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Original Article TRIM25 promotes oncogenic activities through regulation of ZEB1 in breast cancer

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Original Article

TRIM25 promotes oncogenic activities

through regulation of ZEB1 in breast cancer

Fei Cao1*, Da-Peng Li1*, Lei Wang2, Min Li3, Hui Zhang4, Min Tao1

1Department of Oncology, First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China; Departments of 2General Surgery, 3Pathology, 4Oncology, Northern District of Suzhou Municipal Hospital, Suzhou, Jiangsu, China. *Equal contributors.

Received March 6, 2016; Accepted July 19, 2016; Epub October 1, 2016; Published October 15, 2016

Abstract: Tripartite motif 25 (TRIM25) is a kind of E3 ligase of ubiquitin-proteasome pathway. It was found to act in immunologic response, regulation and control of mitotic cycle and tumor cell’s drug resistance. In this study, we found that TRIM25 was increased in breast cancer. Knockdown of TRIM25 inhibited the migration, invasion and epithelial-mesenchymal transitions (EMT) of breast cancer cells. Meanwhile, over-expression of TRIM25 promoted the migration, invasion and EMT of breast cancer cells. Western blot analysis indicated that ZEB-1 may play func-tion as a downstream factor of TRIM25. Knockdown of TRIM25 can inhibit ZEB-1’s expression. And interference of ZEB-1 can also inhibited the cell’s ability of migration, invasion and EMT; even in a cell line who’s TRIM25 was over expressed. Together, these results helped to realize the oncogene functions of TRIM25 in breast cancer cells, and provided a novel direction to treat breast cancer.

Keywords: TRIM25, breast cancer, ZEB-1, EMT

Introduction

Accounting for a 24% of all cancer causes, breast cancer is the most common malignant tumor among women [1]. Although there are

significant advances in diagnosis and treat -ment, breast cancer is still the second leading cause of cancer-related death worldwide [2, 3]. Therefore, there is a great need for develop-ment of novel direction to treat breast cancer. Tripartite-motif protein (TRIM) family is

charac-terized with RBCC multi-domain RING finger,

B-box and coiled-coil [4]. The most of TRIM fam-ily members belongs to a kind of E3 ligase. RING domain is used to recruit E2 enzyme and substrate, and recruit protein which is ubiquiti-noylated or recognized by ubiquitin concurrent-ly to take effects [5]. There have been almost 80 TRIM family members founded in human genome up to the present [6]. It’s also founded that many of them plays an important part in life process, such as apoptosis, mitotic cycle, virus infection and immunologic response [7]. However, the mechanism that TRIM family

pro-teins play their function is still not clearly under-stood at the present time.

Tripartite motif 25 (TRIM25) has typical RBCC multi-domain. It has dominant pleiotropism and plays crucial roles in many pathologic process-es [8, 9]. As a kind of E3 ligase of ubiquitin-pro-teasome pathway, TRIM25 was founded to be associated with immunologic response, regu- lation and control of mitotic cycle and tumor cell’s drug resistance [10, 11]. Nevertheless, the function and mechanism TRIM25 plays in breast cancer have not been well studied. In this study, we investigated the roles of TRIM25 in breast cancer genesis. We found that the expression of TRIM25 increased in a majority of the human breast cancer tissues examined compared with normal tissues. In human breast cancer cell line MCF-7, over-expression of

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these results indicate an important role of TRIM25 in breast cancer cells’ migration, inva-sion and EMT.

Materials and methods

Samples, cells and antibodies

Human normal breast tissue samples and breast cancer tissue samples were provided by First Affiliated Hospital of Soochow

Univer-sity.Human breast cancer cell lines, MCF10A, MDA-MB-468, BT549, MDA-MB-231, HCC14- 28, MCF7 and SK-BR-3 were obtained from ATCC. The cells were maintained in Minimum Essential Medium (MEM) (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine se- rum (FBS) (Invitrogen, Carlsbad, CA).

