Silencing of CPNE1-TRAF2 Axis Restrains the Development of Pancreatic Cancer

Background : Copine 1 (CPNE1) acts as a promoter in the progression of many kinds of cancers with the exception of pancreatic cancer (PC). This research is designed to probe the function of the CPNE1-tumor necrosis factor receptor-associated factor 2 (TRAF2) axis in PC. Methods : In vivo and in vitro models of PC were constructed


Introduction
Pancreatic cancer (PC) ranks fourth among all malignancies in terms of mortality [1,2].Due to the limitations of imaging techniques and specific biomarkers, the diagnostic sensitivity of early PC is unsatisfactory [3].Unfortunately, although chemotherapy, radiotherapy, surgical resection, and other treatments for PC have been developed, PC patients may still exhibit various adverse reactions, tumor resistance, and tumor metastasis [4,5].Insufficient understanding of the complex molecular mechanism of hyperproliferation and apoptotic dysfunction is one of the key reasons for the unsatisfactory therapeutic interventions for PC [6,7].
Copine 1 (CPNE1), belonging to the CPNE family, is a conserved protein-encoding calcium-dependent phospholipid-binding protein that evolved from protozoa to eukaryotes [8,9].CPNE1 consists of a C-terminal A domain and two N-terminal C2 domains, generally functioning in cell signaling transduction via interactions with intracellular proteins [10,11].Research has demonstrated that CPNE1 is actively involved in the tumorigenesis of several types of human cancers [12][13][14][15][16].For instance, increased expression of CPNE1 in non-small cell lung cancer (NSCLC) was associated with poor prognosis [12,13].
Tumor necrosis factor receptor-associated factor 2 (TRAF2) belongs to the TRAF superfamily [17][18][19][20], which has been confirmed to be associated with the occurrence of malignancies [21,22].TRAF2 functions as an oncogene in breast cancer (BC) to accelerate cell proliferation and repress apoptosis [21].The progression of gastric cancer (GC) cells can be enhanced by TRAF2 overexpression [22].Similarly, the positive effect of TRAF2 on PC development has been confirmed [23,24].In addition, TRAF2 generally serves as an adaptor protein [25].Liang et al. [26] indicated that TRAF2 interacts with CPNE1 to affect the tumorigenesis of prostate cancer (PRC).However, the functional interaction between CPNE1 and TRAF2 in PC remains unclear.
In the current research, the function of CPNE1 was determined in PC cells and a tumor xenograft model.The functional interaction between CPNE1 and TRAF2 was further evaluated in PC cells.We concluded that the CPNE1-TRAF2 axis may be a potential therapeutic target for PC.

Cell Viability Assay
The transfected PC cells were plated and incubated for the right time points.Then, 20 µL MTT (Sangon Biotech) was pipetted into each well and incubated at 37 °C for another 4 h.Afterwards, 150 µL DMSO was appended.The optical density was detected at 490 nm using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

Cell Colony Assay
Cells were inoculated in 6-well plates (1000 per well) for 14 days.Then, 4% paraformaldehyde and 0.5% crystal violet were used to fix and stain the cells, respectively.Cell numbers were counted using a microscope (CKX53, Olympus, Tokyo, Japan).

Flow Cytometry Assay
For the cell cycle assay, the transfected PC cells were initially fixed with 75% ethanol for 1 h.After centrifugation at 1000 g, cells were re-suspended in D-Hank's containing RNase (10 mg/mL) and PI (2 mg/mL).The frequencies of the cells were analyzed using a NovoCyte flow cytometer (Agilent Technologies, Santa Clara, CA, USA).The transfected PC cells were re-suspended in an appro-priative buffer and stained with Annexin V-FITC and PI for the apoptosis assay.The apoptotic rate was measured via a NovoCyte flow cytometer (Agilent Technologies).

Mouse Xenograft Model
The BALB/c nude mice used for the tumor xenograft model were obtained from Cavens (Changzhou, China).After acclimatization, mice were divided into control, si-NC, and si-CPNE1-1 groups (n = 6).Si-CPNE1-1 or si-NC was integrated into a lentiviral vector and transfected into PANC/MIA cells.The transfected cells (1 × 10 5 cells/100 µL) were subcutaneously injected into the right flank.After 6 weeks, mice were anesthetized (pentobarbital sodium, 50 mg/kg) and euthanized by cervical dislocation.All operations strictly followed the Guide for the Care and Use of Laboratory Animals and have been approved by the ethical committee of our hospital.

