RasGRP1 promotes the acute inflammatory response and restricts inflammation-associated cancer cell growth

Wang, Cong; Li, Xue; Xue, Binbin; Yu, Changping; Wang, Luoling; Deng, Rilin; Liu, Hui; Chen, Zihao; Zhang, Yingdan; Fan, Suping; Zuo, Chaohui; Sun, Hungyu; Zhu, Haizhen; Wang, Jianli; Tang, Songqing

doi:10.1038/s41467-022-34659-x

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Published: 16 November 2022

RasGRP1 promotes the acute inflammatory response and restricts inflammation-associated cancer cell growth

Nature Communications volume 13, Article number: 7001 (2022) Cite this article

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Abstract

An acute inflammatory response needs to be properly regulated to promote the elimination of pathogens and prevent the risk of tumorigenesis, but the relevant regulatory mechanism has not been fully elucidated. Here, we report that Ras guanine nucleotide-releasing protein 1 (RasGRP1) is a bifunctional regulator that promotes acute inflammation and inhibits inflammation-associated cancer. At the mRNA level, Rasgrp1 activates the inflammatory response by functioning as a competing endogenous RNA to specifically promote IL-6 expression by sponging let-7a. In vivo overexpression of the Rasgrp1 3’ untranslated region enhances lipopolysaccharide-induced systemic inflammation and dextran sulphate sodium-induced colitis in Il6^+/+ mice but not in Il6^-/- mice. At the protein level, RasGRP1 overexpression significantly inhibits the tumour-promoting effect of IL-6 in hepatocellular carcinoma progenitor cell-like spheroids. Examination of the EGFR signalling pathway shows that RasGRP1 inhibits inflammation-associated cancer cell growth by disrupting the EGFR-SOS1-Ras-AKT signalling pathway. Tumour patients with high RasGRP1 expression have better clinical outcomes than those with low RasGRP1 expression. Considering that acute inflammation rarely leads to tumorigenesis, this study suggests that RasGRP1 may be an important bifunctional regulator of the acute inflammatory response and tumour growth.

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Introduction

IL-6 is an important inflammatory cytokine that is expressed during infection and cancer and was identified as a prominent target for clinical intervention^1,2,3,4. Almost all stromal cells and immune cells produce IL-6. Cytokines, bacteria and viruses are major inducers of IL-6 production^5,6. IL-6 expression is relatively low in resting innate immune cells but is rapidly elevated in innate immune cells activated by microbial components such as lipopolysaccharide (LPS)⁷. During the maintenance of IL-6 level under physiological conditions, the microRNA (miRNA) let-7, which is abundantly expressed in resting innate immune cells, represses IL-6 expression by binding to its 3’ untranslated region (UTR)^8,9,10. However, let-7 expression is only slightly downregulated in activated innate immune cells¹⁰. Thus, the rapid inhibitory effect of let-7 on IL-6 in activated innate immune cells, especially during the acute inflammatory response, maybe a potential druggable target in inflammatory diseases.

miRNAs constitute a class of small noncoding RNAs that modulate gene expression by promoting mRNA degradation or inhibiting translation¹¹. An mRNA can sometimes sponge many of its targeted miRNAs via its 3’ UTR. As miRNA sponges, RNA transcripts engage in crosstalk and regulate each other by competing for their shared miRNAs, known as competing endogenous RNAs (ceRNAs)^12,13. Based on competition for targets, ceRNAs exhibit positively correlated and temporal, spatial and disease-specific expression patterns¹³. Recent reports have indicated that mRNAs, long ncRNAs (lncRNAS), pseudogenes and circular RNAs (cirRNAs) can act as ceRNAs to affect the progression of cancer or neurological diseases^{14,15,16,17,18,19}. Interestingly, sponging of an miRNA is competitive after a threshold ceRNA level is reached²⁰. For example, it was recently demonstrated that an miRNA target was derepressed immediately after a threshold of added ceRNA was exceeded²¹. In addition, the expression of inflammatory cytokines, such as IL-6, is substantially upregulated during the acute inflammatory response. However, the crosstalk between ceRNAs and the expression of inflammatory cytokines during the acute inflammatory response remains to be further investigated.

