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Abstract

BACKGROUND:

Immunosuppressive receptor LILRB1 regulates tumors progression by transducing immune inhibitory signals via intracellular immunoreceptor tyrosine-based inhibitory motifs. However, its role in Hepatocellular Carcinoma (HCC) remains vague.

OBJECTIVE:

This study is aimed to disclose the association between LILRB1 and HCC.

METHODS:

Immunoblotting and qRT-PCR were employed to evaluate the level of LILRB1 in hepatocarcinoma cells. LILRB1-positive cells in tissue array were measured using immunohistochemistry staining. The relation among LILRB1, SHP1 and SHP2 and survival rates were analyzed using Gene Expression Profiling Interactive Analysis (GEPIA) and Oncomine database.

RESULTS:

LILRB1 was robustly reduced in hepatocarcinoma cells compared to normal cells. Clinically, LILRB1 was significantly higher in 49 of 75 (65%) paired paracarcinoma tissues than that in paired HCC samples. 48 of 75 (64%) HCC subjects in tissue microarray showed low level of LILRB1, compared to 25 of 75 (33%) in paired-adjacent tissues. Oncomine database and GEPIA analysis confirmed that LILRB1 was lower in HCC than normal tissues. Additionally, lowLILRB1 had a significant association with clinicopathological characteristics and Disease Free Survival, but no association with Overall Survival in HCC patients. Mechanismly, positive correlation between LILRB1 and SHP1, but not SHP2 was observed in HCC.

CONCLUSIONS:

LILRB1 possibly plays an antitumor effect in hepatocarcinoma cells by integrating SHP1, providing evidence that LILRB1 might be involved in the pathologic progression of HCC.

Keywords

Hepatocellular carcinoma LILRB1 Disease Free Survival SHP1 SHP2

1. Introduction

Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide, with a high degree of malignancy and a poor prognosis [1]. Currently, immunotherapy has been developed as an effective therapeutic option for HCC. It has been proved that the complex microenvironment of HCC can activate immunosuppressive receptors [2]. Binding of activated immunosuppressive receptors to its ligands results in a blunt immune response and tolerance of HCC [3]. However, immune checkpoint inhibitors are able to remotivate the immune system through intervening the combination of immunosuppressive receptors and its ligand, resulting in the inhibition of cancer progress [4]. Immune checkpoint inhibitor Opdivo (nivolumab) which targets programmed death-1 (PD-1), has been approved for the treatment of advanced liver cancer patients [5]. Unfortunately, However, a subset of subjects with advanced HCC treated with immune checkpoint inhibitors failed to respond to therapy and presents adaptive resistance to immune checkpoint inhibitors [6]. Therefore, we urgently need to expose new effective immunosuppressive receptors and illuminate their underlying mechanisms in HCC, as this will build a solid foundation for the development of new immune checkpoint inhibitors and offer novel concepts for improving the present situation of patients with advanced HCC

Leukocyte immunoglobulinn (Ig)-like receptor subfamily B (LILRBs) acting as a group of type I transmembrane glycoproteins are mostly expressed on the surface of immune cells such as macrophages, T cells, B cells, and NK cells [7, 8]. Once activated by its’ receptors, LILRBs transduce intracellular inhibitory signals, leading to an immune response silence [9]. LILRB1, also known as ILT2, CD85J and LIR1, is composed of extracellular Ig-like domains, transmembrane domain and intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) [10]. After binding with its specific ligands such as human Leukocyte Antigen-G (HLA-G) [11], UL18 [12], S100A8 and S100A9 [13], the tyrosine residues in the ITIM motif are phosphorylated by Src kinase, resulting in the activation of LILRB1 [14]. It has been demonstrated that the interaction of HLA-G with LILRB1 promotes the expansion of myeloid-derived suppressor cells (MDSCs), which causes the inhibition of the immunocompetence activity and cytotoxicity of T cells [15]. The binding of HLA-G to LILRB1 also inhibits the polarization and cytotoxicity of NK-cells [16]. Besides, LILRB1 also competitively inhibits the combination of HLA I to CD8, leading to the decrease of CD8 ${}^{+}$ T cells activity [17]. Therefore, LILRB1 is an important immunosuppressive receptor through the inhibition of immune response [18, 19]. Importantly, a previous study has indicated that the expression of LILRB1 is not only confined to the immune cell, but also expresses in the solid tumor cells. In gastric cancer tissues, LILRB1 is highly expressed and is closely related to the differentiation degree of gastric cancer [20]. However, whether LILRB1 participates in the development of hepatic carcinogenesis remains unclear.

