Neuropilin1 silencing impairs the proliferation and migration of cells in pancreatic cancer

Abstract Background Neuropilin1 (NRP1) participates in cancer cell proliferation, migration, and metastasis as a multifunctional co‐receptor by interacting with multiple signal pathways, but few studies have addressed the precise function of NRP1 in pancreatic cancer (PACA) cells. We aimed to study whether NRP1 gene silencing involved in the proliferation and migration of PACA cells in vitro. Methods A lentiviral vector expressing NRP1 shRNA was constructed and transfected into human PACA cells (CFPAC‐1 and PANC‐1). The expression of NRP1 protein and mRNA was detected by Western blot and quantitative real‐time polymerase chain reaction (qRT‐PCR) assay, respectively. CCK‐8 assay, wound healing assay, and transwell assay were conducted to examine the effect of NRP1 silencing on cells proliferation and migration capability. Results Results of qRT‐PCR and Western blot showed successfully established, stably transfected shRNA‐NRP1 cells in PACA cells. The proliferation capacity of PACA cells in NRP1 shRNA group was lower significantly than that in the negative control (NC) group (P < .05). The invasion and migration capability of PACA cells in NRP1 shRNA group was lower significantly than that in the NC group (P < .01). Conclusions NRP1‐shRNA lentiviral interference vectors can effectively decrease NRP1 gene expression in PACA cells, thereby inhibiting cells proliferation and migration, which provides a basis for finding a valuable therapeutic target for PACA therapy.


| INTRODUC TI ON
Pancreatic cancer (PACA) is one of the most aggressive human cancer types, accounting for about 2% of all malignant tumors. Owing to the special biological characteristics of the pancreas and its anatomical location, the cancer is associated with high malignancy rate, low early diagnosis rate, and poor prognosis. 1 Globally, the overall 5-year survival rate of PACA patients is <5%. Although radical surgery is potentially the only optimal curative treatment for PACA, a vast majority of patients (80%) have already lost this chance at the time of diagnosis. [2][3][4] In combination with the improvement of standard of living and population aging, we are faced with an increasingly serious challenge of PACA. NRP1 was first found as an axon adhesion protein in the nervous system of frogs, encoding a single type I transmembrane glycoprotein of molecular weight 120-130 kDa. 5 Later research found that NRP1 gene expression is widespread over a variety of cells and tissues, such as endothelial cells, heart, liver, lung, kidney, pancreas, and skeletal muscle. 6 It is a non-tyrosine kinase transmembrane glycoprotein located on the cell membrane, acting as co-receptors for members of the vascular endothelial growth factor (VEGF) family and for secreted Semaphorin3A. In addition, NRP1 also plays a vital role in cardiovascular system, nervous system, immune system, and other aspects of tumor invasion and metastasis through interaction with various ligands, which can promote angiogenesis, neural development, cytoskeleton remodeling, initial immune response, and occurrence and development of tumors. [7][8][9] There is increasing evidence had indicated that higher expression of NRP1 associated with diverse malignant tumors of human, including hepatocellular carcinoma, 10 breast cancer, 11 gastric cancer, 12 and prostatic cancer. 13 NRP1 can promote tumor angiogenesis, cell proliferation, and cell migration, through a variety of mechanisms that play a vital role in the progression of cancer. [14][15][16] The role of NRP1 in malignant tumors of human has become a research hotspot in recent years.
However, to our knowledge, the expression level, biological function, and underlying mechanisms of NRP1 in PACA remain poorly understood. 17 The important role of NRP1 in many malignant tumors has prompted us to study whether NRP1 is a potential therapeutic target for PACA. In this research, we observed CFPAC-1 and PANC-1 cells in vitro for cell proliferation, invasion, and migration in human PACA after NRP1 knockdown by lentivirus-mediated RNA interference. Our study provides an experimental basis for gene-targeted treatment of PACA.

| Human PACA cell lines
CFPAC-1 and PANC-1 cells were purchased from the Shanghai Cell Bank and cultured under standard condition containing PMRI 1640 medium (Gibco) supplemented with 1% Penicillin-Streptomycin (100 IU/mL; Gibco) and 10% fetal bovine serum (Gibco) with 5% CO 2 at 37°C. When the cell density was consistent with the density indicated in the previous article, lentivirus transfection and cell function assays were carried out. 18 All operations were carried out in the biosafety cabinet after ultraviolet sterilization.

