Cyclin-dependent kinase 11p110 (CDK11p110) is crucial for human breast cancer cell proliferation and growth

Cyclin-dependent kinases (CDKs) play important roles in the development of many types of cancers by binding with their paired cyclins. However, the function of CDK11 larger protein isomer, CDK11p110, in the tumorigenesis of human breast cancer remains unclear. In the present study, we explored the effects and molecular mechanisms of CDK11p110 in the proliferation and growth of breast cancer cells by determining the expression of CDK11p110 in breast tumor tissues and examining the phenotypic changes of breast cancer cells after CDK11p110 knockdown. We found that CDK11p110 was highly expressed in breast tumor tissues and cell lines. Tissue microarray analysis showed that elevated CDK11p110 expression in breast cancer tissues significantly correlated with poor differentiation, and was also associated with advanced TNM stage and poor clinical prognosis for breast cancer patients. In vitro knockdown of CDK11p110 by siRNA significantly inhibited cell growth and migration, and dramatically induced apoptosis in breast cancer cells. Flow cytometry demonstrated that cells were markedly arrested in G1 phase of the cell cycle after CDK11p110 downregulation. These findings suggest that CDK11p110 is critical for the proliferation and growth of breast cancer cells, which highlights CDK11p110 may be a promising therapeutic target for the treatment of breast cancer.

progression and are constitutively expressed throughout the cell cycle 13 . CDKs are heterodimeric complexes composed of a catalytic kinase subunit and a regulatory cyclin subunit, regulated by their association with cyclins and endogenous inhibitors, as well as by positive phosphorylation and negative phosphorylation events 14 . In malignant cells, altered expression of CDKs and their modulators, including overexpression of cyclins and loss of expression of CDK inhibitors, result in deregulated CDK activity, providing a selective growth advantage. CDKs are often overexpressed and/or overactive in human cancers owing to various genetic and epigenetic events that affect their regulatory pathways, bringing about loss of checkpoint integrity, and ultimately resulting in uncontrolled cell proliferation [15][16][17][18][19] . Because of the critical roles in cell cycle progression, as well as the association of their activities with apoptotic pathways, CDKs and their associated pathways represent some of the most attractive targets for the development of anticancer therapeutics. CDK11, formerly known as PITSLRE, is encoded by two highly homologous p34cdc2-related genes, CDC2L1 and CDC2L2 (Cell Division Control 2 Like) in humans. These two genes are localized in a genomic region that spans about 140 kb on human chromosome 1 band p36.3 20 . There is only one CDK11 gene, CDC2L1 in mouse. CDK11 involves three major isoforms, CDK11 p110 , CDK11 p58 , and CDK11 p46 , respectively 21 . The larger CDK11 p110 protein kinase isoform is expressed in all human cancer cell lines examined so far, including the cell lines U-2OS, KHOS, Saos, Jurkat, Cem C7, HeLa, HEK 293, K562, HFF, and RNE 21,22 . The CDK11 p58 protein is specifically translated from an internal ribosome entry site and expressed only in the G2/M phase of the cell cycle 23 . CDK11 p58 detection depends primarily on the mitotic characteristics of a particular cell type. Although CDK11 p58 shares the same sequences including the kinase domain as the C terminus of CDK11 p110 , the two isoforms possess different functions. CDK11 p58 is closely related to cell cycle arrest and apoptosis in a kinase-dependent manner [24][25][26] . For human breast cancer, CDK11 p58 has been identified as a negative regulator in the oncogenesis 27,28 . While the larger CDK11 p110 isoform is mainly associated with transcription and RNA processes. Recently, CDK11 p110 has been found to be critical for mesenchymal tissue-originated osteosarcoma cell growth and proliferation by a comprehensive human kinome-wide shRNA screening 22 . Moreover, similar effects of CDK11 p110 on tumor cells have been confirmed in liposarcoma, which also arises from mesenchymal tissues 29 . However, the functional roles and molecular mechanisms of CDK11 p110 in human breast cancer cell proliferation and growth are unknown.