Immunohistochemical staining

The TRIM25 expression of breast cancer tissue samples and normal breast tissue samples was detected by immunohistochemical Envi- sion two-step method. Tissues were blocked

by paraffin, and then sliced so as to obtain 3 μm-thickness paraffin sections. Paraffin sections were deparaffinized by dimethylben -zene, hydrated by alcohol in different con- centrations, washed by PBS one by one, and then incubated with 1:200 TRIM25 anti-bodies at 4°C overnight. Goat-radish peroxi-dase-conjugated secondary antibody (1:500) was then added and further incubated for 1 hour at room temperature. The sections were developed using a 3.3’-diaminobenzidine te- trahydrochloride (DAB) substrate kit at room temperature for 1-5 min and then counter-stained with hematoxylin.

Western blot analysis

Cells were washed and scraped in a lysis bu-

ffer and quantified. Twenty mg of protein were

loaded on a SDS-PAGE and transferred onto a 0.2 mm nitrocellulose membrane. After block-ing with 5% non-fat dry milk in Tris-buffered saline with 1% tween-20 (TBS-T), membranes were incubated with a primary antibody over-night. After washing, the membrane was in- cubated for 1 h with the HRP-conjugated se- condary antibody. Membranes were washed thrice with TBS-T, and proteins were visualized with Super Signal (Pierce, Bonn, Germany) en- hanced chemiluminescence.

RT-PCR

Total RNA was extracted from breast cancer tissues and cell lines using the TRIZOL reagent (Invitrogen). Revert transcription (RT) was per-formed by using the Thermoscript RT System (Invitrogen). PCR conditions were set as fo- llows: 45 s at 94°C, 30 s at 55°C, and 1 min at 72°C for 28-30 cycles (for TRIM25) or 26 cycles [for glyceraldehyde 3-phosphate dehy-drogenase (GAPDH)]. The primers used in the study were: TRIM25: sense 5’-AGAGTTTCATC- CCCAAAGACAA-3’ and antisense 5’-AGTTCAG- GCAGTAGGCAAAGTC-3’; GAPDH: sense 5’-TGC- CTCCTGCACCACCAACT-3’ and antisense5’-CC- CGTTCAGCTCAGGGATGA-3’.

Plasmids construct

Human cDNA of TRIM25 was cloned as previ-ously reported. The full-length cDNAs were sub-cloned into the multiple cloning sites of the vec-tor plasmid forming the vecvec-tor-TRIM25 expres-sion plasmids. Short hair-pin RNA (shRNA) tar-geting TRIM25 (sense: 5’-TCGACGCGAATCTCT- CTTTGGCAAGTTCAAGAGACTTGCCAAAGAGAGA- TTCGTTTTTTGGAAT3’; antisense: 5’-CTAGATT- CCAAAAAACGAATCTCTCTTTGGCAAGTCTCTTG- AACTTGCCAAAGAGAGATTCGCG-3’) was initial- ly inserted into the Sal I and Xba I sites of vector plasmid, forming the vector-shTRIM25 plasmids.

Establishment of stable expression and knock -down cell lines

MDA-MB-231 cell was transfected with the ve- ctor, vector-shTRIM25 plasmid using the Lipo- fectamine 2000 according to the manufactur-er’s instructions (Invitrogen). Meanwhile, MCF-7 cell was transfected with the vector, vector-TRIM25 plasmid similarly use Lipofectamine 2000. Stable transfectants were obtained after

selection by puromycin (Invitrogen; 10 μg/mL)

for 2 weeks. Expression of TRIM25 mRNA and protein in stable cell lines were analyzed by RT-PCR and Western blot, respectively.

