Overexpression of splicing factor poly(rC)-binding protein 1 elicits cycle arrest, apoptosis induction, and p73 splicing in human cervical carcinoma cells CURRENT

Background Splicing factor poly(rC)-binding protein 1 (PCBP1) is a novel tumor suppressor that is downregulated in many cancers thereby regulates tumor formation and metastasis. However, to date, little information has been available on the molecular mechanisms by which PCBP1 evokes apoptosis. Results Here, we explored the molecular mechanism by which PCBP1 triggers apoptosis in human cervical cancer cells. We testified that overexpression of PCBP1 greatly repressed proliferation of HeLa cells in time-dependent manner. It also induced a significant increase in G2 / M phase arrest and apoptosis. Furthermore, it was shown that overexpression of PCBP1 caused p73 splicing, and thus efficiently downregulated the ratio of Bax / Bcl-2, the release of cytochrome c and the expression of caspase-3. Conclusion Our results revealed that PCBP1 played a vital role in cycle arrest, apoptosis induction, and p73 splicing in human cervical carcinoma cells and targeting PCBP1 may be a promising approach in cervical cancer therapy. The were normalized by β-Actin

mRNA, mRNA stability and translation [3]. In recent years, researches focus on the relationship between PCBP1 and tumors. Studies have been found that PCBP1 acts as a tumor suppressor in tumors and the expression is significantly downregulated in various tumors, including gastric cancer [4], acute myeloid leukemia [5], non-small-cell lung cancer [6], cervical cancer [7] et al. In general, PCBP1 plays a multifunctional role in tumor progress. For instance, PCBP1 has an influence on apoptosis in variety cancers [8,9]. It is also involved in alternative splicing which dysregulation usually leads to disease and is increasingly associated with tumorigenesis [10,11]. In addition, PCBP1 negatively regulated the tumor hypoxic microenvironment and inhibited autophagy to further affect the tumor formation and development [8,12,13]. Moreover, PCBP1 prevented the process of EMT to reduce cancer metastasis [6]. The above cases all turn out that PCBP1 is involved in the development of tumors as a tumor suppressor, but little information has been available on the molecular mechanisms by which PCBP1 causes cervical cancer apoptosis. In the present study, we would provide some preliminary data to illustrate the distinct functions of PCBP1 in p73 alternative splicing and the mechanisms of PCBP1 on cervical cancer cells apoptosis. The results suggested that PCBP1 may be an attractive novel target for cervical cancer therapy.

Results
The expression of PCBP1 and its spacial distribution To explore the biologic function of PCBP1, we initially transfected HeLa cells with pEGFP-N1 or pEGFP-N1-PCBP1, and then we verified whether the PCBP1 was successfully transfected and overexpressed in cells. We performed real-time PCR and immunofluorescence experiments. The results demonstrated that PCBP1 mRNA expression was significantly increased in cells transfected with pEGFP-N1-PCBP1 compared to mock group (Fig. 1a) and there was no significant difference in mRNA expression in mock and vector group (Fig. 1a). In addition, further immunofluorescence experiments results showed the PCBP1 protein expression was increased compared with mock and vector group, and the PCBP1 is distributed in both the cytoplasm and the nucleus (Fig. 1b).
Overexpression of PCBP1 and its effects on human cervical carcinoma cells viability In order to understand the effects of overexpressed PCBP1 in HeLa cells, we examined the proliferation of HeLa cells in different time points after transfection. MTS analysis proved that elevated PCBP1 significantly reduced the cell viability of HeLa cells, and the inhibition of proliferation of PCBP1 transfection. The results were significantly time-dependent ( Fig. 2a). Furthermore, the experimental results from the colony formation assay suggested that overexpression of PCBP1 in HeLa cells could significantly repress cell colony formation compared with mock and vector group (Fig. 2b, c).