In the present study, we explore the roles of CDK11 p110 in the proliferation and survival of epithelial tissue-derived human breast cancer cells. Firstly, we detected CDK11 p110 expression in a tissue microarray of human breast tumor samples and analyzed its correlation with the clinical characteristics of the patients. Additionally, we knocked down CDK11 p110 expression with chemically synthetic small interfering RNA (siRNA) and examined the changes in human breast cancer cell proliferation, migration, apoptosis, and cell cycle. Our data show that CDK11 p110 is highly expressed in human breast tumor cells, which correlates with poor prognosis for breast cancer patients. RNAi-mediated knockdown of CDK11 p110 inhibits human breast cancer cell survival and proliferation in vitro. These results indicate that CDK11 p110 plays an important role in the proliferation and growth of human breast cancer cells, and therefore warrants further evaluation as a therapeutic target of breast cancer.

Results
CDK11 p110 is highly expressed in human breast cancer tissues and cell lines. To explore the potential roles of CDK11 p110 in human breast cancer cell proliferation and growth, we first determined the expression of CDK11 p110 in human breast tissues and cell lines. As demonstrated by Western blot, CDK11 p110 was highly expressed in each of the tested human breast tumor tissues compared with the adjacent normal tissues (P < 0.01) (Fig. 1A, Supplementary Figure S1). Additionally, all four human breast cancer cell lines, with diverse histological staining characteristics (Supplementary Table S1), exhibited high levels of CDK11 p110 expression, especially in BT-474, MCF-7, and MDA-MB-468 cells, whereas the expression of CDK11 p110 was tightly regulated in the normal breast cell line (Fig. 1B). To further confirm the expression of CDK11 p110 and determine its subcellular localization in breast cancer cell lines, immunofluorescence was performed in MCF-7 and MDA-MB-468 cells. As shown in Fig. 1C, CDK11 p110 protein was mainly localized in the nucleus of breast cancer cells with some expression in the cytoplasm. For validation of CDK11 p110 expression, we also determined CDK11 p110 expression with a different commercially available CDK11 p110 antibody from Cell Signaling Technology (CST, MA, USA catalog No: #5524) in the breast cancer tissues and adjacent normal tissues. Similar results of CDK11 p110 expression were found in these samples (Supplementary Figure S1A). CDK11 p110 expression levels correlate with the clinicopathological characteristics of breast cancer patients. To further validate the clinical significance of CDK11 p110 expression in patients with breast cancer, we detected CDK11 p110 levels in a breast tumor tissue microarray by immunohistochemistry, and evaluated the correlation of CDK11 p110 expression to the pathological characteristics and clinical prognosis of breast cancer patients. As illustrated in Fig. 2A, CDK11 p110 expression levels were significantly higher in breast tumor tissues than that in their adjacent nontumorous tissues (P = 0.0041). Furthermore, elevated CDK11 p110 expression in breast tumor tissues correlated with poor differentiation of tumor (P = 0.0153) (Fig. 2B). Additionally, CDK11 p110 expression was upregulated in breast tumor tissues in advanced (III) TNM stage compared with that in primary (II) TNM stage, although there was no significant difference (P = 0.3866) (Fig. 2C). Based on data from over 72 months of follow-up, CDK11 p110 expression levels in samples from nonsurvivors were higher than that from survivors, and more importantly, Kaplan-Meier survival analysis showed that the outcome for patients in the CDK11 p110 high-staining (≥ 4) group was worse than for those in the CDK11 p110 low-staining (≤ 3) group (Figs. 2D,E), although the difference was not statistically significant (P = 0.2270 and 0.2372, respectively).

CDK11 p110 knockdown inhibits human breast cancer cell proliferation in vitro.