Cell invasion and motility assay

Invasion of cells was measured in Matrigel (BD,

Franklin Lakes, NJ, USA) -coated Transwell in-serts (6.5 mm, Costar, Manassas, VA, USA) con-taining polycarbonate filters with 8-μm pores

as detailed previously [12]. The inserts were

coated with 50 μl of 1 mg/ml Matrigel matrix

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recommenda-tions. 2×105 Cells in 200 μl of serum-free me-dium were plated in the upper chamber, where-as 600 μl of medium with 10% fatal bovine

serum were added to lower well. After 24 hrs incubation, top cells were removed and bot- tom cells were counted. Cells that migrated to

the lower surface of the membrane were

fix-ed in 4% paraformaldehyde and stainfix-ed with

night at 4°C. After three washes in PBS, the slides were incubated for 1 hour in the dark with FITC-conjugated secondary antibodies (In-

vitrogen, Grand Island, NY, USA). After three

[image:3.612.91.419.76.574.2]

further washes, the slides were stained with DAPI for 5 min to visualize the nuclei, and ex- amined using a Carl Zeiss confocal imaging system (LSM 780) (Carl Zeiss, Jena, Germany).

Figure 1. TRIM25 is high level expressed in human breast cancer samples. A. TRIM25 protein expres-sion was analyzed by immunohistochemistry in 62 breast cancer tissues and 50 normal breast tissues. B. Immunohistochemical results were summarized by statistical software. P<0.001 is based on the Stu-dent t test. Error bars, SD.

Figure 2. Survival rate and TRIM25 expression is in a negative correlation through follow-up visit. Survival analysis of patients with breast cancers was based on the Kaplan-Meier survival analysis.

0.5% crystal violet. For each

membrane, five random fields were counted at ×10 magnifi -cation. The mean was calcu-lated from three independ- ent experiments done in tri- plicate. Motility assays were similar to Matrigel invasion assay except that the Trans- well insert was not coated with Matrigel.

Immunofluorescence staining

Cell lines were plated on cul-ture slides (Costar, Manassas,

VA, USA). After 24 hrs, the

cells were rinsed with PBS

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over-Statistical analysis

Data was described as the mean ± s.d. Comparisons between different groups were undertaken using the Student’s two-tailed t-test. The survival probability was estimated by Kaplan-Meier method, and the comparison of survival curves between groups was done

with the log-rank test. The statistical signifi -cance of the differences between mean va- lues was determined by P<0.05. Statistical analysis was done with SPSS11.0 (SPSS, Inc.,

Chicago, Illinois, USA).

Results

TRIM25 is high level expressed in human breast cancer samples

The protein levels of TRIM25 in the 50 nor- mal breast samples, 62 breast cancer sam- ples (Figure 1A) were analyzed by immuno- histochemistry staining. The data was also analyzed by statistical software (Figure 1B). To investigate the relationship between breast cancer patients’ survival time and their re- spective TRIM25 protein expression, we make a long-term follow-up on 631 patients suffer- ing breast cancer. The correlation of one’s survival time and TRIM25 protein expression

was showed as a curve graph. The survival curves in Figure 2 indicated that the pa- tients with higher TRIM25 level in liver cancer tissues had shorter living length than those with lower TRIM25 expression level, suggest- ing there was a negative correlation between TRIM25 and long survival.

Furthermore, the protein levels of TRIM25 in 8 non-distant metastasis breast cancer sam-ples and 8 distant metastasis breast cancer samples were analyzed by western-blot (Figure 3A) and the data was also analyzed by sta- tistical software (Figure 3B). The mRNA le- vels of TRIM25 in 8 non-distant metastasis breast cancer samples and 8 distant metas- tasis breast cancer samples were analyzed by semiquantitative RT-PCR; the results were showed in the form of statistical analysis (Figure 3C). Compared with normal breast cells,

TRIM25 showed significant high level expres -sion in breast cancer cells, especially in dis- tant metastasized breast cancer cells.

