Molecular Characterization, Expression and Functional Analysis of Yak IFITM3 Gene

Background IFITM3 is interferon-induced transmembrane 3, which plays an extremely key role in anti-proliferation, anti-virus and anti-tumor diseases. To expand our understanding of the role of IFITM3 in yak, this experiment studied its function. Results Firstly, the yak ( Bos grunniens ) IFITM3 ( BgIFITM3 ) gene contained a 5’-untranslated region (UTR) (25 bp), a coding region (441 bp), and a 3’-UTR (115 bp). The expression of BgIFITM3 gene in liver was significantly higher than that in heart, spleen, lung and kidney ( P <0.01). BgIFITM3 protein was localized on the yak hepatocyte membrane, and its expression level was increased first and then stabilized from 1 day to 5 years of age. Moreover, the prokaryotic expression vector of BgIFITM3 protein was constructed and expressed successfully, with a molecular weight of 19.5 kDa. Besides, the activity of yak hepatocyte was significantly inhibited after treating with BgIFITM3 protein (10 and 20 μg/mL) ( P <0.01). The expression levels of ERBB-2, IRS-1, PI3KR-1, AKT-1 and MAPK-3 were significantly lower after treating with 20 μg/mL BgIFITM3 protein ( P <0.05). Finally, the activity of HepG2 cells was significantly inhibited after treating with BgIFITM3 protein (1, 10 and 20 μg/mL) ( P <0.05). While the cloning ability and migration ability of HepG2 cells were significantly inhibited after treating with 10 μg/mL BgIFITM3 protein ( P <0.05). The mitochondria of HepG2 cells were concentrated, cristae widened, and the double film density of mitochondria was increased after


Background
IFITM3 is interferon-induced transmembrane protein 3, also known as fragilis or I-8U is a 15-kDa protein encoded on human chromosome 11 and mouse chromosome Recently, an increasing number of studies have focused on the role of IFITM3 in tumorigenesis and development.IFITM3 was significantly overexpressed in many tumors including colon cancer, astrocytoma, human glioma, myeloid leukemia and prostate cancer, as well as liver cancer [20][21][22][23].
The yak (Bos grunniens) is widespread in the largest and harshest highland in the world, the Qinghai-Tibet Plateau.Yak living at high altitudes.The high-altitude area has the characteristics of low oxygen, low temperature, strong radiation, etc.And these characteristics can cause changes in the body's immune function [24][25][26].Indeed, the yak has become strongly integrated into Tibetan socio-cultural life and plays a significant economic role in the mountainous regions.At present it has become a symbol of plateau animal husbandry [27][28][29].In this study, The BgIFITM3 gene was cloned and identified, and the purified yak IFITM3 protein was obtained by prokaryotic expression, to study the function of BgIFITM3 protein.

Animal and Cell culture
The heart, liver, spleen, lung, and kidney were collected from Maiwa yak (1 day, 15 months and 5 years old) in Sichuan Province, China.Yak was slaughtered under anesthesia and every effort was made to minimize the risk of illness.The excised tissue was frozen in liquid nitrogen for transport and finally stored at -80 ℃.Another part of the yak liver tissue was placed in saline containing penicillin-streptomycin and returned to the laboratory at 4 ℃, and the yak hepatocyte were separated by collagenase digestion.HepG2 cells were donated by Chengdu Medical College.

Gene cloning
Total RNA was extracted from each tissue and cells.The concentration and OD value were measured by an UV-visible spectrophotometer.cDNA samples were synthesized according to the instructions of the PrimeScript TM RT reagent Kit (TaKaRa, Japan).The quality of cDNA was detected by Y-β-actin gene (Table 1).The cDNA was stored at -20 ℃.The primers of BgIFITM3 gene (F: GAAAAGGAAACTTAGGAGAAATC; R: ACTGCCTCGGGCTGTG) were designed in NCBI.PCR conditions were as follows: 98 ℃ for 2 min, then 98 ℃ for 10 s, 54 ℃ for 10 s, 72 ℃ for 10 s, 35 cycles, followed by 72 ℃ for 2 min, 4 ℃ forever.

