Abstract Objective RNA-seq was used to explore the potential mechanism underlying human leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1) inhibition of the proliferation and migration of ovarian cancer cells. Method A LAIR-1–overexpression cell model was established using a LAIR-1-lentivirus. After confirming and identifying the LAIR-1 expression cell clones by flow cytometry and RT-qPCR, the proliferation and migration of the cells were examined by CCK8 and scratch assays, and the differentially expressed genes (DEGs) were searched by RNA-seq and analyzed by GO and KEGG enrichment. String was used for protein interaction network analysis, and Cytoscape was used to identify key proteins. Results LAIR-1 inhibited the proliferation and migration of ovarian cancer cells. LAIR-1 expression caused the upregulation of 83 genes and the downregulation of 80 genes. Among the DEGs, fibronectin 1 (FN1) was a key protein affecting the downstream FAK-MEK-ERK axis. KEGG enrichment analysis identified the MAPK pathway as the most obvious enrichment pathway, followed by PI3K-AKT pathway. Conclusion LAIR-1 downregulates FN1 to inhibit the FAK-MEK-ERK axis, as well as the proliferation and migration of ovarian cancer cells. Supplementary Information The online version contains supplementary material available at 10.1186/s12885-025-13692-1. Keywords: Leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1), Fibronectin 1 (FN1), Ovarian cancer (OC), MAPK, Proliferation, RNA-seq Introduction Ovarian cancer is often diagnosed at an advanced stage due to its insidious onset, absence of specific symptoms in its early stages, and lack of diagnostic markers, making it the fifth leading cause of cancer death in women after lung, breast, colorectal, and pancreatic cancers [[44]1, [45]2]. Current treatments for ovarian cancer are based on surgery combined with chemotherapy, but the ovarian cancer recurrence rate is so high that its survival rate has shown little change in the past 20 years [[46]1]. Thus, an urgent need exists to find new therapeutic targets to improve the prognosis of ovarian cancer. To data, research has indicated that some membrane receptors are aberrantly overexpressed in ovarian cancer cells and are involved in key cellular processes. For this reason, transmembrane tyrosine kinase receptors (EGFR, HER2, VEGFR, IGF-1R, PDGFR), transmembrane glycoprotein receptors (FR, CD44, TFR, CA-125, EpCAM), G protein-coupled receptors (FSHR, LHRHR, Ion Channel-linked Receptors), and other ovarian cancer-associated cell membrane receptors (PD-L1, Integrins) have emerged as potential targets for the treatment of ovarian cancer [[47]3]. One of these receptors, leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1), is widely expressed by solid tumor cells and functions as a tumor suppressor [[48]4, [49]5]. Our previous studies have found that LAIR-1 is expressed in ovarian cancer tumor tissues and in a variety of epithelial ovarian cancer cell lines [[50]6] and that LAIR-1 could inhibit ovarian cancer cell growth by suppressing the PI3K-AKT-mTOR pathway [[51]7]. The aim of the present study was to obtain a comprehensive understanding of the molecular mechanism of LAIR-1 in regulating the biological behavior of ovarian cancer cells. The approach taken was to perform RNA-seq sequencing on SKOV3 cells overexpressing LAIR-1, and to conduct further validation by combinatorial integration of bioinformatics analyses to assess the genetic alterations and using biological experimental findings. Materials and methods Cell lines and cell cultures SKOV3 and A2780 cell lines were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). They were cultured in RPMI 1640 and McCoy’s 5 A medium (supplemented with 10% fetal bovine serum and penicillin-streptomycin), respectively, in a humidified incubator with 5% CO[2] at 37 °C. Reagents and antibodies Reagents and antibodies are listed in Supplementary Tables [52]1 and Table [53]2, respectively. Lentiviral production and establishment of stable expression cell lines LAIR-1-lentivirus and FN1-lentivirus vectors were constructed by GeneChem Company (Shanghai, China). SKOV3 and A2780 ovarian cancer cells were plated on six-well plates at 2 × 10^5 cells/well. The