Study On The Mechanism Of SATB1 Involved In The Carcinogenesis And Detection Of Circulating Tumor Cells In Esophageal Cancer

Esophageal cancer(EC)is a common gastrointestinal tumor originating from the mucosal epithelium of the esophagus and it has been a serious threat to human health worldwide.There are two major pathological types of esophageal cancer,Esophageal Squamous Cell Carcinoma(ESCC)and Esophageal Adenocarcinoma(EAC).China is one of the countries with the highest incidence and mortality rates of esophageal cancer in the world,and more than 90%of esophageal cancer pathology is ESCC,most of the patients are already in advanced clinical stage at the time of diagnosis,and about 40-50%of them develop distant metastasis and have poor prognosis.Therefore,there is an urgent need to elucidate the mechanism of esophageal cancer development,and to search for potential new clinical therapeutic targets and biomarker molecules,so as to improve early screening,diagnosis and treatment,and patient prognosis.SATB1(Special AT rich sequence binding protein,SATB1)is a tissuespecific nuclear matrix binding protein which is localized at 3p23 and encodes 763 amino acids.Studies have shown that SATB1 plays an important regulatory role in biological processes such as cell differentiation and X chromosome inactivation,and is also involved in the development of breast cancer,pancreatic cancer,nasopharyngeal carcinoma,bladder cancer,prostate cancer and other malignant tumors,however,it has been less studied in ESCC,and the detailed mechanism needs further study.First,we used real-time quantitative fluorescence PCR and Western Blot to detect the transcription and translation levels of SATB1 in clinical esophageal squamous cell carcinoma tissues,paraneoplastic tissues and squamous cell lines,and immunohistochemistry to detect the expression of SATB1 in pathological tissue specimens of ESCC patients with clinical pathological correlations between the parameters were analyzed.The results showed that SATB1 showed relatively high expression at transcriptional and translation levels in ESCC tissues and cell lines,and the immunohistochemical results showed that SATB1 was mainly localized in the nucleus closely related to the degree of tumor differentiation.These results suggest that SATB1 may be associated with the development of ESCC;then,we further investigated the effect of SATB1 on the biological behavior of ESCC cells.We found that the proliferation and invasion ability of TE-1 cells and Eca-109 cells decreased and apoptosis level increased after knockdown of SATB1,indicating that SATB1 could promote proliferation and invasion and inhibit apoptosis in ESCC cells in vitro by MTT,flow cytometry and Transwell esperiments.Secondly,TE-1 cells were subjected to high-throughput sequencing before and after knockdown of SATB1 to analyze the changes in their transcriptome levels.Bioinformatics analysis showed a total of 433 differentially expressed genes,of which 150 genes were up-regulated and 283 genes were downregulated,GO and KEGG correlation analyses were performed on the differentially expressed genes.The biofunctional network analysis showed that FN1 and PDGFRB were key genes for the regulatory role of S ATB1 in TE-1 cells;the positive regulation of FN1 and PDGFR-β by SATB1 was confirmed by dual luciferase assay;FN1 and PDGFR-β promoted the proliferation and invasion of ESCC cells in vitro.Subsequently,the mechanism by which SATB1 regulates FN1 and PDGFRβ involved in the development of ESCC was further clarified.In vitro cell level experiments confirmed that SATB1 activates AKT/mTOR signaling pathway to promote cell proliferation and invasion by regulating FN1 and PDGFR-β,and inhibits apoptosis by affecting Bax/Bcl2 expression.The results of the in vivo experiments in nude mouse transplantation tumors were consistent with the in vitro experiments.In addition,more effective adjunctive methods need to be established to improve the early diagnosis and efficacy assessment detection of esophageal cancer.Circulating tumor cells(CTCs)are tumor cells that enter the bloodstream after detaching from the primary lesion or metastatic focus of tumor.The potential for application,however,is limited by the extremely low content of CTCs in peripheral blood and the presence of heterogeneity,which limits the clinical application of most CTCs detection methods.Biological nanopore sensing technology has high sensitivity and versatility,and is expected to be applied in the detection of CTCs in esophageal cancer.In this study,we developed a universal method for the quantitative detection of ESCC CTCs by combining nanopore sensing technology and a multi-stage tandem signal amplification strategy.In this method,the aptamer cross-linked DNA hydrogels was used as carriers to specifically capture specific esophageal cancer cells,and after a cascade amplification reaction,the specific translocation events of triplehelix DNA was measured with the help of M2MspA protein nanopores to finally achieve the quantitative detection of CTCs.The results show that under the driving of voltage,a unique current signal can be generated when the triple-helix DNA passes through the M2MspA nanopore,in which step 2 is generated by the unstranding of triple-helix DNA at the contraction of M2MspA nanopore,and step 3 is generated by the passage or collision of single-stranded DNA at the contraction of M2MspA nanopore,which can realize the quantitative detection of CTCs.The method has a lower limit of detection of 2 cells,which is highly sensitive and has the potential to be applied to clinical samples.In summary,this study explored the mechanism of SATB1 involvement in esophageal squamous cell carcinogenesis and established a method to detect CTCs in esophageal cancer.It was proved for the first time that S ATB1 positively regulates FN1 and PDGFR-β,and then activates AKT/mTOR signaling pathway,affects Bax/Bcl2 to promotes esophageal squamous cell carcinoma proliferation and invasion,and inhibit apoptosis;the new triple-helix DNA-adaptor-based esophageal cancer CTCs detection method is flexible,sensitive and specific,which provides new targets and prognosis for esophageal cancer treatment.