Up-Regulation of RFC3 Promotes Triple Negative Breast Cancer Metastasis and is Associated With Poor Prognosis Via EMT Zhen-Yu He, San-Gang Wu, Fang Peng,

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Up-Regulation of RFC3 Promotes Triple Negative Breast Cancer Metastasis and is Associated With Poor Prognosis Via EMT Zhen-Yu He, San-Gang Wu, Fang Peng, Qun Zhang, Ying Luo, Ming Chen, Yong Bao Translational Oncology Volume 10, Issue 1, Pages 1-9 (February 2017) DOI: 10.1016/j.tranon.2016.10.004 Copyright © 2016 The Authors Terms and Conditions

Figure 1 RFC3 is up-regulated in primary breast cancer. (A) Quantitative real time RT-PCR analysis of RFC3 mRNA from the same four pairs of breast cancer and adjacent non-tumor breast tissues. Error bars represent SDs calculated from triplicate experiments. (B) RT-PCR analysis of RFC3 protein in four pairs of human primary breast tumor tissues (T) and paired adjacent non-tumor breast tissue (N). (C) Western blotting analysis of RFC3 protein in four human primary breast cancer (T) and paired adjacent non-tumor breast tissues (N), with each pair taken from a same patient. Translational Oncology 2017 10, 1-9DOI: (10.1016/j.tranon.2016.10.004) Copyright © 2016 The Authors Terms and Conditions

Figure 2 Up-regulation of RFC3 is associated with poor prognostic phenotype in breast cancer. (A) RFC3 expression level in different differentiation stage (benign tissue, carcinoma in situ, invasive carcinoma) analyzed by immunohistochemistry. (B) Expression levels of RFC3 in 30 paired breast cancer and adjacent non-tumor breast tissues. Alteration of expression is shown as box plot presentations and the mean level of RFC3 expression in breast cancer were significantly higher than that in non-tumor tissues. (P<.0001, independent t test). Expression levels of RFC3 between 127 BCs with and without metastasis. The mean level of RFC3 expression in BCs with metastasis was significantly higher than that in BCs without metastasis (P<.0001, independent t test). (C) Overall survival, disease-free survival and metastasis-free survival rate for cases with high RFC3 expression versus that of cases with low RFC3 expression. Translational Oncology 2017 10, 1-9DOI: (10.1016/j.tranon.2016.10.004) Copyright © 2016 The Authors Terms and Conditions

Figure 3 RFC3 expression is up-regulated in breast cancer cells lines (A) Quantitative real time PCR analysis was performed in 6 breast cancer cell lines and a non-tumor immortalized human breast epithelial cell line MCF-10A. (B) RFC3 protein level in 6 breast cancer cell lines and a non-tumor immortalized human breast epithelial cell line MCF-10A were analyzed by western blotting. Translational Oncology 2017 10, 1-9DOI: (10.1016/j.tranon.2016.10.004) Copyright © 2016 The Authors Terms and Conditions

Figure 4 Inhibition of RFC3 attenuated BC cell proliferation, motility and invasion. (A) The knockdown efficiency of shRNA against RFC3 was examined by Western blotting in MDA-MB-231 and MDA-MB-468 cells. (B) The proliferation ability was measured in MDA-MB-231 and MDA-MB-468 cells using colony formation assay. (C) The migration and (D) invasiveness abilities were analyzed in MDA-MB-231 and MDA-MB-468 cells by Boyden chamber assay (*P<.05, **P<.01). Translational Oncology 2017 10, 1-9DOI: (10.1016/j.tranon.2016.10.004) Copyright © 2016 The Authors Terms and Conditions

Figure 5 Knockdown of RFC3 can relieve xenograph tumor progression in null mice. Xenograft model in nude mice. MDA-MB-231, shVector and shRFC3 cells were inoculated in the fat pat of nude mice (n=5/group). (A) Images of the tumors from all mice in each group. (B) The lung metastasis staining in H&E, (C) tumor volume and (D) mean tumor weight was analyzed. Translational Oncology 2017 10, 1-9DOI: (10.1016/j.tranon.2016.10.004) Copyright © 2016 The Authors Terms and Conditions

Figure 6 Knockdown of RFC3 can reversed epithelial-mesenchymal transition (EMT) The expression of EMT markers of E-Cadherin, ZO-1, Vimentin and N-Cadherin were analyzed by western blotting both in MDA-MB-231 and MDA-MB-468 breast cancer cells. GAPDH was probed as the loading control. Translational Oncology 2017 10, 1-9DOI: (10.1016/j.tranon.2016.10.004) Copyright © 2016 The Authors Terms and Conditions