Inhibiting Effects of Down-regulating of Fascin 1 on Proliferation and Migration in Hepatoma Cells

Objective: To investigate the inhibiting effects of fascin 1 gene knock-down on the proliferation and migration of hepatoma cells by means of small interfering RNA (siRNA). Methods: SiRNA targeting fascin 1 gene (si-fascin) and non-specic sequence siRNA (si-NC(cid:0)were constructed and transfected into human hepatoma cell lines (HepG2 and Huh7) to down-regulate the expression of fascin 1. RT-qPCR, Western blotting, and Immunouorescence technique were used to evaluate the eciency of si-fascin. The proliferation and migration of cells were detected by MTT method and Transwell experiments, and the protein expression of genes related to proliferation and migration in cells were detected by Western blotting. The apoptosis and pseudopodia formation of cells were observed under scanning electron microscope (SEM). Results: Compared with human normal liver cells (LO2), the expressions of fascin 1 mRNA and protein were signicantly higher in HepG2 and Huh7 cells. The expression of fascin 1 was overall inhibited in HepG2 and Huh7 cells transfected by the constructed four si-fascins, among which, fascin_siR3 had the highest inhibitory eciency, therefore was selected in this study. In HepG2 and Huh7 cells transfected by si-fascin signicant knock-down target gene expression, while reducing cell proliferation, migration and the formation of pseudopods, and causes reduced protein expression associated with proliferation and migration. Conclusion: This study further conrmed that fascin 1 gene has the function of promoting hepatoma cells proliferation and migration, suggesting that downregulating the expression of fascin 1 in hepatoma cells may be one of the strategies to intervene in liver cancer.


Intruduction
Hepatocellular carcinoma (HCC) ranks the sixth in the most common malignant tumor and the fourth in cancer mortality in the world [1]. Only 40% of early or local HCC patients are suitable for surgical resection, liver transplantation, and local radiofrequency ablation, and 20% are suitable for transcatheter arterial chemoembolization (TACE) and other treatments. Because of the lack of effective early diagnosis methods, about 80% of patients can only be diagnosed in the advanced stage. The prognosis of these advanced patients is poorer, the overall median survival is about 1 to 2 months [2], the main cause of death is the excessive proliferation, in ltration, and metastasis of the tumor, and the lack of effective intervention means. Therefore, it is of great signi cance for improving the prognosis of HCC patients by exploring the key genes that can regulate tumor proliferation and migration and developing them as intervention targets.
Fascin 1 belongs to the Fascin family. It is an actin-binding protein that can tightly bind to F -action, involved in tumor proliferation, growth, invasion and other malignant processes in a variety of human tumors, and is associated with poorer prognosis [3][4][5][6][7][8]. By using small interfering RNA (siRNA) to knock down the gene [9][10][11] or by inhibiting certain signaling pathways, such as ERK, JNK, rpNK-lysin, PI3K/AKT and STAT3 signaling pathways [5,[12][13][14][15], the expression of fascin 1 was down-regulated, that reducing the invasion and metastasis ability of malignant tumor cells. Therefore, it is considered that fascin 1 is probable to become a therapeutic target for some malignant tumors (from epithelial and mesenchymal origin) [16][17][18][19]. In any case, there are still few studies on fascin 1 gene and protein in tumor tissue and cells, and its mechanism and regulatory factors still not very clear, and further research and veri cation are needed.
In this paper, small interfering RNA (siRNA) was used to down-regulate the expression of fascin 1 in human hepatoma cells (HepG2, Huh7), and to investigate the in uence of fascin 1 on proliferation and migration of hepatocellular cancer cells and its meaning of the intervention.

Cells and reagents
Human hepatoma cells (HepG2, Huh7) and human normal liver (LO2) were purchased from the Liver Cancer Institute of Shanghai Zhongshan Hospital and stored in Biomics Biotechnologies (Nantong) Co., Ltd. DMEM medium and fetal bovine serum (FBS) were purchased from Gibco; Trizol, RNA reverse transcription kit and Lipofectamine TM2000 were purchased from Invitrogen; SYBR Green real-time quanti cation kit was purchased from Roche; rabbit anti-Fascin1 antibody, rabbit anti-hepatocyte growth factor (HGF), rabbit anti-bronectin (FN), rabbit anti-matrix metalloproteinases (MMP7)antibody, rabbit anti-B-actin antibody, goat anti-rabbit IgG-HRP, and Goat anti-Rabbit IgG-TRITC all purchased from Proteintech. MTT staining solution was purchased from Nanjing Shengxing Biological Company; Transwell chamber was purchased from Corning Company; siRNA and Real-time PCR primers were synthesized by Biomics Biotechnologies Co., Ltd.
1.2 Methods 1.2.1 Construction and screening of siRNA targeting fascin1 gene siRNAs targeting fascin 1 gene (si-fascin) including four si-fascin sequences (Fascin_siR1, Fascin_siR2, Fascin_siR3, and Fascin_siR4) and one non-speci c sequence siRNA (si-NC) were designed according to the guideline proposed by Tuschl from GenBank. The sequences of above siRNAs were shown in Table 1. Among four si-fascin sequences, the most effective one in down-regulating target genes was screened out for subsequent experiments.

