Expression Of RPS13 In Triple Negative Breast Cancer And Its Effect And Mechanism On Tumor Biological Behavior

Objective: Triple negative breast cancer(TNBC)is one of the various molecular subtypes of breast cancer,accounting for 12-17% of all breast cancer types.It is characterized by rapid progression,high aggressiveness,easy recurrence and metastasis,and poor prognosis.Chemotherapy remains the main systemic treatment option due to its aggressive nature and lack of clear targeted therapy and endocrine therapy targets.However,less than 30% of patients with TNBC achieve a complete response,and the recurrence and mortality rates remain higher than those of other subtypes.Therefore,it is necessary to explore the expression pattern of genes in TNBC and screen potential targets for diagnosis and treatment of TNBC for early diagnosis of TNBC and to improve the survival rate of patients with TNBC.Ribosomal proteins(RPs)are major components of ribosomes that stabilize specific r RNA structures in mature ribosomal subunits,promote correct folding of r RNA during ribosome assembly,and coordinate interactions between ribosomes and m RNA,as well as initiation and extension factors.Multiple RPs are overexpressed in malignant tumors and associated with tumor progression.In this study,the differentially expressed proteins in TNBC were analyzed by an independent data collection technique based on the digitalization of proteomic samples based on mass spectrometry,and it was found that ribosomal protein S13(RPS13)was highly expressed in TNBC.RPS13 was found to be overexpressed in colon cancer and small cell lung cancer cells.In addition,the expression level of RPS13 was correlated with the growth rate of tumor cells.The expression of RPS13 is up-regulated in multi-drug resistant gastric cancer cells.Overexpression of RPS13 can protect gastric cancer cells from drug-induced apoptosis,promote the growth and tumorigenic potential of gastric cancer cells in vitro,and promote the transformation of gastric cancer cells from G1 to S phase.Knockout of RPS13 leads to G1 arrest of gastric cancer cells.The expression of RPS13 in TNBC and its effect on biological behavior of tumor have not been reported.This study analyzed the expression of RPS13 in TNBC tissues and its correlation with clinicopathological factors and prognosis,and explored its influence on biological behaviors of TNBC cell proliferation,migration,invasion,EMT and apoptosis.In addition,the mechanism by which RPS13 regulates TNBC cell metastasis was also analyzed.In conclusion,this study provides theoretical basis for RPS13 as a potential target for the diagnosis and treatment of TNBC.Methods: Part I: Intraoperative cancer tissue samples were collected from 3 patients with triple-negative breast cancer and 3 patients with luminal A breast cancer who underwent primary surgical treatment in the Department of Breast Surgery of the First Affiliated Hospital of China Medical University.The differentially expressed proteins in TNBC and luminal A breast cancer tissues were screened based on proteomics and bioinformatics.The upregulation proteins identified were analyzed by GO using DAVID Bioinformatics Resources 6.8 software.Disease and pathway analysis using IPA.Part II: The expression of RPS13 in TNBC tissues and adjacent tissues was detected by immunohistochemistry,q RT-PCR and Western blot.The relationship between RPS13 expression and clinical indicators of TNBC patients was further analyzed.The expression of RPS13 in normal breast epithelial cells MCF-10 A and triple negative breast cancer cell lines HCC1937,MDA-MB-231 and MDA-MB-468 were detected by q RT-PCR and Western blot.TNBC cells with stable knockdown of RPS13 were established,and the expression of RPS13 was detected by q RT-PCR and Western blot.Cell proliferation was detected by CCK-8 and plate clonal formation assay.Cell migration ability was detected by scratch assay and Transwell assay.Transwell invasion assay was used to detect cell invasion ability.The apoptosis rate was detected by flow cytometry.Emt-related proteins were detected by Western blot and immunofluorescence.HCC1937 and MDA-MB-231 cells with RPS13 knocked down were injected subcutaneously into nude mice to establish the transplanted tumor model,and tumor volume and mass were detected.Ki-67 expression in the transplanted tumor was detected by immunohistochemistry,and EMTrelated protein expression was detected by Western blot and immunofluorescence.The expression of RPS13 was detected by q RT-PCR and Western blot.Part III: The effect of RPS13 knockdown on the expression of p-NF-κB and NF-κB protein in TNBC cells was detected by Western blot.The effect of RPS13 knockdown on the expression of p-NF-κB protein in TNBC cell nucleus was detected by Western blot and immunofluorescence.The effect of RPS13 