Activated LILRB1 negatively regulates immune cells mainly by activating intracellular ITIMs, which subsequently recruit protein tyrosine phosphatase non-receptor type 6 (PTPN6/SHP-1), PTPN11/SHP-2, or Src homology 2 domain-containing inositol phosphatase (SHIP) [21]. In B-cell lymphoma, LILRB1 curbs the intracellular downstream signal including AKT, mTOR, STAT, c-Raf and GSK3- $\beta$ , which are all regulated by SHP1 or SHP2, resulting in the proliferation inhibition of B lymphoma cells [22]. It has been confirmed that Ptpn11/Shp2 acts as a tumor suppressor in hepatocellular carcinogenesis [23]. In addition, obatoclax derivative, SC-2001, induces cell apoptosis in hepatocellular carcinoma through regulating SHP-1-dependent STAT3 inactivation [24]. SHP-1 also prevents epithelial-mesenchymal transition in HCC by downregulating STAT3 activity [25]. This evidence indicates that SHP1 and SHP2 play anti-tumor roles in the pathogenesis of liver cancer, implying that LILRB1 may function as a negative regulator on tumor cells through regulating SHP1 or SHP2 and SHP1/2-mediated downstream signal transduction in hepatocellular carcinogenesis.

Herein, we demonstrated that LILRB1 was downregulated in HCC in vitro and in human specimens with HCC, and a low level of LILRB1 was closely related to patients’ age, and pathologic grades, tumor diameter, and clinical stages. Further, the expression of LILRB1 was positively correlated with SHP1 but not SHP2 in different liver subtypes and low LILRB1 had a poor DFS. Our data suggested LILRB1 was also expressed in solid liver tumor cells and might act as a tumor suppressor for hepatocellular carcinogenesis involvement with protein tyrosine phosphatase SHP1, providing a new idea for the study of immunosuppressive receptors in solid liver tumors.

2. Materials and methods

2.1 Tissue microarray and Immunohistochemistry

The tissue microarray containing 75 pairs of liver carcinoma tissue and the paired para-carcinoma tissue was purchased from OUTDO Biotechnology co., LTD (Shanghai, China). For immunohistochemistry (IHC) assay, slides were heated in an oven for 30 minutes at 60 ${}^{\circ}$ C. After deparaffinization in xylene and rehydration in gradient ethanol, LILRB1 antigen was retrieved by 0.01 M citrate salt solution (PH 6.0) pressure antigen retrieval. Then, the slides were cleaned by PBST and blocked with 5% BSA for 1 h at room temperature (RT). Primary antibody against LILRB1 (ab185325, abcam, Cambridge, UK) was employed for the incubation with the slides at 4 ${}^{\circ}$ C overnight. The next day, the endogenous peroxidase was neutralized by 30% H ${}_{2}$ O ${}_{2}$ for 15 min. Then sections were incubated with secondary antibody and streptavidin horseradish peroxidase (HRP) for 15 min at RT (PV-9000, ZSGB-BIO, China). Antigen-antibody complexes were visualized after staining with DAB (ZL1-9081, ZSGB-BIO, China). Photograph observation was performed under a Biological inverted microscope (IX51, Olympus, Japan). Comprehensive analysis included measuring staining intensity and the number of positive cells (negative: $-$ ; weakly positive: $+$ , $<$ 20%; middling positive: $++$ , 20%–50%; strong positive: $+++$ , $>$ 50%).

