Gastric cancer cells SGC7901 and AGS were preserved and provided by the Cancer Research Institute, Basic School of Medicine, Central South University. The cells were cultured via adherent growth in an incubator set at 37°C with 5% CO₂ in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco-Life Technologies at Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum (FBS; Gibco-Life Technologies) and 1% Penicillin-Streptomycin Solution.

The plasmids used in this study included: vector pLVX-mCMV-ZsGreen-PGK-Puro-Fra-1 (Fra-1 overexpression), vector pLVX-mCMV-ZsGreen-IRES-Puro-YWHAH (YWHAH overexpression), vector pLVX-mCMV-ZsGreen-PGK-Puro-Fra-1-D1 (expressing Fra-1 aa 1–107), vector pLVX-mCMV-ZsGreen-PGK-Puro-Fra-1-D2 (expressing Fra-1 aa 1–127), and vector pLVX-mCMV-ZsGreen-PGK-Puro-Fra-1-D3 vector (expressing Fra-1 aa 107–271). All plasmids were lentivirus expression vectors carrying an ampicillin resistance gene. Puromycin was used for screening.

After transfection, the cells were cultured in a 100 mm dish for 48 h and then lysed using IP lysis buffer containing a mixture of protease inhibitors and phosphatase inhibitors (Beyotime Biotechnology, P0013, Shanghai, China). Then, 1 µg of primary antibody was incubated with 30 µL of protein A+G beads on a rotator at room temperature for 2 h, the protein lysate was then added and incubated on the rotator at 4°C overnight. Next day, the beads and immune complexes were washed five times with IP lysis buffer by rotating at room temperature for five minutes at a speed (2000 rpm/min) of 20 s per round. The sample was added to the corresponding SDS loading buffer and heated at 95°C for 5 min. The immunoprecipitated samples were detected using western blotting.

The purified protein complex eluent was concentrated using IP beads, separated by SDS-PAGE, and stained with Coomassie brilliant blue (Beyotime Biotechnology). The corresponding protein band was excised from the gel, reduced, alkylated, and digested overnight using trypsin at 37°C (Thermo Fisher Scientific, Waltham, MA, USA). The digested peptides were dried and resuspended in MS compatible buffers, and the mixture was analyzed using an LTQ Orbitrap velos MS instrument (Thermo Fisher Scientific) in combination with a UltiMate RSLC Nano LC system (Dionex, Sunnyvale, CA, USA). Proteome Discoverer 1.4 software (Thermo Fisher Scientific) was used to identify proteins, import files, and search the UniProtKB/Swiss-Prot databases. The mass tolerances of precursors and fragments were set to 10 ppm and 0.8 Da, respectively. Peptide data with an error detection rate of <1% (p < 0.01) were discarded.

The total RNA of GC cells was isolated using the TRIzol reagent (Invitrogen, Waltham, MA, USA), and cDNA synthesis was carried out using the RevertAid First Strand cDNA synthesis kit (CWBio) according to the manufacturer’s facturer’s recommendations. qRT‑PCR was carried out with GoTaq qPCR Master Mix (Promega, Fitchburg, WI, USA). For detection of Fra-1, YWHAH, AKT, PI3K, PDK1, MTOR, and HMGA1 mRNA expression levels, GAPDH was amplified in parallel as an internal control. The sequences of the primers used for qPCR were as follows: Fra-1 forward 5′-CAGTGGATGGTACAGCCTCATTTC-3′, reverse 5′-GCAGTCTCCTGTTCACAAGGC-3′; YWHAH forward 5′-TCAAGAAGGTGGTGAAGCAGG-3′, reverse 5′-TCAAAGGTGGAGGAGTGGGT-3′; AKT forward 5′-ACACCAGGTATTTTG ATGAGGAG-3′, reverse 5′-TCAGGCCGTGCCGCTGGCCGAGTAG-3′; PI3K forward 5′-AGCTGGTTTGGATCTTCGGA-3′, reverse 5′-CAGGTC ATCCCC AGAGTTGT-3′; PDK1 (encoding pyruvate dehydrogenase kinase 1) forward 5′-AGTTCATGTCACGCTGGGTA-3′, reverse 5′-CAGCTTCAGGTCTCCTTGGA-3′; MTOR forward 5′-GCAACCCTTCTTTGACAACATTTTT-3′, reverse 5′-ATTTCTTCTCTCAGACGCTCTCC-3′; HMGA1 forward 5′-TGCGAAGAAACTGGG AGAGA-3′, reverse 5′-TGCGAAGAAACTGGGAGAGA-3′; GAPDH (encoding glyceraldehyde-3-phosphate dehydr-ogenase) forward 5′-TCAAGAAGGTGGTGAAGCAGG-3′, reverse 5′-TCAAAGGTGGAGGAGT GGGT-3′. The expression of mRNA was assessed by evaluated threshold cycle (CT) values. The CT values were normalized to the expression levels of GAPDH and the relative amount of mRNA specific to each of the target genes was calculated using the 2^−ΔΔCT method. qPCR was carried out using the Bio‑Rad CFK96™ Real‑Time System (Bio‑Rad, Hercules, CA, USA). The data were analyzed by Bio‑Rad CFK Manager software (Bio‑Rad).

