The Regulation Mechanism Of Small Nucleolar RNA SNORA51 On Breast Cancer Stem Cell-like Properties Through RPL3/NPM1/c-MYC Pathway

Objective:Breast cancer has surpassed lung cancer as the most common malignant tumor in women all over the world.Breast Cancer stem cells（BCSCs）account for a very small proportion of cells in breast cancer who can result in the heterogeneity in breast cancer.BCSCs can play an important role in the occurrence,development,drug resistance and recurrence in breast cancer.Therefore,exploring the regulation mechanism of breast cancer stem cell-like properties has far-reaching significance in screening of effective therapeutic targets in breast cancer.Small nucleolar RNAs（sno RNAs）are non-protein-coding RNAs with transcription length of 60-300 nucleotides,which are widely existing in eukaryotes and have a conserved secondary structure.According to their structures and functions,sno RNAs can be divided into C/D box sno RNAs,H/ACA box sno RNAs and sca RNAs.The classical biological functions of sno RNAs are to participate in the splicing processing and post-transcriptional modification of ribosomal RNA（r RNA）.In addition,sno RNAs can also participate in RNA alternative splicing,RNA translational regulation and oxidative stress response.Therefore,sno RNAs have gradually become the new hotspots in the oncology related researches.Sno RNAs can take part in the development of non-small cell lung cancer,ovarian cancer,head and neck squamous cell carcinoma and liver cancer by regulating the stem-like characteristics and affecting stemness maintenance.In breast cancer,sno RNAs only had been proved to affect the stem cell-like properties through the process of epithelial-mesenchymal transition（EMT）.Further exploration of the relationship between sno RNAs and breast cancer stem cell-like properties may open new ideas for the diagnosis and treatment of breast cancer.In addition to being part of the translation mechanism in eukaryotes,ribosomal proteins（RPs）are nucleolar proteins which can also play a variety of extra-ribosomal functions on regulating the biological processes in various diseases.RPs can be involved in the external ribosomal mechanisms independent of the p53 pathway as well as participate in a wide range of cellular stress events include influencing nucleolar structure and involvement in p53-dependent nucleolar stress.Along with the developing of the researches,it is found that sno RNAs can influence cancer progression by regulating RPs and its downstream signaling pathways,which may provide a new research direction for exploring the regulatory mechanism between sno RNAs and RPs in cancer beyond their classical pathways.In this study,the relationship between SNORA51 and stem cell-like properties in breast cancer was screened by high-throughput sequencing based on the mammosphere（MS）cells and breast cancer cells.The expression of SNORA51 in breast cancer and its relationship with prognosis and clinicopathological factors were analyzed.Based on the analysis of SNORA51 promoting and maintaining effects on breast cancer stem cell-like properties,the molecular mechanism of SNORA51 affecting breast cancer stem cell-like properties by regulating RPL3/NPM1/c-MYC pathway was verified on the basis of subcellular separation experiments.This study may provide a new idea for the regulation mechanism of sno RNAs in breast cancer and even other malignant tumors.Also,this study may be expected to provide more theoretical and experimental basis for the influence of sno RNAs on the occurrence and development of cancers.Methods:1.Induction and identification of mammosphere cells with breast cancer stem cell-like properties:Breast cancer cells MCF-7 and T47D were suspended and cultured in serum-free medium containing growth factors.After 6-8 days,the cell morphology showed obvious changes to spherical-like form.The stemness of mammosphere cells was confirmed by flow cytometry,q RT-PCR,Western Blot,Transwell and mammosphere cells differentiation analysis.2.Screening of SNORA51 in MCF-7 MS and MCF-7 cells based on high-throughput