Bioinformatics analysis
PCR products were sequenced, and the sequence of BgIFITM3 gene was analyzed by BLAST program.Amino acid sequence was predicted by ORF Finder program.The homology was analyzed by DNAMAN software.The evolutionary tree was constructed by MEGA6 software.The nucleotide sequences of IFITM3 from various species were compared by pairwise alignment using the Species Demarcation Tool.The basic physical and chemical properties were analyzed by ProtParam.Protein domains, signal peptides, transmembrane structure, secondary structure and tertiary structure were predicted by Conserved Domains, SignalP 5.0 Server, SOPMA, SWISS-MODEL, respectively.

Construction of prokaryotic expression vector
The BgIFITM3 gene amplification product was recovered by DNA Gel Extraction Kit (OMEGA, United States), and cloned to pMD19-T vector (Takara, Japan).The positive clone colonies were screened for identification.The correct colony was submitted for sequenced.The correct plasmid and pET-28a (Solarbio, China) were digested with Hind-III (TaKaRa, Japan) and EcoR-I (TaKaRa, Japan).T4 DNA ligase (TaKaRa, Japan) was added to ligate overnight at 16 ℃.
The ligation product was named pET28a-BgIFITM3.

Expression and purification of recombinant plasmid
The pET28a-BgIFITM3 was transformed into BL21(DE3) competent cells (Sangon Biotech, China) and cultured overnight in LB agar medium.Monoclonal white plaques were picked for expanding culture.1.4 mM/L IPTG was added to culture 12 h.The fusion expression product was identified by SDS-PAGE.BgIFITM3 protein was purified by nickel column method, and the concentration of purified protein was detected by BCA Protein Assay Kit (TIANGEN, China).

Western Blot
The total liver protein of yak was extracted by Tissue or Cell Total Protein Extraction Kit (Sangon Biotech, China).The mouse anti-His tag MAb (1:4000, CWBIO, China) was used as the primary antibody, and the HRP-conjugated goat anti-mouse IgG (1:10000, CWBIO, China) was used as the secondary antibody to identified purified BgIFITM3 protein.The Rabbit Anti-Fragilis (1:1500, Bioss, China) was used as the primary antibody, and the HRP-conjugated Goat Anti-Rabbit IgG (1:10000, Sangon Biotech, China) was used as the secondary antibody to detected the expression of IFITM3 protein in yak liver tissues of different age.The target protein was separated by 15% SDS-PAGE electrophoresis and transferred to the PVDF membrane (Sangon Biotech, China), and blocked with 5% milk at room temperature for 1 h.The primary antibody was incubated at 4 ℃ for 12 h, and the secondary antibody was incubated at room temperature for 1.5 h.Finally, the PVDF membrane was rinsed with a ECL luminescent reagents (Sangon Biotech, China) for 5 min in dark.

Immunohistochemistry
The yak livers of different growth stages were taken out of 4% paraformaldehyde fixative solution and made into paraffin sections.Rabbit Anti-Fragilis (1:100, Bioss, China) was used as the primary antibody, and polymerized goat anti-rabbit IgG (1:200, ZSGB-BIO, China) was used as the secondary antibody.The relative average optical density value of IFITM3 expression was determined by Image-Pro Plus 6.0.

CCK-8 assay
2×10 3 Cells were plated onto a 96-well culture plate and incubated overnight.Yak hepatocyte were treated with different concentrations of BgIFITM3 protein (0, 10 and 20 μg/mL).While, HepG2 cells were treated with different concentrations of BgIFITM3 protein (0, 1, 10 and 20 μg/mL).Three replicate wells were set for each concentration.The cell viability was detected according to the instruction of CCK-8 kit (Sangon Biotech, China).

