| ?Background?The incidence of colorectal cancer ranks third in China's malignancy.In additon,the incidence rate has increased year by year and the age of onset has gradually become younger.The 5-year survival rate of colorectal cancer patients is 65% in China.But the 5-year survival rate rises to 90% when early detection and early treatment were done.The current clinical examination of colorectal cancer is difficult to find cancer in early stage.At the same time,the specificity and sensitivity of the common serum diagnostic markers CEA and CA199 of colorectal cancer are unsatisfactory.Therefore,it is very important to establish a new prevention and treatment strategy for colorectal cancer.At the same time,screening and identifying new molecular markers for diagnosing colorectal cancer and elucidating the pathological mechanisms that affects the occurrence and development of colorectal cancer is very essential.Mitochondria are important organelles of eukaryotic cells and play a key role in energy generation,ROS production,signal transduction and apoptosis.More and more studies have confirmed that mitochondria play a very important role in the progression of tumors.Mitochondria participate in many malignant phenotypes such as cancer cell proliferation,invasion,metastasis,and apoptosis.Researches focus on the relationship between tumor and mitochondria becomes a hot topic in the field of cancer research.Mitochondrial biogenesis and quality control are usually upregulated in cancer cells to adapt to changes in the external environment.Many studies have shown that mitochondria play essential role in the regulation of malignant phenotype of tumors by regulating mitochondrial biogenesis.Therefore,the mitochondrial biogenesis is expected to become a target for tumor prevention and treatment.Mitochondrial DNA single-stranded binding protein(mtSSB)plays an important role in promoting mitochondrial DNA replication and damage repair and regulating mitochondrial biogenesis.The role of mtSSB in tumors has been paid more and more attention in recent years.Studies suggest that mtSSB plays an important role in the potential impact on tumorigenesis and progression.However,what is the role of mtSSB in the progression of colorectal cancer with abundant mitochondria? It has not been reported yet.We found that the expression of mtSSB in colorectal cancer tissues was significantly higher than that in peritumorous tissues in previous preliminary experiments,and the expression of mtSSB was negatively correlated with the poor prognosis of patients with colorectal cancer.This suggests that mtSSB may play an important role in the progression of colorectal cancer.What is the biological function of mtSSB in the progression of colorectal cancer? What is the possible mechanism of that? What is its mechanism of upstream regulation? This is the scientific question that we need to answer in this study.?Objective?1.To clarify the expression of mtSSB in colorectal cancer tissues and analyze the relationship between the expression of mtSSB and clinical prognosis.2.To elucidate the biological roles and possible mechanisms of mtSSB in colorectal cancer cells.3.To further explore the upstream mechanisms that regulate the expression of mtSSB in the tumor microenvironment.?Methods?1.Tissue samples of colorectal cancer were studied by Western blot,real-time quantitative PCR and IHC to detect the expression of mtSSB in cancer and paraneoplastic tissues.The relationship between the expression of mtSSB and the prognosis of patients was analyzed.2.The colorectal cancer cell lines LoVo and COLO205 with forced overexpression and downexpression of mtSSB respectively.The effects of mtSSB on proliferation of the above cells were examined using cell counts assay,EDU assay,plate clone assay,and xenograft tumor in nude mice.3.Using qPCR,transmission electron microscopy,oxygen consumption assay and ATP generation assay to detect the effect of mtSSB on mitochondrial biogenesis in the clinical samples and the cell lines of colorectal cancer.The experiments of detecting ROS production,telomerase activity and telomere length in the cell lines were used to investigate the mechanism of promoting proliferation of colorectal cancer cells by mtSSB.4.The upstream molecular mechanism of regulating the expression of mtSSB was analyzed by Western blot,dual fluorescein reporter gene and Chip-PCR.?Results?1.Western Blot,real-time quantitative PCR and IHC results showed that the expression of mtSSB in colorectal cancer tissues was significantly higher than that in adjacent tissues.Survival analysis revealed that the higher the expression level of mtSSB predicts the shorter the survival rate of patients.2.In vitro and in vivo experiments have confirmed that mtSSB significantly promotes the proliferation of colorectal cancer cells,which in turn promotes the progression of colorectal cancer.The results of transmission electron microscopy and real-time quantitative PCR results showed that up-regulation of mtSSB significantly promoted the increase of mitochondrial number and mtDNA copy number in colorectal cancer.Oxygen consumption experiments and ATP production experiments further found that mtSSB can significantly increase the mitochondrial oxidative phosphorylation function.The detection of ROS content found that mtSSB can promote ROS production.It was found that mtSSB activates telomerase activity and enlongates telomere length via ROS and this effect was ROS-dependent by interfering with ROS production.The Results of Western blot experiments revealed that pro-inflammatory cytokine IL-6 in the tumor microenvironment significantly promoted the expression of mtSSB in colorectal cancer cells.The dual fluorescein reporter gene assay and Chip assay suggest that the IL-6/STAT3 pathway activates the transcription factor FOXP1 in the upstream promoter region of mtSSB,which thereby promoting the transcription of mtSSB and upregulating the expression of mtSSB.At the same time,it also confirmed that IL-6 can promote the proliferation of colorectal cancer cells by up-regulating mtSSB in vivo and in vitro.?Conclusion?In this study,we found that IL-6,a pro-inflammatory cytokine in the tumor microenvironment of colorectal cancer,activates the transcription factor FOXP1 of the upstream promoter of mtSSB,which leads to the upregulation of mtSSB expression in colorectal cancer cells.Increased expression of mtSSB significantly promoted mitochondrial biogenesis in colorectal cancer,which in turn increased ROS levels.Elevated ROS can further activate the telomerase activity and lengthen the telomere length,which in turn causes proliferation of colorectal cancer cells and promotes tumor cell development.Therefore,this study may provide an important theoretical basis for further understanding of the progression of colorectal cancer,and may provide a new target for the treatment of colorectal cancer. |
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