| It has been reported that 1.8 million people worldwide are diagnosed with lung cancer and 1.6 million people die from the disease every year.Although the 5-year survival rate of lung cancer has increased in China,it is still less than 20%.Despite of the increased early diagnosis rate of lung cancer in recent years,a large number of lung cancer patients are still in the advanced stage,therefore it is necessary to explore new treatment options.Mesenchymal Stem Cells(MSCs),early undifferentiated cells in the development of mesoderm,are widely distributed in differentiated tissues and are.They have multiple differentiation potentials and strong proliferative capacity.During the process of inflammation,damaged organ tissues can secrete related chemokines to promote the migration of MSCs to the injury site,and induce their differentiation to participate in tissue repair.The tumor tissue has a local inflammatory reaction due to its strong metabolism,and its process is similar to tissue damage.Tumors and their microenvironment mainly rely on various growth factors,inflammatory cytokines,chemokines and other factors to induce MSCs to homing.Initially,it was thought that MSCs exert their beneficial effects through their ability to locally engraft and differentiate into a variety of cell types and,subsequently,replace injured tissues.However,accumulating evidence has hsown that that the most implanted cells do not survive,and that the beneficial benefits of MSC-based therapy may be attributed to a plethora of bioactive molecules secreted by the cells that play an essential regulatory role in major biological processes.Because of its innate migration capacity and the characteristics of homing tumors,MSCs have been designed to provide specific anti-cancer agents for tumor cell-specific targeting,thus providing a new cell-mediated gene therapy option.MSCs have been recognized as a potential tool for cancer treatment,especially because the anti-tumor ability observed in different tumors including lung cancer.However,several studies have found that MSCs are involved in cancer progression,the role of MSCs in tumors remains controversial.It has been revealed that that MSCs can migrate into solid tumors and then promote tumor growth and migration.Human bone marrow MSCs can promote the growth of pancreatic cancer cells.Human adipose stem cells can induce breast cancer cell metastasis through secreted proteins.The same source of MSCs have different effects on different tumors.Human adipose-derived MSCs can inhibit the growth of transplanted tumors of A549 cells,but can promote the transplantation of colorectal cancer HT-29 cells.These findings indicate that the effect of MSCs on tumor proliferation is not only related to its source,but also the type of tumor.Li et al.found that bone marrow-derived MSCs inhibited the proliferation of A549 cells in vitro and promoted the growth of A549 transplanted tumors in vivo,but the potential mechabisms has not been explored.Lin et al.'s showed that MSCs from the same source may have different effects on tumor cells in different environments.Therefore,the mechanism of different effects of MSCs on tumor cell proliferation in different environments need to be explored,thereby making MSCs safer for cancer treatment is of great significance.The response of cells to hypoxia or hypoxic levels is an evolutionarily conserved program driven by a transcription factor called Hypoxia-inducible factor(HIF).Tumor cells can promote tumor proliferation by HIF-1? activation by an enzyme or angiogenesis that promotes glycogen metabolism under hypoxia.Hypoxia is a characteristic of solid tumors,conventional in vitro culture of tumor cells can not reflect the state of the hypoxic microenvironment in solid tumors in vivo.Therefore,we hypothesized that hUCMSCs has opposite effects on the proliferation of lung adenocarcinoma A549 cells in vitro and in vivo is closely associated with the differences of hypoxic microenvironment.Insulin-like growth factor(IGF),named for its structure similar to insulin,is a class of multifunctional cell proliferation regulators that are involved in regulating the function and growth of almost all organs in animals.IGF-1 can promote the synthesis of protein and nucleic acid DNA as well as the metabolism of carbohydrate in the body.,IGF-1 acts on target organs through autocrine,paracrine and endocrine to promote the growth and differentiation of cells under the mediation of receptor and binding protein.Tumor cells can self-synthesize IGF-1 in the process of tumor cell proliferation and stimulate their proliferation via IGF-1 and IGF-1R loop;IGF-1 binds to IGF-1R can up-regulate vascular endothelial growth factor expression in the process of tumor invasion and metastasis.It has been shown that HIF-1? expression in lung cancer cells is not only regulated by hypoxia but also other non-hypoxia factors including