WISP2/CCN5 gene knockdown in vitro and in vivo exhibits proliferation promotion of breast cancer through targeting Skp2 and p27Kip1

Background Emerging evidence has demonstrated that WISP2/CCN5 is critically involved in tumorigenesis. However, the function of WISP2/CCN5 in breast cancer carcinogenesis is largely unclear. Methods we aim to explore the effects and potential mechanisms of WISP2/CCN5 on proliferation of breast cancer cells and carcinogenesis of breast cancer xenograft. Lentivirus vector with WISP2/CCN5shRNA was transfected into MCF-7, and breast cancer cells and xenograft were conducted. Effect of WISP2/CCN5 on growth and carcinogenesis of breast cancer cells and xenografts was evaluated by MTT assay and tumor volume. The relationship between WISP2/CCN5, Skp2 and p27Kip1 was detected in vitro and in vivo by RT-PCR at mRNA level and Western blotting at protein level. Results The result of MTT assay indicated that MCF-7 cell growth viability in WISP2/CCN5 gene knockdown group was significantly higher than negative vector group(P<0.05) or control group (P<0.05). It suggested that knockdown of WISP2/CCN5 gene by shRNA lentivirus plasmid promoted proliferation of MCF-7 cells. The growth curves of breast cancer xenograft showed that xenografts in WISP2/CCN5 knockdown group grew more quickly than negative vector group(P< 0.05) or control group (P< 0.05). Subsequently, the results of RT-PCR and Western blotting revealed that WISP2/CCN5 gene knockdown led to increased Skp2 and decreased p27Kip1 at mRNA and protein levels. WISP2/CCN5 exerts its inhibition on proliferation of MCF-7 cell line and suppressive functions on growth of breast carcinoma via regulation of Skp2 and p27Kip1at mRNA and protein levels. However, WISP2/CCN5 gene knockdown resulted in loss of inhibition effect on MCF-7 and breast cancer. Conclusions Our findings suggest that WISP2/CCN5 could be a useful therapeutic strategy for the treatment of breast cancer through targeting Skp2 and p27Kip1.


Introduction
Breast cancer is one of leading causes of cancer-related death in women worldwide. To explore the mechanism of occurrence and progression of breast cancer, to accurately treat breast cancer is of great significance.
Recently, important roles of CCN5/WISP2 proteins have been recognized in breast cancer progression [1] . WISP-2 (Wnt-1 induced secreted proteins -2, Wnt secretory protein 2) plays an important role in inducing cell apoptosis, inhibiting cell adhesion and metastasis [2] . WISP2/CCN5 was reported to be upregulated in the mammary epithelial cells transformed by the Wnt-1 oncogene [3] . WISP2/CCN5 was found to be directly regulated by the estrogen receptor in human breast cancer cells [4] . One study further identified that WISP2/CCN5 expression was enhanced by serum and correlated with serum-induced cell proliferation in breast cancer cells, demonstrating that WISP2/CCN5 could enhance cell proliferation in breast cancer [5] .
The F-box protein Skp2, a component of the Skp1-Cullin 1-F-box (SCF) E3 ubiquitin-ligase complex, has been shown to regulate cellular proliferation, carcinoma progression and metastasis by targeting several cell cycle regulators for ubiquitinationr [6] . P27 KIP1 is a negative regulator of cell cycle that plays an important role in tumor suppression. Reduction of p27 KIP1 secondary to enhanced Skp2-mediated degradation is involved in atypical hyperplasia and tumorigenesis. [7] .
In present study, we intend to find out the relationship between WISP2/CCN5 gene knockdown and proliferation and progression of breast cancer in vitro and in vivo, and those underlying molecular mechanisms.

Cell line and cell culture
Human breast cancer cell line MCF-7 was purchased from KGI biotechnology co., LTD, Jiang Su, China and cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, USA) containing 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin-streptomycin solution (Gibco, USA) in a incubator at 37°C and a humidified 5% CO 2 atmosphere. MCF-7 passage cell line were prepared using 0.05% trypsin solution (Invitrogen, Carlsbad) and seeded in 96 well tissue culture plates.  weeks, mice were executed, and tumors were taken, the tumor volume were determined.

Xenografts volume measurement
Tumor size was measured daily using a caliper, and the tumor volume was determined with the standard formula: L× W 2 ×0.5 2 , where L is the longest diameter and W is the shortest diameter. The growth curve of xenograft delineated the tendency of WISP2/CCN5 gene knockdown xenograft transformation during 14 days.

Quantitative RT-PCR analysis
In order to find out the mechanism of WISP2/CCN5 regulating MCF-7 proliferation in mRNA level, quantitative RT-PCR was performed to explore mRNA level of WISP2/CCN5, Skp2 and p27Kip1in both MCF-7cells and xenografts.

