The Role of F11R in Pancreatic Cancer Malignancy and Its Clinical Implication as a Therapeutic Target

Background The F11 receptor belongs to the immunoglobulin superfamily and is expressed in epithelial and endothelial cells. F11R mediates the formation of tight junctions between the epithelium and endothelium, and participates in the invasion and metastasis of tumor cells. We have previously shown that the F11R gene is closely related to KRas (P= 0.76), a known therapeutic target for pancreatic cancer (PCa). In recent years, it has been found that F11R is expressed in different tumors and has biological effects.However, according to different tumor cases, different cell lines and experimental conditions, the regulatory results and mechanisms of F11R on tumor are different, even contradictory,and the expression, clinical significance and biological mechanism of F11R in tumor tissues have not been reported in detail. This study used bioinformatics combined with gene chip data to find the gene F11R, which is closely related to KRAS gene, and we used lentivirus to package shRNA plasmid to interfere with the gene F11R in pancreatic cancer panc-1 cells. A series of biobehavioral studies indicated the biobehavioral function and malignancy of panc-1 in pancreatic cancer cells with negative regulation of F11R gene.Based on this, we need to continue to clarify the expression of F11R gene in clinical case samples to determine whether F11R gene can be a new therapeutic target for pancreatic cancer.


Conclusions
This study used bioinformatics combined with gene chip data to find the gene F11R, which is closely related to KRAS gene, and we used lentivirus to package shRNA plasmid to interfere with the gene F11R in pancreatic cancer panc-1 cells. A series of biobehavioral studies indicated the biobehavioral function and malignancy of panc-1 in pancreatic cancer cells with negative regulation of F11R gene.Based on this, we need to continue to clarify the expression of F11R gene in clinical case samples to determine whether F11R gene can be a new therapeutic target for pancreatic cancer.
Keywords : F11R (-/-);pancreatic cancer;genome editing;malignancy degree;cellular behavior Background Pancreatic cancer (PCa) is one of the most aggressive malignant tumors that remains difficult to diagnose and treat. Although progress has been made regarding PCa epidemiology, the mortality rates of PCa patients remain high, and the 5-year survival rates are low (1). The early clinical symptoms of PCa are not obvious, and early detection is rare. Surgical resection with adjuvant chemotherapy remains the mainstay of curative treatment,due to the high degree of malignancy and rapid progression of PCa, the optimal treatment time has often passed upon diagnosis (2).
Early diagnostic and more effective intervention strategies are required to improve the survival rates of PCa patients.
Malignant tumor formation is both multi-stage and progressive. Tumorigenesis is driven by endogenous mutations to genes and exogenous cancer-promoting factors.
Many tumor cells gradually evolve into clinically visible pathological forms. The activation of proto-oncogenes coupled to the inactivation of tumor suppressor genes are known drivers of tumorigenesis (3). Proto-oncogenes are a class of potentially carcinogenic genes that regulate cell growth, differentiation, and apoptosis.
Proto-oncogenes have low levels of expression in normal cells, but when overexpressed promote cancer formationis (4). Like other malignant tumors, PCa is caused by external stimuli that disrupts the balance between proto-oncogenes and tumor suppressor genes.
Human F11R receptor belongs to the immunoglobulin superfamily and promotes tight junction formation between epithelial and endothelial cells is (5). F11R is a 27 KD protein that promotes tumor metastasis and embryogenesis, widely expressed in neutrophils, monocytes, platelets and lymphocytes is (6). F11R regulates epithelial and endothelial cell movement, leukocyte migration, platelet activation, and cell barrier integrity is (7). The regulation of F11R is closely related to its ability to dimerize mediated through intracellular PDZ binding motif is (8).
In the present study, we quantitatively measured the expression of F11R in five pancreatic cancer cell lines(MIA paca-2, bxpc-3, cfpac-1, SW1990, PANC-1) by real-timePCR. F11R level in tumor specimens from PCa was increased significantly compared with level in nonneoplastic tissues.To investigate the role of F11R in carcinogenesis, immunohistochemical studies were done in pancreatic intraepithelial neoplasias.In addition, to evaluate the functional role of F11R and its possible therapeutic implications,we inhibited expression of F11R using RNA interference(RNAi) and investigated its effect on proliferation and invasiveness,at the same time, its effect on cell cycle and apoptosis of pancreatic cancer cells in vitro.
F11R is expressed in a range of tumors but its role in tumorigenesis remains controversialis (9)(10)(11). Histopathological and cytological studies on endometrial carcinoma, prostate cancer, renal cell carcinoma, and some breast cancer cases have shown that low F11R expression positively correlates with tumor cell invasion and motility, tumor grade, stage, and poor prognosis is (12).In contrast, histopathological, cytological, and microRNA analysis suggested that F11R expression in breast and endometrial carcinoma are negatively correlated is (13). The clinical significance and biological roles of F11R in tumor tissue has not been explored in detail. In this study, we silenced F11R in the PCa cell line PANC-1 and assessed its oncogenic potential.Taken together, the data suggest that F11R is a promising therapeutic targets for PCa.

