Expression Clinical Significance And Function Of MicroRNA-485-5p And MicroRNA-381 In Non-small Cell Lung Cancer

Part A.MicroRNA-485-5p suppresses growth and metastasis in non-small cell lung cancer cells by targeting IGF2BP2Background:Non-small cell lung cancer(NSCLC)accounts for 80～85%of all lung cancers.Distal metastasis is the main cause leading to deaths in cancer patients.Epithelial-mesenchymal transition(EMT)is characterized by loss of epithelial phenotypes and acquision of mesenchymal features.EMT is linked to enhanced migration and invasion capacities.Transforming growth factor-β(TGF-β)is an important inducer of EMT.Identification of key regulators of EMT is of significance in developing effective therapies.microRNAs(miRs)are endogenous,non-coding regulatory RNAs involved in the regulatio n of a large number of target genes.miRs can bind to the 3’-untranslated regions(3’-UTR)of target mRNAs,causing mRNA degradation or translational repression.A previous study has demonstrated that deregulation of miR-485-5p contributes to the differentiation betwee n NSCLC patients and healthy controls,suggesting its diagnostic potential.However,the p rognostic significance and biological function of miR-485-5p in NSCLC remain largely un known.Objective:In this study,we aimed to explore the prognostic impact of miR-485-5p on NSCLC.The effects of miR-485-5p overexpression and knockdown on NSCLC growth and metastasis were also investigated.In addition,the molecular mechanism of miR-485-5p-dependent regulation of aggressive phenotype was characterized.Materials and methods:A total of 87 NSCLC and adjacent non-tumor lung tissues were obtained from NSCLC patients who had undergone surgical resection of tumors.Tissue samples were routinely fixed in formalin and embedded in paraffin.All cases were confirmed by pathological examination.No patient received radiotherapy or chemotherapy before surgery.Paraffin was removed from specimens by xylene extraction.Total RNA was extracted from tissue specimens and cell lines with Trizol reagent.Reverse transcription was performed with a specific stem-loop primer for miR-485-5p.Real-time PCR was conducted with the TaqMan microRNA Assay kit.The miR-485-5p level was normalized to that of U6.The fragments of MDM4,NUCB1,DEFA,IGF2BP2,and MMP14 3’-UTR were amplified by PCR and cloned into the pMIR-REPORT firefly luciferase vector.A fragment encoding full-length human IGF2BP2 lacking its 3’-UTR was amplified by PCR and cloned into pcDNA3.1(+)vector.A DNA fragment containing human miR-485-5p precursor was amplified by PCR and inserted into pcDNA3.1(+)vector.Cell transfection was performed using Lipofectamine 3000.To generate miR-485-5p stable expression clones,A549 cells were transfected with miR-485-5p-expressing plasmid or pcDNA3.1(+)empty vector and selected in the presence of G418(800 μg/mL)for 2 weeks.Cells were seeded onto 96-well plates and allowed to grow for 1-5 days.Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide(MTT)method.Cell invasive ability was evaluated by Transwell invasion assay.For quantification of cell cycle distribution,cells were fixed with 70%ethanol and incubated with staining solution containing 50 μg/mL propidium iodide(PI).Stained cells were analyzed by a flow cytometer.Cells were plated at 1 × 105 cells/well in 6-well plates and treated with 5 ng/mL TGF-β for 48 h.Cell morphology was evaluated under a phase-contrast microscope.Cells were lysed with radioimmunoprecipitation assay buffer.Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.E-cadherin and vimentin protein expression was assessed by immunoblotting.HEK293T cells were co-transfected with an individual 3’-UTR reporter construct together with miR-485-5p mimic or control oligonucleotides using Lipofectamine 3000.The pRL-TK vector that encodes Renilla luciferase was used to control for transfection efficiency.Luciferase activity was measured 48 h after transfection.A549 stably expressing miR-485-5p or control miR were implanted s.c.into the flank of male BALB/c nude mice(2 × 106 cells/mouse;n =5).Tumor growth was monitored weekly by measuring tumor volume.Mice were sacrificed 4 weeks after cell implantation,and xenograft tumors were resected and weighed.Tumor tissues were lysed and measured for IGF2BP2 protein expression using Western blot analysis.For assessment of tumor metastasis,miR-485-5p-or control miR-transfected A549 cells(2 ×106 cells/mouse;n = 4)were injected into nude mice via the tail vein.Seven weeks later,the animals were sacrificed to harvest lung tissues for pathological examination.The number of microscopic metastatic nodules in the lung was determined.Mean data were analyzed by the Student’s t-test or one-way analysis of variance followed by the post hoc Tukey’s test.Median levels of miR-485-5p between cancer and non-cancerous tissues were compared using the Mann-Whitney U test.Survival curves were plotted by the Kaplan-Meier method,and the difference was determined by the log-rank test.Statistical significance was defined at