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Abstract：
BACKGROUND: The tumor microenvironment (TME) is an important factor that regulates the progression of colorectal cancer (CRC). Cancer-associated fibroblasts (CAFs) are the main mesenchymal cells in the TME and play a vital role in tumor progression; however, the specific underlying mechanisms require further study.
METHODS: Multiple single-cell and transcriptome data were analyzed and validated. Primary CAFs isolation, CCK8 assay, co-culture assay, western blotting, multiple immunofluorescence, qRT-PCR, ELISA, immunoprecipitation, ChIP, double luciferase, and animal experiments were used to explore the potential mechanism of MYL9 regulation in CRC.
RESULTS: Our findings revealed that MYL9 was predominantly localized and expressed in CAFs rather than in CRC cells, and bioinformatics analysis revealed that high MYL9 expression was strongly associated with poor overall and disease-free survival in various tumors. In addition, high MYL9 expression is closely associated with M2 macrophage infiltration, which can lead to an immunosuppressive microenvironment in CRC, making it insensitive to immunotherapy. Mechanically, MYL9 can regulate the secretion of CAFs on CCL2 and TGF-β1, thus affecting the immune microenvironment and progression of CRC. In addition, MYL9 bounded with IQGAP1 to regulate CCL2 and TGF-β1 secretion through the ERK 1/2 pathway, and CCL2 and TGF-β1 synergistically promoted CRC cells progression through the PI3K-AKT pathway. Furthermore, MYL9 promotes epithelial-mesenchymal transition (EMT) in CRC. During the upstream regulation of MYL9 in CAFs, we found that the EMT transcription factor ZEB1 could bind to the MYL9 promoter in CAFs, enhancing the activity and function of MYL9. Therefore, MYL9 is predominantly expressed in CAFs and can indirectly influence tumor biology and EMT by affecting CAFs protein expression in CRC.
CONCLUSIONS: MYL9 regulates the secretion of cytokines and chemokines in CAFs, which can affect the immune microenvironment of CRC and promote CRC progression. The relationship between MYL9 expression and CRC clinical staging and immunotherapy is closer in CAFs than in tumor cells; therefore, studies using CAFs as a model deserve more attention when exploring tumor molecular targets in clinical research.
METHODS: Multiple single-cell and transcriptome data were analyzed and validated. Primary CAFs isolation, CCK8 assay, co-culture assay, western blotting, multiple immunofluorescence, qRT-PCR, ELISA, immunoprecipitation, ChIP, double luciferase, and animal experiments were used to explore the potential mechanism of MYL9 regulation in CRC.
RESULTS: Our findings revealed that MYL9 was predominantly localized and expressed in CAFs rather than in CRC cells, and bioinformatics analysis revealed that high MYL9 expression was strongly associated with poor overall and disease-free survival in various tumors. In addition, high MYL9 expression is closely associated with M2 macrophage infiltration, which can lead to an immunosuppressive microenvironment in CRC, making it insensitive to immunotherapy. Mechanically, MYL9 can regulate the secretion of CAFs on CCL2 and TGF-β1, thus affecting the immune microenvironment and progression of CRC. In addition, MYL9 bounded with IQGAP1 to regulate CCL2 and TGF-β1 secretion through the ERK 1/2 pathway, and CCL2 and TGF-β1 synergistically promoted CRC cells progression through the PI3K-AKT pathway. Furthermore, MYL9 promotes epithelial-mesenchymal transition (EMT) in CRC. During the upstream regulation of MYL9 in CAFs, we found that the EMT transcription factor ZEB1 could bind to the MYL9 promoter in CAFs, enhancing the activity and function of MYL9. Therefore, MYL9 is predominantly expressed in CAFs and can indirectly influence tumor biology and EMT by affecting CAFs protein expression in CRC.
