| Objective:Lung cancer is the malignant tumor with the highest morbidity and mortality in the world,and there is still a lack of effective treatment.Human umbilical cord mesenchymal stem cells(huc MSCs)and huc MSC-derived exosomes(huc MSC-Ex)are similar in biological functions,and can both be used for the treatment of lung cancer.Small-molecule drug resveratrol(Res)can enhance the biological functions of huc MSCs.We speculate that the exosomes secreted by Res-pretreated huc MSCs(Res-huc MSC-Ex)may have a better therapeutic effect on lung cancer.In this paper,we explored the influence of Res on the biological characteristics of huc MSCs,and analyzed the effect of Res-huc MSC-Ex on lung cancer,and studied the relevant mechanisms involved.Methods:(1)The effect of Res on the biological characteristics of huc MSCs(1)Primary culture and identification of huc MSCs.huc MSCs were isolated by adherent culture of umbilical cord tissue;Osteogenic and adipogenic diferentiation of huc MSCs were induced by special media;Flow cytometry was used to detect the surface antigen(CD44,CD105,CD45,CD34)expression of huc MSCs.(2)The effect of Res on huc MSC proliferation and differentiation.After treatment with Res,Cell counting Kit-8(CCK8)and clone formation assays were adopted to detect huc MSC proliferation;huc MSCs pretreated with Res were cultured with osteogenic and adipogenic induction media,and the differentiation potential was analyzed.(3)The influence of Res on the expression of huc MSC cytokines(IL-6,IL-8,VEGF,MCP-1).After treatment with Res,Luminex assay was performed to screen the significant cytokines of huc MSCs;Enzyme-linked immunosorbent assay(ELISA)and quantitative reverse transcription polymerase chain reaction(q RT-PCR)were used to detect cytokines(IL-6,IL-8,MCP-1,VEGF)secretion and m RNA expression,respectively.(4)The mechanism of Res affecting the proliferation and differentiation of huc MSCs(Wnt/?-catenin signal).Res were used alone or in combination with ?-catenin agonist CP21 to treat huc MSCs;western blot or immunofluorescence was adopted to detect the expression of ?-catenin and Wnt signaling down stream proteins(Cyclin D1 and Cyclin D3);The proliferation and differentiation potential of huc MSCs were also analyzed.(2)The effect of Res-huc MSC-Ex on the proliferation,apoptosis and metastasis of lung cancer cells(1)Purification and identification of Res-huc MSC-Ex.Collecting the supernatant of Res-preconditioned huc MSCs and extracting exosomes by ultracentrifugation;The morphology of exosomes was analyzed by transmission electron microscope;Nanosight Visible Nanoparticle Analyzer was adopted to measure the particle size distribution,quantity and membrane potential of the exosomes;CD9 and CD63 expression of exosomes were detected by western blot.The content of Res in Res-huc MSC-Ex was determined by high performance liquid chromatography.(2)Uptake of Res-huc MSC-Ex by lung cancer cells.A549 cells were transfected with a lentivirus expression vector to construct a cell line of GFP stable expression(GFP-A549);Di R-labeled huc MSC-Ex and Res-huc MSC-Ex co-cultured with GFP-A549,and uptake of the exosomes by A549 cells were observed under a laser confocal microscope.(3)Effect of Res-huc MSC-Ex on migration,proliferation and apoptosis of lung cancer cells.Lung cancer cell lines(A549,H1299)were treated with huc MSC-Ex and Res-huc MSC-Ex,and then Transwell migration assay and real-time unlabeled cell analysis(RTCA)system were adopted to detect cell migration;CCK8assay and annexin V/PI apoptosis staining were performed to analyze cell proliferation and apoptosis,respectively.(4)Construction of lung cancer model and Res-huc MSC-Ex treatment.C57 mice were injected with a murine lung cancer cell line LLC1/2 through tail veins to establish a lung cancer metastasis model.Res-huc MSC-Ex and huc MSC-Ex were injected intravenously for treatment.(3)The mechanisms involved in inhibition of lung cancer progression by Res-huc MSC-Ex(1)The effect of Res-huc MSC-Ex on the signal pathways of lung cancer.Lung cancer cell lines(A549,H1299)were treated with Res-huc MSC-Ex,and then the expression of PI3 K,p-Akt,p-STAT3,p-NF-?B and Wnt signal related molecules(?-catenin,Cyclin D1,Cyclin D3)were detected by western blot;The expression of?-catenin in the lung cancer cells(A549,H1299)and murine lung cancer tissue were further analyzed by immunofluorescence or immunohistochemistry staining.(2)Screening the key molecules within Res-huc MSC-Ex that suppresses Wnt/?