To test the effect of silencing YWHAH and Fra-1, we selected three small interfering RNAs (siRNAs) targeting different regions of the YWHAH and Fra-1 genes. The three siRNA sequences for YWHAH were:

si-YWHAH-001 5′-GCUGGAGACAGUUUGCAAUTT-3′;

si-YWHAH-002 5′-CCAGAAUGCACCUGAGCAATT-3′;

si-YWHAH-003 5′-GCUGAGCUGGACACACUAATT-3′;

and the control si-NC 5′-ACGUGACACGUUCGG AGAATT-3′.

The three siRNA sequences for Fra-1 were:

si-Fra-1-001 5′-GTCGAAGGCCTTGTGAA-3′;

si-Fra-1-002 5′-GCTCATCGCAAGAGTAGCA-3′;

si-Fra-1-003 5′-GGAAGGAACTGACCGACTT-3′;

and the control si-NC 5′-ACGUGACACGUUC GGAGA ATT-3′.

For siRNA transfection, the riboFECT CP Transfection Kit (Guangzhou RiboBio Co., Ltd., Guangzhou, China) was used according to the manufacturer’s instructions, and then the silencing effect was detected. The most successful siRNA sequence for each gene was used for subsequent experiments.

Cells (1 × 10⁵ to 3 × 10⁶) were inoculated in 6-well plates and cultured at 37°C in a 5% CO₂ incubator to the required cell density. Two hours before transfection, serum-free 1640 medium was used. The transfected cells were divided into groups according to the corresponding experiments. After 6 h, the mixed solution was sucked out and replaced with normal culture medium, and culture was continued for 48 h. Referring to the operating instructions of the EdU-647 cell proliferation test kit (Beyotime biotechnology, C0081S), the samples were processed before flow cytometry [28,29]. Finally, the samples were tested by flow cytometry at a 488 nm excitation wavelength and 520 nm emission wavelength. Cells were analyzed with Moflo XDP High-Performance Cell Sorter (Beckman Coulter). Data were acquired and analyzed with Summit v5.2 software.

Cells were digested with trypsin and then lysed with Radioimmunoprecipitation assay (RIPA) buffer (CWBio, Beijing, China). The sample was centrifuged at 12000 × g at 4°C for 15 min to remove insoluble matter. The protein concentration of the retained supernatant was detected, and protein electrophoresis was carried out using a 10% SDS-PAGE gel at 50 V for 40 min, and then at 120 V for 60 min (PowerPac Universal, Bio-Rad, Hercules, CA, USA). Then, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (HyClone Laboratories, Logan, UT, USA) at 100 V for 90 min, and then incubated in phosphate-buffered saline (PBS)-Tween20 with 5% skimmed milk for 1–2 h. The membrane was then incubated with primary antibody at 4°C overnight.

The main antibodies used in this study include: Fra-1 (YM0122,Immunoway, Plano, TX, USA), YWHAH (ab206292, Abcam, Cambridge, MA, USA), HMGA1 (#7777, Cell Signaling Technology, Danvers, MA, USA), PI3K (AF6241, Affinity Biosciences, Cincinnati, OH, USA), AKT (10176-2-AP, Wuhan Sanying Biotechnology Co., Ltd., Wuhan, China), mTOR (ab32028, Abcam), phosphorylated (p)-mTOR (ab109268, Abcam), GAPDH (AB-P-R001, Hangzhou Xianzhi Biotechnology Co., Ltd., Hangzhou, China). The dilution ratio of the antibodies was 1:1000. Rabbit anti-β-tubulin antibody, mouse anti-β-actin, Rabbit anti-GAPDH, from Affinity Bioscience were used at a dilution of 1:5000; anti-rabbit secondary antibody and anti-mouse secondary antibody were purchased from Lianke company (Shanghai, China), and used at a dilution of 1:3000. The membranes were incubated with the secondary antibodies for 1 h and then the immunoreactive proteins were visualized using Molecular Imager Gel Dox XR System (Bio-Rad Laboratories).

GraphPad Prism 5 software (GraphPad Inc., La Jolla, CA, USA) was used perform the statistical analyses. T-tests were used to analyze whether the difference between any two groups was significant. One way analysis of variance (ANOVA) was used to analyze whether the data of more than two groups were significantly different. All experiments were set with three or more replicates, and p < 0.05 indicated a significant difference.