Methods
The PCa cell line PANC-1,MIA paca-2, bxpc-3, cfpac-1, SW1990 were purchased from the cell bank of the Chinese Academy of Sciences in Shanghai.Tissue samples were obtained from 30 patients who underwent surgery of the pancreas between July, 2013 and July, 2018 at the first affiliated hospital of North Sichuan Medical College,Nanchong, China. Written informed consent was obtained from all patients, and the study was conducted according to the Helsinki Declaration.Fetal Bovine Serum (FBS) was purchased from HyClone, USA. High glucose DMEM was purchased from Gibco, USA. Trypsin-EDTA (0.25%) was purchased from Gibco.
Mouse anti-human F11R monoclonal antibodies and rabbit anti-human GAPDH monoclonal antibodies were purchased from Santa Cruz Corporation. HRP-labeled goat anti-rabbit antibodies and goat anti-mouse antibodies were purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. Penicillin, streptomycin and ampicillin were purchased from Shanghai Shenggong engineering Co., Ltd.

Bioinformatics analysis genechips of PCa cells
Affymetrix expression profiling gene chip of PCa cells were purchase by Shanghai Genechem Co., LTD(Shanghai China).In this study, gene chips of PCa cells were produced to screen genes of importance to PCa development and KRas signaling.
F11R was found to be highly expressed in all PCa samples.In addition to,using the TCGA database, we found that F11R expression varied according to stage. In addition, lower F11R expression was an indicator of prolonged survival.

Immunohistochemical studies
Thirty tumoral tissues were obtained from patients who underwent surgery for pancreatic cancer.Sections of formalin-fixed,paraffin-embedded specimens were

RNAi lentiviruses transduction
Cells were transfected with F11R RNAi lentiviruses synthesized by Shanghai GeneChem Co Ltd. Lentiviral vectors expressing green fluorescence protein (GFP) were used as controls. Silencing was confirmed through two primer pairs: HT-29 cell transductants were selected for 1 week using puromycin (Thermo Fisher Scientific, 2μg/mL).

Western blot analysis
Total proteins from infected cells were extracted and protein expression was determined by western blot analysis. Cell lysates containing 5 x SDS sample buffer were boiled at 95 ℃ for 5 min and 30 ug protein was resolved by SDS PAGE.
Proteins were wet-transferred to 2.5um PVDF membranes at 200 mA for 2 h and blocked in 5% milk in PBS-T for 1 hour. Membranes were probed with primary antibodies overnight at 4°C and labeled with the appropriate secondary antibodies at room temperature for 1 h. Protein bands were visualized using the BeyoECL system and imaged on a fluorescence imager.

Quantitative real-time PCR
Total RNA was extracted from five pancreatic cancer cell lines using RNAiso Plus reagent (Takara). Reverse transcription was performed using the PrimeScript RT reagent kit (Takara). The resulting cDNA arrays (Shanghai GeneChem), were used to examine the mRNA levels of Absorbance's were measured at 450 nm.