PCBP1 induces cell cycle arrest and apoptosis
To detect the effect of overexpressed PCBP1, after transfection, we stained the nuclei and then observed the nuclear morphology. We found the nuclear morphology changed and apoptotic bodies appear, this suggested us that apoptosis took place (Fig. 3a).
In order to further verify the mechanism of PCBP1 in inhibiting the growth of HeLa cells, we used flow cytometry to detect the cell cycle and apoptosis. The results showed that overexpressed PCBP1 would cause cell cycle arrest (Fig. 3b), and the ratio of G2 / M cells in HeLa cells were significantly increased (Fig. 3d). Then we detected cell apoptosis to prove that whether the decrease in viability of HeLa cells after transfected with pEGFP-N1-PCBP1 was caused by apoptosis. The results showed cells transfected with pEGFP-N1-PCBP1 for 48 h performed more apoptosis than the mock and vector group (Fig. 3c, e). The results of the apoptosis experiment obtained by flow cytometry analysis were statistically analyzed, and the results were also verified. PCBP1 upregulates Tap73 and downregulates ΔNp73, indicating activation of apoptosis In order to demonstrate whether p73 is involved in PCBP1 induced cell cycle arrest and apoptosis in human cervical carcinoma cells, western blot and immunofluorescence were used to detect the level of Tap73 and ΔNp73 proteins. We found the level of ΔNp73 was significantly reduced in HeLa cells at 24 h after transfected with pEGFP-N1-PCBP1 compared with the mock group. On the contrary, the level of Tap73 was increased compared with the mock group (Fig. 4a). Quantitative results showed, PCBP1 induced an increase in the Tap73 / ΔNp73 ratio (Fig. 4b). Next, we used immunofluorescent to further verify its spatiotemporal distribution in cells (Fig. 4c). ΔNp73 expression clearly reduced but Tap73 expression obviously upregulated. Moreover, Tap73 mainly detected in the cytoplasm, but ΔNp73 mainly detected in the nucleus. These findings suggested that p73 splicing is involved in PCBP1 induced cell cycle arrest and apoptosis in HeLa cells.

PCBP1 regulates apoptosis via mitochondrial pathway
In order to further verify whether PCBP1 induced apoptosis is associated with mitochondrial apoptosis pathway, we used western blot to examine several key proteins in the mitochondrial apoptosis signalling pathway. As shown in Fig. 5, overexpression of PCBP1 upregulated the ratio of Bax / Bcl-2 to promote cell apoptosis (Fig. 5b). In addition, there was a substantial increase in the expression of cytochrome c at 24 h after transfected with pEGFP-N1-PCBP1 (Fig. 5d). Furthermore, decreased procaspase-3 and increased cleaved caspase-3 both indicated the occurrence of apoptosis (Fig. 5c). Thereby confirming that overexpression of PCBP1 upregulated Bax / Bcl-2 ratio, promoted cytochrome c release and activated caspase-3 to induce apoptosis.