Stable cell lines of down-regulated and up-reg -ulated expression of TRIM25 are constructed

[image:4.612.95.519.80.209.2]

TRIM25 protein expression in six different bre- ast cancer cell lines (MCF10A, MDA-MB-468, BT549, MDA-MB-231, HCC1428, MCF7, and

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SK-BR-3) was detected by western blot assay (Figure 4A). The expression of TRIM25 was knockdown by shRNAs in MDA-MB-231 cells and the TRIM25 expression in

MDA-MB-231-shTRIM25s cell line constructed was verified by

western blot (Figure 4B). TRIM25 mRNA’s low expression in MDA-MB-231- shTRIM25s cell

line constructed was verified by quantitative

RT-PCR (Figure 4C). The expression of TRIM25 was up-regulated in MCF7 cells and the TRIM25 expression in MCF7-TRIM25 cell line

construct-ed was verificonstruct-ed by western blot assay (Figure 4D). TRIM25 mRNA’s high expression in

MCF7-TRIM25 cell line constructed was verified by

quantitative RT-PCR assay (Figure 4E).

increase in cell migratory activity than its con-trol cells, i.e. MCF7-vector, (Figure 6A). Mean- while, the statistical column diagram showed more intuitively (Figure 6B). As showed in Matrigel invasion assay, MCF7-TRIM25 cells exhibited more ability of invasion than which of control cells (Figure 6C). At the same time, the statistical column diagram showed us in- tuitively (Figure 6D).

Knockdown of TRIM25 inhibits epithelial-mes -enchymal transitions

Microscope was used to intuitively detect EMT progression between MDA-MB-231-shTRIM25

Figure 4. Stable cell lines of down-regulated and up-regulated expression of TRIM25 are constructed. A. TRIM25 protein expression in seven differ-ent breast cancer cell lines was detected by western blot assay. B. TRIM25 protein’s low expression in MDA-MB-231-shTRIM25 cell line constructed was verified by western blot assay compared with the control one. C. TRIM25 mRNA’s low expression in MDA-MB-231-shTRIM25 cell line constructed was verified by quantitative RT-PCR assay compared with the control one. D. TRIM25 protein’s high expression in MCF-7-TRIM25 cell line constructed was verified by western blot assay compared with the control one. E. TRIM25 mRNA’s high expression in MCF-7-TRIM25 cell line constructed was verified by quantitative RT-PCR assay compared with the control one. P<0.001 is based on the Student t test. Error bars, SD.

Knockdown of TRIM25 in -hibits migratory and invasive capacities of breast cancer cells

To determine whether

knock-down of TRIM25 would influ -ence cell migratory and inva-sive capacities, Transwell mi- gration and Matrigel invasion assays were performed us- ing the established MDA-MB- 231 and MCF-7 stable trans-fectants. The results showed that MDA-MB-231-shTRIM25

cells exhibited more signifi -cant decrease in cell migra- tory activity than its control cells, i.e. MDA-MB-231-vector, (Figure 5A). And the statisti-cal column diagram showed more clearly (Figure 5B). As showed in Matrigel invasion assay, MDA-MB-231-shTRIM- 25 cells exhibited less ability of invasion than which of con-trol cells (Figure 5C). At the same time, the statistical column diagram showed us clearly (Figure 5D).

Over-expression of TRIM25 promotes migratory and in -vasive capacities of breast cancer cells

MCF7-TRIM25, which expre- ssed over-expression TRIM25 protein, with the same as-

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cell line and its control one (Figure 7A). The results showed that EMT progression trend-

ed to be less significant in

MDA-MB-231-shTRIM25 cell line than which of its control one.

[image:6.612.91.379.67.545.2]

25 cell line than which of its control one, while N-cadherin was expressed more in MCF-7-TRIM25 cell line than which of its control one (Figure 8B). Western blot assay was applied to

Figure 5. Knockdown of TRIM25 inhibits breast cancer cells’ migration and invasion in vitro. A. MDA-MB-231-shTRIM25 and its control cells were sub-jected to Transwell migration. B. MDA-MB-231-shTRIM25 and its control cells were subjected to Transwell migration, quantification of migrated cells through the membrane are shown as proportions of their vector controls in a column diagram. C. MDA-MB-231-shTRIM25 and its control cells were sub-jected to Matrigel invasion assay. D. MDA-MB-231-shTRIM25 and its control cells were subjected to Matrigel invasion assay, quantification of invaded cells through Matrigel are shown as proportions of their vector controls in a column diagram. P<0.001 is based on the Student t test. Error bars, SD.