2.2 Cell culture and treatment

The human immortalized normal hepatocyte cell line-LO2 and three HCC cell lines including HepG2, Huh7, and MHCC97L were purchased from Shanghai cell bank, Chinese Academy of Sciences. These cells were cultured with complete Dulbecco’s modified Eagle’s medium (DMEM; 11320-033, GIBCO, USA) supplemented with 10% fetal bovine serum (10091-148, GIBCO, USA), 100 U/ml penicillin and 100 $\mu$ g/ml streptomycin (Sigma, USA) in a 37 ${}^{\circ}$ C humidified incubator with 5% CO ${}_{2}$ .

2.3 Data collection, correlation and survival analysis

The expressions of LILRB1 mRNA and Shp1 mRNA in normal liver tissues or precancerous tissue were collected from Oncomine database (Wurmbach Liver Statistics) and analyzed by GEPIA version 2. In Wurmbach Liver Statistics database, 10 normal, 30 liver cancer precursor and 35 HCC patients were enrolled in our correlation analysis between LILRB1 and pathological types. Expression value of LILRB1 which was indicated as the value of Log2 median-centered intensity in these specimens were used to assess the Pearson correlation of LILRB1 and SHP1 or SHP2 expression by SPSS software in different liver tissues. GEPIA was used to analyze the Overall Survival and Disease Free Survival.

2.4 Western blotting

Total protein extracts were prepared using RIPA protein lysate (P0013B, Beyotime, China). 40 $\mu$ g protein was separated by 10% SDS-PAGE and transferred to an activated PVDF membrane (IPVH00010, Millipore, Thermo Scientific, USA). After blocking with 5% fat-free milk at room temperature for 1 h, the membrane was incubated with primary antibodies against LILRB1 (ab185325, abcam, Cambridge, UK), SHP1 (ab227503, abcam, Cambridge, UK) and SHP2 (ab110194, abcam, Cambridge, UK) at 4 ${}^{\circ}$ C overnight. Actin (BM0627, Boster, China) served as the internal control. Next day, the membrane was incubated with Goat anti-Rabbit Secondary Antibody, HRP (BA1054, BOSTER, China) or Goat anti-Mouse Secondary Antibody, HRP (BA1051, BOSTER, China) for an additional 1 h at RT. After washing with TBST for 3 times, the proteins were visualized using the Imagequant LAS 4000 mini machine (GE Healthcare Life Sciences, USA) after incubating with ECL (NCI5079, Thermo Scientific, USA). All samples were subjected to at least 3 independent experiments.

2.5 Co-immunoprecipitation

Figure 1.

The expression of LILRB1 in liver cancer cells and tissue array. (A) LILRB1 protein levels in LO2, MHCC97L, Huh7 and HepG2 cells as determined by Western blotting. (B) The expression of LILRB1 mRNA in LO2, MHCC97L, Huh7 and HepG2 cells as analyzed by qRT-pCR. (C) Representative IHC images of LILRB1 expression in the microarray that contained 75 pairs of HCC and paired adjacent liver tissues. Brown color indicates positive staining of LILRB1. Multiple images were taken and representative one was presented. Scale bar: 200 and 50 $\mu$ m. (D) The expression difference between Stage I, II, III and IV of liver cancer as analyzed by GEPIA 2. ** indicates liver tumor cells vs human normal liver cells. Data represent the means $\pm$ SD of three experiments. **, $p<$ 0.01.

Total protein lysates extracted from LO2 cells were incubated with antibodies against SHP1 (ab227503, abcam, Cambridge, UK) and isotype control IgG (ab172730, abcam, Cambridge, UK) overnight. And the complexes were incubated with protein A/G beads (88802, Thermo Scientific, USA) for another 3 h at room temperature. Then the immunoprecipitate was resuspended in 2x SDS loading buffer were boiled for 5 minutes and subjected to western blotting assay using LILRB1 (ab185325, abcam, Cambridge, UK) and SHP1 antibodies according to the above method.