Colony formation assay
HepG2 Cells were plated onto a 6-well culture plate.Then, HepG2 cells were treated by BgIFITM3 protein (0, 1 and 10 μg/mL), and incubated in 37 ℃, 5% CO2 incubator.HepG2 cells were fixed with 4% paraformaldehyde (biosharp, China) for 20 min and stained with crystal violet for 15 min.HepG2 cells were rinsed with double distilled water and taken pictures count.

Scarification test
HepG2 Cells were plated onto a 24-well culture plate.When the cell fusion reached 70%, each well was scratched and observed by using 10 μL pipette tip.After 24 h of treatment with BgIFITM3 protein (0, 1 and 10 μg/mL) in the serum-free medium, the scratches of each well were photographed and the cells were measured scratch width after migration.

Transmission electron microscope observation
HepG2 Cells were plated onto a 6-well culture plate and incubated overnight.10 μg/mL BgIFITM3 protein were added to each well.The HepG2 cells were collected by trypsinization.Then the HepG2 cells were fixed, dehydrated, infiltrated, embedded, sectioned, stained, and observed by the JEM-1400PLUS transmission electron microscope.
The cells were digested and collected after BgIFITM3 protein treating.The transcription level of each genes was detected by qRT-PCR.With GAPDH as the internal reference, and transcription levels were calculated by 2 -ΔΔCt method.The reaction system was 10 μL: 5.2 μL TB Green® Premix Ex Taq TM II (TaKaRa, Japan), 0.8 μL of the upstream and downstream primers (10 mmol•L -1 ), 1 μL sample, and ddH2O 2.2 μL.qRT-PCR conditions were as follows: 95 ℃ for 2 min, then 95 ℃ for

Statistical analysis
All data were analyzed by SPSS 26.0 and presented as mean ± SD of three independent experiments.GraphPad Prism 8.0 was used to plot gene expression profiles.One asterisk and two asterisks indicated P <0.05 and P <0.01 between groups, respectively.

Cloning and Bioinformatics Analysis of BgIFITM3 Gene
A 581 bp clear target band was obtained and sequenced by PCR amplification (Fig. 1a).The complete coding sequence (CDS) of BgIFITM3 was 441 bp in length, encoding for a putative protein of 146 amino acids.The BgIFITM3 gene had the 98.9% homology with common cattle, and had the 53.7% homology with frogs.Phylogenetic tree analysis results showed that the BgIFITM3 gene was the closest to the evolutionary distance of the ordinary cattle, and the furthest from the amphibians (Fig. 1b-c).Collectively, these results suggested that IFITM3 was highly conserved in the same genus.
The BgIFITM3 protein had a molecular weight of 36.957kDa, a theoretical isoelectric point pI of 5.15.The instability index of BgIFITM3 protein was 60.23, and the total average hydrophilicity was 0.928.The BgIFITM3 protein was a hydrophilic protein and transmembrane protein, but it had not a signal peptide.The BgIFITM3 protein had 25 Thr sites and a highly conserved structural functional domain CD225.

Transcription levels of BgIFITM3 gene in yak tissues
The transcription level of BgIFITM3 gene in the liver had the highest in 15 months old, followed by higher in 5 years old, and lower in 1 day old, and the difference was extremely significant between 1 day and 15 months, 1 day and 5 years old (P <0.01).The transcription level of BgIFITM3 gene in the spleen had the highest in 1 day old, followed by higher in 5 years old, and lower in 15 months old, and the difference was extremely significant between 1 day and 15 months, 1 day and 5 years old (P <0.01).The transcription level of BgIFITM3 gene in the lung had the highest in 15 months old, followed by higher in 1 day old, and lower in 5 years old, and the difference was significantly between 1 day and 5 years old, 15 months and 5 years old (P <0.05).
The transcription level of BgIFITM3 gene in the kidney had the highest in 1 day old, followed by higher in 15 months old, and lower in 5 years old, and the difference was significantly between 1 day and 5 years old (P <0.05).There was no obvious difference of the transcription level of BgIFITM3 gene in the heart between different ages.(Fig. 2a-e).
The transcription level of BgIFITM3 gene in the liver was all extremely significantly higher than the heart, spleen, lung and kidney (P <0.01).While, the transcription level of BgIFITM3 gene was higher in the heart, and lower in the yak spleen, lung and kidney (Fig. 2f-h).Collectively, these results suggested that the transcription level of BgIFITM3 gene had tissue differences.

