The Study On The Effect And Mechanism Of Nucleolar Small RNA SNORA68 On The Proliferation And Migration Of Triple Negative Breast Cancer

Background:Breast cancer is currently the most prevalent malignancy worldwide.It is also one of the major threats to the life and health of the female population.Among them,triple-negative breast cancer has always been difficult to treat because of its lack of therapeutic targets and its close association with poor prognosis,high metastasis rate and early onset.Therefore,studying the specific molecular mechanisms of triple-negative breast cancer development,metastatic pathways and finding mature and effective therapeutic targets is of profound significance for patients with true triple-negative breast cancer.Small nucleolar RNA(snoRNA),a class of RNAs without protein-coding function,is one of the most characteristic classes of non-coding RNAs in the nucleus,with a length of 60-300 nucleotides.Sno RNAs include C/D box snoRNA and H/ACA box snoRNA.Sno RNAs have post-transcriptional modification functions in ribosomal RNAs and some spliceosomal RNAs,and it is also involved in the translation process as well as in the oxidative stress response.There is growing evidence that dysregulation of snoRNAs can play a role in controlling cells and regulating tumorigenesis and progression,and is a new research hotspot for oncological diseases.Sno RNAs have also been found to play an important role in a variety of malignancies such as gastric cancer,glioma,ovarian cancer,liver cancer,and colorectal cancer.Ribosomal proteins(RPs)are essential for ribosome assembly and function,and their expression is regulated by multiple mechanisms,mainly influenced by sophisticated regulatory actions related to ribosome biosynthesis.In some studies ribosomal proteins were found to be overactivated in cancer.In addition to conventional ribosomal functions,multiple ribosomal proteins have extra-ribosomal functions,including activation of p53-dependent or p53-independent pathways in response to stress,leading to cell cycle arrest and apoptosis.Defects in ribosome biogenesis,translation,and function of individual RPs,including mutations in RPs,have been associated with various human diseases.In this study,the high expression of SNORA68 in triple-negative breast cancer was identified and validated based on bioinformatics analysis and clinical tissue sample detection.We also analyzed the relationship between SNORA68 expression and clinicopathological factors in triple-negative breast cancer,and confirmed the regulatory role of SNORA68 on the proliferation,migration and stemness characteristics of triple-negative breast cancer cells through a series of biological experiments.This study further proposed and validated in vitro and in vivo that SNORA68 affects the proliferation and migration of triple-negative breast cancer cells through the RPL23/c-Myc pathway after forming a complex with U2AF2 protein.This experiment is expected to provide new ideas for the regulatory role of snoRNAs in triple-negative breast cancer and provide new theoretical basis for the development and clinical treatment of malignancies.Methods: 1.To study the expression and clinical significance of nucleolar small RNA SNORA68 in breast cancer based on bioinformatics analysis and clinical tissues: the relationship between SNORA68 expression in breast cancer and prognosis was analyzed based on an online database.The expression of SNORA68 in paraffin sections of 20 pairs of fresh triple-negative breast cancer tissues and 73 triple-negative breast cancer patients was detected by qRT-PCR and in situ hybridization assay.The correlation between SNORA68 expression and clinicopathological factors was analyzed in conjunction with clinical data.2.To analyze the regulatory effect of SNORA68 on triple negative breast cancer: We constructed SNORA68 high expression and knockdown cell models in breast cancer cells MDA-MB-231 and BT-549,and examined the effect of SNORA68 on triple negative breast cancer by CCK-8 assay,plate cloning assay,Transwell assay,scratch assay,apoptosis assay and cycle assay.The effect of SNORA68 on stemness index expression was examined by Western Blot.3.The molecular mechanism of SNORA68 regulation in triple negative breast cancer: Based on bioinformatics,we screened the proteins that may bind to SNORA68,and verified that SNORA68 binds to U2AF2 protein to form a complex by RNA pull down assay and RIP assay,and confirmed that SNORA68 binds to U2AF2 protein to form a complex by recovery assay.The complexes were confirmed by recovery assays and the complexes were confirmed to function together.The effect of SNORA68 expression changes on RPL23 expression in the nucleolus of the nucleolus was examined by subcellular structure isolation assay and Western Blot,and the effect of SNORA68 expression changes on the binding of RPL23 to c-Myc in the nucleus was examined by Co-IP assay.The molecular mechanism of SNORA68 regulation of triple-negative breast cancer through RPL23/c-Myc pathway was verified by combining with U2AF2 protein