2.6 Statistical analyses

Differences between clinical information and LILRB1 expression was analyzed by GraphPad 6.0 using chi-square and Fisher’s exact tests. Pearson correlation coefficient was used for analyzing the relation between LILRB1 and SHP1 or SHP2 by SPSS 25.0 software. Different degrees of staining of histological staining were analyzed by image pro plus. Quantitative analysis of protein and RNA was conducted by Quantity One and Graph Pad Prism using one-way ANOVA, followed by Student’s $t$ -tests, respectively. $P<$ 0.05 was considered statistically significant.

3. Results

3.1 The expression of LILRB1 is depressed in liver cancer cells and liver cancer tissue array

LILRB1, as an immunoregulatory receptor, functions mainly as a key regulator in immune cells and hematologic malignant, but is seldom involved in the progress of solid cancer types [21]. To investigate the role of LILRB1 on HCC, we firstly determined its expression pattern in normal liver LO2 cells, and three liver cancer cell lines including MHCC97L, Huh7 and HepG2. As shown in Fig. 1A, the protein level of LILRB1 was drastically reduced in three liver cancer cell lines compared to that in the LO2 cells (Fig. 1A). Next, qRT-PCR was employed to further evaluate the transcription level of the LILRB1 gene in these cell lines. The results showed that relative expression of LILRB1 mRNA was significantly lower in the three cancer cell lines than that in normal liver cells (Fig. 1B).

To further ascertain the expression pattern of LILRB1 in hepatocellular carcinoma, a tissue microarray containing 75 pairs of HCC samples and paired adjacent liver tissue were employed to evaluate the level of LILRB1 in human specimens by immumohistochemical staining. The IHC results showed that the expression of LILRB1 was notably downregulated in most of the HCC samples compared to the paired adjacent liver tissue (Fig. 1C). Through IHC staining, we discovered that a low expression of LILRB1 ( $+$ and $-$ ) was found in 48 of 75 HCC tissues (64%), but we observed only 25 of 75 (33%) in the paracarcinoma tissue. Conversely, only 27 of 75 (36%) HCC specimens presented a high level of LILRB1 ( $++$ and $+++$ ), compared to 50 of 75 (67%) in adjacent normal tissue (Table 1). Based on Multiple-factor analysis, we observed that there was a significant difference between LILRB1 expression and liver histologic subtype ( $p=$ 0.0003) (Table 1). On the basis of IHC results, we also compared the LILRB1 expression in HCC with that in the paired adjacent liver tissue. The results showed that LILRB1 level was significantly higher in 49 of 75 (65%) paired paracarcinoma tissues than that in paired HCC samples. 15 of 75 subjects had no expression difference in LILRB1 both in HCC samples and paired paracarcinoma tissues and only 11 of 75 samples showed more expression of LILRB1 in HCC samples than that in their paired paracarcinoma tissues (Table S1). These data implies that LILRB1 is decreased in liver cancer cells and is possibly participates in the progression of liver cancer.

Table 1
IHC staining of LILRB1 in human HCC and adjacent liver tissues

Types	Positive expression number				$P$ value
	High expression		Low expression
Adjacent	+++	++	+	–
liver tissues	12	38	23	2
HCC	5	22	37	11	$p=$ 0.0003**

–, +, ++, +++ represent different degrees of staining.

3.2 LILRB1 decline is associated with part of clinicopathological characteristics of HCC patients

Next, we conducted the association between LILRB1 expression and clinicopathological characteristics of HCC patients in the tissue microarray. Among these 75 patients, we observed a significant decrease of LILRB1 in male subjects ( $p=$ 0.0002), which was not found in female patients ( $p=$ 0.6776) (Table 2). Though both different age ( $\leqslant$ 50 and $>$ 50) and different tumor diameter ( $\leqslant$ 5 cm and $>$ 5 cm) groups presented a significant correlation for LILRB1 in HCC tissues compared to normal control tissues, a more marked association was observed in aged subjects ( $p=$ 0.0197 vs 0.0099) and large tumors ( $p=$ 0.0422 vs 0.0018) (Table 2). However, only patients with higher pathologic grades (II $-$ III $+$ III, $p=$ 0.0003) and clinical stages (T3 $+$ T4, $p=$ 0.0003) presented a significant association between relative LILRB1 expression and histologic subtype, and this relationship did not appear in patients with low pathologic (I $-$ II $+$ II) and clinical stage (T1 $+$ T2) (Table 2). Besides, combined with Gene Expression Profiling Interactive Analysis (GEPIA version 2), we did not observe the correlation between LILRB1 expression and different cancer stages (Fig. 1D). Possibly, there is only significant correlation between LILRB1 level and high malignant HCC patients. These results indicate that frustrated LILRB1 is associated with part of the patients’ age, and pathologic grades, tumor diameter, and clinical stages.

