Decreased miR- 31 Suppress Cell Migration and Proliferation By Targeting Syncytin-1 in Pancreatic Carcinoma

Background: Pancreatic cancer(PC) is seriously harmful to human health, and the pathogenesis is not clear. The present study aimed to explore the functional role of syncytin-1 in PC. Methods: Syncytin-1 and miR-31 expression was analyzed by qRT-PCR and Western blot analysis in both human PC cell lines and tissuse. The prognostic signicance of syncytin-1 was investigated using the immunohistochemistry(IHC) and Kaplan-Meier survival. The CCK-8 assay and transwell assays were used to determine the role of syncytin-1 and miR-31 in cell proliferation, migration and invasion. Luciferase reporter assays was used to identify possible miRNA targets in tumorigenesis. Results: The results showed that the syncytin-1 level was signicantly decreased in PC cell lines and tissues than normal(P < 0.05), while miR-31 was markedly higher than normal(P < 0.05), and low expression of syncytin-1 have a poor prognosis than high expression(P < 0.05). Overexpression of syncytin-1 signicantly reduced the PC cell proliferation and invasion ability in PANC-1 and BxPC-3 cells(P < 0.05), and miR-31showed contrary results. The Dual-Luciferase reporter gene assay demonstrated that miR-31 binded directly to 3’UTR of syncytin-1 and resulting in the inhibition of syncytin-1. The overexpression of miR-31 promoted migration and proliferation of PC cells through down-regulating the expression of syncytin-1. Conclusion: We veried that syncytin-1 can inhibit proliferation and invasion of PC cell lines by targeting miR-31.


Introduction
PC is a disease that is insidious, rapid, and malignant high, therapeutic and prognosis of digestive tract malignancies [1,2]. Although, new breakthrough in treatment, only 10% ~ 20% of PC patients can be resected surgically, and their 5-year survival rate is only 4% [3]. So, it is very important to further improve the mechanism of PC. In recent years, more and more evidences showed that the expression of syncytin-1 was related to the occurrence of tumor.
Studies have indicated that syncytin-1 and estrogen stimulation induction is closely related, and which is in the cytotrophoblast fusion into the process of syncytium trophoblast key action [15]. It has been con rmed that the expression level of syncytin-1 is related to clinical manifestations in organ cancer patients [16], acute myeloid leukemia [17], breast cancer [18], endometrial carcinomas [19], ovarian cancer and colorectal cancer [20,21]. In this study, we con rmed the role of syncytin-1 in the carcinogenesis of PC. Furthermore, we proved miR-31 is the upstream gene targeting syncytin-1. Which indicated that syncytin-1 may play an anti-cancer role in PC through miR-31 pathway, is a potential target of PC therapy.

Materials And Method
Clinical Samples From January 2015 to January 2017, we collected the pathological tissue samples of 60 patients with PC who were operated in the Binzhou medical University Hospital. All the patients signed the informed consent form. Inclusion criteria: 1) radical surgical resection; 2) Clear pathological results; 3) Standardized completion of adjuvant therapy; 4) The survival time after operation exceeded 1 month.
Western blot was carried out on the BioRad microgel system (Biosharp, USA). The protein of syncytin-1 and GAPDH in the primary antibody was detected by diluting anti syncytin-1 and anti GAPDH antibody (Beyotime, China) overnight at 4 ℃. The strip was detected by colorimetry and exposed to autoradiographic lm. Cell Counting Kit (CCK) − 8 assay and Transwell assay PANC-1 and BxPC-3 cell lines, sow in 96 well plates for 24 h. Then, at different time points, each well is added with analytical reagent (beyotime, China), and then incubated. The absorbance value at 450 nm was recorded, and the results at four time points were averaged. The cell survival rate were calculated as the percentage of untreated control. Each test was conducted in three wells and repeated three times.

Plasmid construct and transfection
Detection of cell migration ability: after digestion and cultivation of logarithmic cells, the concentration was adjusted to 2 × 10 5 /ml. Cells were incubated at 37 ℃ for 24 hours. The cells were washed twice, xed for 30 minutes and stained for 15 minutes. After drying, the number of cells migrating or invading was analyzed under microscope from 5 randomly selected elds at a magni cation of × 100.

DNA constructs and luciferase reporter assays
In order to construct miR-31-luc report plasmid, the 3′-UTR fragment of syncytin-1 containing miR-31 binding site was cloned into the modi ed pGL3 promoter vector (Beijing transgen biotech). Using the following primers, PCR was used to amplify the full-length 3 '-UTR of syncytin-1. All luciferase data were expressed as the normalized luciferase / renin ratio.