Immunofluorescence method

was also used to detect the expression of E-cadherin and N-cadherin relevant to EMT, between MDA-MB-231-shTR- IM25 cell line and its con- trol one (Figure 7A). E-cad- herin was expressed more in MDA-MB-231-shTRIM25 cell line than which of its con- trol one, while N-cadherin was expressed less in MDA-MB-231-shTRIM25 cell line than which of its control one. Western blot assay was ap- plied to detect expression of

four proteins (E-cadherin,

α-cadherin, N-catenin and Vi- mentin) which are related to EMT between MDA-MB-231-shTRIM25 cell line and its control one. Knockdown of TRIM25 up-regulated the ex- pression of E-cadherin and

α-cadherin, while it down-reg -ulated the expression of N- cadherin and Vimentin com-pared with the control one (Figure 7B).

Over-expression of TRIM25 promotes epithelial-mesen -chymal transitions

Microscope was used to in- tuitively detect EMT progres-sion between MCF-7-TRIM25 cell line and its control one (Figure 8A). The results sh- owed that EMT progression

trended to be more

signifi-cant in MCF-7-TRIM25 cell line than which of its control one.

Immunofluorescence method

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detect expression of four proteins (E-cadherin,

α-catenin, N-cadherin and Vimentin) which are

related to EMT, between MCF-7-TRIM25 cell

[image:7.612.92.376.72.283.2]

was down-regulated after ZEB-1 was interfered in MCF-7-TRIM25 cell lines (Figure 10A). The expression of proteins which is related to EMT

Figure 6. Over-expression of TRIM25 promotes breast cancer cells’ migration and invasion in vitro. A. MCF-7-TRIM25 and its control cells were subjected to Transwell migration. B. MCF-7-TRIM25 and its control cells were subjected to Transwell migration, quantification of migrated cells through the membrane are shown as proportions of their vector controls in a column diagram. C.

MCF-7-TRIM25 and its control cells were subjected to Matrigel invasion as-say. D. MCF-7-TRIM25 and its control cells were subjected to Matrigel inva-sion assay, quantification of invaded cells through Matrigel are shown as proportions of their vector controls in a column diagram. P<0.001 is based on the Student t test. Error bars, SD.

Figure 7. Knockdown of TRIM25 inhibits epithelial-mesenchymal transitions (EMT) in vitro. A. Microscope was used to intuitively detect EMT progression between MDA-MB-231-shTRIM25 #3 cell line and its control one. Immuno-fluorescence method was used to detect the expression of E-cadherin and N-cadherin relevant to EMT, between MDA-MB-231-shTRIM25 #3 cell line and its control one. B. Western blot assay was applied to detect expression of four proteins which are related to EMT between MDA-MB-231-shTRIM25 #3 cell line and its control one.

line and its control one. Over-expression of TRIM25 down-regulated the expression of

E-cadherin and α-cadherin,

while it up-regulated the ex- pression of N-cadherin and Vimentin compared with the control one (Figure 8B). TRIM25 can regulate the ex -pression of ZEB-1