Figure 2.

Expression pattern of LILRB1 in human HCC specimens. (A) The analysis of LILRB1 transcription activity in normal, liver cancer precursor and HCC based on the online Oncomine database. (B) The transcript of LILRB1 in normal and liver tumor as analyzed by GEPIA 2. *, $p<$ 0.05. **, $p<$ 0.01.

Table 2

Correlation between LILRB1 expression and clinicopathological characteristics of HCC patients

Characteristics	Relative LILRB1 expression				$P$ value
	Adjacent liver tissues		HCC tissues
	Low (25)	High (50)	Low (48)	High (27)
Gender
Male	16	45	37	24	$p=$ 0.0002**
Female	9	5	11	3	$p=$ 0.6776
Age
$\leqslant$ 50	8	23	18	13	$p=$ 0.0197*
$>$ 50	17	27	30	14	$p=$ 0.0099**
Pathologic grade
I $-$ II $+$ II	18	26	26	18	$p=$ 0.1352
II $-$ III $+$ III	7	24	22	9	$p=$ 0.0003**
Tumor diameter (cm)
$\leqslant$ 5	9	20	18	13	$p=$ 0.0422*
$>$ 5	16	30	30	14	$p=$ 0.0018**
Clinical stages
T1 $+$ T2	17	23	24	16	$p=$ 0.1793
T3 $+$ T4	8	27	24	11	$p=$ 0.0003**

Figure 3.

Survival rates analysis in low and high LILRB1 group. (A) Overall survival and (B) Disease Free survival in HCC patients with low LILRB1 ( $n=$ 91) and high LILRB1 ( $n=$ 91) as determined by GEPIA 2.

3.3 LILRB1 is downregulated in HCC patients from tumor database

Combined with the online Oncomine database, we further explored the expression profile of LILRB1 mRNA in HCC. The results showed that the expression of LILRB1 mRNA was much lower in HCC tissues ( $n=$ 35) than that in liver cancer precursor tissues ( $n=$ 30) and normal liver ( $n=$ 10) (Fig. 2A). The decline of LILRB1 was also verified utilizing GEPIA. Once matched the TCGA normal data with Genotype-Tissue Expression (GTEx) data, we found that the expression of LILRB1 showed significantly lower in liver cancer subjects ( $n=$ 369) than that in normal subjects ( $n=$ 160) (Fig. 2B).

3.4 HCC patients with low LILRB1 have a short period of Disease Free Survival

To further prove the crucial determinant of LILRB1 in the development of HCC, we analyzed the impact of LILRB1 on survival rates. As shown in Fig. 3, among the 182 HCC patients including low LILRB1 group ( $n=$ 91) and high LILRB1 group ( $n=$ 91), median overall survival was similar and had no difference among both group (Fig. 3A). However, in the same population group, the median DFS was much higher in patients with low LILRB1 than that in HCC patients with high LILRB1 (Fig. 3B). These findings demonstrate that LILRB1 level is associated with Disease Free Survival of HCC patients, implying the key role of LILRB1 in the treatment of HCC.

Figure 4.