Our previous research results showed that Fra-1 was upregulated in GC tissue and is involved in its occurrence and development. To systematically explore the role and possible mechanism of Fra-1 in GC, we first used Co-IP combined with LC-MS/MS to identify proteins that interact with Fra-1 in GC cells. After the Fra-1 interaction proteins were separated by SDS-PAGE, stained with Coomassie blue, and decolorized, we observed obvious difference bands at 25~40, 40~70, and 70~180 kDa on the gel (Fig. 1A). The bands were then analyzed by endogel enzymolysis and mass spectrometry. Among them, 25 specific proteins of the FLAG lane were identified at 25~40 kDa, among which 6 protein molecules belonged to the 14-3-3 protein family, including YWHAE, YWHAH, YWHAG, YWHAZ, and YWHAQ, suggesting that the 14-3-3 protein family might interact with Fra-1 (Suppl. Table 1). To verify the interaction between the 14-3-3 protein family and Fra-1, we performed IP experiments for YWHAE, YWHAH, YWHAG, YWHAZ, and YWHAQ with Fra-1, respectively. The results showed that Fra-1 interacted only with YWHAH in SGC7901 GC cells (Fig. 1B). To further explore the interaction domain between YWHAH and Fra-1, we constructed vectors expressing Fra-1 3-segment domains D1 (1–107 aa), D2 (1–127 aa) and D3 (107–271 aa) with HA tags (Fig. 1C), and conducted IP experiments to verify the domain of interaction. The results showed that YWHAH interacted with full length Fra-1 and all three segment domains; however, the interaction between YWHAH and D2 (1–127 aa) was stronger (Fig. 1D), suggesting that the Bag domain of Fra-1 plays an important role in the interaction between the two proteins. In conclusion, we identified YWHAH as a interacting protein of Fra-1 in SGC7901 GC cells.

To clarify the mutual regulation relationship between YWHAH and Fra-1, we first designed three siRNA sequences for the YWHAH gene, and transiently transfected the three siRNAs into SGC7901 GC cells. Western blotting showed that si-YWHAH-01 had the best effect in reducing the protein level of YWHAH (Suppl. Fig. 1); therefore, si-YWHAH-01 was used in subsequent experiments. Then, we overexpressed and silenced YWHAH, separately, in SGC7901 GC cells. Western blotting and qRT-PCR assays showed that the mRNA and protein levels of Fra-1 were upregulated after YWHAH overexpression (Figs. 2A and 2B) but downregulated after YWHAH silencing (Figs. 2B and 2C), suggesting that YWHAH further affected the translation level after regulating the transcription of Fra-1. To further verify our experimental results, we repeated the above experiments in AGS cells, and the results were consistent with those in SGC7901 cells (Figs. 2D–2F). The above results showed that YWHAH interacted with and positively regulated Fra-1, and affected its protein level by regulating Fra-1 transcription.

Based on the discovery that YWHAH interacts with and positively regulates the mRNA and protein levels of Fra-1, we assessed whether YWHAH could affect the proliferation of GC cells by regulating Fra-1. Cell proliferation assays combined with flow cytometry showed that the cell proliferation increased after the stable overexpression of YWHAH in SGC7901 GC cells. However, when Fra-1 was silenced in SGC7901 cells stably overexpressing YWHAH, cell proliferation was inhibited (Fig. 3A), suggesting that YWHAH might affect GC cell proliferation by regulating Fra-1. When we silenced with YWHAH in SGC7901 cells, their proliferation ability was inhibited. However, in cell silenced for YWHAH, Fra-1 overexpression restored cell proliferation (Fig. 3B). The above results showed that YWHAH affected the proliferation of GC cells by regulating Fra-1. To further verify our experimental results, we repeated the above experiments in AGS cells, and the results were consistent with those in SGC7901 cells (Figs. 3C and 3D).

To explore the possible mechanism by which YWHAH regulates Fra-1 to affect the GC cell proliferation, the whole proteome was analyzed after vector (blank control) and Fra-1 plasmids were transfected into SGC7901 GC cells, separately. The results showed that 206 protein molecules were upregulated (Suppl. Table 2) and 146 proteins were downregulated after overexpression of Fra-1 in GC cells SGC7901 (Fig. 4A, Suppl. Table 3). Based on the analysis of these differentially abundant proteins, we performed functional classification, functional enrichment, and cluster analysis for all differentially abundant proteins. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of upregulated proteins showed that the PI3K/AKT signaling pathway, the mitogen activated protein kinase (MAPK) signal transduction pathway, and the human papillomavirus infection signal transduction pathway were three main signal transduction pathways affected by Fra-1 (Fig. 4B). It has been reported that Fra-1 regulates the expression of HMGA1, and HMGA1 is a regulator of the PI3K/AKT/mTOR signaling pathway. Combined with our previous research results, we clarified whether YWHAH could promote the proliferation of GC cells by positively regulating Fra-1 to activate the HMGA1/PI3K/AKT/mTOR signaling pathway.

