Aberrant Ki‐67 expression through 3′UTR alternative polyadenylation in breast cancers

Ki‐67 (MKI67) is a marker of cellular proliferation of cancer. Here, we show that Ki‐67 is post‐transcriptionally regulated through alternative polyadenylation (APA) and microRNAs in breast cancer. We show that shortening of the Ki‐67 3′UTR results in the loss of the binding sites for the suppressive miRNAs and thus renders the transcript with a shortened 3′UTR insusceptible to miRNA‐mediated suppression. This APA‐mediated shortening of the Ki‐67 3′UTR contributes to increased mRNA stability and enhanced translational efficiency. In summary, our results not only highlight the post‐transcriptional regulation of Ki‐67 involving APA and microRNAs but also suggest that Ki‐67 3′UTR disruption could serve as a molecular marker in breast cancer.

Ki-67 (MKI67) is a marker of cellular proliferation of cancer. Here, we show that Ki-67 is post-transcriptionally regulated through alternative polyadenylation (APA) and microRNAs in breast cancer. We show that shortening of the Ki-67 3 0 UTR results in the loss of the binding sites for the suppressive miRNAs and thus renders the transcript with a shortened 3 0 UTR insusceptible to miRNA-mediated suppression. This APA-mediated shortening of the Ki-67 3 0 UTR contributes to increased mRNA stability and enhanced translational efficiency. In summary, our results not only highlight the post-transcriptional regulation of Ki-67 involving APA and microRNAs but also suggest that Ki-67 3 0 UTR disruption could serve as a molecular marker in breast cancer.
Breast cancer is the most common malignancy in women [1,2]. However, the molecular mechanisms of breast cancer are unclear. Prognostic factors, such as tumor size, nodal status, and histological grade, are the clinicopathological variables associated with the final outcome that are used to estimate the risk of death in breast cancer [3,4].
Ki-67 (MKI67) is a marker of cellular proliferation [5] and can predict the prognosis of patients with cancer [6], including breast cancer [7]. In breast cancer, Ki-67 is an established prognostic and predictive biomarker [8][9][10]. The International Ki-67 in Breast Cancer Working Group set standards for the staining, scoring, and analysis of Ki-67 in breast cancer to ensure the reproducibility, reliability, and accuracy of studies using Ki-67 as their primary measure of outcome [11]. In addition, Ki-67 is an independent prognostic factor in early breast cancer [9] and in neoadjuvant therapy [12,13]. High expression of Ki-67 is associated with a poor prognosis in breast cancer [14]. However, the mechanisms regulating Ki-67 expression are largely unknown.
Alternative polyadenylation (APA) is a widespread phenomenon in the human genome [15][16][17]. Over half of the human genes have APA signals [18]. The importance of APA in human diseases, including cancer, has been emphasized by recent studies [19]. The stability and translation efficiency of messenger RNA isoforms with shorter 3 0 UTRs, generated from APA, were increased due to the loss of miRNA-mediated repression [20]. Recently, studies have shown that the shortening of the 3 0 UTR may serve as a prognostic marker in cancer [21]. Transcriptome-wide studies have indicated a widespread shortening of the 3 0 UTR, which was frequently observed in various cancers, including glioblastoma, liver, lung, and breast cancer [22][23][24].
In the present study, we carried out 3 0 rapid amplification of cDNA ends (3 0 RACE) and qRT-PCR experiments to demonstrate the existence of three 3 0 UTRs of varying length in Ki-67 transcripts. Our results showed that the percentage of Ki-67 mRNA isoforms with shorter 3 0 UTRs was higher in the breast cancer clinical tissues compared to the matched noncancerous breast clinical tissues. Reporter assays revealed that the translation efficiency of shorter transcripts was higher than that of the full-length transcripts, partly through the evasion of miRNA suppression. Taken together, our data highlight a novel regulatory mechanism of Ki-67 and may lead to a deeper understanding of the role of Ki-67 in cancer.

Breast tissue specimens
Twenty consecutive surgical breast cancer specimens and the corresponding adjacent nontumorous breast samples were obtained from Chinese patients at the First Affiliated Hospital of Anhui Medical University between 2006 and 2008. All specimens were confirmed as breast cancer based on histopathological evaluations. Tumor and normal specimens were snap-frozen in liquid nitrogen and stored at À80°C immediately after resection. Patients who had undergone chemotherapy or radiation therapy before surgery were excluded. The protocol of this study was approved by the Ethics Committee of Anhui Medical University. The work undertaken conforms to the provisions of the Declaration of Helsinki. Informed consent was obtained from all patients.

