The Effect Of CSN6 On Growth And Invasion Of Breast Cancer And Network Analysis Of CSN6 Co-expressed Genes In Breast Cancer

Objective:Breast cancer is one of the most common malignancies in women worldwide.With the deepening understanding of breast cancer,important biomarkers such as estrogen receptor(ER),progesterone receptor(PR)and human epidermal growth factor receptor 2(HER2)have been found.These biomarkers help clinicians to make a more accurate judgment on the prognosis of breast cancer patients.In addition,some targeted therapeutic approaches(such as hormone therapy and anti-HRE2 targeted therapy)are gradually introduced,which effectively prolong the patients’ survival time.However,there are still some patients can not benefit from the existing targeted therapy,and with the emergence of drug resistance,to find more effective targets for breast cancer treatment are urgent needed.As the main protein degradation pathway in human body,dysregulated proteasome pathway can promote the development of tumor by enhancing the stability of oncoprotein or promoting the degradation of tumor suppressor protein.Exploring the ubiquitin-proteasome pathway,looking for a new therapeutic target for cancer is becoming a hot spot.The constitutive photomorphogenesis 9(COP9)signalosome subunit 6(CSN6)is involved in the biological processes of cell cycle regulation and DNA damage repair through being a regulator of ubiquitination.E3 ubiquitin ligases determine the specificity of the proteasomal pathway to degrade substrate proteins.Mechanism studies found that CSN6 can interact with various types of E3 ubiquitin ligase and be involved in the development and progression of the tumor.The role of CSN6 in breast cancer still lacks direct and systematic evidence.This study aims to investigate the correlation between CSN6 and the prognosis of breast cancer patients and to explore the effect of CSN6 on the growth,migration and invasion of breast cancer cells,construct network analysis of CSN6 co-expressed genes in breast cancer and thus provide more evidence for the molecular mechanism of breast cancer development and progression.Methods:1.Examine the expression of CSN6 in breast cancer(1)Immunohistochemistry was used to examine CSN6 protein expression in surgical breast cancer specimens;(2)Oncomine and Kaplan-Meier plotter public data platform was used to examine CSN6 m RNA expression in breast cancer.2.The relationship between CSN6 expression and clinicopathological characteristics and prognosis in breast cancer patients.(1)SPSS 18.0 statistical analysis software chi-square test was used to analysis the relationship between CSN6 protein expression and clinicopathological characteristics.Kaplan-Meier method and Cox proportional hazard regression model was used to make survival analysis;(2)Oncomine public data platform was used to to verify the relationship between CSN6 m RNA expression and clinicopathological characteristics;(3)Kaplan-Meier plotter public data platform was used to verify the relationship between CSN6 m RNA and prognosis of breast cancer patients.3.Construct the breast cancer cell lines which were silenced CSN6 gene with lentivirus.(1)Using inverted fluorescence microscope to observe cell morphology and lentiviral transfection efficiency;(2)Western blot was used to confirm knockdown efficiency.4.To examine the effect of CSN6 on the growth and invasion of breast cancer cells(1)Clone formation experiment,MTS experiment was used to observe the effect of CSN6 on the proliferation of breast cancer cells;(2)Transwell assay was used to detect the effect of CSN6 on the invasion and migration of breast cancer cells.5.Bioinformatics analysis of CSN6 signal regulation network in breast cancer(1)Download GEO dataset GSE25066 and acquire the CSN6-related differentially expressed genes;(2)GO analysis and KEGG analysis was used to analyze the potentially biological processes and signal pathways which CSN6 involved;(3)String database was used to analyze co-expression gene set and build CSN6-related co-expression network.Results:1.Elevated CSN6 expression is correlated with high-risk clinicopathological characteristics in breast cancer.Expression of CSN6 protein was positively correlated with tumor size(P = 0.041),lymph node metastasis(P = 0.01)and advanced TNM stage(P = 0.002).CSN6 m RNA was significantly higher in metastasis site,compared with primary site in breast cancer(fold change = 2.154,P = 0.022).In molecular subtypes,HER2-positive breast cancer has a tendency of high CSN6 