Ras guanine nucleotide-releasing protein 1 (RasGRP1), a member of the RasGRP family, is a guanine nucleotide exchange factor associated with Ras activation^22,23. The catalytic region of RasGRP1 consists of a Ras exchange motif (REM) and a cell division cycle 25 (CDC25) domain, and the regulatory region of RasGRP1 contains two EF-hands (calcium-binding domains) and a C1 domain (a DAG-binding domain)^22,24. RasGRP1 is highly expressed in T cells and can also be detected in B cells, mast cells, natural killer cells, neuronal cells and activated macrophages^{25,26,27,28,29,30}. Previous studies have shown that RasGRP1 is critical for mediating Ras/Erk activation, thus promoting the positive and negative selection of T cells^31,32,33. Recently, increasing evidence has shown that RasGRP1 is involved in many human diseases, especially inflammatory diseases such as systemic lupus erythematosus, type 2 diabetes, and cancer^{34,35,36,37,38}. Although the function of RasGRP1 has been extensively investigated in T cells, its role in innate immune cells, especially in macrophages, is still unclear. Elucidating the function of RasGRP1 in innate immunity will provide further insight into the pathophysiology and underlying mechanisms of inflammatory diseases.

Here, we show that RasGRP1 is an important regulator of acute inflammation that promotes the production of the proinflammatory cytokine IL-6 by competing with let-7a and decreases the probability of inflammation-associated cancer in a protein-independent and protein-dependent manner, which may explain why acute inflammation rarely leads to tumorigenesis.

Results

The expression of both RasGRP1 and IL-6 is regulated by let-7a

Previous studies have shown that let-7 can directly inhibit the expression of IL-6 by binding the IL6 3’ UTR^8,9. We examined the expression profiles of let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, let-7i and let-7k in mouse peritoneal macrophages and found that let-7a showed the highest expression level in these cells (Fig. 1a). In addition, the results of RNA immunoprecipitation (RIP) experiments in which miRNA was used to pull down mRNA showed that let-7a binds Il6 mRNA in a dose-dependent manner (Fig. 1b), suggesting that let-7a may be the major let-7 family member that regulates IL-6 expression in peritoneal macrophages. To confirm that let-7a inhibits IL-6 expression, we cotransfected Il6 or Tnf expression plasmids and synthetic let-7a mimics and/or let-7a inhibitors into HEK293 cells. The results showed that the let-7a mimics inhibited the expression of IL-6 and TNF by binding the Il6 3’ UTR and that this effect was reversed by the let-7a inhibitor (Fig. 1c and Supplementary Fig. 1a, b). To investigate whether let-7a is a critical mediator for inhibiting IL-6 expression in macrophages, we overexpressed or silenced let-7a in macrophages by transfecting the cells with synthetic let-7a mimics or a let-7a inhibitor. Quantitative RT‒PCR analysis showed that Il6 mRNA levels exhibited negligibly changes in LPS-stimulated macrophages transfected with let-7a mimics or a let-7a inhibitor (Fig. 1d). After stimulation of the cells with LPS, IL-6 protein levels decreased in the let-7a mimic-transfected cells and increased in the let-7a inhibitor-transfected cells compared with the control cells (Fig. 1e). To explore the mechanism that let-7a regulates IL-6 production, luciferase reporter plasmids containing the wild-type Il6 3’ UTR or a mutant Il6 3’ UTR were constructed (Supplementary Fig. 1c). Luciferase reporter system analysis indicated that let-7a mimics disrupted Il6 3’ UTR reporter gene activity but did not disrupt the luciferase activity of the mutant Il6 3’ UTR reporter gene (Fig. 1f). These results suggested that IL-6 expression in macrophages was inhibited by let-7a via translational inhibition but not via mRNA degradation.