Immunohistochemistry (IHC)
Formalin-fixed, paraffin-embedded tissue was cut into 4 lm sections, deparaffinized in xylene, rehydrated through graded ethanol, quenched for endogenous peroxidase activity in 3% hydrogen peroxide, and processed for antigen retrieval by heating in 10 mM citrate buffer (pH 6.0) at 90-100°C. Sections were incubated at 4°C overnight with Ki-67 (1 : 500; Santa Cruz Biotechnologies, Santa Cruz, CA, USA). Immunostaining was performed using the UltraSensitive S-P Detection Kit (KIT-9720; Maixin, Fuzhou, China), and then, the color was developed using a DAB kit (DAB-0031; Maixin). Subsequently, the sections were counterstained with hematoxylin. Quantification of immunohistochemical stain intensity was performed as previously described [25].

Quantitative real-time RT-PCR
RNA was transcribed using the TaqMan Reverse Transcriptase Kit (Applied Biosystems, Foster City, CA, USA), and the resulting cDNA was used for real-time quantitative PCR (Applied Biosystems 7900) using SYBR green PCR master mix from SensiMix SYBR Ò from Bioline (Taunton, MA, USA). The TaqMan probes and primers were purchased from Shenggong (Shanghai, China). Human GAPDH was used as an endogenous control.

Luciferase reporter assay
Luciferase reporter assays were performed using the psi-CHECK2-Ki-67-3 0 UTR vector. The Ki-67 short, medium, and long 3 0 UTRs were amplified and cloned downstream of Renilla luciferase in a psiCHECK2 vector (Promega, Madison, WI, USA). Cells were grown to approximately 60% confluence in 24-well plates and transfected with 100 ng plasmid or empty plasmid using Lipofectamine 3000. Forty-eight hours after transfection, cells were lysed and analyzed with the dual-luciferase assay (Promega) according to the manufacturer's instructions. Renilla/firefly luciferase readouts from the constructs were normalized to those of empty psi-CHECK2, which was set to 1. Three independent experiments were performed in triplicate.
3 0 RACE and quantitative assessment of APA One microgram of total RNA was used to generate cDNA with Superscript III reverse transcriptase (Invitrogen) according to the manufacturer's instructions using 3 0 RACE-RT as a primer. The PCR was performed with a Ki-67-specific forward primer and 3 0 RACE-R as a reverse primer. Primer pairs are as follows:

Statistical analysis
Student's t-test (two-tailed) was used to compare two experimental groups; differences were considered statistically significant at P < 0.05.