sequencing and the bioinformatics analysis related to SNORA51:MCF-7 and MCF-7 MS cells with stable and well condition were used to screen the sno RNAs which were significantly differently expressed according to the high-throughput sequencing technology,and SNORA51 was defined as closely related to breast cancer stem cell-like properties.The relationship between SNORA51 and stemness biomarkers and prognosis was analyzed based on TCGA database.3.The expression and clinical significance of SNORA51 in breast cancer:The expression of SNORA51 in 30 pairs of fresh breast cancer tissues and 158 paraffin sections of breast cancer patients was detected by q RT-PCR and in situ hybridization,and the expression of OCT4 in 30 pairs of fresh breast cancer tissues and 158 paraffin sections of breast cancer patients was detected by q RT-PCR and immunohistochemistry.Spearman correlation analysis was used to explore the correlation between SNORA51and OCT4 expression.Kaplan-Meier analysis was used to analyze the relationship between SNORA51 and prognosis in 158 breast cancer patients.Pearson Chi-square test,Fisher’s exact test and Logistic regression analysis showed the association between SNORA51 and clinicopathological factors4.To analyze the promoting and maintaining effects of SNORA51 on stem cell-like properties in breast cancer:Cells with high expression of SNORA51 were established in MCF-7 and T47D cells.Flow cytometry,q RT-PCR,Western Blot,CCK-8 assay,cloning formation assay,Transwell assay and Wound-healing assay were used to detect the effects of high expression of SNORA51 on breast cancer stem cell-like properties.Antisense oligonucleotides of SNORA51 were transfected into MCF-7 MS and T47D MS to inhibit the expression of SNORA51.Flow cytometry,q RT-PCR,Western Blot,CCK-8 assay,Transwell assay and mammosphere formation assay were used to detect the effect of SNORA51 low-expression on breast cancer stem cell-like properties.5.The molecular mechanism of SNORA51 on regulating the stem cell-like properties in breast cancer:According to the bioinformatics analysis,the expression of RPL3 was significantly and negatively related to SNORA51.Subcellular structure separation experiment and Western Blot were used to detect the effect of SNORA51 on nucleolus RPL3 expression and NPM1 expression in nucleolus and nucleoplasm.Co-IP assay was used to detect the influence of SNORA51 on the binding of RPL3-NPM1 and NPM1-c-MYC in nucleus.Combined with the detection of the expression of NPM1 in both nucleolus and nucleoplasm and the expression level of c-MYC in nucleoplasm,we verified that SNORA51 could regulate the stem cell-like properties through the RPL3/NPM1/c-MYC pathway in breast cancer.6.Experiments in vivo:The mechanism of SNORA51 regulating the stem cell-like properties in breast cancer via RPL3/NPM1/c-MYC pathway was verified by the nude mouse transplanted tumor model in vivo.Results:1.Induction and identification of mammosphere cells with breast cancer stem cell-like properties:Adherent breast cancer cells grew into mammosphere cells after serum-free suspension culture.Cells with CD24^-/lowCD44⁺were significantly increased in mammosphere cells（p<0.0001）.SOX2,OCT4 and Nanog were significantly increased in mammosphere cells（p<0.05）.The mammoshere cells of breast cancer had stronger migration ability（p<0.001）and has differentiation potential.2.High-throughput sequencing results showed that SNORA51 was significantly higher expressed in mammosphere cells than in breast cancer cells.Analysis based on TCGA showed that SNORA51 was significantly and positively associated with the expression of OCT4,SOX2,MYC and HIF-1α（p<0.05）.Also,high expression of SNORA51 was closely related to poor prognosis in breast cancer（p=0.0034）.3.Expression and clinical significance of SNORA51 in breast cancer tissues:The expression of SNORA51 in 30 breast cancer tissues was significantly higher than that in adjacent normal tissues（p<0.0001）,and was positively correlated with the expression of OCT4（p=0.0003,r=0.6135）.Among 158 patients with breast cancer,94 patients（59.49%）had