To identify the possible targets of let-7a that might compete with Il6 during the innate immune response, the gene expression profiles of mouse peritoneal macrophages stimulated with LPS were further analysed. We focused on the expression of 1046 putative let-7 target genes predicted by the TargetScan algorithm (http://www.targetscan.org), and 23 putative let-7 target genes, including 10 genes with upregulated expression, showed an expression change of 5-fold or greater compared to LPS-unstimulated macrophages (Fig. 1g). To verify the accuracy of the gene expression profiles, the expression of 10 upregulated genes was further quantified by qPCR, and the results showed that among these 10 genes, Rasgrp1 exhibited the most strongly upregulated expression (Fig. 1h), indicating that Rasgrp1 might effectively sponge let-7a. To confirm that Rasgrp1 was regulated by let-7a, we cotransfected Rasgrp1 expression plasmids and synthetic let-7a mimics and/or a let-7a inhibitor into HEK293 cells and found that the let-7a mimics inhibited the expression of RasGRP1 by binding the Rasgrp1 3’ UTR and that the let-7a inhibitor reversed the effect of the let-7a mimics (Fig. 1i, j and Supplementary Fig. 1d). Luciferase reporter analysis also showed that the let-7a mimics reduced Rasgrp1 3’ UTR reporter gene activity but did not reduce the luciferase activity of the mutant Rasgrp1 3’ UTR reporter gene (Fig. 1k and Supplementary Fig. 1e). In summary, these results indicated that both IL-6 and RasGRP1 were regulated by let-7a.

Rasgrp1 is coexpressed with Il6, and RasGRP1 silencing selectively inhibits IL-6 protein expression during the acute inflammatory response

To explore the relationship of Rasgrp1 and Il6 during the acute inflammatory response, we treated mouse peritoneal macrophages with a range of doses of LPS and observed that the expression of Rasgrp1 and Il6 was significantly upregulated in a dose-dependent manner (Fig. 2a). A similar phenomenon was observed when peritoneal macrophages were stimulated by a range of doses of polyinosinic-polycytidylic acid (poly(I:C)) (Fig. 2b) or CpG oligodeoxynucleotide (ODN) (Fig. 2c). In addition, we stimulated mouse bone marrow-derived macrophages with a range of doses of LPS, poly (I:C) or CpG ODN. We found that the expression of Rasgrp1 and Il6 was significantly upregulated in a dose-dependent manner (Supplementary Fig. 2a–c). These results suggested that Rasgrp1 may have been coexpressed with Il6.

**Fig. 2: *Rasgrp1* is coexpressed with *Il6*, and *Rasgrp1* silencing selectively inhibits IL-6 protein expression in an acute inflammatory response.**

To confirm the coexpression of Rasgrp1 and Il6, we treated mouse peritoneal macrophages with 100 ng/ml LPS for different durations and found that the expression levels of both Rasgrp1 and Il6 were quickly and significantly upregulated in a time-dependent manner (Fig. 2d). Similar results were obtained when peritoneal macrophages were stimulated with 10 μg/ml poly (I:C) (Fig. 2e) or 5 μg/ml CpG ODN (Fig. 2f) for different durations. In addition, we stimulated mouse bone marrow-derived macrophages with 100 ng/ml LPS, 10 μg/ml poly (I:C) or 5 μg/ml CpG ODN for different durations. We observed that the Rasgrp1 and Il6 expression levels were quickly and significantly upregulated in a time-dependent manner (Supplementary Fig. 2d–f).

Next, we examined the coexpression of Rasgrp1 and Il6 in intestinal epithelial cells isolated from inflamed colons of C57B/6 mice. We found that Rasgrp1 and Il6 expression was significantly upregulated in these cells in the days after the mice were fed 2.5% dextran sulfate disodium (DSS; Fig. 2g). We also examined the coexpression of Rasgrp1 and Il6 in cells isolated from the lung, kidney, colon, heart and liver of C57B/6 mice challenged with LPS. We observed that the expression levels of both Rasgrp1 and Il6 were significantly upregulated during LPS-induced systemic inflammation (Fig. 2h). Together, these data indicated that Rasgrp1 is coexpressed with Il6 in acute inflammation and inflammation-associated diseases.

