| Objective: Breast cancer is the most common malignant cancer in women.It is estimated that more than one million women worldwide suffer from the disease each year,accounting for about 25% of all tumors.Breast cancer is also the second cause of death from cancer in women.The incidence of breast cancer in China is rising rapidly,especially in coastal cities.Therefore,breast cancer has become one of the most serious cancer problems that severly dangers women's health.Many abnormal genes expression or functional changes in tumor are closely related to the occurrence and development of breast cancer.Therefore,in-depth study of the function and mechanism of these tumor-related factors provides a new target for the treatment of breast cancer and early prevention of this disease.The Rab protein is the largest branch of the small molecule G protein family(Ras superfamily).It is a family of GTP-binding proteins with a molecular weight of 20,000 to 30,000.It has more than 60 subunits and is composed of about 200 amino acids.They are highly conserved in the process of biological evolution,exist in almost all eukaryotes,and exhibit functional and quantitative diversity in different species.Currently,about 70 Rab proteins have been discovered in humans.Most of them are widely distributed in tissues and mainly localized to specific organelles and vesicles.Rab protein is involved in the formation,transport,adhesion and fusion of vesicles,which regulates the level and localization of a large number of proteins.Therefore,Rab protein regulates cell proliferation,metabolism,adhesion and migration through membrane transport.Rab13,a member of its family,is located on 1q21.3,and its gene sequence is 61%homologous to Rab8 and less than 47% similar to other Rab proteins.With the deep understanding of Rab13,it has been found that it not only regulates vesicle transport,but also plays an important role in the occurrence and development of malignant tumors.However,despite that,only three related studies have reported Rab13 in breast cancer.Therefore,this study provides a novel thinking for the treatment of breast cancer by studying the role of Rab13 in the development and progression of breast cancer.Through in vivo and in vitro experiments,we explored the mechanism of breast cancer malignant biological behavior.The new findings might provide a potential therapeutic target for the treatment of breast cancer in the future.Methods: 1.Western blot,immunohistochemistry,RT-PCR and other methods were applied to compare the difference of Rab13 expression between breast cancer tissues and normal tissues far away from cancer.Eighty patients with breast cancer who were treated in our hospital from 2011 to 2012 were selected.Paraffin sections were organized and the expression of Rab13 was detected by immunohistochemistry and correlated with clinicopathological parameters.These patients were followed up and the Kaplan-Meier survival analysis was used to compare the effect of Rab13 expression on survival and prognosis.2.Breast cancer MCF7 and MDA-MB-231 cell lines with Rab13 silencing were constructed.In vitro plate colony formation assay,Cell Counting Kit-8(CCK-8)assay and nude mice tumorigenesis was utilized to detect the proliferative activity of Rab 13 knockdown on breast cancer.The effect of cell invasive activity was detected by Transwell assay.The apoptosis rate of breast cancer cells after Rab13 knockdown was detected by flow cytometry.3.Gene Expression Omnibus(GEO)breast cancer dataset GSE54002 was collected for this study.After data normalization,the differential gene expression analysis of Rab13 high expression group and low expression group was carried out.In order to explore the biological processes and signaling pathways of Rab13-related differentially expressed genes,GO analysis and KEGG analysis were performed.Furthermore,Western blot and RT-PCR were applied to detect fold changes in EMT-related markers E-cadherin,N-cadherin and Vimentin after knockdown or overexpression of Rab13.Phosphorylation proportion of ERK1/2 and EMT were also detected by Western blot after Rab13 knockdown.Finally,the protein level of EMT-related markers including E-cadherin,N-cadherin and Vimentin were detected in ERK1/2 inhibitor treat group and Rab13 knockdown +ERK1/2 agonists group.Results: 1.The protein expression level of Rab13 was significantly higher in breast cancer than that in adjacent normal breast tissue(65% vs 10%,P <0.001).It presents a negative correlation with age(?50 years old or >50 years old,P =0.037)and ER(P =0.045),Rab13 was overexpressed in lymph node metastasis group(P = 0.040)and Ki67 overexpression group(P = 0.017);while no significant correlation with TNM stage,tumor size,PR,Her-2(P > 0.05).Breast cancer patients with high Rab 13 expression level manifested shorter survival time and poorer prognosis(P = 0.026).2.In MDA-MB-231 and MCF-7 cell lines,the proliferation activity of breast cancer cells was also significantly inhibited after knocking down Rab13,and the growth of tumors in nude mice was slowed down.The invasive ability of breast cancer cells was reduced and the proportion of apoptosis was increased significantly.3.Through GO analysis,we confirmed the hypothesis that Rab13-related differentially expressed genes are mainly involved in biological processes such as cell adhesion,migration and proliferation,which suggests that Rab13 plays an important role in breast cancer cell proliferation,adhesion and migration.Knockdown of Rab13 inhibits EMT and phosphorylation of ERK1/2 pathways in breast cancer cells;overexpression of wild-type Rab13 and mutant Rab13-GTP promote phosphorylation of ERK1/2 pathway and EMT.Rab13 promotes breast cancer EMT via the ERK1/2 pathway.Conclusion: The expression of Rab13 in breast cancer tissues is higher than that in adjacent tissues,and it is significantly correlated with age,lymphatic metastasis,ER expression and Ki67.Patients with breast cancer in the Rab13 high expression group had short OS.Knockdown of Rab13 can significantly inhibit the proliferation of breast cancer cells,promote the apoptosis of breast cancer cells,and inhibit the invasion of breast cancer cells.Rab13 promotes EMT in breast cancer cells via the ERK1/2 pathway.This study explored the role of Rab13 in the development of breast cancer,and provided a theoretical basis for its molecular mechanism,providing a potential therapeutic target for the treatment of breast cancer. |
PDF Full Text Request