To further evaluate the functional role of CDK11 p110 in human breast cancer cells proliferation in vitro, increasing concentrations of synthetic human CDK11 p110 siRNA were transfected into MCF-7 and MDA-MB-468 cells. 72 hours later, the morphological changes were observed using phase contrast microscopy. As shown in Fig. 3A,B, CDK11 p110 knockdown notably decreased the percentage of adherent cells, accompanied by dose-dependent death of transfected cells, which was not observed in the control or nonspecific siRNA transfected cells. Furthermore, the MTT assay also demonstrated that downregulation of CDK11 p110 inhibited breast cancer cell viability in a dose-dependent manner (Figs. 3C,D). Additionally, the expression of CDK11 p110 mRNA and protein in siRNA transfected cells was measured by RT-PCR and Western blot, respectively. As shown in Fig. 3,F, dose-dependent inhibition of CDK11 p110 mRNA and protein expression were observed, which demonstrates that the suppressed cell viability was associated with the knockdown of CDK11 p110 in breast cancer cells.
The role of CDK11 p110 expression on breast cancer cell proliferation was further evaluated by immunofluorescence. Consistent with the results of the MTT assay, after transfection with 40 nM of CDK11 p110 siRNA, MCF-7 and MDA-MB-468 cell viability decreased significantly, accompanied by reduced expression of CDK11 p110 protein (Figs. 3G,H). As expected, there was no significant change in the cell proliferation and CDK11 p110 expression in the breast cancer cells transfected with the same dose of nonspecific siRNA. The alteration of human breast cancer cell proliferation after CDK11 p110 knockdown was also assessed by a cell colony formation assay. As demonstrated in Fig, 3I-K, 40 nM of CDK11 p110 siRNA transfection significantly inhibited the colony formation efficiency in both MCF-7 and MDA-MB-468 cells (both P < 0.01), which was not observed in cells transfected with the same dose of nonspecific siRNA.

CDK11 p110 knockdown suppresses human breast cancer cell migration.
To investigate the effect of CDK11 p110 knockdown on human breast cancer cell migration ability, a wound healing assay was performed after CDK11 p110 siRNA transfection. Because 40 nM of CDK11 p110 siRNA transfection resulted in pronounced cell death, we choose a siRNA dose of 20 nM for the wound healing assay. As illustrated in Fig. 4, after 72 hours of incubation, the migration activities of both MCF-7 and especially of MDA-MB-468 cells were significantly repressed in CDK11 p110 siRNA transfected groups, as compared with nonspecific siRNA transfected and control groups (both P < 0.01).

CDK11 p110 knockdown induces cell apoptosis in human breast cancer cells.
To explore the underlying mechanisms that inhibit breast cancer cell proliferation by CDK11 p110 knockdown, we examined cell apoptosis using double independent experiments, including flow cytometry analysis and apoptosis-associated protein measurement. As demonstrated by flow cytometry analysis, increased apoptosis rates were observed in both MCF-7 and MDA-MB-468 cells transfected with 20 nM of CDK11 p110 siRNA for 72 hours (Figs. 5A-D) (both P < 0.01). Meanwhile, as shown by Western blot, 72 hours of depletion of CDK11 p110 by siRNA resulted in a dose-dependent decrease in the expression of anti-apoptotic proteins, including Survivin, Bcl-X L , cyclin D1, and CDK11 p110 -paired protein cyclin L1 in both MCF-7  date the potential mechanism underlying breast cancer cell apoptosis by CDK11 p110 knockdown, flow cytometry analysis was used to determine cell cycle phase distributions in human breast cancer cells after CDK11 p110 knockdown for 72 hours. As demonstrated in Fig. 6, after 20 nM of CDK11 p110 siRNA transfection, a significant G1 cell cycle arrest accompanied by reductions in the fraction of cells in S phase was observed in both MCF-7 and MDA-MB-468 cells (both P < 0.01), suggesting that CDK11 p110 knockdown was able to induce human breast cancer cell cycle arrest in G1 phase and inhibit DNA synthesis.

Discussion
Cancer is characterized by uncontrolled proliferation and aberrant division of mammalian cells. CDKs and their related pathways control the cell cycle progression by maintaining ordered exit and entry to the different phases of the cell cycle. Aberrant expression or altered activity of CDKs results in escape of cells from the cell cycle control and leads to malignant transformation 12,14,15,[30][31][32][33] . Therefore, inhibition of CDKs offers a promising therapeutic strategy in the defense against human malignancies.