high expression of SNORA51 and 64 patients（40.51%）had low expression of SNORA51.There was also a positive correlation between SNORA51 and OCT4 expression in 158 patients（p<0.0001,r=0.411）.Kaplan-Meier analysis found that the high expression of SNORA51 in 158 breast cancer patients was significantly correlated with shorter overall survival（p=0.0021）and shorter disease-free survival（p=0.0168）.SNORA51 was significantly correlated with lymph node status（p=0.002）,ER expression level（p=0.024）,Ki-67 index（p=0.033）,histological grade（p=0.002）and TNM stage（p=0.043）.Multivariate Logistic regression analysis showed that lymph node status（p=0.021）,Ki-67 index（p=0.023）and histological grade（p=0.021）were independent factors affecting SNORA51 expression.4.Analysis of SNORA51 on promoting and maintaining the stem cell-like properties in breast cancer:The CD24^-/lowCD44⁺subpopulations of MCF-7 and T47D cells with SNORA51 high expression were significantly increased（p<0.0001）.SOX2,OCT4 and Nanog were significantly increased in MCF-7 and T47D cells with SNORA51overexpression（p<0.05）.In the cells with high expression of SNORA51,the proliferation ability and clonal formation ability were significantly enhanced（p<0.01）,and the migration ability of the cells was also significantly enhanced（p<0.05）.In MCF-7 MS and T47D MS cells with SNORA51 silenced,the subpopulation of CD24^-/lowCD44⁺cells were significantly decreased（p<0.0001）.SOX2,OCT4 and Nanog were significantly decreased in SNORA51 silenced mammosphere cells（p<0.05）.The proliferation ability and the spherical formation ability were significantly decreased in SNORA51 silenced mammosphere cells（p<0.01）,and the cell migration ability was also significantly decreased（p<0.001）.5.The regulation mechanism of SNORA51 on breast cancer stem cell-like properties through RPL3/NPM1/c-MYC pathway:Based on bioinformatics analysis,the expression of SNORA51 was significantly and negatively related with ribosomal protein RPL3（p=0.0021）.In the MCF-7 and T47D cells with overexpressed of SNORA51,the expression of RPL3 in nucleolus was significantly decreased（p<0.05）.The expression of NPM1 was decreased in the nucleolus and increased in the nucleoplasm at the same time（p<0.05）.Co-IP results showed that the high expression of SNORA51 could decreased the binding of RPL3 to NPM1 and increased the binding of NPM1 to c-MYC at the same time,also could enhanced the expression of c-MYC in nucleoplasm（p<0.05）.After bidirectional intervention of SNORA51 and RPL3,the influence on breast cancer stem cell-like properties caused by the overexpression of SNORA51 could be reversed by overexpression of RPL3.6.The verified of SNORA51 can regulate breast cancer stem cell-like properties through RPL3/NPM1/c-MYC pathway in vivo:In the nude mouse transplanted tumor model,it was also verified that the high expression of SNORA51 could promote tumor growth,and its effect could be reversed by the overexpression of RPL3.The promoting effects of SNORA51 on the expression of OCT4 and Ki-67 could be observed in transplanted tumors,and these influences could also be reversed by the high expression of RPL3.The detection of SOX2,OCT4 and Nanog expression in transplanted tumors,the expression of RPL3 and NPM1 in nucleolus and the expression of NPM1 and c-MYC in the nucleoplasm had the consistent results with the experiments in vitro.Thus,we verified that SNORA51 could regulate breast cancer stem cell-like properties through RPL3/NPM1/c-MYC pathway in vivo.Conclusion:1.SNORA51 is closely related to breast cancer stem cell-like properties.The high expression of SNORA51 is significantly and positively correlated with poor prognosis in breast cancer.2.SNORA51 can promote and maintain the biological function of breast cancer stem cell-like properties.3.The high expression of SNORA51can reduce the expression of RPL3 in the nucleolus,promote the expression changes of NPM1 in the nucleolus and nucleoplasm,then enhance the expression of c-MYC in nucleoplasm.SNORA51 can promote the stem cell-like properties of breast cancer through the RPL3/NPM1/c-MYC pathway.