CONCLUSIONS: MYL9 regulates the secretion of cytokines and chemokines in CAFs, which can affect the immune microenvironment of CRC and promote CRC progression. The relationship between MYL9 expression and CRC clinical staging and immunotherapy is closer in CAFs than in tumor cells; therefore, studies using CAFs as a model deserve more attention when exploring tumor molecular targets in clinical research.
摘要：
背景：肿瘤微环境（TME）是调节结直肠癌（CRC）进展的重要因素。癌相关成纤维细胞(CAFs)是TME中的主要间质细胞,在肿瘤进展中起着至关重要的作用;具体的潜在机制需要进一步研究。
方法：分析和验证多个单细胞和转录组数据。主CAF隔离,CCK8测定，共培养试验，西方印迹,多重免疫荧光，qRT-PCR,ELISA,免疫沉淀,ChIP,双荧光素酶,并通过动物实验探讨MYL9调控CRC的潜在机制。
结果：我们的研究结果表明，MYL9主要在CAF中定位和表达，而不是在CRC细胞中。和生物信息学分析显示,MYL9高表达与各种肿瘤的总体生存率和无病生存率低密切相关.此外,MYL9高表达与M2巨噬细胞浸润密切相关,这可能导致CRC中的免疫抑制微环境，使其对免疫疗法不敏感。机械上,MYL9可以调节CAFs在CCL2和TGF-β1上的分泌，从而影响免疫微环境和CRC的进展。此外,MYL9与IQGAP1结合，通过ERK1/2通路调节CCL2和TGF-β1的分泌，CCL2和TGF-β1通过PI3K-AKT途径协同促进CRC细胞进展。此外，MYL9促进CRC中的上皮-间质转化(EMT)。在CAF中MYL9的上游调节期间，我们发现EMT转录因子ZEB1可以与CAFs中的MYL9启动子结合，增强MYL9的活性和功能。因此,MYL9主要在CAFs中表达,并且可以通过影响CRC中的CAFs蛋白表达来间接影响肿瘤生物学和EMT。
结论：MYL9调节CAFs中细胞因子和趋化因子的分泌，影响CRC的免疫微环境,促进CRC进展。MYL9表达与CRC临床分期和免疫治疗之间的关系在CAFs中比在肿瘤细胞中更接近；因此，在临床研究中探索肿瘤分子靶标时，使用CAFs作为模型的研究值得更多关注。
方法：分析和验证多个单细胞和转录组数据。主CAF隔离,CCK8测定，共培养试验，西方印迹,多重免疫荧光，qRT-PCR,ELISA,免疫沉淀,ChIP,双荧光素酶,并通过动物实验探讨MYL9调控CRC的潜在机制。
结果：我们的研究结果表明，MYL9主要在CAF中定位和表达，而不是在CRC细胞中。和生物信息学分析显示,MYL9高表达与各种肿瘤的总体生存率和无病生存率低密切相关.此外,MYL9高表达与M2巨噬细胞浸润密切相关,这可能导致CRC中的免疫抑制微环境，使其对免疫疗法不敏感。机械上,MYL9可以调节CAFs在CCL2和TGF-β1上的分泌，从而影响免疫微环境和CRC的进展。此外,MYL9与IQGAP1结合，通过ERK1/2通路调节CCL2和TGF-β1的分泌，CCL2和TGF-β1通过PI3K-AKT途径协同促进CRC细胞进展。此外，MYL9促进CRC中的上皮-间质转化(EMT)。在CAF中MYL9的上游调节期间，我们发现EMT转录因子ZEB1可以与CAFs中的MYL9启动子结合，增强MYL9的活性和功能。因此,MYL9主要在CAFs中表达,并且可以通过影响CRC中的CAFs蛋白表达来间接影响肿瘤生物学和EMT。
结论：MYL9调节CAFs中细胞因子和趋化因子的分泌，影响CRC的免疫微环境,促进CRC进展。MYL9表达与CRC临床分期和免疫治疗之间的关系在CAFs中比在肿瘤细胞中更接近；因此，在临床研究中探索肿瘤分子靶标时，使用CAFs作为模型的研究值得更多关注。
