Part ?:Molecular Mechanism Of SNORD-X Mediated Immune Escape Of Esophageal Squamous Cell Carcinoma By Regulating T Cell Activity Through ILF2 Part ?:Molecular Mechanism Of MiR-3653-5p Regulating Autophagy To Inhibiting Breast Cancer Cells Proliferation,I

Molecular Mechanism of SNORD-X in Regulating The Immune Function of Esophageal Squamous Cell Carcinoma through ILF2Esophageal cancer is one of the most common malignant tumors.Cancer statistics in China showed that esophageal cancer has become the sixth most common tumor and the fourth most lethal tumor in China.Among them,esophageal squamous cell carcinoma(ESCC)is the main tissue type of esophageal cancer cases in China's mainland.The prognosis of advanced ESCC is poor,the therapeutic effect of chemotherapy is limited and the long-term curative effect is not ideal.The mechanism of occurrence and development of ESCC is still unclear.Clinical trial data showed the key role and great potential of PD-1 immunotherapy in ESCC.However,there are only a few patients that sustained durable clinical benefit from PD-1/PD-L1 immune checkpoint blockade in clinical practice.Currently,there were no biomarkers that accurately identified whether ESCC patients could benefit from PD-1/PD-L1 inhibitor before treatment or at the early stage of treatment.Therefore,it is significant to explore biomarkers that predict PD-1/PD-L1 immunotherapy for esophageal cancer and clarify its mechanism.Studies have shown that snoRNAs play a key role in the occurrence and development of malignant tumors.SnoRNAs are stably expressed in peripheral blood and easy to detect,and could be used as biomarkers for tumor screening and efficacy prediction.Our study aimed to find biomarkers that predicted the efficacy of PD-1 treatment in ESCC patients and to clarify its molecular mechanism.First,it was found that SNORD-X was involved in disease progression through gene ChIP analysis of the serum of ESCC patients receiving PD-1 immunotherapy.The expression of SNORD-X in the serum of patients was significantly higher than that of patients with complete remission and partial remission,and the expression of SNORD-X in the tissues and serum of patients with ESCC was higher than that of adjacent tissues and healthy human serum.The expression of SNORD-X in ESCC was similar to lymph node metastasis is positively correlated.The TCGA database copy number variation data shows that the region where SNORD-X is located is the region of ESCC gene copy number amplification.The cell phenotype experiments showed that SNORD-X promoted the malignant phenotype of ESCC cells in vitro.Animal experiments showed that SNORD-X promoted tumor growth and lung metastasis in vivo.In order to explore the mechanism of SNORD-X in ESCC,we used RNA pulldown combined with mass spectrometry technology,and verified the specific binding of SNORD-X to ILF2 through RIP.Further research found that SNORD-X bound with ILF2 to stabilize ILF2 in the nucleus,preventing it from entering the cytoplasm and undergo ubiquitination degradation,thereby enhancing ILF2 protein stability.IL2 is a key factor for T cell activation.It has been reported that ILF2 forms a complex with ILF3,which acted as a transcription factor of IL2 in the nucleus to promote IL2 transcription,and the complex bound with IL2 mRNA in the cytoplasm to enhance IL2 mRNA stability.Our study found although SNORD-X stabilized ILF2 in the nucleus,overexpressed SNORD-X inhibited the transcriptional activity of IL2 in ESCC cells.The decrease of ILF2 in the cytoplasm resulted in declined stability of IL2 mRNA,which ultimately led to down-regulation of IL2 protein level.Because ILF2 mainly plays a role as a transcription factor in the nucleus,we have confirmed that ILF2 could be used as a transcription factor of PD-L1 to promote the transcription of PD-L1,leading to up-regulated PD-L1 expression by ChIP-seq combined with the dual-luciferase reporter gene experiment and other technical methods.SNORD-X could be detected in the serum of ESCC patients,and the expression of SNORD-X could also be measured in the exosomes produced by ESCC cells.Therefore,we speculated that SNORD-X in ESCC cells could play a role by being encapsulated and secreted into the tumor microenvironment by exosomes.Because SNORD-X is highly expressed in the disease progression group of PD-1 treatment,and PD-1 treatment is closely related to T cell activity,we first considered the effect of SNORD-X on T cell activity in the tumor microenvironment.We treated human peripheral blood leukemia T cell Jurkat with exosomes produced by ESCC cells with high expression of SNORD-X,which could promote the expression levels of ILF2 and decrease the expression levels of IL2 in Jurkat cells.It suggested that SNORD-X in exosomes