MGP Promotes CD8~+T Cell Exhaustion By Activating The NF-κB Pathway Leading To Liver Metastasis Of Colorectal Cancer

Background:Colorectal cancer is a common malignant gastrointestinal tumor.Several studies have shown that about 14-25% of colorectal cancer patients have developed synchronous colorectal cancer liver metastases when the primary tumor is diagnosed,and 20-33% of CRC patients have developed synchronous liver metastasis.Metachronous colorectal cancer liver metastases occurred after the primary tumor was diagnosed and treated,and eventually 40-50%of CRC patients died of colorectal cancer liver metastases.Therefore,in-depth exploration of the specific molecular mechanism of colorectal liver metastasis is of great significance for reducing colorectal liver metastasis and improving subsequent treatment.Matrix gamma protein(MGP)is a multifunctional protein,which is mostly involved in the construction and biological activity of organisms as a matrix protein.Some studies have shown that it participates in the occurrence and development of tumors by directly promoting the proliferation and migration of tumor cells.However,the relationship between MGP and tumor immune microenvironment,immune cell response,and whether it is involved in the process of colorectal cancer liver metastasis is still unclear.Aims:This project intends to explore the effect of MGP on immune cell responses in the immune microenvironment of colorectal cancer,and then to study the function of MGP in the progression of colorectal cancer liver metastasis,and to further explore the potential mechanism behind the function of MGP in colorectal cancer liver metastasis,In addition,the diagnosis and treatment value of MGP was analyzed.Methods:Chapter 1: Single-cell sequencing and subsequent analysis of human colorectal cancer tissue samples and combined with GEO database to evaluate the expression status and potential function of MGP in CRC tumor cells.Subsequently,the expression of MGP m RNA in 5 pairs of CRC tissues was preliminarily verified by real-time quantitative polymerase chain reaction(q RT-PCR)technology,and the Expression of MGP protein in 4 pairs of CRC tissues was preliminarily verified by western blotting(WB)and immunohistochemistry(IHC).q RT-PCR and WB experiments were used to verify the expression of MGP m RNA and protein at the level of CRC cell lines.The q RT-PCR experiment was used again to expand the scale to detect 57 CRC tissue samples.The KM survival curve and TCGA data were used to jointly analyze the relationship between MGP and prognosis,and the correlation between MGP and clinicopathological data was analyzed by univariate and multivariate.Chapter 2: MGP knockdown and overexpression CRC stable cell lines were constructed by lentivirus,and then the effect of MGP on the proliferation of CRC cells was detected by CCK8、EDU and clone formation experiments,the apoptosis of CRC cells was detected by flow cytometry.The effect of MGP on the invasion ability of CRC cells was detected by transwell assay,and then a co-culture model was constructed using CRC stable cell lines and extracted CD8+T cells,and CD8+T exhaustion-related markers were detected by flow cytometry.Chapter 3: Perform whole-transcriptome sequencing on MGP knockdown CRC stable cells to find potential downstream regulatory units.q RT-PCR was used to detect the m RNA expression level of MGP downstream regulator PD-L1,and the TISIDB database,WB,IHC,and immunofluorescence(IF)were used to predict and detect the protein expression level of PD-L1.GSEA was used to analyze the possible regulatory pathway of MGP on PD-L1,calcium ion probes were used to detect the changes of intracellular calcium ion after MGP knockdown and overexpression,and WB was used to detect the changes of NF-κB pathway after MGP knockdown and overexpression as a whole.The CRC cell model of knockdown of NF-κB pathway core factor p65 was constructed,and the expression changes of PD-L1 after p65 knockdown were detected by q RT-PCR and WB.JASPAR was then used to predict the potential binding sites of p65,and the GSE131710 data,co-immunoprecipitation experiment(CHIP)and q RT-PCR experiments were used to jointly verify.Finally,the dual luciferase reporter gene experiment was performed for the binding site of p65 to verify p65 again.Specific regulatory sites for PD-L1.Chapter 4: Construction of PD-L1 knockdown CRC stable transfected cells,and combined with MGP knockdown stable transfected cells to construct a co-knockdown CRC cell model,and then used CCK8,EDU experiments,and clone formation experiments to detect PD-L1 knockdown and combined MGP In the case of knockdown,the proliferation ability of CRC cells changed.Flow cytometry was used to detect the level of CRC apoptosis,transwell assay was used to detect their invasion ability,and finally CD8+T exhaustion-related markers were detected by flow cytometry.Chapter 5: For in vivo experiments,the spleen and liver metastasis model was firstly constructed to observe the effect of MGP knockdown and combined use of PD1 m Ab on CRC liver metastasis.WB was used to detect MGP knockdown efficiency,and H&E and IHC assays were used to detect metastases、Pathological characteristics、markers of proliferation,apoptosis,MGP,CD8,PD-L1,etc,finally,mass cytometry was used to detect the overall impact of MGP knockdown and combined use of PD1 m Ab on the immune microenvironment of