Colony forming assays
Cells that can divide and form clones are both adherent and proliferative. The clone formation rate reflects two important characters of the cell population, namely dependence and proliferation ability. NC and RNAi-F11R cells were plated into 600-well plates containing DMEM plus 10% FBS. Colonies were visible after~1 week. The media was removed, cells were washed in PBS, and fixed with 4% paraformaldehyde for 20 min. Colonies were stained in 0.1% crystal violet for 2 h and imaged using Carestream Molecular Imaging software. Colony formation rates were calculated as follows: (%) = number of clone's generated/the total number of inoculated cells x 100%.

Flow cytometry
Cells in the logarithmic phase were detached with trypsin and cell suspensions were treated with cell cycle and apoptosis analysis kits (C1052). Stained cells were analyzed on an ACEA NovoCyte.

Apoptosis analysis
After 72 hours of lentivirus transfection, cells in the logarithmic phase were detached with trypsin and cell suspensions were labeled with the Annexin v-PI apoptosis assay kit (C1065M Beyotime Shanghai). Samples were analyzed on an ACEA NovoCyte.

Invasion Assays
For Leica DC 300F microscope, and count by Image J.

Reactive oxygen species (ROS)
Cells were seeded at a density of 1 x 10 4 cells in six-well plates and treated with DCFH-DA after 24 hrs. DCFH-DA is hydrolyzed by intracellular esterases into DCFH, which is oxidized to fluorescent DCF by ROS. The fluorescence intensity of DCF is therefore proportional to ROS production. Cells were treated with DCFH-DA in serum-free medium and cells were washed in PBS. The OD values were measured at

Statistical analysis
Images were analyzed on Image J (National Institutes of Health).Statistical analysis were performed using SPSS 13.0. Data were compared using an unpaired two tailed Student's T-test. A P < 0.05 (*) and P < 0.01 (**) were considered statistically significant.

Bioinformatics analysis of F11R about pancreatic cancer
Upon bioinformatics analysis of F11R expression in 179 pancreatic cancer tissues and 171 normal tissues, the expression of F11R was significantly higher in pancreatic cancer ( Fig 1A).Through bioinformatics analysis of the gene chip data, F11R was found to be highly expressed in PCa. GO enrichment analysis of the differentially expressed mRNAs revealed a high correlation between F11R and KRas (Tab1,p= 0.76).F11R expression in the pancreatic cancer tissue also varied according to stage and grade,the expression of F11R in pancreatic cancer patients of different ages were also assessed ( Fig 1B).Data from the TCGA database showed that low F11R expression can prolong the overall survival rates of pancreatic cancer patients( Fig 1C).

F11R expression in pancreatic cancer cell lines and PCa pathological specimens
To confirm expression of F11R in pancreatic cancer cell lines,the expression of F11R in 5 pancreatic cancer cell lines was analyzed by q-PCR, and F11R was highly expressed (Fig 2A).For subsequent experiments, we selected PANC-1 cells because its expressed moderate levels of F11R. A total of 30 clinical pancreatic cancer paraffin pathological specimens from 2013 to 2018 were assessed, and 300 pathological sections were produced, 5 of which were collected for immunohistochemical staining.
The expression of F11R in the samples reached 86 % (Fig 2B).The results between the groups were consistent, demonstrating that F11R was highly expressed not only in pancreatic cancer cell lines, but also in the pathological tissues of pancreatic cancer patients.

Lentiviral transduction and Confirmation
The transfection efficiency of the lentiviruses reached more than 90% in PANC-1

cells. Western blot analysis of F11R expression in PANC1 cells transfected with
lentiviruses showed that the expression of F11R proteins were significantly lower after lentivirus transfection.

Cell proliferation
Enhanced cell proliferation is a characteristic feature of tumor cells. Cell Counting Kit-8 ( Figure 4A) and colony formation assays ( Figure 4B) were performed to assess the proliferation of PANC-1 F11R -/-cells. Comparable proliferation rates were observed between Control and NC cells. However, cell numbers in the F11R -/group were significantly lower than the control and NC groups, indicating reduced proliferation (P < 0.05).

