| Objective: Oral squamous cell carcinoma(OSCC)is a common malignant tumor in the oral cavity and the most common head and neck squamous cell carcinoma(HNSCC),which seriously threatens people’s health.The treatment method is usually surgery combined with radiotherapy and chemotherapy.However,the five-year survival rate is still low.Therefore,it is urgent to explore the pathogenesis of OSCC and lay a theoretical foundation for improving the survival rate of OSCC patients.In recent years,the concept of tumor microenvironment(TME)has been proposed,which plays an important role in the process of tumor occurrence and development as the "soil" for tumor survival and development.In HNSCC,interstitial hyperplasia is obvious,and there are abundant cancer-associated fibroblasts(CAFs).Because of their complex origin and the influence of heterogeneous tumor cells,CAFs have multiple cell subsets in tumors,which are involved in tumorigenesis,development,infiltration,metastasis and drug resistance.Bone marrow mesenchymal stem cells(BMSCs),as pluripotent stem cells,are considered to be one of the main sources of CAFs.Nowadays,BMSCs are widely used in the field of tissue regeneration due to their multi-differentiation potential and immunomodulatory effects.However,in cancer,BMSCs located in the bone marrow are recruited by a variety of cells in the TME to migrate into the tumor microenvironment and participate in tumor progression.The role of BMSCs in tumors is multi-faceted.It can promote or inhibit tumor in different types of tumors,and even have different effects in the same type of tumor.In OSCC,the biological role of BMSCs is also controversial.Therefore,this study aimed to explore the biological effect of BMSCs on OSCC and its mechanism.Methods:1)Bioinformatics analysis was performed firstly,sc RNA-seq data of OSCC were obtained from GEO database,dimensionality reduction and clustering were performed using Seurat function package,and specific expression genes were obtained,and each cell subset was annotated using Single R function.Gene expression data and survival data of squamous cell carcinoma patients were obtained from the TCGA database,and survival analysis was performed on cell subsets using the Survival function,and the survival analysis curve was drawn.2)The OSCC cell line CAL27,Fa Du cells and BMSCs were recovered,the BMSCs conditioned medium was collected,and BMSC-CM was co-cultured with OSCC cells for 1 d,3 d,and 5 d for subsequent experiments.3)The activity of OSCC cells after co-culture was detected by CCK-8 cell assay,the stemness of OSCC cells was detected by colony formation assay,the proliferation ability of OSCC cells was detected by cell cycle assay,the apoptosis of OSCC cells was detected by cell apoptosis assay.The migration ability and wound healing ability of OSCC cells were tested by Trans-well migration experiment and scratch experiment,respectively.4)Real-time PCR and Western blot were used to detect the m RNA transcription level and protein expression of EMT-related molecular markers and transcription factors in OSCC cells,ELISA was used to detect the content of TGF-β1 in the conditioned medium of BMSCs,and Western blot was used to detect Ras in OSCC cells and phosphorylation of the Raf,ERK signaling pathway.Subsequently,OSCC cells were pretreated with TGF-β1 inhibitor SB431542 and ERK inhibitor U0126,and the proliferation,migration and EMT-related protein expression of OSCC cells were detected by CCK-8 cell assay,Trans-well cell migration assay and cellular immunofluorescence.5)Finally,using CAL27 cells,BMSC-CM co-culture CAL27 cells,CAL27 cells cocultured after pretreatment of SB431542 and U0126 established a nude mouse subcutaneous tumor model,the tumor volume during the growth process was measured,and immunohistochemical staining was used to detect Ki67,Vimentin and Snail expression.Results:1)Bioinformatics analysis showed that all cells in OSCC tissue were divided into 17subgroups(clusters),and the "fibroblast" cell group was extracted for further clustering and difference analysis,showing that 12 subgroups and their marker genes.The MSCs markers were expressed in 6 subgroups of cluster 0,5,6,7,8,and 10,and the scatter plot showed that MSCs were mainly source of fibroblasts.After extracting MSCs for clustering and difference analysis,9 subgroups and their marker genes were obtained.The Kaplan-Meier curve showed that the cluster 5 of MSCs was associated with a worse prognosis,while the cluster 8 had a better prognosis,and other cell subsets have no significant effect on the survival of patients.2)In vitro experiments,the CCK-8 cell assay showed that the number of OSCC cells was significantly increased after BMSC-CM co-culture compared with the control group.The colony formation experiments showed that the number of clonal colonies of OSCC cells increased significantly after co-culture with BMSC-CM.Cell cycle experiments showed that the proportion of cells in S phase of OSCC cells increased significantly after co-culture.Apoptosis experiments found that compared with the control group,the early apoptosis and late apoptosis of co-cultured OSCC cells were significantly reduced.Trans-well cell migration assay found that the number of penetrating cells of co-cultured OSCC cells increased significantly.Scratch experiments showed that co-cultured OSCC cells had faster scratch healing time.3)Real-time PCR results showed that the m RNA expression of E-cadherin in OSCC cells decreased after co-culture,while the m RNA expression of N-cadherin,Vimentin and Snail,Zeb1,Zeb2 and Twist increased.The results of Western blot showed that compared with the control group,the expressions of ZO1 and Ecadherin decreased in the co-cultured OSCC cells,while the expressions of Fibronectin,Vimentin,Snail,Slug and Zeb1 increased.ELISA results showed that a large amount of TGF-β1 growth factor existed in BMSC-CM.Western blot was used to detect the expression of Ras and the phosphorylation of Raf and ERK in OSCC cells.The results showed that compared with the control group,the expressions of Ras,p-Raf and p-ERK in the BMSC-CM co-culture group were increased.After pretreatment of OSCC cells with TGF-β1 inhibitor SB431542 and ERK inhibitor U0126,CCK8 cell assay found that compared with the co-culture group,OSCC cell proliferation decreased after inhibition.Trans-well cell migration assay showed that compared with the co-culture group,the number of penetrating cells of OSCC cells decreased after inhibition.As shown by cellular immunofluorescence,the expression of E-cadherin was increased and the expression of Vimentin was decreased in OSCC cells after inhibition.4)The results of the OSCC xenograft model in nude mice showed that compared with the control group,the tumor volume in the BMSC-CM-treated group was significantly increased,and the inhibitor-treated group was significantly smaller than that in the control and co-culture groups.The results of immunohistochemical staining showed that compared with the control group,the co-culture group was strongly positive for Ki67,Vimentin expression and Snail expression were increased in the cancer nests.However,the positive range of Ki67 in the SB431542 and U0126 inhibitor groups was lower than that in the control group,and Vimentin was positive in the tumor stroma,but negative in the cancer.Snail expression was negative.Conclusion: MSCs in tumor tissues of OSCC patients have multiple cell subsets,which may have different biological effects on OSCC.In vitro and in vivo studies have found that BMSCs,a subset of MSCs,can activate the TGF-β1/Ras/Raf/ERK signaling pathway to promote EMT in OSCC cells,thereby promoting their proliferation and migration. |
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