Prokaryotic expression and identification of BgIFITM3 protein
The recombinant strain pET28a-BgIFITM3/DE3 was induced by IPTG and detected by SDS-PAGE.The results showed that the target recombinant protein band with a size of about 19.5 kDa appeared (Fig. 3a).After purification, SDS-PAGE showed a single band at about 19.5 kDa (Fig. 3b).
Western blot identification results showed that specific bands appeared (Fig. 3c).Recombinant BgIFITM3 protein had been expressed and proven.

Localization and expression of BgIFITM3 protein in liver
BgIFITM3 protein was expressed in the yak livers at different growth stages, and the protein was mainly located on the yak hepatocyte membrane (Fig. 4a-f).Yellow or brownish yellow staining was judged as positive cells.With the increase of age, the relative average optical density value of IFITM3 expression showed the difference was significantly between 1 day and 15 months of age (P <0.05) (Fig. 4g-h).These results were consistent with the transcription level results.The proliferation and PI3K/Akt related genes of yak hepatocyte Purified BgIFITM3 protein was added to culture yak hepatocyte for 24 h, CCK-8 experiment results showed that after adding different concentrations of BgIFITM3 protein, the activity of yak hepatocyte gradually decreased with the increase in concentration.And the yak hepatocyte activity was significantly lower after treating with 20 μg/mL BgIFITM3 protein (P <0.01) (Fig. 5a).The results showed that addition of BgIFITM3 protein had an inhibitory effect on the proliferation of yak hepatocyte.

The proliferation and migration of HepG2 cells
Purified BgIFITM3 protein was added to the cultured HepG2 cells for 24 h.The HepG2 cells activity was significantly lower after treating with 1 μg/mL BgIFITM3 protein (P <0.05), and it was extremely significantly lower after treating with BgIFITM3 protein (10 and 20 μg/mL) (P <0.01) (Fig. 6a).The results of the cell scratch experiment showed that the width of the cells was significantly higher after treating with 10 μg/mL BgIFITM3 protein (P <0.05) (Fig. 6b and e).The experimental results of HepG2 cells clone formation showed that the number of cell clones was 226.67, 149.33 and 55.00 after treating with BgIFITM3 protein (0, 1 and 10 μg/mL).The numbers of cell clones were extremely significant lower after treating with BgIFITM3 protein (1 and 10 μg/mL) (P < 0.01) (Fig. 6c and d).

The ultrastructure of HepG2 cells
The mitochondria of HepG2 cells were concentrated, Cristae widened and the double film density of mitochondria was increased after treating with 10 μg/mL BgIFITM3 protein.The above results indicated that the addition of BgIFITM3 protein decrease HepG2 cell activity may be caused by the ferroptosis mechanism (Fig. 7).