Discussion
Cervical cancer is one of the most diseases threatening women's health, but our understanding of its pathogenesis is still not deep enough. It is imminent to further study its pathogenesis [14]. PCBP1 is an evolutionarily conserved RNA-binding protein that regulates transcription, translation, and alternative splicing of genes [3, 15,16].  [20]. Interestingly, PCBP1 has gained more attention due to its multiple functions in tumor progression, but the real mechanism is relatively unexplored. Therefore, it is worthy to further study the relationship between PCBP1 and cervical cancer. Here, we transfected PCBP1 into HeLa cells and testified that overexpression of PCBP1 greatly repressed proliferation of HeLa cells in time-dependent manner (Fig. 2). It also induced G2 / M phase arrest and significant rise of apoptotic cells (Fig. 3). Overall, these results indicated that elevated PCBP1 is an efficient way to inhibit tumor cell progression, and this will provide a reference for further understanding of the pathogenesis of cervical cancer.
RNA splicing is the key to the pathology of numerous diseases, and experiments have shown that dysregulation of splicing isoforms were increasingly associated with tumor proliferation, metastasis and apoptosis [21,22]. Additionally, PCBP1 is related to alternative splicing. In pancreatic cancer, upregulated PCBP1 reduced tumor metastasis by interacting with integrin β1 to regulate its alternative splicing [11]. Moreover, overexpression of PCBP1 inhibited the tumor invasion and metastasis in HepG2 cells via regulating exon inclusion of CD44 [10]. In our study, we transfected PCBP1 into HeLa cells and indicated that overexpressed PCBP1 obviously enhanced the expression of Tap73 and decreased ΔNp73 expression (Fig. 4). Tap73 and ΔNp73 are two variants of p73, which is a structural homolog of p53 and acts as a tumor suppressor. This gene often encodes two opposing variants: the transcriptionally active TAp73 and the dominant-negative ΔNp73 [23]. ΔNp73 overexpressed in a variety of cancers and it is correlated with poor prognosis [24,25]. Additionally, ΔNp73 can interact with wild-type p53 or Tap73 to efficiently counteract wild-type p53 and TAp73 mediated apoptosis, and growth suppression [26].
Therefore, ΔNp73 has become a novel tumor-specific molecular target for cancer because of its anti-apoptotic functions. TAp73 contains the NH2-terminal domain and plays a similar role to p53 as a tumor suppressor [27,28]. TAp73 is relevant to DNA damage and upregulates proapoptotic Bcl-2 family members and causes apoptosis via the mitochondrial pathway [29]. Recently, some studies elucidated that the ratio between Tap73 and ∆Np73 might contribute to tumorigenesis and resistance to chemotherapy and determined the fate of the cell [30]. Our results showed overexpressed PCBP1 upregulated the ratio of Tap73 / ΔNp73 and caused HeLa cell apoptosis. These data are evidenced by our finding that that PCBP1 is involved in p73 gene splicing and it will induce cell apoptosis via upregulation of Tap73 / ΔNp73 ratio in human cervical cancer (Fig. 4). This may have a certain inspiration for cancer treatment. Taken together, our data indicated that PCBP1 is an important gene and a tumor suppressor in cervical cancer.
To further study the mechanism of PCBP1 induced apoptosis, we examined the expression of some related proteins after transfection of PCBP1. Indeed, our data revealed that overexpressed PCBP1 significantly decreased anti-apoptosis Bcl-2 expression and increased the Bax / Bcl-2 ratio (Fig. 5). It has been confirmed that Bax / Bcl-2 ratio regulated cytochrome c release from mitochondria [31]. In addition, we further detected the expression level of cytochrome c, which can activate the caspase-3 and downstream cell death pathway. We discovered that procaspase-3 expression levels were significantly reduced, while cytochrome c levels were elevated. In light of our previous work, Tap73 / ΔNp73 ratio also plays an important role in regulating apoptosis via mitochondrial pathway [32,33]. PCBP1 may initiate a mitochondria-mediated apoptotic pathway by inducing p73 alternative splicing. As expected from the above results, we believe that PCBP1 plays a pivotal role in arresting cell cycle, inducing apoptosis, regulating p73 splicing in human cervical carcinoma cells, and it induced splice regulation of p73 may be another downstream signaling pathway independent of p53. In conclusion, our results suggested that PCBP1 could be used as a potential candidate for cervical cancer therapy and it has broad prospects as a molecular therapeutic target for cervical cancer. However, this also requires the use of tumor-bearing animal models and clinical trials to further study the effects of PCBP1.

Materials And Methods
Cell culture and transfection

Colony formation assay
The cells were harvested for 24 h after transfection, and seeded into 60 mm dishes. After incubated at 37 °C for 10-14 days, the culture was terminated when macroscopic clones appeared in the dishes. The clone was fixed with paraformaldehyde and stained with crystal violet. Count clones containing more than 50 cells, calculated clone formation rate and collected images. Each experiment was performed in triplicate. Figure 1 The expression of PCBP1 mRNA and protein and its spacial distribution. a Expression of PCBP1 mRNA was determined in HeLa cells, the data was normalized to β-Actin expression. b PCBP1 protein is distributed in both the cytoplasm and the nucleus. All experiments were repeated at least three times.