To better characterize the mechanisms by which TRIM- 25 engaged in the develop-ment and progression of bre- ast cancer cell, western blot assay was performed using cell line MDA-MB-231-sh- TRIM25, MCF-7-TRIM25 and their control cells with the empty vector plasmids re- spectively. Western blot re- sults indicated that a list of

proteins significantly

differ-entially expressed after TR- IM25 down-regulated expres-sion (Figure 9A) or up-regu- lated expression (Figure 9B). It was found that at the time when TRIM25 was low level expressed, the expres-sion of ZEB-1 was reduced compared with the control situation (Figure 9A). Corre- spondingly, ZEB-1’s expres-sion in protein level was high-er than that of the TRIM25 overexpression cells (Figure 9B). These results suggest- ed that TRIM25 regulates breast cancer’s functions and which may be mediated by ZEB-1. To further determine the relationship between TR- IM25 and ZEB-1, we use Snail protein’s expression as an indirect indicator to detect

the TRIM25’s influence on

[image:7.612.91.376.417.577.2]
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progression was down regulated in MCF-7-TRIM25 cell line after interfering ZEB-1 (Figure 10B). Interference of ZEB-1 inhibited migra- tion and invasion in MCF-7-TRIM25 cell lines (Figure 10C).

Discussion

In this study, the role of TRIM25 in breast can-cer was characterized. Increased levels of

[image:8.612.92.377.75.232.2]

lation and control of mitotic cycle and tumor cell’s drug resistance [15]. We detected the protein expression of TRIM25 in breast cancer, and then investigated the relationships be- tween TRIM25 expression and ZEB-1 expres-sion. Our results showed that TRIM25 was high-er expressed in breast canchigh-er tissues than in normal breast tissues, and high-expressed in a majority of the high-grade breast cancer tis-sues examined.

Figure 8. Over-expression of TRIM25 promotes epithelial-mesenchymal

transitions (EMT) in vitro. A. Microscope was used to intuitively detect EMT progression between MCF-7-TRIM25 cell line and its control one. Immuno-fluorescence method was used to detect the expression of E-cadherin and N-cadherin relevant to EMT, between MCF-7-TRIM25 cell line and its control one. B. Western blot assay was applied to detect expression of four proteins which are related to EMT between MCF-7-TRIM25 cell line and its control one.

Figure 9. TRIM25 regulates the expression of ZEB-1. A. A list of signal pro-teins’ expression was detected by western blot assay between MDA-MB-231-shTRIM25 #3 cell line and its control one. B. A list of signal proteins’ expression was detected by western blot assay between MCF-7-TRIM25 cell line and its control one.

TRIM25 mRNA and protein were detected in a majority of the breast cancer tissues examined as compared to the normal breast tissues. Knockdown of TRIM25 was found to inhibit cell migration and invasion as well as to decelerate the EMT progres-sion. We also found that over-expression of TRIM25 in

breast cancer cells signifi -cantly promotes tumor cell’s migration, invasion and EMT progression. The results ana-lyzed from Western blot as- say showed that TRIM25 af- fect the expression of ZEB-1 in breast cancer cells.

So far, there have been more than 80 members of TRIM family protein found in hu- man genome [13]. TRIM pro-teins contain three conser- ved motif in the N terminus, which are RING domain, B- box domain and coiled-coil domain while the C-terminal domain is variable. Based on the existence of the con-served RING domain, many TRIM family members have been reported to function as an E3 ligase in the ubiquitin

modification [14].

TRIM25 protein, also known

as

estrogen-responsive-finger-protein (EFP), is located at human’s 17q23 chromosome [15]. As a kind of E3 ligase

in the ubiquitin modification,

[image:8.612.95.375.344.532.2]
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regu-Secondly, we found that knockdown of TRIM25 by shRNA inhibited breast cancer cell’s ability of migration, invasion and EMT. While over-expression of TRIM25 produced an encourag-ing effect on the same functions detected. Epithelial-mesenchymal transition (EMT) hap-pens gradually in the process of tumor develop-ment and is extremely complex. As a feature of aggressive tumors, epithelial to mesenchymal transition is characterized by reduced E-

cadherin, α-catenin and increased N-cadherin,

vimentin expression [3]. E-cadherin plays an important role in sustaining completeness and polarity of epithelial cell’s structure and shape. As has been shown in many researches, down-regulated of E-cadherin protein expression is in a close relationship with epithelial cell [16]. Meanwhile, cells that EMT progression occurred usually accompanied with up-regulated expres-sion of protein originated from mesenchymal cells, especially vimentin [3]. Contrast with E-cadherin, N-cadherin protein’s up-regulated