To confirm whether YWHAH can affect the proliferation of GC cells by positively regulating Fra-1 to activate the HMGA1/PI3K/AKT/mTOR signaling pathway, we first transfected NC+siNC, OE-YWHAH+siNC, and OE-YWHAH+siFra-1 plasmids into SGC7901 GC cells, and detected the mRNA levels of PI3K/AKT/mTOR signal pathway related molecules (PI3K, AKT, PDK1, MTOR, etc.) using qRT-PCR. The results showed that the mRNA expression levels of PI3K, AKT, PDK1, and MTOR were upregulated after overexpression of YWHAH. However, after Fra-1 silencing in a background of YWHAH overexpression, upregulation of the mRNA expression levels of PI3K, AKT, PDK1, and MTOR molecules was inhibited (Fig. 5A). In addition, western blotting analysis also showed that the protein levels of PI3K and PDK1 were upregulated, as were the levels of phosphorylated (p)-AKT and p-mTOR after YWHAH overexpression. However, after Fra-1 silencing in a background of YWHAH overexpression, the upregulation of PI3K and PDK1 protein levels was inhibited, as were the upregulation of p-AKT and p-mTOR levels (Fig. 5B). The results suggest that YWHAH can activate the PI3K/AKT/mTOR signaling pathway through Fra-1.

At the same time, we silenced the expression of YWHAH, in which siNC+NC, siYWHAH+NC, and siYWHAH+OE-Fra-1 plasmids were transfected into SGC7901 GC cells, and the related molecules were detected using qRT-PCR. The results showed that the mRNA expression levels of PI3K, AKT, PDK1, and MTOR were downregulated after YWHAH silencing. However, overexpression of Fra-1 in a background of YWHAH silencing restored the mRNA expression levels of PI3K, AKT, PDK1, and MTOR (Fig. 5C). In addition, the protein levels of PI3K/AKT/mTOR signaling pathway-related molecules were detected by western blotting. The results showed that after YWHAH silencing, the protein levels of PI3K and PDK1 were downregulated, and the levels of p-AKT and p-mTOR were downregulated. Overexpression of Fra-1 in a background of YWHAH silencing restored the protein levels of PI3K and PDK1, as well as the levels of p-AKT and p-mTOR (Fig. 5D). Our results suggested that YWHAH can promote the proliferation of gastric cancer cells by activating the HMGA1/PI3K/AKT/mTOR signaling pathway through Fra-1.

To further confirm that YWHAH promotes gastric cancer cells proliferation by activating the PI3K/AKT/mTOR signaling pathway, we used rapamycin, an inhibitor of the PI3K/AKT/mTOR signaling pathway to treat gastric cancer cells. Firstly, we divided gastric cancer cells SGC7901 into three groups: OE-YWHAH+siNC+DMSO, OE-YWHAH+siFra-1+DMSO, OE-YWHAH+siFra-1+Rapamycin by transfecting the different plasmids and whether treating cells with rapamycin. Then, we tested the gastric cancer cells proliferation using an EDU proliferation detection kit and flow cytometry. Our results showed that compared to the OE-YWHAH+siNC+DMSO group, OE-YWHAH+siFra-1+DMSO group had a decrease in the proportion of proliferating cells, OE-YWHAH+siFra-1+Rapamycin group had a more significant reduction in the proportion of proliferating cells (Fig. 6A). To further confirm the effect of silencing YWHAH, we divided gastric cancer cells SGC7901 into three groups: siYWHAH+NC+DMSO, siYWHAH+OE-Fra-1+DMSO, siYWHAH+OE-Fra-1+Rapamycin by transfecting the different plasmids and whether treating cells with rapamycin. Then we tested the gastric cancer cells proliferation using an EDU proliferation detection kit and flow cytometry. Our results showed that compared to the siYWHAH+NC+DMSO group, the siYWHAH+OE-Fra-1+DMSO group had an increase in the proportion of proliferating cells. But, the siYWHAH+OE-Fra-1+Rapamycin group had a decrease in the proportion of proliferating cells (Fig. 6B). In AGS cells, we obtained results with the same trend (Figs. 6C and 6D). Above of all, our results suggested that YWHAH could promote the proliferation of gastric cancer cells by activating the PI3K/AKT/mTOR signaling pathway.