al expression were used to infect A549 cells.Infected cells were cultured with cisplatin and the chemo-sensitivity of the cells to cisplatin was measured through MTT assay.Autophagic flux indicators such as P62 and LC3 were detected through immunoblotting to reflect cell's autophagy level.(6)Immunoblotting was used to determine the modulation relationship between AMPK ?1 and Trx.Apoptosis proteins(Bax,Bcl-2)as well as autophagic signaling proteins(mTOR,p-mTOR)were also detected through using immunoblotting.H2DCFH reagent was used to determine the intracellular reactive oxygen species(ROS)levels.Results:(1)qRT-PCR and ISH were all found that miR-137 expression was down-regulated in cancer tissues compared with paired adjacent normal tissues.miR-137 expression negatively correlated with tumor size(p=0.045).However,other characteristics,including age(p=0.599),gender(p=0.144),pathological pattern(p=0.615),tumor location(p=0.615),differentiation degree(p=0.774),N-status(p=0.601),and M stage(p=1.000)showed no significant association.Additionally,patients with high(? median)expression of miR-137 had longer overall survival(OS)than patients with low miR-137expression(p=0.009).(2)Compared with the control group cells,lentivirus that could inhibit miR-137 significantly down-regulated miR-137 expressin in lung cancer A549 cells(p=0.0017),and the lentivirus that could overexpress miR-137 significantly up-regulated miR-137 expressin in lung cancer A549 cells(p=0.0046).Inhibition of miR-137 could decrease chemo-sensitivity of lung cancer A549 cells to cisplatin through increasing autophagic level,while over-expressed miR-137could decrease autophagic level and in turn partially restore chemo-sensitivity of A549 cells to cisplatin.(3)AMPK al was indicated as a direct target gene of miR-137 through using gene target predicting software and luciferase reporter gene system.qRT-PCT and immunoblotting further confirmed that miR-137 could negatively regulate AMPK al.(4)The expression level of AMPK al was up-regulated in NSCLC tissues compared with adjacent normal tissues,over-expressed AMPK al was positively correlated with N-status(p=0.005).Nevertheless,other characteristics,including age(p=0.861),gender(p=0.715),pathological pattern(p=0.240),tumor location(p=0.867),differentiation degree(p=0.326),tumor size(p=0.188),and M stage(p=0.497)showed no significant association.Additionally,patients with high(? median)expression of AMPK al had shorter OS when compared with low AMPK ?1 expression patients(p=0.001).(5)Compared with the control group cells,lentivirus that could over-express AMPK ?1 significantly up-regulated AMPK al expressin in lung cancer A549 cells(p=0.0015),and the lentivirus that could inhibit AMPK al significantly down-regulated AMPK al expressin in lung cancer A549 cells(p=0.0008).Up-regulation of AMPK ?1 could decrease chemo-sensitivity of lung cancer A549 cells to cisplatin through increasing autophagic level,while inhibition of AMPK al could decrease autophagic level and in turn partially restore chemo-sensitivity of A549 cells to cisplatin.(6)Low expression of miR-137 in lung cancer A549 cells colud negatively regulate AMPK al and up-regulate the expression of phosphorylated AMPK al,which could inhibit mTOR phosphorylation and then leading to activition of autophagy.High expression of AMPK ?1 could up-regulate Trx expression,Trx leading to dysfunction of apoptosis through decreasing intracellular ROS levels in lung cancer A549 cells.Conclusion:miR-137 expression correlates with clinicopathological characteristics and prognoses in NSCLC patients,miR-137-AMPK al could influence chemosensitivity of A549 cells to cisplatin through regulating autophagic pathway.The results suggest that miR-137 possess the potential to be a prognostic biomarker and treatment target.