Western blot analysis and antibodies
Western blotting was performed according to the established methods.
The breast tumor tissues were cut into small pieces. Cells were rinsed twice with ice-cold phosphate buffered saline (PBS, pH7.4), lysed in a cooled buffer. Subsequently, PMSF was added into xenograft tissues homogenate or cells, and pulverized. Then the lysates were boiled in a water bath for 10 minutes, and centrifuged at 12,000 g for 10 minutes at

Statistical analysis
The statistical analysis was performed using the Graph Pad Prism 4 (Graph Pad Software, Inc, La Jolla, CA, USA) and PASS 15.0 software.
Results are shown as mean ± S.D. Means between the groups were calculated and compared among or within variants using a two-sided Student's t-test. Pearson Correlation was used to analyze the correlation between WISP2/CCN5 and Skp2 and p27Kip1. P value of <0.05 was considered statistically significant. Consequently, western blot stripe of Skp2 displayed darker and wider in WISP2/CCN5 gene knockdown group than negative vector group and control group, while lighter and thinner stripe of p27Kip1 was showed in WISP2/CCN5 gene knockdown ( Figure 5A).

Knockdown of
The results of correlation analysis between WISP2/CCN5 and Skp2 and p27Kip1 were showed in Table 1 and Table 2. There was significant correlation between WISP2/CCN5 and Skp2 and p27Kip1 at mRNA and protein levels in three groups, including WISP2/CCN5 gene knockdown group, negative vector group and control group. Especially, correlation in WISP2/CCN5 gene knockdown group was remarkable between WISP2/CCN5 and Skp2 and p27Kip1 at mRNA and protein levels in WISP2/CCN5 gene knockdown group.

Knockdown of WISP2/CCN5 gene resulted in abnormal level in mRNA and protein of Skp2 and p27Kip1 in vivo.
In the animal experiment, knockdown of WISP2/CCN5 gene elevated the  Figure 5D). Similar changes were observed in western blot stripes ( Figure 5C). Skp2 protein blot appeared darker stripe in WISP2/CCN5 gene knockdown group than negative vector group and control group. However, p27Kip1 protein blot presented lighter stripe in WISP2/CCN5 gene knockdown group.
The results of correlation analysis between WISP2/CCN5 and Skp2 and p27Kip1 were showed in Table 3 and Table 4. There was significant correlation between WISP2/CCN5 and Skp2 and p27Kip1 at mRNA and protein levels in three groups, including WISP2/CCN5 gene knockdown group, negative vector group and control group. Especially, correlation in WISP2/CCN5 gene knockdown group was remarkable between WISP2/CCN5 and Skp2 and p27Kip1 at mRNA and protein levels in WISP2/CCN5 gene knockdown group.

Discussion
Up to now, the role of WISP2 in ESCC is unelucidated, although the function of WISP2/CCN5 was explored in a variety of human cancers S-phase kinase-associated protein 2 (SKP2) is an oncogene and cell cycle regulator that specifically recognizes phosphorylated cell cycle regulator proteins and mediates their ubiquitination, which is overexpressed in lymphoma [14] ,prostate cancer [15] ,melanoma [16] , nasopharyngeal carcinoma [17] , breast cancer [18] . P27Kip1, as one of the best known protein substrates of Skp2, is the cyclin-dependent kinase (CDK) inhibitor 1B (CDKN1B) [15] . Moreover, over expression of Skp2 and low expression of p27Kip1 are strongly associated with aggressive tumor behavior and poor clinical outcomes in a variety of cancers [16][17][18] . WISP2/CCN5 has been reported to govern several gene expressions in human cancer cells. For instance, deficiency of WISP2/CCN5 promotes mesenchymal to epithelial transition (MET) [21] . Moreover, MET is required for the growth of micrometastatic breast cancer [22] . Our study reported that WISP2/CCN5 gene knockdown may promote the proliferation of breast cancer cells and development of breast carcinoma. It was ever reported that loss of WISP2/CCN5 in MCF-7 breast cancer cells can also promote the emergence of a cancer stem-like cell phenotype characterized by high expression of CD44 [23] . That demonstrated that loss of WISP2/CCN5 play an important role in tumorigenesis, in another world WISP2/CCN5 inhibit the carcinogenensis.
Simultaneously, some researchers also hold the same opinion as us that loss of WISP2/CCN5 protein in MCF-7 promotes a stem-like cell, that means deficiency of WISP2/CCN5 have closed relationship with monoclonal proliferation of breast cancer cells [24][25][26] . Consequently, the role of WISP2/CCN5 in development of breast cancer has received widespread attention [27][28][29] .
Finally, we believed that underlying mechanisms of MCF-7 cells proliferation and tumorigenesis may be results of decreased WISP2/CCN5 through aberrant levels of Skp2 and p27Kip1. There was ever research reported that induced activation of CCN5 in triple-negative breast cancer (TNBC) cells promotes cell growth arrest at the G0/G1 phase, reduces cell proliferation and delays tumor growth in the xenograft model [30] , whereas our research showed that the regulation between WISP-2/CCN5 and Skp2 with p27Kip1 in MCF-7 cell line with ER positive.
We highlighted that WISP2/CCN5 may be a promising therapeutic target and development of WISP2/CCN5 promoter would have a great impact on breast cancer therapy. The relationship between deficiency of WISP2/CCN5 and estrogen dependent MCF-7 has been explored because WISP2/CCN5 is an estrogen response gene in ER-α-positive breast cancer cells, such as MCF-7 cell line, and present studies showed that the estradiol-treatment is able to activate CCN5 transcription [31][32][33] . Therefore, correlation between WISP2/CCN5 and ER, PR and Her-2 still need to be detected in breast cancer.