medium was changed with complete medium without penicillin/streptomycin prior to infection. According to the virus titer, cell count, and MOI of SKOV3 and A2780 cells, the appropriate amount of virus suspension was added. After incubation with the constructed lentivirus for 48 h, the cells were selected with 5 µg/mL puromycin (or 200 µg/mL G418) for 3 days. Green (or red) fluorescent cells were examined by fluorescence microscopy to determine the infection efficiency. Protein expression was further confirmed by flow cytometry or Western blotting. Immunofluorescence Cells were seeded into 24-well plates and incubated overnight until reaching 60–70% density. The medium was aspirated, and the cells were washed three times with PBS, 5 min each. 4% paraformaldehyde was added, and the plates were gently shaken at room temperature for 20 min, followed by three PBS washes. Blocking solution was added and incubated at room temperature for 30 min. After removing the blocking solution, primary antibody was added and incubated overnight at 4 °C, followed by three PBS washes. Fluorescent secondary antibody was added and incubated at room temperature in the dark for 1 h, followed by three PBS washes. DAPI was added and incubated at room temperature in the dark for 8 min, followed by three PBS washes. Finally, 200 µL PBS was added, and the cells were observed and imaged under a fluorescence microscope. Flow cytometry analysis Cells were harvested and resuspended 1 × 10^6 cells/ 100 µL PBS. A total of 5 µL Alexa Fluor^® 647 anti-human LAIR-1 antibody was added to each tube and incubated at 4 °C for 30 min. After two washes with PBS, the stained cells were analyzed by flow cytometry. Western blot analysis Cells were lysed in RIPA lysis buffer containing protease and phosphatase inhibitors at 4 °C for 30 min, followed by centrifugation at 12,000 rpm for 15 min at 4 °C to collect the supernatant. After calculating the protein concentration using the BCA protein assay kit, the protein samples were separated by SDS-PAGE, transferred to PVDF membrane, and blocked with 5% skim milk. The membranes were incubated with the indicated primary antibodies overnight at 4 °C and then with the corresponding secondary antibodies for 1.5 h at room temperature. An enhanced chemiluminescence kit was used to detect the secondary antibody-specific signals. GAPDH was used as an internal protein reference control. Quantitative real time PCR (qRT-PCR) Total cellular RNA was extracted using the Trizol method, and cDNA was obtained by gDNA removal and reverse transcription using a reverse transcription kit. The following primers were used in the study: LAIR-1: forward primer: 5’-TCTCCTCCTGGTCTTCTTC-3’, reverse primers: 5’-GCTCTGCTGCTGCTCTA-3’, FN1: forward primer: 5’- GGCGACAGGACGGACATCTTTG-3’, reverse primer: 5’- GGCACAAGGCACCATTGGAATTTC-3’, GAPDH: forward primer: 5’-GTCTCCTCTGACTTCAACAGCG-3’, reverse primer: 5’-ACCACCCTGTTCTGTAGCCAA-3’. The relative expression of mRNAs was compared using the relative quantitative analysis 2^−ΔΔCT method. Cell proliferation and migration assays For the cell proliferation assay, a 100 µL of 4 × 10^4/mL cells was added to each well in a 96-well plate. Each group was divided into five time points: 0, 24, 48, 72, and 96 h, and cultured in the incubator until the time of detection. A 10 µL CCK8 was added and the absorbance at 450 nm was detected after incubation for 3 h. For the scratch assay, 2 × 10^5 SKOV3 cells/well and 4 × 10^5 A2780 cells/well were seeded into the 12-well plates. After the cells had grown to 80–90% confluence, they were scratched with the tip of a 200 µL pipette, washed with PBS, and replaced with fresh basal medium. At 0, 24, and 48 h of incubation the scratches were photographed using an inverted light microscope. Construction of transcriptome libraries Total RNA was extracted with Trizol, 1 µg of total RNA was hybridized to the probe and digested with RNase H followed by DNase I. RNA was purified using VAHTS RNA purification beads, 18.5 µL Frag/Prime Buffer (1x) was added, mixed by pipetting, and allowed to stand for 2 min at room temperature. After standing on a magnetic stand for 5 min to allow the solution to clear, 16 µL of supernatant was gently aspirated into