| Objective(s):Lung cancer ranks first in the incidence and mortality of cancer in China and is still on the rise.Non-small cell carcinoma is the core type,and Platinum-based chemotherapy is still an important treatment for advanced non-small cell lung cancer.Platinum resistance is the main cause of treatment failure and high mortality in NSCLC.Therefore,it is urgent to elucidate the mechanism of platinum drug resistance and find sensitizing drugsmiRNAs are a group of short sequence RNAs that do not encode proteins.There are many studies have found that miRNAs are not only involved in the proliferation,metastasis and apoptosis of tumor cells,but also involved in the regulation of chemotherapy sensitivity.Our previous miRNAs microarray screening found that miR-424-5p was highly expressed in cisplatin-resistant NSCLC tissues,which may be the driving miRNAs of cisplatin resistance in NSCLC.In NSCLC,studies on miR-424 mainly focus on its expression and prognostic prediction.The relationship between miR-424-5p and chemotherapy resistance and the specific mechanism have not been clarified.After cisplatin is introduced into cells as a cell non-specific anticancer drug,it binds to target DNA to form DDP-DNA adduct,which leads to DNA damage and can induce cell death mediated by transcription factors such as p53.Studies have confirmed that the mutation rate of tumor suppressor p53 in NSCLC patients is significantly higher than that in the general population,and patients with wild-type p53 are more likely to benefit from cisplatin chemotherapy than those with mutant p53.In addition,as a transcription factor,tumor suppressor genes p53 can regulate the expression of multiple miRNAs.Whereas in NSCLC the expression of miR-424 was negatively correlated with wild-type p53 expression.Therefore,it is highly likely that the transcription factor p53 is a miR-424-5p upstream regulator.Exosomes are a type of vesicle membrane with a diameter of 30-150 nm,whose membrane can fuse with the target cell membrane.Exosomes can release the bioactive substances they carry into target cells,thereby modulating the biological functions of the target cells.In many cancers,exosomes can act as miRNAs transporters to transport miRNAs to target cells,thereby regulating the sensitivity of tumor cells.However,the research on the effect of miR-424 on chemotherapy sensitivity of NSCLC through exosomes needs to be continued.This study mainly investigated the effects of miR-424-5p on biological functions and cisplatin resistance of NSCLC cells.And the up and downstream regulatory relationships of miR-424-5p were studied.Finally,the effect of exosome-mediated miR-424-5p on cisplatin resistance of NSCLC cells was investigated.Methods:1.Effects of miR-424-5p on biological functions and cisplatin resistance of NSCLC cells.Using A549 as experimental cell line.After transfection with miR-424-5p mimics/inhibitor,cisplatin IC50 and cell viability were determined by CCK-8 assay and cell cycle and death was measured by flow meter.A549/DDP was used as the experimental cell line to construct a nude mouse transplanted tumor model.A nude mice xenograft tumor model with specific knockdown of miR-424-5p was constructed,followed by administration of cisplatin.Compared the growth of transplanted tumors in nude mice,the proliferation and apoptosis of cells in tumor body were detected by Ki67 and TUNEL.HE staining was used to detect the pathological status of tumor tissues and the expression amount of miR-424-5p was detected by q-PCR.2.Mechanism of miR-424-5p on NSCLC cell biological behavior and cisplatin resistance.A549 was used as the experimental cell line.After transfection with miR-424-5p mimics/inhibitor,JAK2/STAT3 and PI3K/Akt signaling pathway related protein expression and SOCS5/6 protein expression were detected by Western blot,and SOCS5/6 mRNA expression was detected by q-PCR.SOCS5/6 were bioinformatically predicted to be the target genes of miR-424-5p,and dual luciferase reporter gene was used to validate the binding of miR-424-5p to the SOCS5/6 3’-UTR.A549/DDP was used as the experimental cell line to construct a nude mouse transplanted tumor model.A nude mice xenograft tumor model with specific knockdown of mir-424-5p was constructed,followed by administration of cisplatin.The protein expressions of SOCS5/6,JAK2/STAT3 and PI3K/AKT signaling pathways were detected by immunohistochemistry.3.Transcription factor p53 regulates miR-424-5p to induce cisplatin resistance in NSCLCA549 and A549/DDP were used as experimental cell lines.p53 interference vector was constructed.Western blot was used to detect the expression of P53 protein,and q-PCR was used to detect the expression of p53 and miR-424-5p.The binding of p53 to miR-424-5p promoter region was predicted by bioinformatics,and the promoter binding of p53 to miR-424-5p was further verified by dual luciferase reporting.After overexpression/interference of p53,the expression of miR-424-5p was detected by q-PCR,JAK2/STAT3 and PI3K/Akt signaling pathway related protein expression and SOCS5/6 protein expression were detected by Western blot.CCK-8 was used to detect the cell survival rate.A549/DDP was used as the experimental cell line to construct a nude mouse xenograft model.To construct a nude mouse transplanted tumor model capable of overexpressing p53.Ki67 and TUNEL were used to detect the proliferation and apoptosis of cells in the tumor.HE staining was used to detect the pathological status of tumor tissues.The protein expressions of SOCS5/6,JAK2/STAT3 and PI3K/AKT signaling pathways were detected by immunohistochemistry,and the expression of miR-424-5p was detected by q-PCR.4.Effect of exosome-mediated miR-424-5p on cisplatin resistance in NSCLC cells.A549 and A549/DDP were used as experimental cell lines.The expression level of miR-424-5p in cell