Cells transfection and grouping
The experimental cells were stored in Biomics Biotechnologies (Nantong) Co., Ltd. The cells were grown in DMEM medium containing 10% FBS and routinely cultured in a humidi ed incubator at 37°C with 5% CO2. The density of logarithmic growth cells as adjusted to 1×10 6 cells/mL, and the cells were seeded in a 24-well plate (1 ml per well). SiRNAs was respectively transfected into HepG2 and Huh7 cells according to the instructions of Lipofectamine TM2000 (In each well, appropriate amount of siRNA was add to 50 μL Opti-MEM, then mixed gently. The nal concentration of siRNA is 100 nM).
The experiment groups: si-fascin transfected group(si_fascin), si-NC transfected group (si_NC) and untreated group. The latter two groups served as experimental control groups.

Real-time quantitative PCR (RT-qPCR)
Transfected cells and untreated cells at logarithmic growth phase were collected by centrifugation. The total RNA was extracted separately with Trizol reagent and reversely transcribed into cDNA. RT-qPCR ampli cation was performed according to the instructions of the SYBR Green Real-Time Quantitative Kit. The cycle parameters of PCR ampli cation were 95℃ for 20sec, 55℃ for 30sec, 72℃ for 30sec, which repeated 45 cycles. The 2 -△△Ct value method [20] was used to calculate the relative expression of the target gene. Relative messenger RNA levels were normalized to housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase) from the same sample. The primer sequences were shown in Table 2. All samples were run in triplicate. RT-PCR primers were synthesized by Biomics Biotechnologies Co., Ltd. Table 2 The primer sequences and length

Western blotting
The cells were inoculated into 6-well plates and cultured in 37℃ and 5% CO2 incubator for 24 hours. When the fusion degree was 70% ~ 80%, the cells were harvested and lysed with SDS Lysis Buffer, extracted total protein and determined protein concentration by BCA method. After the protein lysate was electrophoretic with SDS-PAGE (5% laminating gel, 8% separating gel) and transferring for 2 h at 200 mA constant Flow using a wet transfer instrument, the proteins in the gels were transferred onto the PVDF membrane. After blocked for 2 hours at room temperature with TBS blocking solution containing 5% skimmed milk powder, the PVDF membrane was incubated at 37°C for 2 hours with primary antibody, including fascin 1 (1:2000), HGF (1:2000), FN (1:2000), and MMP7 (1:2000), then added the secondary antibody (1:1000) and incubated at 37°C for 2 h. Finally, the cleaned membrane was developed by electrochemiluminescence (ECL) method. Result analysis: the internal reference gene β-actin (1:1000) was used as an internal control, the gray value of the target band was analyzed by Image J software. The relative expression of the target gene is equal to the ratio of the gray value of the target band to the gray value of the internal reference of the same sample.

Immuno uorescence Staining
The growth phase cells were collected and plated for 24 hours. When the fusion degree reached 50% ~ 60%, the cells were xed with 4% paraformaldehyde for 30 min, and treated with 0.5% Triton membrane for 15 min, blocked with 1% BSA solution for 30 min, and incubated with a primary fascin 1 antibody (1:50 diluted) at 4°C overnight, then added goat anti-rabbit IgG-TRITC antibody (1:50 diluted) for 30 min at room temperature. The cells were counter-stained with 1 μg/mL Hoechst in the dark, incubated at room temperature for 10 min, mounted with anti-uorescence quenching mounting solution, observed under a uorescent microscope, and captured image.

MTT detection
In each group, the cells at logarithmic growth phase were harvested by centrifugation, plated in 4 parallel wells of 96-well plate at 5×10 4 cells/mL (100 µl /well). Then, 10 μL MTT was added to each well and store at 37°C in the dark for 4 hours, followed by 150 μL/well of DMSO at 37°C for 10 min. After pipetting and mixing, 120 µL was taken out and transferred to another clean 96-well plate, and 120 µl DMSO was taken as a blank control to zero. Then the OD value was measured at 0 h, 24 h, 48 h, 72 h and 96 h after transfection on the microplate reader with a wavelength of 490 nm, and cell proliferation curve was drawn with time as the horizontal axis and OD value as the vertical axis.