protein expression(85.7%),although the results are not statistically significant(P = 0.052).2.CSN6 was correlated with poor prognosis in breast cancer patients.The high CSN6 m RNA expression was correlated with poor relapse-free survival(HR = 1.19,95% CI: 1.07-1.33,log-rank P = 0.0015)and distant metastasis-free survival time(HR = 1.26,95% CI: 1.02-1.54,log-rank P = 0.028).High CSN6 protein expression was associated with poor disease-free survival(log-rank P = 0.028)and overall survival(log-rank P = 0.038)in breast cancer patients and was an independent prognostic indicator for disease-free survival(HR = 3.83,95% CI: 1.14-12.79,P value = 0.029).To further investigate the effect of CSN6 on the prognosis of different molecular subtypes of breast cancer,we explored CSN6 m RNA in all four subtypes of breast cancer patients.CSN6 expression correlated with a shorter relapse-free survival in all four molecular subtypes.The results in Luminal B subtype(HR = 1.53,95% CI: 1.25-1.88,log-rank P = 2.9e-05)and basal like subtype(HR= 1.48,95% CI: 1.15-1.91,log-rank P = 0.0023)breast cancer patients were more obvious,with statistical differences.3.Silencing CSN6 inhibits the proliferation of breast cancer cells.MTS assay was used to detect the effect of CSN6 silencing on the proliferation of MCF7,T47 D and MDA-MB-231 breast cancer cells at different time points.The results showed that the proliferation rate of MCF7 / KD cells was 209.22 ± 9.76% and the proliferation rate of MCF7 / NC-KD cells was 306.85 ± 35.36%(P = 0.014)after 120 hours of culture.The proliferation rate of T47 D / KD cells was 284.43 ± 1.65 %,and the proliferation rate of T47 D / NC-KD cells was 356.59 ± 14.22%(P = 0.012).The proliferation rate of MDA-MB-231 / KD cells was 310.30 ± 11.76%,and the proliferation rate of MDA-MB-231 / NC-KD cells was 348.52 ± 19.25%(P = 0.043).Colony formation assay showed that,after 14 days of culture,the number of colonies of MCF7 / KD was 66.67 ± 13.65 and the number of colonies of MCF7 / NC-KD was 115 ± 15.72(P = 0.016).The number of colonies of KD cells was 141 ± 40.04,and the number of colony of MDA-MB-231 / NC-KD cells was 374.67 ± 28.31(P = 0.001).4.Silencing CSN6 inhibits the invasion and migration of breast cancer cells.Transwell assay was used after 24 hours culture,the number of migrated cell in MDA-MB-231 / KD(matrigel-free transwell group)was 26.89 ± 4.33 and the number of migrated cell in MDA-MB-231 / NC-KD cells was 24.4 ± 6.30(P = 0.007).The number of migrated cell in MDA-MB-231 / KD(transwell group with matrigel)was 16.89 ± 8.76 and the number of migrated cell in MDA-MB-231 / NC-KD was 41.22 ± 7.41(P= 0.021).5.CSN6 in breast cancer may be involved in tumor immune response and cell adhesion process.According to the biological process enrichment results of GO analysis,immune regulation and cell adhesion occur multiple times.A similar result can be observed in the KEGG pathway enrichment analysis.6.Establishment of CSN6 co-expression gene network and interaction network in breast cancer.We demonstrate that CSN6 and ILF2 have a significant overlap of coexpressed genes.Moreover,several Oncomine datasets indicate a positive correlation between CSN6 and ILF2.Conclusion: 1.Elevated CSN6 expression is associated with tumor size,lymph node metastasis,and distant metastasis in breast cancer.2.CSN6 overexpression is associated with poor survival breast cancer patients,and CSN6 protein can be used as an independent prognostic indicator for disease-free survival in breast cancer.3.Elevated CSN6 m RNA in Luminal B subtype and basal-like subtype breast cancer patients is more obviously correlated with poor relapse-free survival.4.Silencing CSN6 inhibits the viability of breast cancer cell MCF7,T47 D and MDA-MB-231.5.Silencing CSN6 inhibits the clone formation of breast cancer cell MCF7 and MDA-MB-231.6.Silencing CSN6 inhibits the invasion and migration of breast cancer cell MDA-MB-231.7.CSN6-related differentially expressed genes are enriched in cell adhesion process,suggesting that CSN6 play a more significant role in the invasion and metastasis of breast cancer.8.CSN6-related differentially expressed genes are enriched in immune response process,suggesting that CSN6 may be involved in the process of tumor immunity in breast cancer.9.Some tumor-related genes E2F1,HRAS,Caspase 9 and WBSCR22,which highlighted as nodes,play important regulatory role in CSN6 co-expression network in breast cancer.10.ILF2 may be involved in the process of CSN6 induced breast cancer progression.