Immunohistochemistry
Tumors were fixed in 4% paraformaldehyde and cut into 4 µm sections.Sections were dewaxed in conventional xylene and dehydrated in gradient ethanol.After incubation with anti-Ki67 (Proteintech, China) and horseradish peroxidase-labeled goat anti-rabbit IgG (antibodies-online, Germany), sections were stained with DAB solution, antistained with hematoxylin, dehydrated, and sealed.The results were observed using a microscope and analyzed via Image J software (1.44, LOCI, University of Wisconsin, Madison, WI, USA).

Statistical Analysis
All experiments were repeated at least three times.Data were presented as mean ± standard deviation (SD) and performed using SPSS 22.0 (IBM Corp., Chicago, IL, USA).One or two-way ANOVA followed by Tukey's test were used to compare groups.If the value of p was less than 0.05, the difference in means was considered statistically significant.

CPNE1 was Up Regulated in PC Cells
The expression of CPNE1 in PC cell lines (MIA and PANC) was initially determined.We found a relative overexpression of CPNE1 in both MIA and PANC cells compared to that in H6C7 cells (Fig. 1A, p < 0.01).Si-CPNE1 was silenced in order to explore the function of CPNE1 on PC cells in vitro.The mRNA (p < 0.01) and protein (p < 0.001) expression of CPNE1 were decreased by si-CPNE1-1/-2 transfection in both MIA and PANC cells (Fig. 1B,C), which suggested CPNE1had been successfully silenced.

Silencing of CPNE1 Inhibits PC Cell Proliferation
We explored the influence of CPNE1 downregulation on cell proliferation in MIA and PANC cells.The results demonstrated that compared with the si-NC group, si-CPNE1-1/-2 significantly repressed the viability of two PC cell lines (Fig. 2A, p < 0.01) and downregulated the number of cell colonies (Fig. 2B, p < 0.05).

Silencing of CPNE1 Promotes G1/S Cell Cycle Arrest and Apoptosis
The influence of CPNE1 silencing on the cell cycle was further studied in MIA and PANC cells due to its inhibiting effect on cell proliferation.Compared to the si-NC group, the cell frequency of the G1 phase increased, whereas the cell frequency of the S phase reduced in the si-CPNE1-1/2 groups (Fig. 3A,B, p < 0.05), suggesting that the downregulation of CPNE1 led to the G1/S arrest in PC cells.Flow cytometry analysis also demonstrated that the apoptosis rate was elevated in si-CPNE1-1/2-transfected PC cells compared with the si-NC group (Fig. 4A,B, p < 0.05).

Silencing of CPNE1 Represses the Growth of Tumor Xenografts
The influence of CPNE1 silencing on tumor growth was further determined in a mouse xenograft model.The intervention of si-CPNE1-1 in MIA and PANC cells led to a remarkably reduced tumor growth rate in mice (Fig. 5A,B, p < 0.05).The percentage of Ki67-positive cells was also remarkably reduced after the intervention of si-CPNE1-1 in MIA and PANC cells (Fig. 6A,B, p < 0.05).These results demonstrated that CPNE1 knockdown repressed the growth of tumor xenografts in vivo.

CPNE1 Interacts with TRAF2 in PC Cells
Previous reports have confirmed an oncogene role of TRAF2 in PC [23,24] and have also indicated that TRAF2 acts as an adaptor protein to interact with CPNE1 in PRC [26].Therefore, the relationship between CPNE1 and TRAF2 was then investigated in PANC cells.As presented in Fig. 7A, compared with the si-NC + pcDNA-NC group, the mRNA expression of CPNE1 and TRAF2 was inhibited in the si-CPNE1 + pcDNA-NC group, whereas it was promoted in the si-NC + pcDNA-TRAF2 group (p < 0.01).Interestingly, pcDNA-TRAF2 reversed the inhibiting effect of si-CPNE1 on CPNE1 expression, and si-CPNE1 reversed the positive effect of pcDNA-TRAF21 on TRAF21 expression (p < 0.01).Similar results were observed in protein levels of CPNE1 and TRAF2 in PANC cells (Fig. 7B, p < 0.01).

Silencing of CPNE1 Suppresses the Proliferation and Accelerates the Apoptosis of PC Cells via Interacting TRAF2
Relevant cellular function experiments in PANC cells further validated the interaction between CPNE1 and TRAF2 in PC progression.The results indicated that si-CPNE1 + pcDNA-TRAF2 reversed the promotion of TRAF2 overexpression and the inhibition of CPNE1 silencing on cell proliferation (Fig. 8A,B, p < 0.01).On the con- trary, the inhibiting effect of TRAF2 overexpression and the promoting effect of CPNE1 silencing on G1/S arrest and apoptosis were also reversed by si-CPNE1 + pcDNA-TRAF2 transfection (Fig. 8C,D, p < 0.05).