a new nuclease-free centrifuge tube, and the samples were fragmented using a PCR instrument and sequenced using an Illumina sequencer. RNA-seq data analysis Reads were compared to the reference genome using Subjunc v2.0 and then quantified using featureCounts v2.0.3. Differential analysis was performed using the DEseq2 R package, and differential genes were screened for fold change greater than 2-fold and q-values less than 0.05. GO and KEGG enrichment analyses of differential genes were performed using the OmicShare Cloud platform ([54]https://www.omicshare.com/). Protein interaction network analysis was performed using String ([55]https://cn.string-db.org/), and Cytoscape was used to perform key gene searches for protein interaction networks. Survival analysis of key genes was performed using Kaplan-Meier Plotter ([56]https://kmplot.com/analysis/). Statistical analysis All the statistical analyses were performed using GraphPad Prism 8 ([57]http://www.graphpad.com/) statistical analysis software. Data are presented as the mean ± SD. T-test was used to analyze the statistical difference between two independent groups and one-way ANOVA was used to analyze differences more than two groups for a single variable. P value < 0.05 was considered statistically significant. Results LAIR-1 inhibits ovarian cancer cell proliferation and migration in vitro We successfully established LAIR-1 overexpressing SKOV3 and A2780 cell lines (Fig. [58]1A, B). As determined by qRT-PCR, the LAIR-1 mRNA expression levels were significantly higher in the LAIR-1–overexpression (OE) SKOV3 and A2780 cell groups than in the negative control (NC) groups (Fig. [59]1C, E). In parallel, the LAIR-1 protein expression levels were also higher in the OE groups than in the NC groups, as assessed by flow cytometry and western blot (Fig. [60]1D, F, G and H). Subsequent CCK-8 results showed significantly greater inhibition of cell proliferation in the OE groups than in the NC groups (Fig. [61]1I, L). The results of scratch assays showed that, in SKOV3 cells, the 24 and 48 h migration rates were significantly lower in the OE groups than in the NC groups (Fig. [62]1J, K). In the A2780 cells, the 48 h migration rate was also substantially lower in the OE groups than in the NC groups, but no significant difference was detected in the 24 h migration rates between the OE and NC groups (Fig. [63]1M, N). Fig. 1. [64]Fig. 1 [65]Open in a new tab LAIR-1 inhibits the proliferation and migration of SKOV3 and A2780 ovarian cancer cells. (A and B). Detection of LAIR-1 expression level after lentivirus infection by immunofluorescence assay. (C and E). Detection of LAIR-1 mRNA expression level after lentivirus infection by qRT-PCR; data are presented as mean ± SD (D and F). Detection of LAIR-1 protein expression level after lentivirus infection by flow cytometry. (G and H). Detection of LAIR-1 protein expression level after lentivirus infection by Western blot; data are shown as mean ± SD (I and L). CCK-8 detects the effect of LAIR-1 on the proliferation of ovarian cancer cells; data are shown as mean ± SD (J, K, M and N). Scratch assay to detect the effect of LAIR-1 on the migration of ovarian cancer cells; data are shown as mean ± SD The DEGs were identified by RNA-seq and were enriched by GO and KEGG We performed RNA-seq analyses on the LAIR-1–overexpressing and the corresponding control vector–transduced null SKOV3 cell lines that we established. We found 83 upregulated genes and 80 downregulated genes by comparing the OE groups to the NC groups (Fig. [66]2A, B). Clustering analysis of the DEGs revealed a high degree of concordance of LAIR-1–mediated transcription between the NC and OE groups (Fig. [67]2C). GO enrichment analysis further confirmed that the DEGs were enriched in a variety of biological processes, including developmental processes, localization, negatively regulated biological processes, cell proliferation, motility, biological attachment, reproduction, reproductive processes, and growth pathways. The GO enrichment data were consistent with our obtained phenotypic results. Subsequent KEGG pathway enrichment analysis observed that the most significant enrichment was in the mitogen-activated protein kinase (MAPK) signaling pathway, followed by the phosphatidylinositol 3-kinase-protein kinase (PI3K-AKT) pathway, supporting our previous demonstration that LAIR-1 inhibits the PI3K-AKT signaling pathway [[68]7]. Fig. 2. [69]Fig. 2 [70]Open in a new tab DEGs as well as GO and KEGG enrichment analyses. (A and B). DEGs alterations in SKOV3 cells caused by LAIR-1. (C). Heat map of differentially expressed genes. (D). GO enrichment analysis of differentially expressed genes. (E). KEGG enrichment analysis of the differentially expressed genes Search for key genes We performed protein-protein interaction (PPI) network analysis of the DEGs using the String website (Fig. [71]3A). After screening the PPI network using Cytoscape, we identified fibronectin 1 (FN1) as a key node in the PPI network (Fig. [72]3B), suggesting that FN1 play a pivotal role in the DEG alterations caused by LAIR-1. We further evaluated the role of FN1 in ovarian cancer by analyzing the effect of FN1 on the survival of ovarian cancer patients using the Kaplan-Meier Plotter website. The survival rate was lower for patients with high FN1 expression than with low FN1 expression (Fig. [73]3C), suggesting that FN1 expression may be a high risk factor for ovarian cancer. FN1 was downregulated in the LAIR-1 OE groups (Fig. [74]2B), and the expression level of FN1 was significantly lower in the LAIR-1 OE groups than in the NC groups, as verified by qRT-PCR (Fig. [75]3D). This suggests that LAIR-1 expression may suppress the malignant behavior of ovarian cancer cells by decreasing the expression of FN1. Fig. 3. [76]Fig. 3 [77]Open in a new tab Protein-Protein Interaction Networks of the Key Gene Screen. (A). Protein-protein interaction networks analysis. (B). Screening of key genes. (C). Survival analysis of FN1 high and low expression groups in ovarian cancer patients. (D). Detection of FN1 mRNA expression levels in SKOV3 cells overexpressing LAIR-1 groups by qRT-PCR; data are shown as mean ± SD LAIR-1 overexpression inhibits FN1 expression and phosphorylation levels of FAK, MEK, and ERK We verified the effect of LAIR-1 on FN1 expression by western blotting experiments to compare FN1 expression in SKOV3 and A2780 cell lines showing differential expression of LAIR-1 (i.e., OE vs. NC groups). The FN1 expression levels were significantly lower in the OE groups than in the NC groups (Fig. [78]4A, B, D, E). Since alterations in FN1 in a variety of cancers affect the phosphorylation levels of Focal Adhesion Kinase (FAK) [[79]8, [80]9] and because alterations in FAK can cause changes in the MAPK pathway [[81]10, [82]11], we also examined the expression levels of FAK, MEK, ERK, and their corresponding phosphorylated proteins by western blotting. In both SKOV3 and A2780 cell lines, the expressions of the phosphorylated FAK, MEK1/2, and ERK1/2 proteins were significantly lower in the OE groups than in the NC groups, although no significant changes were detected in the total proteins in the NC and OE groups (Fig. [83]4A-F). Fig. 4. [84]Fig. 4 [85]Open in a new tab Effect of LAIR-1 on the protein expression level of SKOV3 and A2780 cell lines. (A, B and C). LAIR-1 caused changes in FN1, p-FAK, FAK, p-MEK, MEK, p-ERK, and ERK protein expression levels in the SKOV3 cell line; data are shown as mean ± SD. (D, E and F). LAIR-1 caused changes in FN1, p-FAK, FAK, p-MEK, MEK, p-ERK, and ERK protein expression levels in the A2780 cell line; data are shown as mean ± SD Establishment and validation of FN1 backfilling expression in LAIR-1–overexpressing cell lines We further verified the role of FN1 in LAIR-1 inhibition of ovarian cancer cell proliferation and migration by constructing FN1 back-supplemented cell lines using FN1overexpressing lentivirus and by constructing SKOV3 and A2780 human ovarian cancer cell lines stably overexpressing LAIR-1. Fluorescence microscopic examinations showed that the FN1 transfection efficiency greater than 80% in LAIR-1–overexpressing SKOV3 and A2780 cell lines (Fig. [86]5A, B). The protein expression levels of FN1 and LAIR-1 in the LAIR-1 and FN1 null groups (NC + NC groups), the LAIR-1–overexpressing groups (LAIR-1 + NC groups), and the FN1 backfill groups (LAIR-1 + FN1 groups) were detected by western blotting. In the