tumor suppressor genes p53,pVHL and PTEN7 viral oncogenes such as EBV and HPV-16,growth factor IGF-123-25,etc.Therefore,IGF-1,which is an upstream regulatory signal of HIF-1?,may regulate the proliferation of A549 cells under hypoxic conditions.miRNA,a class of non-coding RNA consisting of about 22 nucleotides,inhibit gene expression at the transcriptional level.It form a hairpin structure that is a non-coding region derived from cell chromosome.miRNAs have specific expression and can distinguish tissue sources.Their expression or structural abnormalities are closely related to tumorigenesis,mediating tumor proliferation,invasion,metastasis,staging and prognosis.Therefore,abnormal miRNA expression in tumor tissue has the potential to aid in the diagnosis and development of targeted therapeutic drugs.A human non-contact co-culture model of human umbilical cord derived MSCs (hUCMSCs) and A549 cells and A549 cell xenograft model were established to investigate HIF-1? and IGF-1 in hUCMSCs under hypoxic conditions in this study.The effect of hUCMSCs on the proliferation of A549 cells and its molecular mechanism has been investigated.The effect of hUCMSCs on the miRNA expression profile of A549 cells was detected by Exiqon miRNA oligonucleotide microarray.The qPCR was used to verify the biological functions of differentially expressed miRNAs.The relationship between IGF-1 and HIF-1? provides a preliminary laboratory basis for further exploring the mechanism by which hUCMSCs promote the proliferation of A549 cells and finding effective target genes for the treatment of lung cancer.PART I The role and mechanism of HIF-1? in promoting the proliferation of lung adenocarcinoma A549 cells under hypoxia ObjectiveThis study was to observe the effect of hUCMSCs on the proliferation of A549 cells under hypoxia and to explore the mechanism of HIF-1 a in hUCMSCs promoting the proliferation of A549 cells..Methods1.A non-contact co-culture model of A549 cells and hUCMSCs were established and the expression of HIF-la gene in A549 cells were examined.MTT assay,cell scratch assay and flow cytometry were used to detect the proliferation,migration and apoptosis of A549 cells induced by hUCMSCs under hypoxic conditions.Real-time fluorescence quantitative PCR(RT-PCR)and western blot were used to detect the expression of HIF-la,survivin,Bcl-2,Caspase3,JAK1,JAK2 and STAT3 genes and proteins in A549 cells.2.,The effect of hUCMSCs on the growth of transplanted tumors of A549 cells was observed in a transplanted tumor model with co-injecting hUCMSCs and A549 cells.After the expression of HIF-la gene in A549 cells interfered and the expression levels of HIF-la,survivin,Bcl-2,Caspase3,JAK1.JAK2,STAT3 was detected.Results1.hUCMSCs inhibited proliferation(P<0.01)and migration(P<0.01)of A549 cells under normoxic conditions after cultured for 72 hours.The proliferative and migration ability of A549 cells were greatly enhanced under hypoxic conditions compared with normoxic conditions(P<0.01).hUCMSCs further enhanced their proliferation and migration ability(P<0.01).The proliferation and migration ability of A549 cells were decreased significantly after HIF-la koncldown in A549 cells under hypoxic conditions(P<0.01).The proliferation and migration ability of A549 cells did not reverse after co-culture with hUCMSCs(P>0.05).2.The apoptosis rate of A549 cells in the co-culture group was higher than that in the culture group after cultured for 72 hours(P<0.01).The apoptotic rate of A549 cells was significantly decreased under hypoxic conditions compared with the group cultured under normoxic conditions(P<0.01).The apoptotic rate of A549 cells was further decreased after co-culture with hUCMSCs.The apoptotic rate of A549 cells was significantly increased after knocking down the expression of HIF-la in A549 cells(P<0.01).There was no significant change in the apoptosis of A549 when co-cultured with hUCMSC(P>0.05).3.The expression levels of HIF-la,survivin,Bcl-2,JAK1,JAK2,STAT3 genes and proteins in A549 cells were markly increased under hypoxic conditions compared with normoxic conditions(P<0.01),and the expression levels of Caspase3 gene and protein were down-regulated(P<0.01).hUCMSCs greatly upregulated HIF-la,survivin,Bcl-2,JAK1,JAK2,STAT3 gene and protein expression and down-regulated the expression of Caspase3 gene and protein in A549 cells under hypoxic conditions.The expression of HIF-la,survivin,Bcl-2,JAK1,JAK2,STAT3 genes and protein levels were down-regulated in A549 cells after knocking down IGF-1 gene in A549 cells under hypoxic conditions(P<0.01),and the expression levels Caspase3 was up-regulated(P<0.01).There was no significant change in the expression of these genes in A549 cells which HIF-la gene interfered after co-culture with hUCMSCs(P>0.05).4.hUCMSCs promoted the growth of A549 cell xenografts in vivo and the expression levels of HIF-la,survivin,Bcl-2,JAK1,JAK2,STAT3 