The functions of CDK11 have been proved to be linked with the regulation of cell cycle, RNA transcription and processing, neuronal function, and apoptosis 21,34-37 . The potential for CDK11 to regulate these diverse cellular activities is unique in the CDK family and highlights that CDK11 may exert critical regulatory roles in human tumorigenesis, cancer cell growth and proliferation. These kinases have been renamed CDK11 p110 and CDK11 p58 when cyclins L1 and L2 were identified as regulatory subunits of CDK11 p110 . The smaller CDK11 p58 isoform is expressed specifically in G2/M via an internal ribosome entry site (IRES) located within the CDK11 p110 mRNA, and is closely related to cell cycle arrest and apoptosis upon binding to cyclin D3 partner 35 . In contrast, the larger CDK11 p110 isoform, which is ubiquitously express throughout the cell cycle, is mainly associated with transcription and RNA processes by interacting with its regulatory partner cyclin L 21,38-44 . Moreover, both CDK11 p110 and cyclin L1 proteins demonstrate increased expression in various cancers, and amplification of CCNL1 is associated with poor prognosis [45][46][47] . Recent research on mesenchymal tissue-originated tumors indicated that CDK11 p110 plays important roles in osteosarcoma and liposarcoma cell survival 22,29 . However, the biological functions of CDK11 p110 in human breast cancer cell growth and proliferation remain unclear. In the present study, we aimed to extend our study by investigating the roles of CDK11 p110 in human breast cancer   cell proliferation and growth. Our results demonstrated that CDK11 p110 was highly expressed in human breast tumor tissues, which was significantly associated with poor differentiation, and also correlated with advanced TNM stage and poor clinical prognosis for breast cancer patients. In vitro knockdown of CDK11 p110 expression by siRNA inhibited cell growth and migration, and induced apoptosis and cell cycle arrest in human breast cancer cells.
Firstly, we determined CDK11 p110 expression in human breast tumor tissues and adjacent tissues, as well as in a normal human breast cell line and four breast cancer cell lines by Western blotting. The results indicated that CDK11 p110 was highly expressed in human breast tumor tissues and cell lines. To further confirm the expression and subcellular localization of CDK11 p110 in human breast cancer cells, immunofluorescence was performed on MCF-7 and MDA-MB-468 cells. The morphological images showed that CDK11 p110 was highly expressed and mainly localized in the nucleus of both MCF-7 and MDA-MB-468 cells, with some cytoplasmic expression. To further validate the correlation of CDK11 p110 expression with the pathological characteristics and clinical prognosis in breast cancer patients, we analyzed CDK11 p110 protein levels by using a human breast tissue microarray. The results demonstrated that CDK11 p110 expression levels are significantly higher in breast tumor tissues than that in their adjacent nontumorous tissues, Furthermore, CDK11 p110 expression levels in breast tumor tissues positively correlated with the histological grade and clinical TNM stage of tumor. More importantly, CDK11 p110 expression levels in samples from nonsurvivors were higher than that from survivors, and the outcome for patients in the CDK11 p110 high-staining group was worse than for those in the CDK11 p110 low-staining group, although there was no statistical significance. These data are likely confounded by the small, heterogeneous sample size, and larger studies with similar breast cancer patient samples are required. Nonetheless, these results provide a rationale for further evaluation of CDK11 p110 as a marker for prognosis of breast cancer.
Recently, Chi and co-workers have evaluated the expression of a smaller CDK11 isoform, CDK11 p58 , in a breast tissue array by immunohistochemical staining 27 . The data showed that the disease-free survival (DFS) was significantly poorer in breast cancer patients with low CDK11 p58 expression. Although CDK11 p58 is structurally located within the C-terminal region of CDK11 p110 , their functions diverge distinctly due to their different crystal conformations 24 . For example, CDK11 p58 , in association with cyclin D3, was reported to negatively affect androgen receptor transcriptional activity, whereas CDK11 p110 positively affected the transcription activity of androgen receptor 24 . Furthermore, it has been reported that treatment of the Fas-activated T cells with a serine protease inhibitor prevented apoptotic death and led to the accumulation of CDK11 p110 isoform, but not the CDK11 p58 isoform 48 . These discrepancies highlight that CDK11 p110 and CDK11 p58 may exert opposite functions on cancer cell growth and proliferation via different molecular mechanisms, which deserves further investigation.