Correlation Analysis of Syncytin-1 Expression and Clinicopathological Parameters and Overall Survival of PC
We analyzed the expression of syncytin-1 in PC and its relationship with clinicopathological factors by IHC. The results showed that syncytin-1 was mainly distributed in the cytoplasm (Fig. 1A, B). Syncytin-1 was highly expressed in 21 out of 60 (35.00%) cases of adjacent tissues and in 11 out of 60 (18.33%) cases of PC with signi cant difference (χ2 = 4.261, P = 0.039). Statistical analyses were performed to explore the correlation between syncytin-1 expression and the clinical characteristics of PC as detected by immunohistochemical staining. The result of Chi-square test showed that syncytin-1 expression was positively correlated with grade (P = 0.041) and tumor T stages (P = 0.021)(  Table 1 Correlation between syncytin-1 expression and clinicopathological characteristics Kaplan-Meier analysis was used to assess the overall survival according to syncytin-1 expression and to further explore the correlation between syncytin-1 expression and PC survival. The 1-year, 3-year cumulative survival rate was 63.6%, 27.3% in patients with PC and high syncytin-1 expression; and 38.8%, 18.4% in those with low expression of syncytin-1. The patients with PC and low expression of syncytin-1 have a poor prognosis (Fig. 1C, χ 2 = 4.079, P = 0.043). Therefore, syncytin-1 expression is associated with survival and prognosis. Further, univariate and multivariate Cox regression analyses were performed to determine whether the expression of syncytin-1was an independent prognostic factor of patients' outcomes. The results revealed that the M stage and TNM stage was identi ed as independent prognostic factor, the expression of syncytin-1was not identi ed as independent prognostic factor ( The Expression of Syncytin-1 in PC Tissues The expression levels of syncytin-1 was evaluated in human PC samples by qPCR and western blot analysis. The data from qPCR analysis showed that the expression of syncytin-1 was expressed at low levels in PC samples compared with normal tissues (P < 0.01; Fig. 1D). In addition, western blot analysis in 3 pairs of PC samples also revealed low syncytin-1 expression level in PC tumors (P < 0.01; Fig. 1E,F).

The Expression of Syncytin-1 in PC cell lines
We evaluated the expression of syncytin-1 in PC cell lines PANC-1, BxPC-3, AsPC-1 and normal pancreatic epithelial cell line HPC-Y5. In the present study, the expression detection of syncytin-1 was subjected to western blot analysis. We found that the expression of syncytin-1 was signi cantly decreased in PANC-1, BxPC-3, and AsPC-1 cells, while the expression of syncytin-1 was obviously increased in normal HPC-Y5 cells, and the expression of endogenous syncytin-1 in PANC-1 cells was signi cantly higher than that in other cancer cells, while BxPC-3 cells is the lowest (Fig. 2A, B). This result provides a basis and premise for the subsequent exogenously highly expressed adenovirus syncytin-1 or sh-Syncytin-1 as a tool to study the effects of syncytin-1 on the function of both cells PANC-1 and BxPC-3.

Syncytin-1 inhibits PC cell proliferation, migration and invasion in vitro
To investigate whether syncytin-1 is essential for PC cell proliferation, We generated stable syncytin-1overexpression BxPC-3 cell lines, syncytin-1-knockdown PANC-1 cell line, as well as corresponding control cell lines. Compared with the vector control group using a CCK8 assay and Plate cloning experiment overexpression of syncytin-1 signi cantly suppressed BxPC-3 tumor cell growth ( P < 0.01, Fig. 2C-E).

Overexpression of syncytin-1 inhibits PC cell migration and invasion
Because we found that overexpression of syncytin-1 inhibited PC cell proliferation, we further assessed the effect of syncytin-1 on migration and invasion, the data showed that overexpression of syncytin-1 markedly suppressed the migration and invasion capacities of BxPC-3 (P < 0.01, Fig. 3A-D). While the shsyncytin-1 in PANC-1 cells showed that markedly promoted the migration and invasion capacities (P < 0.01, Fig. 3E-H).
The 3′-UTR of syncytin-1 is a possible target of miR-31 We would like to know if miRNAs are involved in syncytin-1-induced PC generation. In order to solve this problem, we used Diana, targetscan, and pita database to analyze bioinformatics, and constructed the ceRNA network of syncytin-1. We found that there were three miRNAs (including miR-31, mir-202 and mir-108) in the network. In addition, Starbase program was used to predict the potential miRNA targets of syncytin-1. Among these miRNAs, only the expression of miR-31 was negatively correlated with the expression of syncytin-1 in PC samples (P < 0.01), miR-31 binds to the syncytin-1 mRNA 3'UTR and downregulates the synthesis of syncytin-1 protein to potentially regulate the biological processes of cancers (Fig. 4A). Luciferase report analysis showed that miR-31 mimic signi cantly inhibited the activity of syncytin-1 wild-type reporter plasmid, indicating that syncytin-1 directly affected miR-31 (Fig. 4B).