binding to form a complex.4.Animal experiments: In vivo validation of the mechanism of SNORA68 regulation of triple-negative breast cancer by binding to U2AF2 protein to form a complex and then through the RPL23/c-Myc pathway was performed by constructing a nude mouse transplantation tumor model.Results: 1.Based on bioinformatics analysis,SNORA68 expression was found to be higher in breast cancer than in normal breast tissue,and high SNORA68 expression was significantly associated with poor prognosis of breast cancer(P=0.038).Based on the detection of SNORA68 expression in breast cancer cell lines,SNORA68 expression was found to be higher in triple-negative breast cancer cell lines than in normal breast epithelial cells and higher in triple-negative breast cancer cells than in non-triple-negative breast cancer cells.2.SNORA68 expression and clinical significance in triple-negative breast tissues: SNORA68 expression was significantly higher in 20 pairs of fresh triple-negative breast cancer tissues than in normal tissues adjacent to the cancer(P<0.0001).Based on the expression of SNORA68 in paraffin sections of 73 triple-negative breast cancers,it was found that 46 patients(53.01%)had high expression and 27 patients(36.99%)had low expression.In the correlation analysis with clinicopathological factors,SNORA68 was significantly correlated with tumor size(P=0.038),lymph node status(P=0.002),ki-67 expression level(P=0.021)and TNM stage(P=0.011),but not with age and histological grade.3.The effects of SNORA68 on proliferation,migration,apoptosis,cell cycle and stemness characteristics of triple-negative breast cancer cells were analyzed: SNORA68 promoted the proliferation of MDA-MB-231 and BT-549,and also promoted the migration of MDA-MB-231 and BT-549,inhibited the proportion of apoptosis in triple-negative breast cancer cells and regulated the proportion of apoptosis in triple-negative breast cancer cells.SNORA68 also promoted the expression of SOX2,OCT4 and Nanog proteins,which are stemness indicators of triple-negative breast cancer.4.SNORA68 formed a complex with U2AF2 protein and then regulated the proliferation,migration of triple-negative breast cancer through the RPL23/c-Myc pathway: Based on bioinformatics screening of U2AF2,a protein that may bind to SNORA68,it was confirmed by RNA pull down assay and RIP assay.Also by cell function assay,after bidirectional intervention of SNORA68 and U2AF2,the increased expression of stemness index and enhanced proliferation and migration ability caused by high expression of SNORA68 could be reversed by knockdown of U2AF2,which confirmed that SNORA68 combined with U2AF2 to form a complex and promoted the proliferation,migration of triple negative breast cancer.Using bioinformatics analysis again,SNORA68 may be associated with the ribosomal protein pathway.Subcellular isolation assays and Western Blot experiments revealed and verified that RPL23 expression was significantly decreased in the nucleoplasm while significantly increased in the nucleolus in triple negative breast cancer cells MDA-MB-231 and BT-549 with high SNORA68 expression.Co-IP results suggest that high expression of SNORA68 in cells can reduce the binding of RPL23 to c-Myc and enhance the expression of c-Myc.5.Animal experiments demonstrated the mechanism by which SNORA68 forms a complex with U2AF2 protein and then regulates the proliferation,migration and stemness characteristics of triple-negative breast cancer through the RPL23/c-Myc pathway: in a nude mouse transplantation tumor model,high expression of SNORA68 promoted tumor growth,and its promoting effect could be reversed by knocking down the U2AF2 expression level.In transplanted tumors with high SNORA68 expression,the expression of ki-67,which is an indicator of proliferation,was significantly increased,and its upward trend could be reversed by knocking down U2AF2.The expression of SOX2,OCT4,Nanog and RPL3 in the nucleolus and nucleoplasm of transplanted tumors was determined by extracting total proteins from transplanted tumors,and the results were consistent with in vitro cellular experiments.The mechanism by which SNORA68 regulates the proliferation,migration and stemness characteristics of triple negative breast cancer through the RPL23/c-Myc pathway after forming a complex with U2AF2 protein was verified in vivo.Conclusion:1.SNORA68 is highly expressed in breast cancer and associated with poor prognosis;2.SNORA68 can regulate proliferation,migration,apoptosis,cell cycle and stemness characteristics of triple-negative breast cancer;3.SNORA68 can bind to U2AF2 protein to form a complex to regulate triple-negative breast cancer;4.High expression of SNORA68 can promote the translocation of RPL23 in the nucleolus and nucleoplasm.At the same time,it affected the reduction of binding between RPL23 and c-Myc,which in turn increased the expression of c-Myc.This study clarifies the mechanism by which SNORA68 regulates triple-negative breast cancer through binding to U2AF2 protein to form a complex and then through the RPL23/c-Myc pathway.