gene promoters through the zinc finger domains

to repress genes transcription. This signaling has emerged in recent years as an attractive target for anticancer therapy because its aber-rant activation is implicated in several cancers [3, 17]. In cell lines whose TRIM25 was knock-down, the expression level of ZEB-1 decreased

significantly compared with the control empty

vector plasmid cells. Over-expression of TRIM25 can increase the expression of ZEB-1

protein. It has been verified that Snail is

upstream of ZEB-1. Via detecting Snail expres-sion in MCF-7-TRIM25 cell line where ZEB-1

was interfered, it was confirmed that TRIM25

regulates oncogenic activities through ZEB-1. In conclusion, we have demonstrated that TRIM25 is high level expressed in breast can-cer tissues, and the knockdown of TRIM25 inhibited breast cancer cells’ ability of

[image:9.612.92.377.73.336.2]

migra-tion, invasion and EMT. Therefore, these find -ings may shed light to potential targets in breast cancer prevention and therapy.

Figure 10. TRIM25 regulates breast cancer cells’ function through modifying ZEB-1. A. Snail protein expression was down regulated in MCF-7-TRIM25 cell line after interfering ZEB-1 through western blot assay. B. The expression of proteins which is related to EMT progression was down regulated in MCF-7-TRIM25 cell line after interfering ZEB-1 through western blot assay. C. Inter-ference of ZEB-1 inhibited migration and invasion in MCF-7-TRIM25 cell lines (left) and the results from Transwell migration and Matrigel invasion assays of MCF-7-TRIM25, whose ZEB-1 was interfered were summarized by statisti-cal software (right). P<0.001 is based on the Student t test. Error bars, SD.

expression was found to be

in a positive of EMT.

α-cate-nin can modify cell adhesion activity, whose down-regula-tion could lead to E-cadherin’s deprivation of function, and the contact inhibition among epithelial cells immediately

followed [3]. Thus, α-catenin

protein’s down-regulated ex- pression is in a positive cor- relation with EMT progre- ssion.

To investigate the mechan- ism of TRIM25 modifying the development of cancer, west-ern blot assay was carried

out. We identified zinc finger

E-box binding 1 (ZEB-1) as an effective mediator of these TRIM25 induced phenomena. ZEB-l, as one member of ZEB

family is a zinc finger home -odomain transcription factor in vertebrates [3]. The ZEB family has the same struc-ture: one C2H2 zinc finger

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Acknowledgements

This work was supported by National Science Foundation of China Grant no. 81402193 and the China Postdoctoral International Exchange Program 2015.

Disclosure of conflict of interest None.

Address correspondence to: Min Tao, Department of Oncology, First Affiliated Hospital of Soochow University 188 Shizi Road, Suzhou 215006, Jiang- su, China. Tel: 86-512 6522 3637; Fax: 86-512 6522 3637; E-mail: zysdoctor@126.com

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[16] Wang Y, Ma G, Wang Q, Wen M, Xu Y, He X, Zhang P, Wang Y, Yang T, Zhan P and Wei G. Involvement of CUL4A in regulation of multi-drug resistance to P-gp substrate multi-drugs in breast cancer cells. Molecules 2013; 19: 159-176.

Figure

Figure 1. TRIM25 is high level expressed in human breast cancer samples. A. TRIM25 protein expres-sion was analyzed by immunohistochemistry in 62 breast cancer tissues and 50 normal breast tissues
Figure 3. TRIM25 is high level expressed in distant metasta-sis breast cancer tissues
Figure 5. Knockdown of TRIM25 inhibits breast cancer cells’ migration and invasion in vitro
Figure 7. Knockdown of TRIM25 inhibits epithelial-mesenchymal transitions fluorescence method was used to detect the expression of E-cadherin and (EMT) in vitro
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References

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