| Lentivirus vector and transfection
Based on the results of bioinformatics, we selected three short hairpin RNA (shRNA) targeting human NRP1 se-

| Quantitative real-time polymerase chain reaction (qRT-PCR)
In this work, qRT-PCR was performed according to the previously reported method. 18

| Protein extraction and Western blot (WB) analysis
In this study, protein extraction and WB assays were carried out according to the previous method. 18 Briefly, the protein extraction reagents and enhanced chemiluminescence kit were purchased from Thermo, Israel. The NRP1 antibody was purchased from Abcam company. Finally, the relative proteins were quantified by Image-Pro Plus 6.0 (Media Cybernetics). All experimental details were given in Supporting information.

| Cell proliferation, invasion, and migration assays
To determine cell proliferation activity, we performed CCK-8 assay, experiments in 6 replicates. To determine cell invasion and cell migration capability, we performed transwell assay and wound healing assay, experiments in 3 replicates. The above assays were carried out according to the previous method. 18,20 All experimental details were given in Supporting information.

| Statistical analysis
All data were analyzed using SPSS 20.0 software (IBM Corp.), and were summarized and presented as the mean ± SD. Student's t test and ANOVA (Tukey's multiple comparison test) were used to compare statistical significance in the various groups. P < .05 was considered statistically significant difference.

| Detection of NRP1 mRNA expression in the target cells after transfection with qRT-PCR
In order to detect the transfection efficiency of sh1 NRP1, sh2 NRP1, and sh3 NRP1, on the third day after transfection, mRNA was extracted from all types of cells. Then, the expression level of NRP1 mRNA was detected by qRT-PCR. Results showed that NRP1 mRNA level in three shRNA targeting groups reduced significantly compared to that in the negative control (NC) group (P < .01; Figure 1).
Meanwhile, as shown in the Figure 1, the sh1 NRP1 interference worked best.

| Determination of transfection efficiency
In order to further confirm the transfection efficiency of sh1 NRP1, on the third day after transfection, the lentivirus-trans-

| NRP1 silencing impairs proliferation in PANC-1 and CFPAC-1 cell lines in vitro
In the CCK-8 assay, the proliferation ability of NRP1 shRNA group was apparently worse than that of NC and blank groups in the PANC-1 cells, especially in the 6 and 7 days (P < .01). CFPAC-1 cells showed the same trend, and the proliferation ability of NRP1 shRNA group was apparently worse than that of NC and blank groups in the 7 day (P < .05), hence suggesting that the proliferation capacity of NRP1 shRNA group was apparently lower. The results revealed that eliminating NRP1 can continuously and effectively impair proliferation in PANC-1 and CFPAC-1 cells (Figure 4).

| NRP1 silencing impairs invasion and migration in PANC-1 and CFPAC-1 cell lines in vitro
For the PANC-1 transwell migration assay, the average number of transmembrane cells was 46.5 ± 3.8 in the NRP1 shRNA group, 228.4 ± 6.7 in the NC group, and 256.1 ± 5.6 in the blank group.

| D ISCUSS I ON
Currently, the expression and role of NRP1 in tumors have attracted the attention of researchers. Much evidence has confirmed NRP1 is overexpression in a range of human malignant tumors and is also closely related to the degree of malignancy. [21][22][23] Previous published studies have indicated that NRP1 protein is highly expressed in human PACA tissues, and it is closely related to angiogenesis, tumor stage, and poor postoperative overall survival. 24 Although there are some reports about the relationship between NRP1 and PACA, research related to proliferation and migration of NRP1 in PACA has been rarely reported. [24][25][26][27] Therefore, we were interested in the effect of NRP1 on PACA cells function.
To confirm the effect of NRP1 in human PACA cells, RNA interference (RNAi) technology was used to explore the relationship between silencing NRP1 and the ability of proliferation, invasion, and

| CON CLUS IONS
This study has shown that NRP1-shRNA lentiviral interference vectors can effectively decrease NRP1 gene expression in PANC-1 and CFPAC-1 cells, thereby inhibiting cell proliferation and migration.
These findings contribute in several ways to our understanding of NRP1 and provide a basis for finding a valuable potential therapeutic target for PACA therapy.

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest with respect to the research, authorship, and publication of this article.

DATA AVA I L A B I L I T Y S TAT E M E N T
The analyzed data and materials during the study can be obtained from the corresponding author on reasonable request. E-mail address: doctor1909@163.com.