P<0.05.Results:NSCLC tissues showed a significantly lower level of miR-485-5p than adjacent non-cancerous lung tissues(P = 0.0217).According to the median level of miR-485-5p,the tumor specimens were dichotomized into high and low miR-485-5p groups.Low miR-485-5p expression was significantly associated with advanced TNM stage(P = 0.0216)and lymph node metastasis(P = 0.0037).Patients with low tumoral expression of miR-485-5p had significantly shorter overall survival(P = 0.0136)and disease-free survival(P = 0.0085)than those with high expression of miR-485-5p.Transfection with miR-485-5p mimic significantly decreased the proliferation and invasion of A549 and NCI-H1299 cells,compared to transfection with control mimic(P<0.05).After culturing for 72 h,miR-485-5p suppressed the proliferation of A549 and NCI-H1299 cells by 74%and 56%,respectively.Overexpression of miR-485-5p significantly promoted a G0/G1 cell-cycle arrest and blocked TGF-β-induced EMT in A549 cells.In addition,knockdown of miR-485-5p with anti-miR-485-5p inhibitors led to an enhancement of cell proliferation and invasion.miR-485-5p overexpression significantly(P<0.05)decreased the activity of luciferase reporter with IGF2BP2 3’-UTR,but spared the reporters with the 3’-UTRs of MDM4,NUCB1,DEFA,and MMP14.When miR-485-5p mimic was transfected to A549 and NCI-H1299 cells,the endogenous expression of IGF2BP2 was significantly downregulated.Similar to the findings in A549 cells transfected with miR-485-5p mimic,depletion of IGF2BP2 with specific small interfering RNA led to inhibition of A549 cell proliferation and invasion.Enforced expression of IGF2BP2 with a miR-resistant variant(lacking the 3’-UTR)significantly rescued A549 cells from miR-485-5p-mediated suppression of growth and invasion.To further validate the function of miR-485-5p in vivo,we subcutaneously injected A549 cells stably overexpressing miR-485-5p into the flank of nude mice.It was found that overexpression of miR-485-5p remarkably restrained the growth of A549 xenograft tumors 1 week after tumor cell injection.Final tumor weight in the miR-485-5p group was reduced to 30%of that in the control group(P<0.05).The levels of IGF2BP2 were significantly reduced in the miR-485-5p-overexpressing group(P<0.05).We also checked the anti-metastatic potential of miR-485-5p in vivo.Notably,ectopic expression of miR-485-5p significantly(P<0.05)impaired the ability of A549 cells to form tumor nodules in the lung after intravenous injection.Conclusions:Our data shed light on the miR-485-5p/IGF2BP2 axis in the aggressive phenotype of NSCLC.miR-485-5p downregulation is a poor prognosis factor in NSCLC.Overexpression of miR-485-5p blocks the growth and invasion of NSCLC cells through repression of IGF2BP2.These findings suggest the prognostic and treatment significance of miR-485-5p in NSCLC.Part B.microRNA-381 suppresses the growth and increases cisplatin sensitivity in non-small cell lung cancer cells through inhibition of nuclear factor-KB signalingBackground:Despite treatment improvement in the past decades,the 5-year overall survival rate for NSCLC is low.The therapeutic efficacy of chemotherapy is limited due to development of drug resistance.microRNAs(miRs)are a large class of endogenous,non-coding regulatory RNAs of-22 nucleotides.Several miRs have been found to modulate the chemosensitivity of NSCLC cells to cisplatin.A previous study has shown that miR-381 is downregulated in human lung adenocarcinoma and overexpression of miR-381 significantly decreases cell migration and invasion through repression of inhibitor of differentiation 1(ID1).However,the role of miR-381 in the growth and survival of NSCLC cells remains unknown.Objective:In this study,we explored the effects of restoration of miR-381 on the proliferation and apoptosis of NSCLC cells and examined its impact on cisplatin sensitivity.The molecular mechanism underlying the action of miR-381 was also checked.Materials and methods:A cisplatin-resistant A549 cell line(A549/CDDP)was generated via exposure to increasing concentrations(0.5,1,2,4,and 10 μM)of cisplatin.Briefly,A549 cells were exposed to 0.5 μM cisplatin for 3 days and recovered for 4 days.This treatment was repeated one more cycle at each concentration of cisplatin.The cells were then treated with increasing concentrations of cisplatin up to 10μM.The cisplatin-resistant cell lines were maintained in culture medium supplemented with 4 μM cisplatin.Plasmid transfection was carried out using Lipofectamine 2000.For stable expression of miR-381,A549 and NCI-H460 cells transfected with the miR-381-expressing plasmid or control miR were selected in the presence of G418(800μg/mL)for 2 weeks.Cells were plated in 96-well plates at a density of 5 × 103 cells per well.At 24,48,and 72 h after transfection,cells were collected and the cell number was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide(MTT)assay.For assessment of cisplatin cytotoxicity,A549/CDDP cells transfected with indicated constructs were exposed to different concentrations of