Association analysis of LILRB1, SHP1 and SHP2 in HCC. (A) Protein expression of LILRB1, SHP1 and SHP2 in different cells as determined by western blotting assay. (B) Grayscale quantitation of LILRB1, SHP1 and SHP2 protein expression in different cells. (C) Correlation analysis between LILRB1 and SHP1 as evaluated by GEPIA 2 in liver, normal, tumor and the three combination. (D) Correlation analysis between LILRB1 and SHP2 as evaluated by GEPIA 2 in liver, normal, tumor and the three combination. R represent Pearson’s correlation coefficient. (E) Evaluation of the interaction between LILRB1 and SHP1 in liver LO2 cells using co-immunoprecipitation assay. The antibody against SHP1 was used to immunoprecipitate specific complexes. Then the immunoprecipitate was subjected to SDS-polyacrylamide gel electrophoresis for protein detection. *, $p<$ 0.05. **, $p<$ 0.01.

3.5 LILRB1 is positively correlated with SHP1 in liver cancer

The SH2 domain of SHP1/2 phosphatases constitutively bind to the cytoplasmic ITIMs of LILRB1, which play a pivotal role in cellular homeostasis and tumor development [26]. We therefore extensively searched the expression association of LILRB1, SHP1 and SHP2 in different liver subtypes. First, we determined the protein expression of SHP1, SHP2 and LILRB1 in different liver cell lines. We observed a moderate expression of SHP1, SHP2 and LILRB1 in normal liver cells. However, both the protein levels of SHP1 and LILRB1 were robustly declined in liver cancer cell lines such as MHCC97L, Huh7 and HepG2 cells (Fig. 4A and B). Though a moderate increase in the expression of SHP2 was observed in MHCC97L cells compared to LO2, no alteration appeared in Huh7 and HepG2 cells compared with LO2 cells (Fig. 4A and B). Additionally, based on search results from the online Oncomine database, we observed that transcription levels of LILRB1 had a significantly positive correlation with SHP1 in normal liver tissues ( $n=$ 10, $p=$ 0.028 and $R=$ 0.686) (Fig. S1A), liver cancer precursor ( $n=$ 30, $p<$ 0.001 and $R=$ 0.704) (Fig. S1B), and HCC specimens ( $n=$ 35, $p=$ 0.002 and $R=$ 0.512) (Fig. S1C). However, there was no association between the expression of LILRB1 mRNA and SHP2 among normal liver tissues ( $n=$ 10, $p=$ 0.30 and $R=$ 0.365) (Fig. S1D), liver cancer precursor ( $n=$ 30, $p=$ 0.30 and $R=$ $-$ 0.396) (Fig. S1E), and HCC tissues ( $n=$ 35, $p=$ 0.634 and $R=$ 0.083) (Fig. S1F). Similarly, utilizing GEPIA, there was significant correlation between LILRB1 and SHP1 (PTPN6) in liver ( $R=$ 0.58), normal ( $R=$ 0.77), tumor ( $R=$ 0.66) and the three combination ( $R=$ 0.42) (Fig. 4C). Though the moderate correlation between LILRB1 and SHP2 (PTPN11) was discovered in liver ( $R=$ 0.21) and normal ( $R=$ 0.41), there was a weak link between them in tumor ( $R=$ 0.18) and this three combination ( $R=$ 0.10) (Fig. 4D). Thus, it may be SHP1 but not SHP2 that transduces the LILRB1-evoked downstream signal which possibly takes part in the progression of HCC. To further prove the direct interaction between LILRB1 and SHP1 in liver cells, the antibody against SHP1 was used to immunoprecipitate specific complexes. After subjecting SDS-polyacrylamide gel electrophoresis using the immunoprecipitate, as shown in Fig. 4E, LILRB1 was immunoprecipitated using specific antibody against SHP1 but not the IgG antibody (Fig. 4E). The data proved that there was fairly strong interactions between LILRB1 and SHP1 in liver cells. And the correlation between LILRB1 and SHP1 might contribute to the progression of HCC.