To ascertain the function of Rasgrp1 in the acute inflammatory response, we silenced Rasgrp1 expression in mouse peritoneal macrophages (Fig. 2i) and found that short interfering RNA (siRNA) knockdown of Rasgrp1 did not affect the mRNA expression of cytokines or chemokines in peritoneal macrophages stimulated with LPS, poly (I:C) or CpG ODN (Fig. 2j–l and Supplementary Fig. 3a–o). Consistent with the mRNA expression of cytokines and chemokines, the activation of ERK, JNK and P38 MAPKs, as well as that of the IKKα/β-IκBα pathway, was not affected by Rasgrp1 expression knockdown after LPS treatment (Supplementary Fig. 3p). Interestingly, IL-6 secretion was significantly decreased when we stimulated Rasgrp1-silenced peritoneal macrophages with LPS, poly (I:C) or CpG ODN (Fig. 2m–o). In contrast, no difference in TNF or IL-1β secretion was observed between the Rasgrp1-silenced macrophages and the control macrophages (Supplementary Fig. 3q–v). Taken together, these results showed that Rasgrp1 selectively increased IL-6 protein levels during the acute inflammatory response.

We also examined the coexpression of RasGRP1 and IL6 in human peripheral blood monocyte-derived macrophages (MDMs). We found that RasGRP1 and IL6 expression levels were also significantly upregulated in MDMs stimulated with a range of doses of LPS (Supplementary Fig. 4a), poly (I:C) (Supplementary Fig. 4b) or CpG ODN (Supplementary Fig. 4c). Similar results were obtained when MDMs were stimulated with 100 ng/ml LPS (Supplementary Fig. 4d), 10 μg/ml poly (I:C) (Supplementary Fig. 4e) or 5 μg/ml CpG ODN (Supplementary Fig. 4f) for different durations.

Next, we silenced RasGRP1 expression in human MDMs (Supplementary Fig. 4g) and found that siRNA knockdown of RasGRP1 did not affect the mRNA expression of cytokines or chemokines in human MDMs stimulated with LPS, poly (I:C) or CpG ODN (Supplementary Fig. 4h–m). Consistent with the effects observed in mouse peritoneal macrophages, IL-6 secretion was significantly decreased when we silenced RasGRP1 expression in human MDMs stimulated with LPS, poly (I:C) or CpG ODN (Supplementary Fig. 4n). However, TNF secretion was not affected (Supplementary Fig. 4o). Taken together, these results showed that RasGRP1 also selectively promoted IL-6 protein levels in human MDMs during the acute inflammatory response.

The Rasgrp1 3’ UTR enhances IL-6 protein expression levels by sponging let-7a

To verify that Rasgrp1 competes with Il6 for let-7a binding, we cotransfected an Il6 expression vector and let-7a mimics with the Rasgrp1 coding sequence (CDS) and/or a 3’ UTR expression vector into HEK293 cells and found that the Rasgrp1 3’ UTR, but not the Rasgrp1 CDS, promoted IL-6 expression by competing with let-7a for Il6 binding (Fig. 3a). RIP results also showed that the Rasgrp1 3’ UTR, but not the mutant Rasgrp1 3’ UTR, competed with Il6 for binding to let-7a (Fig. 3b). To identify the sequence in the Rasgrp1 3’ UTR that binds to let-7a, three candidate let-7a-sponging RNAs that were each 30 bp in length were synthesised on the basis of the sequence proximal to the potential let-7 binding site in the Rasgrp1 3’ UTR (Fig. 3c). Then, the effects of the three let-7a-sponging RNA candidates were examined by quantitative RT‒PCR, and the results showed that the let-7a-sponging RNAs had no significant effect on Il6 mRNA levels in LPS-stimulated macrophages in the presence or absence of the let-7a mimic (Fig. 3d and Supplementary Fig. 4p). ELISA showed that only the sponging RNA candidate containing a sequence complementary to the seed sequence (Supplementary Fig. 5a), which is important for the binding of an miRNA to a mRNA, increased IL-6 protein levels by competing with Il6 in macrophages stimulated with LPS (Fig. 3e). Confocal microscopy showed that only the sponging RNA candidate containing a sequence complementary to the seed sequence bound let-7a (Fig. 3f and Supplementary Fig. 6). RIP results also showed that the sponging RNA candidate containing a sequence complementary to the seed sequence competed with Il6 for let-7a binding and that this competition was dose-dependent (Fig. 3g). These data suggested that the sponging RNA candidate containing a sequence complementary to the seed sequence positively regulated the production of the IL-6 protein in macrophages during an acute inflammatory response.

