Inhibitory Effect And Mechanism Of Mesenchymal Stem Cells Cultured In 3D System On NSCLC Cell Lines

Objective:Non-small cell lung cancer(NSCLC),with its high mortality and poor prognosis,remains a worldwide problem.Mesenchymal stem cells(MSCs)have the ability of homing to tumor sites and low immunogenicity,so they are ideal seed cells for tumor therapy.Compared with traditional two-dimensional cell culture,three-dimensional cell culture can make MSCs grow in a three-dimensional state and better simulate the in vivo microenvironment.At the same time,the previous study of our group showed that compared with the two-dimensional cultured MSCs(2D-MSCs),the gene expression profile of three-dimensional cultured MSCs(3D-MSCs)changed significantly,and interleukin-24(IL-24)was the most up-regulated gene,up to 20.6 times,which has inhibitory effects on a variety of tumors.However,the inhibitory effect of IL-24 secreted by 3D-MSCs on NSCLC cell lines and its molecular mechanism is still unknown.Therefore,the objective of this thesis was to explore the inhibitory effect of MSCs cultured in the 3D system on NSCLC cell lines,and further study the inhibitory molecular mechanism of IL-24 secreted by 3D-MSCs,to provide an experimental basis for the application of 3D-MSCs in tumor treatment.Methods:NSCLC cell lines from two types of tissue were used in this thesis,including large cell lung cancer cell line NCI-H460 and squamous cell lung cancer cell line SK-MES-1.Firstly,the effects of 3D-MSCs and 2D-MSCs on the proliferation and migration of NSCLC cell lines were studied by MTT,colony formation assay and scratch experiments,respectively.Reverse transcription polymerase chain reaction(RT-PCR)was used to detect the expression of IL-24 receptor IL-22R1 and IL-20R2 in NSCLC cells.Then the primers were designed according to the sequence of IL-24 in GenB ank.The IL-24 cDNA was obtained from MSCs by RT-PCR,and the overexpression plasmid pIRES2-EGFP-IL-24 was constructed.The overexpression plasmid pIRES2-EGFP-IL-24 and the silencing plasmid pGPHl-EGFP-IL-24 were transfected into MSCs to obtain MSCs-O(MSCs overexpression IL-24)and MSCs-S(MSCs silencing IL-24)respectively.The expression of IL-24 in MSCs,MSCs-O and MSCs-S was detected by Western blot.Finally,RT-qPCR and Western blot were used to investigate the inhibitory mechanism of 3D-MSCs on NSCLC cell lines.Results:(1)MTT results showed that 3D-MSCs had a strong inhibitory effect on the proliferation of NCI-H460 and SK-MES-1 with the inhibition rate of 44.6%and 39.6%respectively.(2)Colony formation assay showed that 3D-MSCs had a strong inhibitory effect on the colony-forming capacity of NCI-H460 and SK-MES-1.(3)Scratch assay showed that 3D-MSCs CM had a strong inhibitory effect on the migration of NCI-H460 and SK-MES-1 with migration inhibition rate of 44.6%and 39.6%respectively.(4)RT-PCR results showed NCI-H460 and SK-MES-1 cells expressed IL-24 receptot IL-22R1/IL-20R2.(5)By constructing MSCs-O(MSCs overexpressing IL-24)and MSCs-S(MSCs silencing IL-24)to verify that IL-24 is the key cytokine for 3D-MSCs to improve the inhibition of NSCLC cell lines significantly.MSCs-O and MSCs-S enhanced and weakened the inhibitory effect of NSC LC cell proliferation and migration respe ctively(6)Western blot was used to further verify the inhibitory mechanism,The results showed that IL-24 could affect p38 MAPK and CXCR4/AKT signaling pathways.In p38 MAPK signiling pathway,IL-24 could upregulate p-p38 MAPK expression and then upregulated Bax/Bcl-2 to induce apoptosis.In CXCR4/AKT signaling pathway,IL-24 could downregulate the expression of CXCR4 and AKT by inducing the expression of IL-24 mRNA in NSCLC cells,which could downregulate the expression of proliferation-related protein c-Myc and migration-related protein MMP2 to inhibit proliferation and migration of NSCLC cell lines.Conclusion:This study proved that 3D-MSCs had a stronger inhibitory effect on the proliferation and migration of NCI-H460 and SK-MES-1 cell lints than 2D-MSCs.The inhibitory effect may be mediated by the p38 MAPK and CXCR4/AKT signaling pathways regulated by IL-24 secreted by 3D-MSCs,so as to play a tumor inhibitory role.It provides basis basis for the clinical of 3D-MSCs in tumor inhibition.