Ferroptosis-related genes and migration-related in HepG2 cells
The expression of ferroptosis-related genes (FTH-1, VDAC-2, VDAC-3, GPX-4 and p53) in HepG2 cells were detected by qRT-PCR.The results showed that the expression level of the FTH-1 gene showed an upward trend with the increased of BgIFITM3 protein concentration (Fig. 8a).
The expression levels of VDAC-2, VDAC-3 and p53 genes were significantly higher after treating with 10 μg/mL BgIFITM3 protein (P <0.01) (Fig. 8b-d).The expression level of GPX-4 gene was significant lower after treating with BgIFITM3 protein (1 and 10 μg/mL) (P <0.05) (Fig. 8e).The expression of MMP2 and MMP9 mRNA level was extremely significant lower after treating with At present, there were a few reports about the transcription level of IFITM3 gene in various animal.IFITM3 was induced by interferon (IFN), which participated in the signal transduction pathway under hypoxia stress conditions and acted on a variety of immune cells to regulate the balance of the body [36].Zebrafish IFITM3 gene had the higher transcription level in the ovary, and lower in the liver, kidney, brain, muscle and testis [37].Swine IFITM3 gene had the highest transcription level in the spleen and lung, followed by higher in the kidney, and lower in the heart and liver [38].Goose IFITM3 gene had the highest transcription level in the lungs, followed by higher in the spleen and kidney, and lower in the heart and liver [39].Rat IFITM3 gene had the highest transcription level in the liver, followed by higher in the spleen and heart, and lower in the lung and kidney [40].In this study, BgIFITM3 gene was also the highest expressed in liver tissue.
The transcription level of the IFITM3 gene in each tissue has undergone significant changes with the increase of age except the heart.Therefore, it was speculated that the differential expression of the BgIFITM3 gene in various tissues might be participates in regulating the body's immune balance in a hypoxic environment.The reasons for these differences of above reports may be due to the species differences and living environment.

Conclusion
In summary, we found that the BgIFITM3 protein mainly expressed in the yak liver.It could inhibit the proliferation of hepatocyte by down-regulating the PI3K/Akt pathway, and it also could inhibit the activity of HepG2 cells by regulating ferroptosis-related genes.
7 and is induced by type I, II and III IFNs [1-3].IFITM3 is mainly located on endolysosomal membrane, and placed an inhibitory role at an early stage of viral replication [4].It can inhibit the entry of viruses by preventing virus fusion with endolysosomal membrane [5-8].Among all IFITM family proteins, IFITM3 has the highest antiviral activity and is one of the most popular proteins in human antiviral research [9-12].IFITM3 can resist viral infection in various ways, including: changing the characteristics of cell membrane and interacting with multiple viral receptors on cell membrane [13, 14].Overexpression of human IFITM3 restricts the infection, replication and proliferation of vaccinia virus, mainly by interfering with the entry of virus in a low pH-dependent manner [15].Recently, IFITM3 can regulate γ-secretase in human Alzheimer's disease [16].Swine IFITM3 had an inhibitory effect for PRRS virus, Japanese B type encephalitis virus and FMDV [17-19].Avian IFITM3 had a significant inhibitory effect on avian influenza virus [18-19].

Fig. 1
Fig. 1 Analysis of BgIFITM3 Gene. a Result for PCR amplification of BgIFITM3.Note: M. DL2000 DNA Marker; IFITM3.BgIFITM3 gene.b Phylogenetic tree based on IFITM3 gene.c The nucleotide sequences of IFITM3 from various species were compared by pairwise alignment

Fig. 2
Fig. 2 The expression levels of BgIFITM3 in different tissues of the yak.a The expression level of BgIFITM3 gene in heart.b The expression level of BgIFITM3 gene in liver.c The expression level of BgIFITM3 gene in spleen.d The expression level of BgIFITM3 gene in lung.e The expression level of BgIFITM3 gene in kidney.f The expression level of BgIFITM3 gene in 1 day old.g The expression level of BgIFITM3 gene in 15 months old.h The expression level of BgIFITM3 gene in 5 years old.Values are expressed as mean ± SD of n = 3. * P <0.05, ** P <0.01

Fig. 4
Fig. 4 Detection of IFITM3 expression distribution in yak liver tissues at different growth stages.a-f Cell localization of BgIFITM3 protein.a 1-day-old yak liver (200×).b 15-month-old yak liver (200×).c 5-year-old yak liver (200×).d Group a negative control.e Group b negative control.f Group c negative control.g Relative average optical density.h The expression level of IFITM3 protein in yak at different ages.HCs: Hepatocyte.Values are expressed as mean ± SD of n = 3. * P <0.05