-catenin signaling pathway.huc MSCs were treated with Res,and then q RT-PCR was performed to screen the key molecule with significant changes,which could inhibit Wnt/?-catenin signaling pathway(DKK3).ELISA and western blot were used to detect DKK3 expression in huc MSC supernatant and exosomes;The expression of DKK3 in the murine lung cancer tissues was further analyzed by immunohistochemistry staining;The expression of DKK3 in lung cancer cells(A549,H1299,H460)and normal cells(human embryonic lung fibroblast MRC-5,huc MSCs)were compared by western blot;H1299 cell was transfected with a lentivirus Dkk3 interference vector(sh DKK3)to construct a DKK3-deficient H1299 cell line(sh DKK3-H1299);the GFP expression in H1299 cell was observed under fluorescence microscope to evaluate the efficiency of lentivirus transfection,and the DKK3 m RNA expression was detected by q RT-PCR to assess the interference efficiency of sh DKK3;Transwell migration assay was used to display the migration ability of the DKK3-deficient H1299.(3)The effect of the key molecule(DKK3)within Res-huc MSC-Ex on lung cancer metastasis and Wnt/?-catenin signaling pathway.huc MSCs were transfected with a lentivirus DKK3 interference vector(sh DKK3)to construct a DKK3-deficient cell line(sh DKK3-huc MSC),and then preconditioned with Res.The supernatant of the sh DKK3-huc MSCs was collected for exosome extraction.Transwell migration assay and murine lung cancer metastasis model were used to evaluate the effect of DKK3-deficient Res-huc MSC-Ex on lung cancer metastasis.Western blot and immunohistochemical staining were adopted to analyze the influence of DKK3-deficient Res-huc MSC-Ex on the expression of ?-catenin,Cyclin D1 and Cyclin D3 in the lung cancer(tissues / cells).Results:Huc MSCs adhered to the surface of the culture dish and displayed a population of cells with spindle shape.huc MSCs presented the ability of differentiating into either osteocytes or adipocytes,displayed by positive staining of Alizarin Red S and Oil Red O.In addition,huc MSCs were positive for CD44 and CD105 but negative for CD45 and CD34.Res inhibited the m RNA expression and protein secretion of IL-6,IL-8 and MCP-1;Res promoted huc MSC proliferation,induced osteogenic and adipogenic differentiation,and inhibited the expression of ?-catenin(total / nuclear proteins)and downstream molecules of Wnt signal(Cyclin D1,Cyclin D3),and restoring the expression of ?-catenin by CP21 impaired the effect of Res on the proliferation and differentiation of huc MSCs.Res-huc MSC-Ex is about 110 nm in diameter and appear in a cup-shaped morphology,and express CD9 and CD63;Res stimulated huc MSCs to secret exosomes,enhanced the membrane potential of the exosomes.;Res-huc MSC-Ex could be absorbed by lung cancer cells,displayed better effect on the inhibition of lung cancer metastasis than huc MSC-Ex,and did not affect lung cancer cell proliferation and apoptosis.Res-huc MSC-Ex did not affect the inflammatory signaling pathway(STAT3,NF-?B,Akt,PI3K),but inhibited the expression of Wnt/?-catenin signaling pathway related proteins(?-catenin,Cyclin D1,Cyclin D3)in lung cancer cells or murine lung cancer tissues.CP21 was used to restore the expression of ?-catenin,which could reverse the expression of Cyclin D3 and abolish the inhibitory effect of Res-huc MSC-Ex on the migration ability of lung cancer cells;Res promoted huc MSC DKK3 m RNA expression and protein secretion,and enriched DKK3 in Res-huc MSC-Ex;DKK3 expression in lung cancer cell lines(A549,H1299,H460)is significantly lower than that in MRC-5 and huc MSCs,and DKK3 knockdown in H1299 promoted cell migration;DKK3-deficient huc MSC-Ex and Res-huc MSC-Ex could not inhibit lung cancer metastasis,and could not induce DKK3 expression in murine lung cancer tissues;In addition,DKK3-deficient Res-huc MSC-Ex were unable to reduce the expression of Wnt/?-catenin signaling pathway related proteins(?-catenin,Cyclin D1,Cyclin D3)in lung cancer cells or murine lung cancer tissues.Conclutions: Res reduced the expression of inflammatory factors(IL-6,IL-8,MCP-1)of huc MSCs,and promoted huc MSC proliferation and differentiation by inhibiting Wnt/?-catenin signaling pathway.In addition,Res stimulated huc MSC-Ex secretion and DKK3 expression,and Res-huc MSC-Ex could transport DKK3 to suppress lung cancer metastasis by inactivating Wnt/?-catenin signaling pathway. |
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