Transwell experiment
The logarithmic growth phase cells were taken to inoculate in 24-well plates, 100μL and 600 μL DMEM complete medium were added to the upper and lower chambers of the Transwell chamber, respectively, then incubated overnight at 37°C with 5% CO2. After 48 h of transfection, the cells were resuspended in DMEM basal medium and the cell density was adjusted to 1×10 6 /mL. After 24 h, the medium in the upper and lower chambers was aspirated and discarded. The cells in the upper chamber were gently wiped with a cotton swab. After washing with PBS, the cells were xed with 10% methanol for 30 sec. The lower chamber was immersed in 0.2% crystal violet solution for 5 min. The membrane of the small chamber was cut off along the edge, and the cells that passed through the membrane and moved to the lower chamber were counted under an inverted microscope, and each membrane was counted with 5 different elds (×250) and photographed.

Preparation of electron microscope samples
The sterilized small round slides were placed in 24-well plates, and cells were respectively seeded on slides, and transfection was performed when the cell climbing density reaches about 60%, then added 2.5% glutaraldehyde and xed at 4℃ for 24 h. The preparation of electron microscope samples was assisted by the Electron Microscope Room of Nantong University.

Statistical analysis
Each group of experiments should be repeated at least 3 times. All data are expressed as mean±SD, and GraphPad Prism 8 software was used for statistical analysis. The comparison between groups of samples used t test. Differences were considered statistically signi cant when P<0.05.

Expression of fascin 1 in HepG2 and Huh7 cells
The expression of fascin 1 in HepG2 and Huh7 cells were detected by RT-qPCR, Western blot and immuno uorescence staining, respectively, and LO2 cells were used as a control (Fig. 1). Figure 1 showed that, compared with LO2, the expression of fascin 1 mRNA and protein in HepG2 and Huh7 cells were signi cantly upregulated (P < 0.01). Immuno uorescence Staining results showed that fascin 1 protein was ne granules diffusely distributed in the cytoplasm of HepG2 and Huh7 cells, while coarse granules were scattered in the nucleus. But almost no expression was seen in LO2 cells (Fig. 1c). These results suggest that the gene and protein of fascin 1 were signi cantly overexpressed in HepG2 and Huh7 cells.

Si-fascins Selection
After four si-fascins were transfected into HepG2 and Huh7 cells, respectively, the effects of si-fascin on target gene knockdown were detected and screened by RT-qPCR (Fig. 2). As shown in Fig. 2, the mRNA level of fascin 1 was signi cantly down-regulated in si-fascin cells (p < 0.05), compared with the untreated cells and si-NC cells. Fascin_siR3 has the best inhibitory e cient in the four si-fascins. Therefore, Fascin_siR3 was used in subsequent experiments.
As shown in Fig. 3

si-Fascin inhibited the migration of HepG2 and Huh7 cells
The results of Transwell migration experiment showed that number of migration cells in si-Fascin treating HepG2 (115 ± 7.23) and Huh7 (125 ± 10.02) were signi cantly less than the si-NC cells (273 ± 10.02) and the untreated cells (247 ± 19.30) (all p < 0.001) (Fig. 4). The results suggested that down-regulating fascin 1 can suppresses the migration of hepatoma cells. 2.6 Morphological changes related to cell growth and migration were observed by scanning electron microscope (SEM) The surface morphology related to cell growth and migration of HepG2 and Huh7 cells transfected by si-fascin was observed by SEM (Fig. 6). As shown in the Fig. 6, the HepG2 and Huh7 transfected by si-fascin were shrinkage, size decreased, and the connection with neighboring cells reduces, showing apoptotic changes, while the cell surface pseudopodia branches decrease and the shorter, loss of some inter-cell connection. On the contrary, si-NC treated cells and untreated cells were normal size and had no obvious tendency of apoptosis; the pseudopodia branches of the cells were more and rich, and abundant inter-cellular connections exist.