Discussion
PC is a malignant endocrine and digestive system tumor with a high incidence rate and mortality.According to incomplete statistics, approximately 448,000 new pathologies and 430,000 deaths occur yearly [27].In addition to a late diagnosis, limitations still exist despite developing therapies for PC [4,5].Therefore, an in-depth exploration of the molecular mechanisms will contribute to better PC treatment.
CPNE1 is a key member of the CPNE family, which is up regulated in a series of cancers, such as NSCLC [12,13], OS [14], CRC [15], and TNBC [16].This study demonstrated that CPNE1 was also up regulated in PC cell lines.This result suggests that CPNE1 may be an oncogene in PC.Until now, multiple studies have shown that CPNE1 depletion could inhibit the proliferation and induce the apoptosis of cancer cells.Research has found that the downregulation of CPNE1 attenuates the viability but triggers the apoptosis of CRC cells [15].Moreover, the proliferation is repressed, and apoptosis is enhanced in TNBC cells by the transfection of sh-CPNE1 [16].In this study, we found that the transfection of CPNE1 silencing remarkably reduced proliferation and expedited apoptosis of PC cells.The inhibition of hyperproliferation caused by CPNE1 silencing may be useful for blocking PC progression.Further studies confirmed that silencing of CPNE1 increased PC cells in the G1 phase and reduced those in the S phase.This result was consistent with a previous NSCLC study [13] and suggests that the downregulation of CPNE1 can lead to G1/S arrest in PC cells.In summary, silencing CPNE1 may inhibit PC progression by blocking malignant proliferation in vitro.Consistent with the experimental results in vitro, si-CPNE1-1 repressed the growth of tumor xenografts.Therefore, we conclude that the knockdown of CPNE1 can retard PC development by inhibiting tumor growth.
Increasing evidence has confirmed that the C2 domains of CPEN1 are crucial for interacting with intracellular proteins, especially for phospholipid and calciumrelated proteins [11,28,29].TRAF2 is an oncogene that has been confirmed to modulate cell proliferation and apoptosis in PC [23,24].TRAF2 generally functions as an adaptor protein to link upstream receptors to downstream molecules [20].CPEN1 can bind to different intracellular proteins through its C-terminal A structural domain, and TRAF2 is an adaptor protein [26].Notably, a previous study by Liang et al. [26] found that TRAF2 affects the tumorigenesis of PRC by interacting with CPNE1.Based on the above phenomena, we speculated that TRAF2 may also interact with CPNE1 in the tumorigenesis of PC.As expected, TRAF2 overexpression reversed the effect of si-CPNE1 on CPNE1 expression, and CPNE1 silencing reversed the effect of pcDNA-TRAF21 on TRAF21 expression.These results indicate that TRAF2 positively interacted with CPNE1 in PANC cells.Furthermore, the following functional validation experiments also confirmed our expectations.Taken together, we conclude that silencing of CPNE1 may repress cell proliferation and induce apoptosis in PC through interacting with TRAF2.
There were several limitations of this study.First, other malignant behaviors such as migration, invasion, and drug resistance in PC cells must be studied except for excessive proliferation.Second, the interaction between CPNE1 and TRAF2 is not verified in vivo.In future studies, an indepth study of the direct mechanistic relationship between CPNE1 and TRAF2 should be performed.Third, the downstream mechanism of CPNE1/TRAF2 requires further exploration.However, this study verified CPNE1-TRAF2's role in PC development and provided novel targets for clinical treatment.

Conclusions
The current study illustrated an increased expression of CPNE1 in PC cells.Silencing of CPNE1 repressed cell proliferation and induced apoptosis via interaction with TRAF2 in PC.Both CPNE1 and TRAF2 may be potential molecular targets for the treatment of PC. and YZ performed the data collection.All authors contributed to editorial changes in the manuscript.All authors read and approved the final manuscript.All authors have participated sufficiently in the work to take public responsibility for appropriate portions of the content and agreed to be accountable for all aspects of the work in ensuring that questions related to its accuracy or integrity.

Fig. 6 .
Fig. 6.Silencing of CPNE1 represses the proliferation of tumor xenografts in mice.(A) The percentage of Ki67-positive cells in mice after injection of MIA cells transfected with si-CPNE1-1/NC.(B) The percentage of Ki67 positive cells in mice after injection of PANC cells transfected with si-CPNE1-1/NC.*p < 0.05, **p < 0.01 vs. the si-NC group.