| Objective: SP1（specific protein 1）,as a multifunctional transcription factor,can promote the occurrence and development of colorectal cancer.However,the heterogeneity of SP1 expression have not been reported in colorectal cancer and its function in colorectal cancer.Therefore,this study will preliminarily explore the characteristics of intratumoral heterogeneity of Sp1 in colorectal cancer and its potential role in antitumor therapy.Methods: In this study,we first analyzed the expression level of SP1 in different tumor cell subsets of colorectal cancer by using the single-cell sequencing data of cancer sea and IHC（immunohistochemistry）data in HPA database,GEPIA database was used to analyze the correlation between SP1 expression and clinical stage and prognosis of colorectal cancer patients;Further,IHC technology was used to detect the protein expression level and pattern of SP1 in clinical specimens of patients with colorectal cancer.Based on the protein expression level and distribution pattern of Sp1 in patients with colorectal cancer,we quantified the heterogeneity level of SP1 protein expression,and the correlation between the heterogeneity level and clinicopathological characteristics of patients was analyzed;In addition,based on RNAi technology and lentivirus mediated,SP1 stable silencing expression（sw620shsp1#2）and control group（sw620shcon）colorectal cancer cell lines were constructed,and the effects of SP1 expression silencing on colorectal cancer cell proliferation and antitumor therapy were detected by CCK8 experiment.Results: By searching the single cell sequencing data（Exp ID:EXP0051）,we found that the number of SP1 negative expression（expression=0.0）cells in colorectal cancer cells（n= 290）was 163（56.2%）and the number of cells with positive expression of SP1（expression > 0）was 127（43.8%）,the results showed that in the expression level of SP1 in different colorectal cancer cells there were significant differences;And consistent with the results of single-cell sequencing,HPA database and the IHC results of SP1 in this subject show that based on the protein expression level of SP1,colorectal cancer cells in the same patient can be clearly divided into SP1 negative cell subsets and SP1 positive cell subsets,suggesting that in colorectal cancer patients the expression of SP1 has significant intratumor heterogeneity;In addition,we quantified and analyzed its intratumoral heterogeneity level,based on the heterogeneity of SP1 expression.It was found that the clinical stage（P =0.038）and lymph node metastasis（P=0.044）was significantly correlated with the high heterogeneity level of SP1;Finally,we successfully constructed the stable silencing expression of SP1（sw620shsp1#2）and control（sw620shcon）colorectal cancer cell lines,and confirmed that the silencing of SP1 expression in SW620 cells can significantly induce the inhibition of cell proliferation;However,in colorectal cancer cell line SW620,compared with control cell line sw620 shcon,when SP1 expression was silenced（sw620shsp1#2）,it showed significant resistance to both autophagy inhibitor CQ（chloroquine）and programmed cell necrosis induced by SM-164 combined with TNF-a.Conclusions: In patients with colorectal cancer,the expression of SP1 has significant intratumoral heterogeneity;SP1 high level of heterogeneity was significantly associated with patient stage and lymph node metastasis;Although SW620 proliferation can significantly inhibit by SP1 silencing,SP1 expression silencing can significantly resist CQ induced autophagy and SM-164 combined with TNF-a induced programmed cell necrosis,suggesting that the SP1 negative cancer cell subsets in colorectal cancer patients may be a group of tumor cells with slow proliferation but resistance to potential antitumor drugs,Although it is only a small number of cell subsets in the tumor tissue of patients with colorectal cancer,it may play an important role in the occurrence,development and drug resistance of colorectal cancer. |
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