might inhibit T cell activation by regulating the expression of IL2 through ILF2,and the related molecular mechanisms needed to be further studied.In addition,due to the good conservation of SNORD-X among species,we constructed transgenic mice with knockout SNORD-X as an animal model to subsequently study SNORD-X that regulated immune escape of ESCC cells in vivo.In summary,our study first discovered that SNORD-X mediated the immune escape of ESCC cells by regulating T cell activity through ILF2.SNORD-X was expected to be a biomarker for predicting the efficacy of PD-1 inhibitors,and targeted SNORD-X combined with PD-1 therapy might become a new strategy for immunotherapy of ESCC.Molecular Mechanism of miR-3653-5p in inhibiting Breast Cancer Cell Proliferation,Migration and InvasionBreast cancer is the most common malignancy among Chinese women.Although there is a breakthrough in the diagnosis and treatment of breast cancer in recent years,it is still the leading cause of death among women worldwide.The classification of breast cancer is complex,and the pathogenesis is still not clear,which greatly hinders the research progress of molecular targeted therapy and chemotherapy drugs for breast cancer.Autophagy is an important process of material catabolism in cells.Autophagy is related to many steps in the occurrence and development of malignant tumors.Many chemotherapeutic drugs induce autophagy in tumor cells.Therefore,it is expected to become a new strategy for cancer treatment to take relevant interventions against autophagy.As a lysosomal inhibitor,chloroquine,an old antimalarial drug,inhibits the fusion of autophagosome and lysosome,resulting in the decrease of autophagy,thus enhancing the sensitivity of tumor cells to anti-tumor drugs.Chloroquine is an autophagy-targeted compound entering clinical research.Preliminary clinical studies have been carried out in colon cancer,glioma and melanoma.However,the role of chloroquine in the treatment of breast cancer and the clinical benefit population are still indistinct.MicroRNAs(miRNAs)are endogenous non-coding small RNA molecules,which regulate a series of biological processes and participate in the occurrence and development of tumors by promoting the degradation or inhibiting the translation of target gene mRNA.miRNAs are stable in the body fluid of tumor patients and easy to detect and reflect tumor size,metastasis and drug sensitivity to a certain extent.miRNAs are ideal markers for tumor diagnosis.It has been reported that miR-3653-5p plays an oncogene role in a variety of tumors,but its mechanism in breast cancer is still unclear.RT-qPCR was used to detect the expression of miR-3653-5p in 100 cases of breast cancer and paired adjacent tissues.Cell function experiment showed that miR-3653-5p inhibited the proliferation,invasion,migration and clone formation of breast cancer cells.Animal experiments were conducted to construct breast cancer cells with stable and high expression of miR-3653-5p,and the results showed that the metastasis of breast cancer cells overexpressing miR-3653-5p was reduced.TargetScan and other websites predicted the target genes of miR-3653-5p and verified them in combination with the dual-luciferase reporter genes experiment.The results showed that miR-3653-5p bound 3'UTR of autophagy-related genes ATG12 and AMBRA1 and inhibited their expression.The expression of ATG12 and AMBRA1 in 41 pairs of breast cancer tissues and paired adjacent tissues was detected by RT-qPCR.It was found that the expression of ATG 12 and AMBRA1 in breast cancer tissues was significantly increased,and negatively correlated with the expression of miR-3653-5p.Further studies showed that miR-3653-5p inhibited autophagy level and epithelial-mesenchymal transition of breast cancer cells by targeting ATG 12 and AMBRA1.Because ATG12 and AMBRA1(the target genes of miR-3653-5p)are key molecules in autophagy,and high expression of miR-3653-5p inhibited autophagy level in breast cancer cells.We found that knockdown of miR-3653-5p in breast cancer cells resulted in increased autophagy level and higher sensitivity to the autophagy inhibitor chloroquine;however,over-expression of miR-3653-5p resulted in decreased autophagy level and lower sensitivity to chloroquine.It suggested that breast cancer patients with low expression of miR-3653-5p had more breast cancer cells in autophagy state,which might be more sensitive to chloroquine treatment.In conclusion,our study proposed for the first time that miR-3653-5p participates in the occurrence and development of breast cancer through autophagy,and miR-3653-5p could be used as a marker to distinguish benefit population with breast cancer of CQ treatment,which provided a new idea for the treatment of breast cancer.