metastases.Subsequently,a mouse subcutaneous tumor model was constructed to observe the effect of MGP knockdown on the proliferation ability of CRC cells.H&E and IHC experiments were used to detect the pathological characteristics of the metastases and markers such as proliferation,apoptosis,MGP,CD8,and PD-L1.Results:Chapter 1: Human single-cell sequencing analysis showed that MGP was significantly highly expressed in CRC tumor cells with certain specificity.The same trend was observed in transcriptome data for mice.RC tissues and CRC cell lines preliminarily suggested that MGP m RNA(5 pairs)and protein levels(4 pairs)were highly expressed.Subsequently,57 CRC tissue expanded specimens verified the trend of high MGP expression.KM Survival analysis and TCGA prediction both indicated that the higher the MGP expression,the worse the patient’s prognosis.Univariate analysis showed that the MGP expression was correlated with the number of lymph node metastasis,TNM stage,and tumor size,and multivariate analysis showed that MGP was significantly associated with tumor size.Chapter 2: In vitro experiments,CCK8,EDU,and clone formation experiments showed that MGP knockdown significantly reduced the proliferation of CRC cells.Flow cytometry showed that after MGP knockdown,CRC cell apoptosis was significantly enhanced.Transwell experiments showed that MGP After knockdown,the invasion ability of CRC cells was significantly weakened.After co-culture of CRC knockdown stable cell line and extracted CD8+ T cells,CD8+ T ex-related markers such as PD1,LAG3,TIGIT,and TIM3 were significantly reduced,and the depletion state was as follows.alleviated.Chapter 3: Whole transcriptome sequencing analysis showed that the expression of PD-L1 also showed a downward trend after MGP knockdown,and functional enrichment analysis suggested that MGP was closely related to immune response and cytokine secretion.Subsequent experiments such as q RT-PCR,TISIDB database,WB,IHC,and immunofluorescence(IF)also proved this trend.GSEA analysis showed that MGP may regulate PD-L1 through the calcium ion-NF-κB pathway.Calcium ion probe detection showed that intracellular calcium ion was significantly reduced after MGP knockdown,while MGP overexpression showed the opposite trend.WB experiments showed that NF after MGP knockdown-κB pathway was significantly inhibited,and overexpression showed an activated state.PD-L1 expression was significantly decreased after knockdown of NF-κB pathway core factor p65.Subsequently,JASPAR was performed to predict the potential binding sites of p65p1-5,and the GSE131710 data,co-immunoprecipitation experiments(CHIP)and q RT-PCR experiments jointly suggested that p65 regulates PD-L1 through the p5 site,and finally The dual-luciferase reporter gene experiment confirmed that the p5 site was a key position for p65 regulation of PD-L1.Chapter 4: In the PD-L1 knockdown CRC stable transfected cells and the combined MGP knockdown stable transfected cells to construct a co-knockdown CRC cell model,CCK8,EDU,and clone formation experiments suggest that CRC cells proliferate after PD-L1 knockdown The ability of CRC cells decreased.In the case of combined MGP knockdown,the proliferation ability of CRC cells was more inhibited.flow cytometry showed that the apoptosis of CRC cells increased after PD-L1 knockdown.In the case of combined MGP knockdown,the level of CRC cell apoptosis was higher.Transwell experiments showed that the invasion ability of CRC cells decreased after PD-L1 knockdown.In the case of combined MGP knockdown,the migration ability of CRC cells was more inhibited.Finally,flow cytometry showed that after PD-L1 knockdown,CD8+T depletion-related markers such as PD1,LAG3,TIGIT,and TIM3 were significantly reduced,and in the case of combined MGP knockdown,the markers related to CD8+T depletion in CRC cells were even lower.Chapter 5: For in vivo experiments,in the spleen and liver metastasis model,it was observed that after MGP knockdown,the metastases were significantly reduced,and the combination of PD1 m Ab reduced the degree to a higher degree.WB experiments verified the efficiency of MGP knockdown,and H&E showed that MGP knockdown After MGP knockdown,the pathological state was improved,and the combined use of PD1 monoclonal antibody was even more improved.IHC experiments showed that after MGP knockdown,the proliferation ability was reduced,the apoptosis level was increased,the level of CD8 was increased,and markers such as MGP and PD-L1 were decreased.The above trend was significantly enhanced after PD1 m Ab.Finally,mass cytometry was used to find that MGP was knocked down,PD1 expression decreased,and the overall immune microenvironment showed a trend of improvement.The above trend was further strengthened after the combined use of PD1 m Ab.In the mouse subcutaneous tumor model,it was observed that after MGP knockdown,the proliferation ability of CRC cells was significantly decreased,and the pathological state was significantly improved.Markers such as MGP、PD-L1 showed a decline.Conclusion:1.MGP promotes the functional exhaustion of CD8+ T cells and leads to the occurrence and development of colorectal cancer liver metastasis;2.MGP promotes the functional exhaustion of CD8+ T cells by activating the NF-κB pathway with P65 as the core,which leads to liver metastasis of colorectal cancer;3.Interfering with MGP can increase the efficacy of PD1 m Ab.