knockdown on the expression of p-NF-κB and NF-κB protein in TNBC cell grafts was detected by Western blot.The effect of RPS13 knockdown on the expression of p-NF-κB protein in the cell nuclei of transplanted HCC1937 and MDA-MB-231 cells was detected by immunofluorescence.TNBC cells with RPS13 knocked down were treated with NF-κB agonist LPS,and the expressions of p-NF-κB and NF-κB proteins were detected by Western blot.The expressions of p-NF-κB protein and EMT-related protein were detected by Western blot and immunofluorescence.Cell migration ability was detected by scratch assay and Transwell assay.Cell invasion ability was detected by Transwell invasion assay.Snail si RNA was transfected with NF-κB agonist LPS to detect the expression of p-NF-κB,NF-κB and Snail proteins in TNBC cells,and the expression of EMT-related proteins was detected by Western blot and immunofluorescence.Cell migration ability was detected by scratch assay and Transwell assay.Cell invasion ability was detected by Transwell invasion assay.The tumor volume and quality of the transplanted tumor were detected by subcutaneous injection of TNBC cells that overexpressed Snail and reduced RPS13.Ki-67 expression in the transplanted tumor was detected by immunohistochemical method,and RPS13 and Snail expression in the transplanted tumor were detected by q RT-PCR and Western blot.Emt-related proteins were detected by Western blot and immunofluorescence.Results: Part I: Compared with Luminal Type A BC patients,533 DEPs were screened in TNBC patients,among which 207 were up-regulated and 326 were downregulated.RPS13,dissociation stimulator like protein 2,RGL2(ral guanine nucleotide dissociation stimulator like protein 2,RGL2),keratin 15,KRT15),HEAT repeat containing protein 1(HEATR1)and exosome component 2(EXOSC2)were significantly up-regulated.DEPs are mainly involved in the regulation of biological processes such as reaction to bacteria,negative regulation of catalytic activity,negative regulation of endogenous peptidase activity,adaptive immune response and negative regulation of hydrolase activity.It is involved in the cell composition of endoplasmic reticulum compartment,endoplasmic reticulum,organelle compartment,epicontinurousendoplasmic reticulum network,cytoplasmic ribosome and cell surface.It plays the molecular functions of immunoglobulin complex circulation,intermediate fiber formation,blood microparticle formation and immunoglobulin complex formation.Significantly activated diseases or functions include tumor-related tumor cell death and gastrointestinal tumors,while significantly inhibited diseases or functions include infectious disease-related tumor cell line infection and RNA virus infection.Part II: RPS13 expression was up-regulated in TNBC tissues compared with paracancer tissues,and positive RPS13 was significantly correlated with larger tumor,higher histological grade,and lower 5-year survival rate,regardless of patient age.The expression of RPS13 in TNBC cell lines HCC1937,MDA-MB-231 and MDA-MB-468 was up-regulated compared with that in MCF-10 A cells.Knockdown of RPS13 downregulates the expression of RPS13 in TNBC cells,inhibits cell proliferation,clonogenesis,migration and invasion,increases cell apoptosis rate,decreases the expression of Ncadherin,Vimentin,Snail and Slug proteins.RPS13 knockdown also decreased tumor volume and mass,decreased Ki-67 expression,decreased N-cadherin,Vimentin,Snail and Slug protein expression in TNBC cell grafts.Part III: RPS13 knockdown decreased the expression of p-NF-κB in TNBC cells and transplanted tumors and in the nucleus.The administration of NF-κB agonist increased the expression of p-NF-κB protein and p-NF-κB in the nucleus of RPS13 knockdown TNBC cells,increased the expression of N-cadherin and Vimentin protein,and increased the ability of cell migration and invasion.Snail silence decreased the expression of Ncadherin and Vimentin proteins in NF-κB agonist-treated RPS13 knockout TNBC cells,and decreased the cell migration and invasion ability.Snail overexpression increased the tumor volume and mass of RPS13-impaired TNBC cell grafts,up-regulated Ki-67 expression,and increased N-cadherin and Vimentin protein expression.Conclusion: RPS13,RGL2,KRT15 and HEATR1 are all up-regulated in TNBC tissues,and are expected to become potential targets for diagnosis and treatment of TNBC.RPS13 is highly expressed in both TNBC tissues and cells,and its high expression is associated with larger tumors,lymph node metastasis,and lower 5-year survival rate in TNBC patients.RPS13 promotes TNBC cell proliferation,migration,invasion and EMT in vitro,inhibits cell apoptosis,and promotes tumor growth and EMT in TNBC cell transplantation tumors.By activating NF-κB to up-regulate Snail expression,RPS13 induces EMT and promotes TNBC cell migration and invasion.