4. Discussion

Compared with traditional surgery, radiation therapy, and chemotherapy, immunotherapy focuses on the activation of immunogenicity of tumor cells, which improves the recognition of tumor cells by immune cells and enhances anti-tumor immune response [27]. The well-known targets of tumor immunotherapy include cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), PD-1 and its ligand PD-L1, and lymphocyte activation gene 3 (LAG-3) [28]. However, owing to the complex mechanism of tumor progression, the anti-tumor effects against these targets is not sufficient to satisfy the clinical demand. In the present study, we found that immunosuppressive receptor LILRB1 was depressed in hepatocellular carcinoma cells and had a positive correlation with SHP1 that is a key switch in the progression of hepatocellular carcinoma, implying that LILRB1 may act as a potential therapeutic target for hepatocellular carcinoma immunotherapy.

Previous studies have shown that LILRB1 is not only expressed on immune cells, but also in tumor cells, in which LILRB1 is responsible for the regulation of tumor growth, drug resistance and cancer stem cell activities [29]. It has been proved that LILRB1 is expressed on acute myeloid leukaemia (AML) cells [30], neoplastic B cells (including B-cell leukemia, B-cell lymphoma, and multiple myeloma cells) [22], T cell leukemia and lymphoma cells [31]. However, LILRB1 plays a dual role in the occurrence and development of hematological tumors. LILRB1 can inhibit the cytotoxicity effects of immune cells and promote tumorigenesis. On the other hand, LILRB1 is able to ameliorate the progression hematological carcinoma by inhibiting the proliferation of lymphoma cells [32]. It is demonstrated that the expression of LILRB1 in NK cells was negatively correlated with the function of NK cells, and LILRB1 depletion could revive the immune function of NK cells in triple-negative breast cancer patients [33, 34]. In our study, LILRB1 was downregulated in liver cancer cells and HCC patients with different ages, especially in male HCC patients. But whether LILRB1 expression in liver cancer was associated with patients’ gender needed to further explore based on large scale clinical trials. Moreover, the low expression of LILRB1 was more significant when occurring in patients with high pathological grades and clinical stages. These results indicate that the declined LILRB1 may be involved in the occurrence of liver cancer. However, the decrease of LILRB1 in HCC tissue is not limited to hepatic parenchymal cells. It may be due to the downregulation of LILRB1 in many other cell types such as stellate cells, macrophages and other inflammatory cells, and this still needs further investigation.

Paired immunoglobulin-like receptor B (PirB), as the ortholog of LILRB2, participates in the process of immune response by regulating SHP1 or SHP2 [35, 36]. Removal of PirB leads to a decrease of phosphorylation of SHP-1 and SHP-2 in acute myeloid leukemia [37]. LILRB2 inhibition in non-small cell lung cancer (NSCLC) cells blunts the proliferation and colony formation viability of A549 cells by targeting the SHP2/CAMKI/CREB signal pathway [38]. Besides, Leukocyte-associated Ig-like receptor (LAIR1), also containing the immunoreceptor tyrosine-based inhibition motif (ITIMs), is crucial for the development of AML through activating SHP-1/CAMKI/CREB signal transduction [39]. LILRB1 recruiting SHP2 but not SHP1, as an anti-tumor regulator, significantly arrests the cell-cycle of human-activated T cells [40]. In our data, we detected that LILRB1 was positively correlated with SHP1 but not SHP2 in both HCC tissues and normal liver tissues. There had strong interaction between LILRB1 and SHP1 in liver cells. Therefore, the interaction between LILRB1 and SHP1 in liver cells may be involved in the pathogenesis of liver cancer. Besides, we confirmed that high LILRB1 had a good prognosis through affecting the period of DFS of HCC patients. Given that SHP1 had been proved to affect the prognosis of several cancer types [41, 42], we thought that the LILRB1-mediated improvement of prognosis was involved with SHP1. However, we can’t deny that SHP2 is also very important for HCC progress. SHP2 facilitates HCC cell differentiation and cancer stem cell expansion by promoting $\beta$ -catenin signaling [43]. However, there is also research indicating that SHP2 acts as a tumor suppressor gene which is significantly associated with the progression and prognosis of HCC [44]. Hence, based on our results, SHP2-mediated progression of HCC is not dependent on LILRB1. Additionally, as a nonclassical HLA class I, the binding to HLA-G of LILRB1 on natural killer (NK) cells and T lymphocytes leads to the inhibition of proliferation and cytotoxicity of T lymphocyte or NK cell, inducing immunosuppression function [45]. In liver cancer cells, high level of HLA-G expression allows escape of tumoral cells from immune response in inhibiting the properties of immune cells [46, 47]. In the present study, LILRB1 was lowly expressed in HCC tissues and liver cancer cells. On the basis of the promotive role on immunosuppression of the binding of HLA-G to LILRB1, we thought that low expression of LILRB1 in liver cancer cells might impair the interaction between HLA-G and LILRB1, leading to the inhibition of tumor immune evasion which facilitated the restoration of immune cell cytotoxicity to HCC cells. On the contrary, low LILRB1 presented a poor prognosis of HCC patients which was possibly related with weak proliferation inhibition signals mediated by SHP1. In other words, low LILRB1 might play a dual role in HCC including immune activation and proliferation inhibition. However, the regulation role in HCC of HLA-G through LILRB1 was still obscure. Therefore, LILRB1’s homeostatic control should contribute to the treatment of liver cancer. However, to determine whether there is an anti-tumor role of LILRB1 on HCC development also needs further investigation of the biological function of LILRB1 in in vivo and in vitro experiments.