To further characterize the functional roles of CDK11 p110 in breast cancer cell survival and proliferation, we investigated the phenotypic alterations of MCF-7 and MDA-MB-468 cells after CDK11 p110 knockdown by using chemically synthetic siRNA. The results showed that CDK11 p110 knockdown inhibits human breast cancer cell proliferation in a dose-dependent manner accompanied by reduced expression of CDK11 p110 mRNA and protein. Interestingly, after exposure to the same dose of CDK11 p110 siRNA, MDA-MB-468 cell proliferation was repressed more significantly compared with MCF-7 cells, whereas the levels of downregulation of both CDK11 p110 mRNA and protein had no significant difference. This phenotypic diversity may result from the heterogeneous genetic and biochemical background between MCF-7 and MDA-MB-468 cells, which changes the downstream signal transduction of CDK11 p110 . For example, MCF-7 cells express wild-type p53, while p53 in MDA-MB-468 cells is mutated [49][50][51][52][53][54] . Similarly, the wound healing assay illustrated that the migration activities of both MCF-7 and MDA-MB-468 cells were suppressed after CDK11 p110 knockdown, with MDA-MB-468 cell migration more significantly inhibited. This altered phenotypic change may be caused by the intrinsic diversity in migration activity between these two breast cancer cell lines. Notably, MDA-MB-468 cells without CDK11 p110 siRNA transfection migrated so quickly that they became confluent after 72 hours of incubation, while MCF-7 cells migrate slowly even without CDK11 p110 knockdown.
To investigate how CDK11 p110 sustains tumor cell survival and proliferation, we performed apoptotic assays after CDK11 p110 knockdown in human breast cancer cell lines. As expected, cell apoptosis was observed in both MCF-7 and MDA-MB-468 cells after CDK11 p110 siRNA transfection. The western blot assay for apoptosis-related proteins revealed that several anti-apoptotic proteins are reduced by CDK11 p110 knockdown in breast cancer cell lines, suggesting that CDK11 p110 is involved in cellular apoptotic signaling pathway. Thus, it can be reasoned that CDK11 p110 knockdown inhibits human breast cancer cell proliferation by inducing cell apoptosis.
Considering that downregulation of CDK11 p110 induces human breast cancer cell apoptosis leading to suppression of cell proliferation and that CDK11 p110 exerts critical role in regulating cell cycle, we further explored the underlying mechanisms of cell apoptosis induction through CDK11 p110 knockdown by determining alterations of the cell cycle. The results showed that cell cycle progression was blocked in G1 phase for both MCF-7 and MDA-MB-468 cells after CDK11 p110 knockdown, with the MDA-MB-468 cell cycle arrested more significantly, which was consistent with the diversity in cell apoptosis induction and proliferation inhibition. Thus, we conclude that knockdown of CDK11 p110 induces human breast cancer cell apoptosis via arresting cells in G1 phase of the cell cycle.
Cell cycle alterations are common in breast cancer. Newly popular targeted agent in breast cancer are CDK inhibitors which are agents inhibiting the function of different CDKs. CDK inhibitors have been Scientific RepoRts | 5:10433 | DOi: 10.1038/srep10433 tried as monotherapy and combination therapy in breast cancer clinical trials 55,56 . For example, CDK4 and CDK6 inhibitor palbocyclib is designed for a phase III trial for estrogen receptor (ER) positive breast cancer after showing favorable results in progression free survival in a phase II trial 57 . Flavopiridol, a pan-CDK inhibitor and targeting CDK2, CDK4, CDK6 and CDK9, synergizes with sorafenib to induce cytotoxicity and potentiate antitumorigenic activity in EGFR/HER-2 and mutant RAS/RAF breast cancer model systems 58 . In addition, CDK7/CDK9 inhibitors have also been evaluated in different stages of clinical trials in breast cancer. CDK7/9 inhibitor in ER-positive breast cancer cells has showed to prevent activating phosphorylation of ER-α 59 . More recent study revealed a synergistic effect existd between inhibitions of CDK 4/6 and PI3K in PIK3CA mutant breast cancer. CDK 4/6-PI3K inhibition is very effective in several PIK3CA mutant xenograft tumor models 60 . These ongoing clinical trials appeared promising and our current study suggests that CDK11 p110 may be another potential therapeutic target for breast cancer.