Fig. 5
Fig. 5 Effects of different concentrations of BgIFITM3 protein on yak hepatocyte a The yak hepatocyte activity.b Expression of ERBB-2.c Expression of IRS-1.d Expression of PI3KR-1.e Expression of AKT-1.f Expression of RAF-1.g Expression of MAPK-3.Values are expressed as mean ± SD of n = 3. * P <0.05, ** P <0.01

Fig. 6
Fig. 6 The proliferation and migration of HepG2 cells.a HepG2 Cells Activity.b The scratch width of HepG2 cells.c The Number of Clones Formed in HepG2 Cells.d HepG2 cell clone formation.e HepG2 cell migration.Values are expressed as mean ± SD of n = 3. * P <0.05; ** P <0.01

Fig. 8
Fig. 8 The expression of related genes in HepG2 cells.a Expression of FTH-1.b Expression of VDAC-2.c Expression of VDAC-3.d Expression of P53. e Expression of GPX-4.f Expression of MMP2.g Expression of MMP9.Values are expressed as mean ± SD of n = 3. * P <0.05; ** P <0.01 Discussion So far, IFITM3 gene has been cloned in humans, mice, pigs, cattle, monkeys and other mammals [30-34].The cattle and chicken IFITM3 protein contained a highly conserved structuralfunctional domain CD225 [33, 35].In this study, the BgIFITM3 protein also contained the domain CD225.These reports showed that the IFITM3 gene was highly conserved in different species.The above results indicated that the structure and conserved regions of IFITM3 may play a very important role in its function.
With the deepening of research on antiviral natural immunity, it has been found that interferoninduced transmembrane proteins can inhibit a variety of viral infections [1].IFITM3 has been shown to restrict replication of around seventeen, mostly enveloped, RNA viruses including influenza A virus (IAV), HIV-1, Ebola, SARS coronavirus, and Dengue virus [41].The research of human IFITM3 has made great progress, and it had inhibitory effects on a variety of viruses, but mainly using gene over expression or silencing [42-45].The down-regulation of IFITM3 significantly reduced c-myc expression and inhibited the proliferation of liver cancer in vitro and in vivo [46].IFITM3 played a key role in the regulation of malignant tumor cell proliferation, invasion, and bone migration by TGF-β-Smads Signaling Pathway [47].IFITM3 promotes hepatocellular carcinoma invasion and metastasis by regulating MMP9 through p38/MAPK signaling [48].IFITM3 may a crucial mediator in TGF-β-induced glioma cells invasion and promote TGF-β-mediated glioma cells invasion through STAT3 activation [49].IFITM3 knockdown led to reduced expressions of CCND1 and CDK4 and reduced RB phosphorylation, leading to inhibition of OSCC cell growth [50].IFITM3 can anti-proliferation, anti-virus and anti-tumor.Overexpression of the human IFITM1 gene can significantly inhibit the growth of normal hepatocyte [51].Pig HDCA suppressed intestinal epithelial cell proliferation through PI3K/Akt signaling pathway [52].LncRNA FER1L4 suppressed cell proliferation and metastasis by inhibiting the PI3K/Akt signaling pathway in lung cancer [53].Our study was the same as the above reports.Moreover, the BgIFITM3 protein can cause the ferroptosis of HepG2 cells.Ferroptosis is a newly discovered form of regulated cell death.The knockout of GPX-4 causes cell death in a pathologically relevant form of ferroptosis in mice [54].The p53 expression was increased in PC12 cells will lead to ferroptosis [55].The FANCD2 was knockout will be decreased FTH-1 and GPX-4 expression to induced ferroptosis [56].Erastin can regulate the increasing of VDAC2/3 expression and inducing ferroptosis [57].The results of this are basically consistent with the above results.

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