In summary, this study demonstrated that LILRB1 was expressed on liver cells and little expression in HCC patients and liver tumor cells. Low levels of LILRB1 had a significant positive association with SHP1 level and with part of clinicopathological data of HCC patients. LILRB1 level also affected the period of DFS. These data implied that LILRB1 might participate in tumorigenesis and therapy of HCC. Possibly, LILRB1 acts as an anti-cancer factor by interacting with SHP1 in the pathological process of HCC.

Footnotes

Acknowledgments

We thank the support of The Youth Innovation Team of Shaanxi Universities and Gene Expression Profiling Interactive Analysis (GEPIA). This work was supported by the Research Foundation of Xi’an Medical University (Grant No 2017DOC18 and 2018GJFY04), Natural Science Basic Research Plan in Shaanxi Province of China (Grant No. 2018JQ 8043), Young Talent Fund of University Association for Science and Technology in Shannxi (Grant No. 20190309) and Natural Science Foundation of China (81802332).

Conflict of interest

The authors declare that they have no competing interests.

Abbreviations

Supplementary data

Supplement Table S1
Differential expression of LILRB1 in human HCC and adjacent liver tissues
	Higher expression in adjacent liver tissues	No variance	Higher expression in HCC tissues
Number of samples	49 (49/75, 65%)	15 (15/75, 20%)	11 (11/75, 15%)

Supplementary Fig. S1.

Association analysis ofLILRB1, SHP1 and SHP2 mRNA in human HCC samples. (A) Correlation analysis between LILRB1 mRNA and SHP1 as evaluated by SPSS in normal liver tissues, (B) liver cancer precursor tissues (C) and HCC tissues. (D) Correlation analysis between LILRB1 mRNA and SHP2 as determined by SPSS software in normal liver tissues, (E) liver cancer precursor tissues (F) and HCC tissues. Data represent the means $\pm$ SD of three experiments. R represent Pearson’s correlation coefficient. *, $p<$ 0.05; **, $p<$ 0.01.

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Immunosuppressive receptor LILRB1 acts as a potential regulator in hepatocellular carcinoma by integrating with SHP1

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1. Introduction

2. Materials and methods

2.1 Tissue microarray and Immunohistochemistry

2.2 Cell culture and treatment

2.3 Data collection, correlation and survival analysis

2.4 Western blotting

2.5 Co-immunoprecipitation

3. Results

3.1 The expression of LILRB1 is depressed in liver cancer cells and liver cancer tissue array

Table 1 IHC staining of LILRB1 in human HCC and adjacent liver tissues

3.4 HCC patients with low LILRB1 have a short period of Disease Free Survival

4. Discussion

Footnotes

Acknowledgments

Conflict of interest

Abbreviations

Supplementary data

References

Table 1
IHC staining of LILRB1 in human HCC and adjacent liver tissues