Taken together, our current study demonstrated that CDK11 p110 is highly expressed in human breast tumor tissues and cell lines, which correlates with the clinicopathological characteristics of breast cancer patients. In vitro knockdown of CDK11 p110 by RNAi inhibits human breast cancer cell survival and proliferation by apoptosis induction via G1 cell cycle arrest. Our results suggest that CDK11 p110 is critical for the proliferation and growth of human breast cancer cells, and may be a promising therapeutic target for the treatment of breast cancer patients.

Methods
Human breast tumor specimens. Eighteen pairs of breast tumor and their adjacent normal tissues were collected immediately after surgical resection at the First Affiliated Hospital of Zhengzhou University (Zhengzhou, China). No patients recruited in this study received any preoperative treatment. All diagnoses were confirmed histologically. Access to the materials was approved by the ethics committee of Zhengzhou University, Henan, China, and written informed consents were obtained from all patients. The collected tissues were frozen in liquid nitrogen until Western blot analysis for CDK11 p110 expression levels. All experiments were performed in accordance with relevant guidelines and regulations.
Tissue microarray and immunohistochemistry. A commercially available human tissue microarray, containing 40 breast cancer tumor tissues and 9 paired normal breast tissues, was purchased from Imgenex Corporation (Catalog number: IMH-364, CA, USA). Clinical data of patients for the microarray are detailed in Supplementary Table S2. CDK11 p110 expression was examined with a SABC Immunohistochemistry Staining Kit (Boster, Wuhan, China) according to the manufacturer's instructions. In brief, the paraffin-embedded slide was deparaffinized with xylenes and rehydrated with ethanol. After antigen retrieval in heated citrate buffer, the slide was incubated with 3% hydrogen peroxide solution to quench the endogenous peroxidase, followed by blocking with 5% BSA blocking buffer. Subsequently, the slide was incubated with rabbit polyclonal antibody to human CDK11 p110 (SC-928, 1:50 dilution, Santa Cruz Biotechnology, CA, USA.) at 4 °C overnight, followed by incubation with biotin-conjugated secondary antibody at room temperature for 30 min. The slide was then incubated with Avidin-Biotin Complexes for 30 min, followed by coloration with DAB Chromogen Solution for 5 min. Finally, the tissue array was counterstained using hematoxylin (Boster, Wuhan, China), dehydrated in ethanol, and mounted with gelatine. The slide was imaged using a LeicaMicrosystems (Wetziar, Germany).
Immunostaining of the whole slide area was viewed and scored separately by three independent pathologists who were blinded to tumor characteristics and patient details of the samples. CDK11 p110 staining patterns were categorized into 6 groups: 0, no positive staining; 1 + , < 10% of positive cells; 2 + , 10%-25% positive cells; 3+ , 26%-50% positive cells; 4 + , 51%-75% positive cells; and 5+ , > 75% of positive cells. Tumors with a staining score of ≥ 4 were defined as high expression and ≤ 3 were defined as low expression of CDK11 p110 . Cell proliferation assay. 72hours after CDK11 p110 siRNA or nonspecific siRNA transfection, the morphological changes of MCF-7 or MDA-MB-468 cells were observed with a ZEISS microscope (Oberkochen, Germany). Meanwhile, in vitro cell viability of transfected cells was determined using the MTT assay. Briefly, at the end of CDK11 p110 siRNA treatment, 20 μ L of MTT (5 mg/mL, Sigma-Aldrich) was added to each well and the 96-well plates were incubated at 37 °C in a humidified 5% CO 2 atmosphere for 4 hours. Finally, the resulting formazan product was dissolved with 150 μ L of DMSO and the absorbance at a wavelength of 490 nm was measured on a Multiskan photometer microplate reader (Thermo Scientific, MA, USA).