Results
Ki-67 mRNA with a shorter 3 0 UTR is upregulated in breast cancer Initially, we compared Ki-67 expression in breast cancer tissues and matched noncancerous breast tissues using IHC. The expression of Ki-67 was significantly higher in all breast cancer tissues compared to the adjacent noncancerous breast tissues (Fig. 1A). Moreover, the expression of Ki-67 mRNA also increased in the breast cancer tissues compared to the adjacent noncancerous breast tissues (Fig. 1B). Recent studies have shown widespread shortening of the 3 0 UTR in cancer cells. We observed that the 3 0 UTR of the Ki-67 gene contains three alternative polyadenylation signals (PASs; Fig. 1C). To determine the relative abundance of the Ki-67 mRNA isoforms terminated at the different PASs in the breast cancer cells, we performed RT-qPCR using primers flanking different regions of the Ki-67 3 0 UTR (F1/R1, F2/R2, F3/R3, F4/R4, F5/R5, and F6/R6; Fig. 1C). Interestingly, an increase in the short 3 0 UTR isoform of Ki-67 was detected in five of the twenty breast cancer tissues compared to adjacent noncancerous breast tissues (Fig. 1D). Additionally, the 3 0 RACE assay was carried out to verify the relative abundance of the different Ki-67 mRNA isoforms (Fig. 2A). The Ki-67 mRNA isoforms terminated at PAS1 and PAS2 were designated 3 0 UTR-short (3 0 UTR-S) and 3 0 UTR-medium (3 0 UTR-M), respectively (Fig. 2B,C). In the 3 0 RACE assay, the percentage of the Ki-67 mRNA isoforms terminated at PAS1 (3 0 UTR-S) and PAS2 (3 0 UTR-M) were significantly higher in the breast cancer tissues compared to the adjacent noncancerous breast tissues (Fig. 2D).
Ki-67 mRNAs with a shorter 3 0 UTR have greater stability and produce more protein Recent studies have revealed that the production of mRNA isoforms with a shorter 3 0 UTR via APA results in increased mRNA stability and increased protein production. Thus, we examined the stabilities of the shorter and the longer Ki-67 mRNAs in the MCF7 and T47D cell lines. The total RNAs of the MCF7 and T47D cell lines were extracted after treatment with actinomycin D, and the expression of the mRNA was investigated by qRT-PCR. The results showed that the shorter mRNA was more stable than the longer mRNA (Fig. 3A,B). In addition to mRNA destabilization, to determine whether different isoforms produced different amounts of protein, we cloned each of the three 3 0 UTR isoforms of Ki-67 downstream of a luciferase reporter gene into the psiCHECK2 vector. The reporter mRNA isoform with the shortened Ki-67 3 0 UTR exhibited a higher luciferase reporter activity compared to the Ki-67 3 0 UTR-medium and Ki-67 3 0 UTR-long reporter mRNA isoforms (Fig. 3C). These results suggest that the Ki-67 3 0 UTR is shortened in breast cancer cells through APA, resulting in an increased Ki-67 translation efficiency.
After miR-128-3p mimics the transfection of MCF7 and T47D cells, a significant decrease in luciferase activity of all forms of 3 0 UTRs was observed (Fig. 4D-F). After miR-133a-3p mimics the transfection of MCF7 and T47D cells, a significant decrease in luciferase activity of the long and medium 3 0 UTRs was observed, while the short variant was not affected (Fig. 4D-F). Moreover, after miR-140-3p mimics the transfection of MCF7 and T47D cells, a significant decrease in luciferase activity of the long 3 0 UTRs was observed, while the short and medium variants were not affected. These data show that miR-128-3p, miR-133-3p, and miR-140-3p not only target Ki-67 but also target the position of the 3 0 UTR.
To determine whether the miRNAs contributed a greater translational repression for the longer 3 0 UTR, we mutated the miRNA complementary sites of miR-140-3p, downstream to the proximal polyA sites. The two miR-140-3p sites in the Ki-67 3 0 UTR were mutated in the context of the long 3 0 UTR. The results showed that loss of the miRNA sites led to significant increases in luciferase activity in four breast cancer cell lines (Fig. 4G).
However, the luciferase activity was slightly increased in T47D (Fig. 4G). Thus, the results suggest that the expression of endogenous miR-140-3p may be low in T47D. In addition, we examined the expression of miR-140-3p in the tumor samples by qRT-PCR and found that the expression of miR-140-3p in the tumor samples was not significantly different   Ki-67 mRNA levels at the different time-points were measured by qPCR, using GAPDH as the input control. (C) Luciferase reporter activities from a reporter containing the 3 0 UTR of the short or medium Ki-67 isoform, compared to that from the reporter containing the 3 0 UTR of the long Ki-67 isoform. The luciferase activity was measured by the dual-luciferase reporter assay and is presented as Rluc/ Fluc (renilla/luciferase firefly). The Rluc/Fluc value of the control was set as 1. (D) Graphical representation of the approximate position of the reported miRNA target sites are shown on long, medium, and short 3 0 UTRs. *P < 0.05. from the expression of miR-140-3p in the adjacent nontumor samples (Fig. S1). The results indicated that Ki-67 transcript isoforms with short 3 0 UTRs generated by alternative cleavage and polyadenylation exhibit increased stability and produce more protein due to the loss of miRNA-mediated repression, but not because of the decreased expression of miR-140-3p.

Discussion
The mechanisms to explain the increased expression of the Ki-67 protein in cancer cells are poorly understood. Recent studies have shown that a change in 3 0 UTR length could alter the expression of many genes during cancer development [15,20].
Recent studies report that messenger RNA isoforms with short 3 0 UTRs, generated by APA, exhibit increased stability and produce more protein by the loss of miRNA-mediated repression. The longer 3 0 UTR harbors target sites for regulation by miRNAs. We hypothesized that truncations in Ki-67 mRNA exist in cancer cell lines and alter miRNA binding sites. Part of the Ki-67 oncogene upregulation observed in this study was explained by the evasion of miRNA-mediated repression by the shorter isoform. We found that the longer Ki-67 3 0 UTR harbors more potential binding sites than the shorter 3 0 UTR, such as hsa-miR-140-3p. Hsa-miR-140-3p can suppress breast cancer proliferation and migration, and increased expression of hsa-miR-140-3p is predicted to improve breast cancer survival [28,29].
Overall, our data identified a novel post-transcriptional mechanism, involving APA and miRNA, that underlies the elevated expression of Ki-67 in breast cancer. The results have shown that breast cancer cells preferentially express Ki-67 mRNA isoforms with short 3 0 UTRs, and the expression of shorter Ki-67 mRNAs leads to an increase in Ki-67 mRNA stability and translational efficiency. Our study presented here highlights the importance of APA in the regulation of Ki-67.

Supporting information
Additional Supporting Information may be found online in the supporting information tab for this article: Fig. S1. Expression levels of miR-140-3p were examined by qRT-PCR in tumor samples and their adjacent nontumor samples.