SKOV3 and A2780 cell lines, FN1 expression levels were lower and LAIR-1 expression levels were higher in the LAIR-1 + NC groups than in the NC + NC groups. The FN1 expression levels were increased, whereas the LAIR-1 expression levels were not significantly changed, in the LAIR-1 + FN1 groups compared to the LAIR-1 + NC groups (Fig. [87]5C, D). These findings indicated that the LAIR-1 stably overexpressing SKOV3 and A2780 cell lines were successfully backfilled with FN1; however, the addition of FN1 did not alter LAIR-1 expression. Fig. 5. [88]Fig. 5 [89]Open in a new tab FN1 backfill in LAIR-1 overexpressing ovarian cancer cell lines. (A and B). Lentiviral transfection rate of SKOV3 and A2780 cells. (C and D). Western blot protein level validation results; the data are shown as mean ± SD LAIR-1 inhibits ovarian cancer cell proliferation and migration by regulating the FN1-FAK-MEK-ERK signaling pathway We investigated whether LAIR-1 inhibits the proliferation and migration of ovarian cancer cells by regulating the FN1-FAK-MEK-ERK axis. We found significantly greater cell proliferation and migration in the LAIR-1 and FN1 co-overexpression groups than in the groups overexpressing LAIR-1 alone (Fig. [90]6A-F). To date, an effect of FAK on the MAPK pathway has not been reported in ovarian cancer. We explored whether FAK can cause downstream MAPK alterations and subsequently affect biological functions in ovarian cancer, and we found that the addition of FAK inhibitor could inhibit cell proliferation and migration in the LAIR-1 and FN1 co-overexpression groups (Fig. [91]6A-F). Transfection of FN1 into the LAIR-1–overexpressing ovarian cancer cell line, increased the expression levels of p-FAK, p-MEK, and p-ERK, according to the western blotting results, indicating that FN1 has a critical influence on the FAK-MEK-ERK axis. The phosphorylation levels of FAK, MEK, and ERK were significantly decreased by the FAK inhibitor treatment (Fig. [92]6G-K), suggesting that the phosphorylation of FAK can affect the phosphorylation of the MEK-ERK axis in ovarian cancer. Taken together, these results indicate that LAIR-1 may play an inhibitory role in ovarian cancer cell proliferation by affecting the FN1-FAK-MEK-ERK axis. Fig. 6. [93]Fig. 6 [94]Open in a new tab FN1 and FAK inhibitors affect the proliferation and migration of ovarian cancer cells. (A and B). Effects of FN1 and FAK inhibitors on the proliferation of ovarian cancer cell line; data are presented as mean ± SD. (C-F). Effect of FN1 and FAK inhibitors on the migration of ovarian cancer cell lines; data are shown as mean ± SD. (G-K). Changes in protein expression levels of LAIR-1 overexpressing ovarian cancer cell lines by FN1 and FAK inhibitors; data are presented as mean ± SD Discussion LAIR-1, a type I transmembrane glycoprotein of the immunoglobulin superfamily, was first discovered by Meyaard et al., who demonstrated its role in inhibiting NK cells-mediated cytotoxicity [[95]12]. In addition to its expression in NK cells, LAIR-1 was also expressed and showed inhibitory functions in many other peripheral blood mononuclear cells, such as B cells [[96]13], naive T cells [[97]14], and inflammation-infiltrated neutrophils [[98]15]. In some tumors [[99]16], including cervical cancer [[100]17], liver cancer [[101]18], and osteosarcoma [[102]5], LAIR-1 was also constitutively expressed. We previously demonstrated the expression of LAIR-1 in ovarian cancer tissues and ovarian cancer cell lines, and we found a correlation between its expression level and the tumor histological grade [[103]6]. Further exploration of the biological functions and regulatory mechanisms of LAIR-1 revealed that LAIR-1 could inhibit ovarian cancer cell growth by suppressing the PI3K-AKT-mTOR axis and by regulating mitochondrial bioenergy metabolism [[104]7, [105]19]. In the present study, we used RNA-seq technology and GO and KEGG pathway enrichment analysis as bioinformatics tools to map and investigate the genetic alterations controlled by LAIR-1, aiming to obtain a more comprehensive understanding of the molecular mechanism of LAIR-1 and its downstream molecules that regulate the altered biological behavior of ovarian cancer cells. RNA-seq sequencing data obtained