Cell colony formation assay. MCF-7 or MDA-MB-468 cells were transfected with 40 nM of CDK11 p110 siRNA or nonspecific siRNA and seeded at a density of 100 cells per well in 6-well plates. After 10 days, when a macroscopic cell colony had formed, the cells were fixed in 4% paraformaldehyde (Solarbio, Beijing, China) and stained with Crystal Violet Staining Solution (Beyotime, Haimen, China), and the colonies were counted on a Zeiss microscope (Oberkochen, Germany) only if they contained more than 50 cells. The typical macroscopic images were photographed by a common Canon camera (Tokyo, Japan). Colony formation rates (%) were calculated using the following formula: (number of colonies / number of seeded cells) × 100%.
Cell migration assay. Cell migration activity was detected by the wound healing assay. In brief, after transfection with 20 nM of CDK11 p110 siRNA or nonspecific siRNA for 12 hours, the adherent MCF-7 or MDA-MB-468 cell layer was scraped in three parallel lines with a sterile 10 μ L tip. After starved incubation with low-serum medium containing 3% FBS for an additional 72 hours, the cells were photographed under a Zeiss microscope (Oberkochen, Germany) equipped with a Zen Imaging software. The wound width was evaluated by measuring the distance between the two edges of the scratch at 5 sites in each image. Cell migration distance was determined using the following formula: (wound width at the starting time point -wound width at the end time point)/ 2. Semiquantitative reverse transcription-PCR. After 48 hours of CDK11 p110 siRNA or nonspecific siRNA transfection, total RNA was extracted from MCF-7 and MDA-MB-468 cells with the TRIzol reagent (Invitrogen, USA). Reverse transcription of 100 ng of total RNA was performed using PrimeScript RT-PCR Kit (TaKaRa, Japan), and cDNA was subjected to PCR amplification. The sequences of CDK11 p110 primers were CDK11-F: 5′ -CGGGAAGTCAGAAATCGA-3′ , and CDK11-R: 5′ -CGTGGTGGTAAGGTGGA A-3′ . As an internal reference, a GAPDH gene segment was amplified with primers GAPDH-F: 5′ -GACCACAGTCCATGCCATCAC-3′ and GAPDH-R: 5′ -GTCCACCA CCCTGTTGCTGTA-3′ . Finally, the gene-specific PCR products were subjected to electrophoresis on 2% agarose gel and imaged using a BioSpectrum Imaging System (UVP, CA, USA) for CDK11 p110 mRNA semiquantitative analysis.

Flow cytometry analysis.
After 72 hours of transfection with 20 nM of CDK11 p110 siRNA or nonspecific siRNA, MCF-7 and MDA-MB-468 cells were subjected to flow cytometry analysis to examine the cell apoptosis and cell cycle changes. For cell apoptosis analysis, the cells were collected by trypsinization and resuspended in Binding Buffer, followed by staining with Annexin V-FITC and Propidium Iodide (KeyGEN BioTHCH, Nanjing, China) for 30 min, and then subjected to flow cytometry (FACSCantoll, BD, NJ, USA). For cell cycle analysis, the collected cells were fixed in 70% ethanol at 4 °C overnight, incubated in RNase A at 37 °C for 30 min, followed by dyeing with Propidium Iodide (KeyGEN BioTHCH, Nanjing, China) for 30 min. The DNA content was determined by flow cytometry (Beckman Coulter, CA, USA) and the population of cells in each cell cycle phase was analyzed by the equipped MultiCycle software (Phoenix Flow Systems, CA, USA). Statistical analysis. Statistical analysis was performed using the GraphPad PRISM 4 software (GraphPad Software, San Diego, CA, USA). Data are expressed as mean ± SD. Student's t-test was used to determine the statistical significance of differences between groups. Survival analysis was performed using the Kaplan-Meier method, and significance was determined by the log-rank test. A P value of ≤ 0.05 was considered as statistically significant.