Up-regulation of miR-31 in human PC
The expression levels of miR-31 were evaluated in human PC samples. Our results show that the expression of miR-31 was (3.72 ± 0.51) in PC tissues of patients, markedly higher than (2.09 ± 0.27) in adjacent tissues, with a signi cant difference (t = 17.632, P = 0.012; Fig. 4C). Indicating miR-31 might be involved in the pathological development of human PC.
Overexpression of miR-31 result in promotion of cell migration and proliferation, reduce the protein levels of the syncytin-1 To explore the role of miR-31 involved in PC, we transfected BxPC-3 and PANC-1 cell lines with miR-31 mimics to upregulate their expression. The transwell assay and CCK8 assay showed that the migration and proliferation rate of BxPC-3 and PANC-1 cells were signi cant increased after overexpression of miR-31 (Fig. 4D). The downregulation of the syncytin-1 protein levels were also detected by western blot assay after the transfection ( Fig. 4E).

Discussion
More and more studies show that the critical roles of syncytin-1 in human diseases [22][23][24][25][26][27][28]. However, the functional role of syncytin-1 is complicated. The integrin of syncytin-1 vary signi cantly among different cancers [29][30][31][32][33][34][35]. For example, in colorectal cancer, the methylation level of syncytin-1 promoter is higher than the normal tissues [36], but is lower in the testis and endometrial cancer [37,38], which is related to tissue types and their signaling pathways. Nevertheless, the signal pathway of syncytin-1 in PC is not clear. In order to clarify the role and pathway of syncytin-1, we analyzed the effect of syncytin-1 on cell proliferation and cell transfer, as well as the relationship between syncytin-1 and upstream miRNA through PC cells.
On the basis of our experimental data in vitro, the expression of syncytin-1 was downregulated either in PC tissues and cell lines, compared with normal tissues and cells. Further study showed that the expression of syncytin-1 was related to T stage and pathological grade. In CCK8 and Transwell experiments, it could signi cantly inhibit the proliferation and invasion of PC cell lines, indicating that syncytin-1 may play an anti-cancer role in the process of PC development.
MiRNAs are a kind of noncoding RNAs, which have been proved to play a role in the post-transcriptional level by targeting the 3'-untranslated region (3'-UTR) of the downstream gene [39][40][41][42][43]. We use variety of online tools to predict the potential RNAs for syncytin-1, including DIANA, TargetScan, and PITA. Among these miRNAs, only the expression of miR-31 was negatively correlated with the expression of syncytin-1 in PC samples (P < 0.01), miR-31 binds to the syncytin-1 mRNA 3'-UTR and down-regulates the synthesis of syncytin-1 protein to potentially regulate the biological processes of cancers, and miR-31 mimic signi cantly inhibited the activity of syncytin-1 wild-type reporter plasmid, indicating that syncytin-1 directly affected miR-31.

Conclusion
To sum up, we con rmed the antitumor effect of syncytin-1 in the development of PC by its expression in tissues and cell lines. In addition, miR-31 can play a regulatory role by targeting the 3'-UTR of syncytin-1 mRNA, further clarifying the mechanism of syncytin-1.

Declarations
Ethics approval and consent to participate: Ethical issues (Including plagiarism, informed consent, misconduct, data fabrication and/or falsi cation, double publication and/or submission, redundancy, etc.) have been completely observed by the authors.

Consent for publication:
Authors grant World Journal of Surgical Oncology to publish the article.
Availability of data and materials: The author states that the data and materials are authentic.
Competing interests:   Comparison with the shNC group using a CCK-8 assay and Plate cloning experiment con rmed that shsyncyntin-1 signi cantly promotes the proliferation in PANC-1 tumor cell growth (*P < 0.01). Comparison with the shNC group using a CCK-8 assay and Plate cloning experiment con rmed that shsyncyntin-1 signi cantly promotes the proliferation in PANC-1 tumor cell growth (*P < 0.01).   Overexpression of miR-31 result in promotion of cell migration and proliferation, reduce the protein levels of the syncytin-1 (A) miR-31 directly targets the 3'UTR of syncytin-1 mRNA. The predicted target site of miR-31 (middle) in the syncytin-1-3'UTR region (bottom) was detected by three software. (B) The dualluciferase activity assay was used to analyze the relative luciferase activities, miR-31 mimics reduced the uorescence intensity of syncytin-1-3'-UTR cells in a dose-dependent manner, while the miR-NC did not change the uorescence intensity of syncytin-1-3'-UTR cells. Each bar represents the mean ± SEM of three independent experiments. *P < 0.001, compared with miR-NC. (C) qRT PCR showed that the expression of miR-31 was signi cantly higher in the PC tissue than that in the tissue adjacent to carcinoma, with a signi cant difference (P=0.012). (D) The transwell assay and CCK8 assay showed that the migration and proliferation rate of BxPC-3 and PANC-1 cells were signi cant increased after overexpression of miR-31.**P < 0.001, compared with Control. (E) Overexpression of miR-31 result in downregulation of the syncytin-1 protein levels of BxPC-3 and PANC-1 cells were detected by western blot. *P < 0.001, compared with miR-NC.