cisplatin ranging from 0 to 40 μM for 48 h.Cell viability was measured by MTT assays as described above.The half maximal inhibitory concentration(IC50)for cisplatin was estimated.Cells were trypsinized,counted,and seeded in triplicate onto 6-well plates at a density of 600 cells per well.After incubation for 2 weeks,cells were fixed and stained with crystal violet for 5 min.The number of colonies consisting of>50 cells were counted.Cells were trypsinized,counted,and seeded in triplicate onto 6-well plates at a density of 600 cells per well.After incubation for 2 weeks,cells were fixed and stained with crystal violet.The number of colonies consisting of>50 cells were counted.Apoptosis was assessed using using the Annexin-V FITC Apoptosis Detection Kit following the manufacturer’s protocol.Cells were collected at 48 h after transfection,fixed in 70%ethanol,and labeled with fluorescein isothiocyanate-conjugated annexin-v and PI.Cells were analyzed by a flow cytometrer.For preparation of cytoplasmic/nuclear lysates,a commercially available kit was used.In brief,cells were lysed in lysis buffer containing protease inhibitors and pelleted by centrifugation.The supernatant was collected as cytoplasmic extracts.Protein samples(50μg per lane)were separated by sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes.After blocking non-specific binding sites with 5%fat-free milk,the membranes were incubated with primary antibodies.Horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG was used as secondary antibodies.Immunoreactive protein was visualized by enhanced chemiluminescence.Cellular total RNA was extracted using TRIzol reagent and then reverse transcribed using the Taqman miRNA Reverse Transcription kit.Real-time PCR analysis of miR-3 81 was conducted using the TaqMan miRNA assay system.NF-KB-dependent transcriptional activity was measured using luciferase reporter assay.A NF-κB-Luc reporter plasmid was co-transfected with a Renilla luciferase-expressing pRL-CMV plasmid and miR-381 precursor or control miRNA.After 48 h of transfection,cells were collected and lysed for luciferase assays.A549 stably expressing miR-3 81 or control miR(2 × 106 cells/mouse)were injected s.c.into the flanks of male BALB/c nude mice(6 weeks old).Each group consisted of 5 mice.Tumor volumes were measured weekly for 4 weeks,and growth curves were plotted.Results:MTT assay showed that the delivery of miR-381 precursor significantly(P<0.05)inhibited the proliferation of A549 and NCI-H460 cells,compared to control miR-transfected cells.miR-381 re-expression led to an 80%decline in colony formation ability.In vivo tumorigenic studies further confirmed that overexpression of miR-3 81 retarded the growth of A549 xenograft tumors.Compared to control miR-transfected A549 cells,miR-381 overexpression caused a significant increase in the percentage of cells at the G0/G1 phase and decrease in the percentage of cells at the S phase(P<0.05).Similar results were observed in NCI-H460 cells transfected with miR-381 precursor or control miR.Western blot analysis confirmed that miR-381-overexpressing A549 and NCI-H460 cells had significantly(P<0.05)greater protein levels of p21 and p27 and lower levels of cyclin D1 and CDK4 than control miR-transfected counterparts.Compared to A549 parental cells,cisplatin-resistant counterparts(A549/CDDP)had 2.8-fold lower levels of miR-381(P<0.05).Notably,delivery of miR-381 rendered A549/CDDP cells more sensitive to cisplatin,decreasing the IC50 values by 6-fold.Moreover,overexpression of miR-381 significantly induced apoptotic response in A549/CDDP cells,increasing the percentage of apoptosis from 7.4 ± 1.4%in the control group to 28.2 ± 2.1%(P<0.05).Western blot analysis demonstrated that restoration of miR-381 decreased nuclear accumulation of NF-κB p65 and increased the level of IκBα in parental and cisplatin-resistant A549 cells.Luciferase reporter assay validated that miR-381 overexpression caused a significant decline in NF-κB p65-dependent transcriptional activity(P<0.05 vs.control miR-transfected cells).Consistent with the suppression of NF-κB activation,overexpression of miR-381 reduced the expression of Bcl-2 and Bcl-xL.Rescue experiments showed that overexpression of ID1 restored the nuclear translocation of NF-κB p65 and induced the expression of Bcl-2 and Bcl-xL in miR-381-overexpressing A549 cells.ID1 overexpression significantly(P<0.05)restored the growth of miR-381-overexpressing A549 cells.Co-expression of ID1 almost completely prevented miR-381-induced reduction in the IC50 value for cisplatin in A549/CDDP cells.Moreover,miR-381-mediated apoptosis was significantly abolished by overexpression of ID1.Conclusions:In conclusion,we demonstrate that miR-381 overexpression inhibits cell proliferation and tumorigenesis and increases cisplatin sensitivity in NSCLC cells.Inactivation of NF-κB signaling via downregulation of ID1 accounts for the tumor suppressive activity of miR-381.Restoration of miR-381 may provide a potential therapeutic approach for NSCLC.