Discussion
Fascin was found in the cytoplasm of sea urchin oocytes by Bryan et al. in the 1970s [21]. It was named as fascin because it could bind tightly to F-action and stabilized into bundles. Fascin have 3 forms in humans, named as fascin 1 (also known as fascin), fascin 2 and fascin 3, locating on chromosomes 7p22, 17q25, and 7q31, respectively. The human fascin 1 gene encodes a cytoskeletal protein with a molecular weight of 55 kDa, which is located in cytoplasmic tension bers and laments, lamellar pseudopods, and core actin bundles of microspikes at the edges of cell membrane folds [22]. In normal tissues and organs, fascin-1 is expressed in mesenchymal tissues and nervous system, mainly in endothelial cells, dendritic cells and nerve cells, and plays an important role in cell migration, cell adhesion, and communication between cells [23]. However, there is almost no expression in most normal epithelial tissues. But in human tumors, such as dephosphorylation at fascin S39 [24], activated HIF1α [25], leucine aminopeptidase 3 (LAP3) [26], epidermal growth factor [5] and TGFβ [27], etc, these factors can cause the activation of NF-К, JAK-STAT, ERK-JNK and other signal pathways [12][13]28], which further induced the up-regulation of fascin expression in cancer, and promote the proliferation and invasion of cancer tissues. Especially in metastatic and recurrent lesions, fascin has a higher expression [8, [29][30]. So fascin is even considered as a crucial protein that promotes tumor cells to participate in the invasion function [6]. Similar to the literature report [31], the results of RT-qPCR, Western blot and immuno uorescence staining in HepG2 and Huh7 cells, showed that the expression levels of fascin 1 mRNA and protein were signi cantly up-regulated compared with normal liver LO2 cells. And the four siRNAs speci cally targeting fascin 1 constructed in this article all down-regulated the mRNA expression of fascin 1 gene in HepG2 and Huh7 cells, among them, Fascin_siR3 had the best inhibitory e ciency and was used in subsequent experiments.
Lin et al [32] found that the proliferation of Lewis lung cancer cells lacking fascin 1 was signi cantly slower.
Maria et al [13] found that down-regulation of fascin 1 inhibited the migration and invasion of more aggressive glioblastoma cells. Similar to reports in the literature, MTT assay showed that compared with the transfected si-NC cells and the untreated cells, the cell proliferation ability of the si-fascin cells decreased. Similarly, the results of the Transwell experiment showed that number of migration cells in si-Fascin treating HepG2 and Huh7 were signi cantly less than the si-NC cells and the untreated cells. These suggested that the proliferation and migration ability of hepatoma cells was inhibited after knockdown of fascin 1. This is because fascin participates in the regulation of focal adhesion transition and nuclear translocation in migrating cells [33,34], plays an actin-bundling independent role [35]. Scanning electron microscopy showed that after HepG2 and Huh7 cells transfected with si-fascin, the cellular size was reduced, the surface was wrinkled, and there was a tendency of apoptosis, the cell surface pseudopodia branches decrease and the shorter, loss of some inter-cell connection. can promote metastatic expansion independent of its role in cell motility [32] . Similarly, Western blot detection showed that not only signi cantly decreased Fascin 1 expression, but also signi cantly decreased proliferationrelated genes HGF, migration and invasion related genes FN and MMP7 protein expression in si-fascin treated cells, which may be one of the mechanisms for cancer suppression by si-fascin. The results of this paper are further indicating that the down-regulation of fascin 1 gene may also inhibit the proliferation and migration of hepatoma cells by reducing the expression of proliferation-related genes and migration-related genes. Based on this speculation, the highly expressed fascin 1 may increase the protein expression of FN, MMP7 and HGF, which is bene cial to the metastasis and spread of cancer cells.
In summary, in hepatoma cells, constructed si-fascin (especially by Fascin_siR3) effectively down-regulated the mRNA and protein levels of fascin 1, and inhibited the proliferation and migration, increased cell apoptosis, and reduced the formation of pseudopodia of hepatoma cells. Its inhibitory mechanism on proliferation and migration may partly be due to the low expression of fascin 1 which weakens the protein expression of gene related to proliferation and migration. The research of this project will be a deeper understanding of the invasion and metastasis mechanism of HCC, which will help guide the clinic to nd more effective new targeted treatment sites, block the malignant process of HCC, and thereby improve the clinical e cacy and prognosis of HCC patients. But in any case, the detailed inhibitory mechanism remains to be further studied.
In conclusion, fascin 1 gene can regulate the proliferation and migration of hepatoma cells, fascin 1 can be used as an promising target for intervention in hepatoma.

Funding
This study is supported by the Innovation and Entrepreneurship Training Program for College Students in Jiangsu Province (No.201913993018Y), and the Natural Science Project of Medical School, Nantong University (TDYXY2019012).

Con icts of interest
There is no con ict of interest in this research work.

Ethics approval
Not applicable     Si-fascin down-regulated the expressions of fascin 1, FN, MMP7 and HGF in HepG2 and Huh7 cells The Western blot images showed that, compared with si-NC cells and untreated cells, the protein expression of fascin 1, FN, MMP7 and HGF was signi cantly reduced in HepG2 and Huh7 cells transfected with si-fascin.