genes and protein were increased(P<0.01),while the expression of Caspase3 gene and protein levels were down-regulated(P<0.01).The growth of transplanted tumors was decreased after HIF-la konckdown in A549 cells and the expression levels of HIF-la,survivin,Bcl-2,JAK1,JAK2,STAT3 genes and protein in transplanted tumor tissues were decreased(P<0.01),while the expression levels of Caspase3 gene and protein was up-regulated(P<0.01).hUCMSCs could not reverse the growth ability of A549 cell xenografts and the expression levels of the above genes.Conclusion1.hUCMSCs inhibited the proliferation and migration of A549 cells and promoted apoptosis under normal oxygen culture conditions.However,hUCMSCs promoted proliferation and nigration of A549 cells and inhibited apoptosis under hypoxia or in vivo.2.hUCMSCs upregulated the expression of HIF-la,survivin,Bcl-2,JAK1,JAK,STAT3 gene and protein levels in A549 cells under hypoxia or in vivo,while down-regulated the expression of Caspase3,3.In vivo or under hypoxic conditions,hUCMSCs promoted proliferation and migration of A549 cells in combination with hypoxia.HIF-la expression played an important role in this process.PART ? The role and mechanism of IGF-1 in promoting the proliferation of lung adenocarcinoma A549 cells by hUCMSCs under hypoxiaObjectiveThis study was to explore the mechanism of IGF1 in hUCMSCs promoting the proliferation of lung adenocarcinoma A549 cells and the effect of hUCMSCs on EMT in A549 cells under hypoxia.Methods:1.The effect of hUCMSCs on the expression of IGF-1 in A549 cells under hypoxia and A549 cell transplanted tumors was observed by establishing a non-contact co-culture model of hUCMSCs and transplanted tumors of A549 cells in nude mice.2.The effects of hUCMSCs on the proliferation,migration and apoptosis of A549 cells in vitro were observed after IGF-1 knockdown in A549 cells.3.The expression levels of IGF-1,Bcl-2,HIF-la,survivin,JAK/STAT3,Caspase3 genes and proteins in A549 cells and transplanted tumors were detected by RT-PCR and Western blot,respectively..4.The growth of transplanted A549 cells in nude mice was observed after IGF-1 knockdown in A549 cells,and the expression levels of EMT-related genes including E-cadherin,?-catenin,N-cadherin,vimentin and Twist in transplanted tumors were detected by qPCR and western blot,respectively.Results:1.The expression of IGF-1 in A549 cells of the co-culture group was decreasedunder normal oxygen condition(P<0.05),while the expression of IGF-1 in A549 cells of co-culture group was increased under hypoxia(P<0.01).2.The proliferation and migration ability of A549 cells was decreased after IGF-1 knockdown in A549 cells under hypoxia in vitro(P<0.01).The expression levels of Bel-2,HIF-la,survivin,JAK1,JAK2 and STAT3 was decreased(all P<0.01),while the expression level of Caspase3 was increased(all P<0.01).Furthermore,hUCMSCs partially reversed the expression levels of Bcl-2,HIF-la,survivin,JAK1,STAT3 and Caspase 3 in A549 cells after IGF-1 interfered.3.The apoptotic rate of A549 cells was increased after IGF-1 knockdown under hypoxia in vitro(P<0.01)and it did not change significantly when co-cultured with hUCMSCs(P>0.05).4.The growth of transplanted A549 cells in nude mice was inhibited after IGE-1 knockdown in A549 cells.The expression levels of Bcl-2,HIF-la,survivin,JAK1,JAK2 and STAT3 were all down-regulated(all P<0.01)and the expression of Caspase 3 was up-regulated(P<0.01).hUCMSCs partially reversed the expression of Bcl-2,HIF-1a,survivin,JAK1,JAK2,STAT3 and Caspase 3 in A549 cells after IGF-1 knockdown.5.Under hypoxic conditions in vitro or in vivo,hUCMSCsenhanced the expression of EMT-related genes including beta-catenin,N-cadherin,vimentin and Twist in A549 cells(all P<0.01),and decreased the expression of E-cadherin(At the protein level in vivo,p<0.05;All the restP?<0.01).After knocking down with the expression of IGF-1 gene in A549 cells in vivo,the expression of ?-catenin,N-cadherin,vimentin and Twist in A549 cells was decreased,and the expression of E-cadherin was increased.The effect of EMT induced by hUCMSCs was decreased in A549 cells.Conclusion:1.hUCMSCs up-regulated the expression of HIF-la by promoting IGF-1 expression in A549 cells under hypoxia or in vivo and simultaneously activated JAK/STAT3 pathway,mediating the up-regulation of anti-apoptotic genes Bcl-2,survivin and inhibit Caspase3 expression.Therefore,the up-regulation of IGF-1 expression in A549 cells 1s one of the mechanisms by which hUCMSCs promoted proliferation,invasion and inhibited apoptosis of A549 cells under hypoxia or in vivo.2.hUCMSCs induced EMT in A549 cells in vivo.The effect of hUCMSCs on inducing EMT in A549 cells was decreased when IGF-1 knocked down in A549 cells.The mechanism of EMT induced by hUCMSCs in A549 is related to the up-regulation of IGF-1 expression.PART ? Effect of huMCMSCS on the expression profile of microRNAs