Flow cytometry
Flow cytometry (FCM) was used to assess the cell cycle distribution of PANC-1 F11R -/-cells. Compared to control and NC groups, the F11R -/-group showed higher levels of GO/G1 arrest, and lower S-phase progression ( Figure 5). The G2 phase remained unchanged by F11R silencing.

Apoptosis assays
Cancer cells are typically refractory to apoptotic stimuli. Annexin v-PI apoptosis assay kits (C1065M Beyotime Shanghai) were used to assess apoptotic induction in control, NC, and F11R -/-cells by flow cytometry (Figure 6).

PANC-1
RNAi-GFP RNAi-F11R Figure 6. Apoptosis assessments. The number of apoptotic cells significantly increased following For early PCa detection, surgery can remove the tumor and other pathological tissue, but the lack of early PCa symptoms means most patients are diagnosed during late disease stages,the postoperative survival rates of PCa are low (15% to 25%) (24)(25)(26).
Recent advances in gene therapies offer novel opportunities for treatment, based on previous studies of PCa genes (27)(28)(29)(30)(31).KRas is implicated in PCa development and its reduced expression can reduce the degree of malignancy (32). The production of KRAS specific inhibitors is challenging, and many inhibitors lack specificity and affinity (33)(34)(35). In addition, the RAS protein lacks small molecules that can target natural binding sites is.After 30 years of in-depth research, no Ras inhibitors are clinically available (36). We previously investigated dysregulated genes in PCA using bioinformatics and gene chip data,and discovered that F11R, a gene closely related to KRAS (p= 0.76), was significantly upregulated in PCA tissue.This provides a new idea for this study --to solve the problem of KRAS as a gene therapy target in the application of PCa by interfering with F11R.
The F11R receptor belongs to the immunoglobulin superfamily and is expressed in epithelial and endothelial cells (37)(38)(39)(40). Bioinformatics strategies were based on the interconnectivity of intracellular signaling networks, which may also be applicable to other malignant tumors (42)(43).
Compared to surgery, radiotherapy, and chemotherapy, gene therapy has many advantages, including low cytotoxicity, low side effects, and high tolerance. There are already reports that,F11R antagonists as a therapeutic agents for the prevention and treatment of thrombosis, atherosclerosis, heart attacks, stroke and other clinical disorders (44)(45),but whether F11R antagonists can be used in treating malignant tumors is still unknown.The identification of F11R as a novel anti-PCa target therefore holds interest from the perspective of gene therapy and precision treatment.
Goetsch found that F11R monoclonal antibodies could reduce MCF-7 breast cancer cell proliferation is (46). Phosphoinositide 3-kinase (PI3K) and protein kinase C (PKC) inhibitors F11R-induced migration providing mechanistic insight into its oncogenic effects (47). The effects of F11R antibodies in PCa have not been investigated. The behavioral changes in PCa cells caused by the low expression of F11R remain unclear, but may be related to the immunologic responses of F11R.This will be the focus of our future PCa studies.

Additional files
Additional file 1: Table 1

Availability of data and materials
All the data presented in this study is provided free and open to be used,included in the Supplementary Files that are quoted and described alongthe manuscript.

Consent for publication
Not applicable. Figure 1 Bioinformatics analysis of F11R about pancreatic cancer. A: Bioinformatics analysis showed that the expression of F11R in pancreatic cancer was higher than that of normal tissue (P < 0.05). B: Data from the TCGA database showing the variable expression of F11R in pancreatic cancer tissue from different stages , different grades and different age. C:Data from the TCGA database showed that low expression of F11R prolongs the OS of pancreatic cancer patients.     PANC-1 cell migration and invasion in vitro.In the F11R -/-group, the number of cells Transwell assay signi cantly decreased, indicating a loss of invasion. Cell numbers were calculated using ImageJ.

Figures
Transwell assays showed that the number of migrating cells signi cantly decreased following F11R silencing.(* represent P<0.05) Figure 8 ROS production in PANC-1 cells. Fluorescence intensity represents intracellular ROS levels. ROS production was assessed through comparison of the uorescent intensities of DCFH-DA staining in Control, NC and F11R groups.(* represent P<0.05)