for LAIR-1–overexpressing SKOV3 cells in the OE vs. NC groups revealed that the genes altered by LAIR-1 expression included 83 upregulated and 80 downregulated genes. Subsequent GO and KEGG enrichment analysis of the DEGs revealed that the enriched pathways were broadly related to many biological processes, including developmental processes, localization, negatively regulated biological processes, cell proliferation, motility, biological adhesion, reproduction, reproductive processes, and growth. The GO enrichment of these pathways was generally associated with cell proliferation and migration, further confirming that LAIR-1 expression affects the proliferation and migration of ovarian cancer cells. The KEGG enrichment results showed that the enriched pathways included the MAPK signaling pathway, transcriptional dysregulation in cancer, the PI3K-AKT signaling pathway, adherent spots, the Rap1 signaling pathway, and the Ras signaling pathway, etc., among others with the MAPK signaling pathway being the most obviously enriched. The MAPK signaling pathway can regulate various cellular processes such as proliferation, differentiation, and apoptosis, through ERK overexpression and activation, with the Ras-Raf-MEK-ERK axis as the most important pathway [[106]20]. Elucidation of PPI networks and key gene screening allowed us to identify FN1 as the most critical gene responsible for DEG alterations in LAIR-1–induced ovarian cancer cells, and KM survival analysis demonstrated that FN1 is a high-risk factor for ovarian cancer. It is reported that FN1 can enhance the angiogenesis and vascularization of multicellular aggregates (MCA)by increasing the expression of integrin, thus promoting the metastasis and growth of ovarian cancer [[107]21–[108]23]. Integrin-FAK system is also considered to be an important way to regulate the growth, invasion and metastasis of ovarian tumor cells [[109]24, [110]25]. FN1 expression can promote carcinogenesis by activating FAK phosphorylation [[111]9, [112]26]., and altered FAK phosphorylation can affect both the MAPK and PI3K-AKT signaling pathways [[113]10, [114]27]. Therefore, we speculated that LAIR-1 overexpression might exert an inhibitory effect by downregulating FN1 and thereby affecting the FAK-MEK-ERK axis. Examination of protein expression in SKOV3 and A2780 cell lines by western blotting revealed that FN1 was downregulated in the LAIR-1–overexpression groups compared with the null NC groups, and that the phosphorylation levels of FAK, MEK, and ERK were also reduced. We further investigated whether the alteration in FAK was caused by the downregulation of FN1 and whether the reduced phosphorylation of FAK in ovarian cancer could inhibit the MER-ERK axis by examining the alteration of FAK, MEK, and ERK by backfill experiments with FN1 overexpression and by intervention with FAK inhibitors. We found that the upregulation of FN1 was correlated with the LAIR-1-induced inhibition of ovarian cancer cell proliferation, migration, and FAK, MEK, and ERK phosphorylation, and that the complementary effect of FN1 could be inhibited by treatment with a FAK inhibitor. Taken together, our results suggest that LAIR-1 exerts an inhibitory effect on ovarian cancer cell proliferation by inhibiting the FAK-MEK-ERK axis through the downregulation of FN1. Apart from FN1, the key genes (e.g., KDR, PDGFB, FIGF, and ANGPT1) screened by the PPI network and Cytohubba are associated with angiogenesis. FN1 can enhance angiogenesis and vascularization [[115]28], suggesting that the changes in genes related to angiogenesis induced by LAIR-1 may also be caused by changes in FN1. In the tumor microenvironment, tumor angiogenesis or vasculogenesis provides a favorable microenvironment for tumors and promotes cancer growth, progression, and metastasis [[116]29]. Our follow-up studies will investigate the specific mechanism by which LAIR-1 changes FN1 expression and the role of LAIR-1 in regulating the ovarian cancer microenvironment. Electronic supplementary material Below is the link to the electronic supplementary material. [117]Supplementary Material 1^ (12.6KB, docx) [118]Supplementary Material 2^ (17.7KB, docx) [119]Supplementary Material 3^ (33.3MB, pptx) Acknowledgements