in lung adenocarcinoma A549 cells in vivo and bioinformatics analysisObjectiveThis study was to investigate the effect of hUCMSCs on the niRNA expression profile of A549 cells in vivo,and analyze its expression characteristics by bioinformatics.MethodsThe differential expression of miRNAs in the transplanted tumors of A549 cells and A549 cells in the hUCMSCs+A549 co-injected group was detected by Exiqon miRNA microarray.The top up-regulated or down-regulated 10 miRNAs was identified by qPCR.The gene function and regulatory pathways of miRNAs with up-regulated and down-regulated differential expression was analyzed by the DAVID database(GO and KEGG pathways enrichment analysis).miRNAs associated with lung cancer and mirDB and IGF-1 in HMDD or the HIF-1? gene-related miRNAs were also analyzed,and then intersected with the chip results to find miRNAs associated with IGF-1 or HIF-la and lung cancer.Result:1.A total of 337 differentially expressed miRNAs was detected between hUCMSCs co-injection group and A549 cells group.The number of up-regulated miRNAs was 154,and the number of up-regulated miRNAs was 63.The number of genes down-regulated was 183,of which 108 were significantly down-regulated(differential multiple>2,P<0.05).2.The result of qPCR showed that among the top 20 genes,12 were up-regulated,7 were down-regulated,and 1 was no change.The coincidence rate between miRNA microarray and qPCR was 70%.3.Bioinformatics analysis showed that abnormal expression of miRNAs with differential expression may be related to abnormal regulation of DNA transcription,gene expression,apoptosis,cell cycle,etc.,involving cancer-related microRNA,MAPK signaling pathway,PI3K-Akt signaling pathway,Multi-functional stem cell signaling pathway regulation,ErbB signaling pathway,etc.There are 6 miRNAs associated with lung cancer,9 miRNAs associated with HIF-la,and 19 miRNAs associated with IGF-1.miR-1827 is associated with lung cancer and IGF-1.Conclusions:1.hUCMSCs regulates the expression of a part of miRNA in A549 cells in vivo.2.Differentially expressed microRNAs are associated with cell proliferation,apoptosis and signaling pathways.HIF-la and IGF-1 are potential target genes of miRNAs.SUMMARYWe found that hUCMSCs inhibited the proliferation of A549 cells under normoxic conditions by a non-contact co-culture model in vitro,while hUCMSCs promoted the proliferation of A549 cells under hypoxic conditions.By interfering with the expression of HIF-1? in A549 cells,we found that hUCMSCs promoted the proliferation of A549 cells under hypoxic conditions,which was similar to as in vivo experiments.We also found that hUCMSCs induced EMT in A549 cells in vivo or under hypoxia.Therefore,hypoxic microenvironment was the major cause of different proliferation effects of hUCMSCs on A549 cells in vitro and in vivo.In terms of molecular mechanism,our study showed that hUCMSCs up-regulated HIF-la,Bcl-2 and Survivin expression,down-regulated expression of Caspase 3 and activated STAT3 pathway in A549 cells under hypoxic environment.Further studies showed that hUCMSCs up-regulated the expression of IGF-1 in A549 cells under hypoxia or in vivo.,However,the proliferation ability of A549 cells was decreased when the expression of IGF-1 gene interfered in A549 cells.The expression of HIF-1?in A549 cells was decreased,indicating that IGF-1 regulated the expression of HIF-1?.Meanwhile,the expression of Bcl-2 and Survivin was down-regulated,Caspase 3 was up-regulated and JAK/STAT3 pathway was inhibited in A549 cells after the expression of IGF-1 gene interfered.In addition,hUCMSCs induced EMT in A549 cells in vivo,and the effect of hUCMSCs induced EMT in A549 cells was decreased when the expression of IGF-1 interfered in A549 cells.The results of microarray and bioinformatics analysis showed that hUCMSCs regulated the expression of microRNAs in A549 cells in vivo.The accuracy of the microarray in this study was 70%verified by qPCR.Bioinformatics analysis showed that these differentially expressed microRNAs were related to cell proliferation,apoptosis and signaling pathways,and some of them were related to HIF-laor IGF-1.This study confirmed that hypoxic microenvironment was responsible for the difference of proliferation ability of hUCMSCs on A549 cells in vitro under hypoxia or in vivo,and further revealed the molecular mechanism of hUCMSCs promoting the proliferation of A549 cells and inducing EMT by up-regulating the expression of IGF-1 in A549 cells under hypoxic microenvironment.It revealed that hUCMSCs regulated the expression of some microRNAs in A549 cells in vivo.The differentially expressed microRNAs associated with IGF-1 and HIF-1 alpha was explored by bioinformatics analysis,which would provide a preliminary laboratory basis for finding new and effective target genes for the treatment of lung cancer by hUCMSCs. |
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