supernatant and exosomes was detected by q-PCR.Western blot was used to detect the expression levels of Exosome related marker proteins in supernatant.q-PCR was used to detect the expression of exosomal miR-424-5p after overexpression/interference of p53 in A549 cells.Exosomes from A549/DDP cells were co-incubated with A549 cells.DAPI counterstaining was used to detect exosome uptake.Proliferation was measured by CCK-8.Period and death by flow meter.The expression of miR-424-5p was detected by q-PCR.JAK2/STAT3 and PI3K/Akt signaling pathway related protein expression and SOCS5/6 protein expression were detected by Western blot.Results:1.Effects of miR-424-5p on biological functions and cisplatin resistance of NSCLC cellsCompared with the control group,miR-424-5p mimics promoted the proliferation of A549 cells,inhibited cell apoptosis and promote cell G1/S transformation,Cell survival rate increased at 12h,24h,48h and 72h after IC50 cisplatin administration(P<0.01).miR-424-5p inhibitor insignificantly inhibited the proliferation of A549 cells,promoted cell apoptosis and arrested the cell cycle at G1 phase(P<0.01).Nude mice transplanted tumor experiments showed:Compared with the control group,miR-424-5p knockdown combined with cisplatin could inhibit cell proliferation,promote cell apoptosis,restore the sensitivity of drug-resistant cells to cisplatin,and thus reduce tumor size(P<0.05).2.Mechanism of miR-424-5p on NSCLC cell biological behavior and cisplatin resistanceCompared with the control group,the expression levels of p-JAK2,p-STAT3,p-PI3K and p-AKT proteins were increased(P<0.01),and the expression levels of SOCS5/6 protein and SOCS5/6 mRNA were decreased(P<0.01)after transfection with miR-424-5p mimics.After transfection with miR-424-5p inhibitor,the protein expressions of p-JAK2,p-STAT3,p-PI3K,and p-AKT were decreased(P<0.01),and the protein expression of SOCS5/6 was increased(P<0.01),and SOCS5/6 mRNA was also increased(P<0.01).Dual luciferase reporter gene verified that miR-424-5p could target SOCS5/6.Immunohistochemistry of transplanted tumor in nude mice showed:Compared with the control group,the specific knockdown of miR-424-5p combined with cisplatin increased the expressions of SOCS5 and SOCS6,while the expressions of p-JAK2,p-STAT3,p-PI3K and p-AKT decreased.3.Transcription factor p53 regulates miR-424-5p to induce cisplatin resistance in NSCLCThe expression level of miR-424-5p in A549/DDP cells was significantly higher than that in A549 cells(P<0.01),while the expression level of P53 in A549/DDP cells was significantly lower than that in A549 cells(P<0.01).Dual luciferase reporter gene verified that p53 could target miR-424-5p.Compared with the control group,after interfering the expression of P53 the expression of miR-424-5p was significantly increased(P<0.01),the expression of SOCS5 and SOCS6 proteins was significantly decreased(P<0.01),and the expression of p-JAK2,p-STAT3,p-PI3K and p-AKT proteins was increased(P<0.01).The cell survival rate increased at 12h,24h,48h and 72h after IC50 cisplatin administration(P<0.01).However,overexpression of P53 significantly decreased the expression of miR-424-5p(P<0.01),increased the protein expression of SOCS5 and SOCS6(P<0.01),and decreased the protein expression of p-JAK2,p-STAT3,p-PI3K,and p-AKT(P<0.01).Nude mice transplanted tumor experiments showed:Compared with the control group,overexpression of P53 combined with cisplatin could inhibit cell proliferation,promote cell apoptosis,restore cisplatin sensitivity of drug-resistant cells,and achieve tumor volume reduction.At the same time,the expression levels of miR-424-5p were decreased(P<0.01),the expression levels of SOCS5 and SOCS6 were increased,while the expression levels of p-JAK2,p-STAT3,p-PI3K,and p-AKT were decreased.4.Effect of exosome-mediated miR-424-5p on cisplatin resistance in NSCLC cells.The expression of miR-424-5p in the supernatant of A549/DDP was significantly higher than that in A549 cells(P<0.01),and the expression of Exosome marker proteins CD9,CD63 and TSG101 in the supernatant of A549/DDP was higher than that in A549 cells(P<0.01).The expression level of miR-424-5p of A549/DDP-exo was significantly higher than A549-exo(P<0.01).miR-424-5p of A549-exo was increased after interfering the expression of p53(P<0.01),and the expression level of miR-424-5p in exosomes of A549 cells was decreased after overexpressing p53(P<0.01).There was no significant difference in the survival rate of A549 cells incubated with A549/DDP-exo after treated with different concentrations of cisplatin.Compared with the IC50 group,the cell apoptosis was increased and the cell cycle was arrested in G1 phase in the 2-fold IC50 group.The expression of miR-424-5p was decreased(P<0.01),the expression levels of SOCS5 and SOCS6 proteins were increased(P<0.01),and the expression levels of p-JAK2,p-STAT3,p-PI3K,and p-AKT proteins were decreased(P<0.01).In the 0.5-fold IC50 group,the cell apoptosis was decreased and promote cell G1/S transformation(P<0.01).The expression of miR-424-5p was increased(P<0.01),the expression levels of SOCS5 and SOCS6 proteins were significantly decreased(P<0.01),while the expression levels of p-JAK2,p-STAT3,p-PI3K,and p-AKT proteins were significantly increased(P<0.01).Conclusion(s):1.miR-424-5p targeted and inhibited SOCS5/6,activates PI3K/AKT and JAK2/STAT3 pathways,promotes NSCLC cell proliferation,inhibits apoptosis,promote cell Gl/S transformation,and induces cisplatin resistance.2.Transcription factor p53 regulates miR-424-5p to induce cisplatin resistance in NSCLC3.Transcription factor p53 can regulate miR-424-5p and transport it to recipient cells through exosomes,inhibit SOCS5/6,activate PI3K/AKT and JAK/STAT3 pathways,and induce cisplatin resistance of recipient cells. |
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