**Fig. 3: The 3’ UTR of *Rasgrp1* increases IL-6 protein levels by sponging let-7a.**

To provide further evidence, two sgRNAs and three genotyping primers were designed to deplete the let-7a-binding site in the 3’ UTR of Rasgrp1 in RAW264.7 cells via a genome-editing technique (Supplementary Fig. 7a). Five candidate Rasgrp1 3’ UTR mutant clones in RAW264.7 cells were confirmed by PCR screening (Supplementary Fig. 7b). DNA sequencing confirmed two positive clones among the five candidate mutant clones (Fig. 3h and Supplementary Fig. 8). In the two confirmed clones with mutations, we observed that the Rasgrp1 and Il6 mRNA levels were not affected by mutation of the Rasgrp1 3’ UTR (Fig. 3i and Supplementary Fig. 9), but the IL-6 protein levels were significantly decreased after LPS, poly (I:C) or CpG ODN treatment (Fig. 3j). To exclude off-target effects, we transfected the sponging RNA candidate containing a sequence complementary to the seed sequence into the positive clones. We found that the sponging RNA candidate containing a sequence complementary to the seed sequence reversed the effect of Rasgrp1 3’ UTR mutation on IL-6 protein levels after TLR3/4/9 agonist treatments (Fig. 3k). Together, these results suggested that the Rasgrp1 3’ UTR sponges let-7a to increase IL-6 protein levels during an acute inflammatory response.

The Rasgrp1 mRNA 3’ UTR aggravates LPS-induced systemic inflammation in Il6 ^+/+ mice

To investigate the role of the Rasgrp1 3’ UTR in vivo, we synthesised cholesterol-conjugated let-7a-sponging RNA and let-7a mimics. After intraperitoneal injection of the cholesterol-conjugated let-7a-sponging RNA and let-7a mimics, we found that the sponging RNA candidate containing a sequence complementary to the seed sequence effectively bound to the let-7a mimics in vivo (Fig. 4a, b). The mice that had been intravenously injected with the cholesterol-conjugated let-7a mimics (Supplementary Fig. 10a) produced less IL-6 in response to LPS than did the control mice, and the sponging RNA candidate containing a sequence complementary to the seed sequence reversed the effects of the let-7a mimics (Fig. 4c). After lethal challenge with LPS, the Il6^+/+ mice but not the Il6^-/- mice that had been intravenously injected with cholesterol-conjugated let-7a exhibited substantially prolonged survival, whereas the Il6^+/+ mice treated with let-7a-sponging RNA exhibited profoundly reduced survival (Fig. 4d, e). Furthermore, after intraperitoneal injection of the cholesterol-conjugated let-7a mimics into mice, we observed milder inflammatory infiltration in the lungs, but intraperitoneal injection of the cholesterol-conjugated let-7a-sponging RNA caused more severe inflammatory infiltration in the lungs, and the sponging RNA candidate containing a sequence complementary to the seed sequence reversed the effects of the let-7a mimics (Fig. 4f). Immunohistochemistry (IHC) and immunofluorescence (IF) staining showed that the let-7a mimics inhibited IL-6 production (Fig. 4g–j and Supplementary Fig. 10) in the lungs of mice, but the sponging RNA candidate containing the sequence complementary to the seed sequence promoted IL-6 production. All these results suggested that the Rasgrp1 3’UTR promoted LPS-induced systemic inflammation by competing with Il6 for let-7a binding, which led to enhanced IL-6 production. Considering that the uptake of the Rasgrp1−3’ UTR in vivo might not be very efficient, the observed in vivo effects might partly be due to the induction of an interferon response.

**Fig. 4: The 3’ UTR of *Rasgrp1* aggravates lipopolysaccharide (LPS)-induced systemic inflammation in *Il6*^+/+ mice.**

The Rasgrp1 3’ UTR aggravates DSS-induced colitis in Il6 ^+/+ mice

Our data indicated that the Rasgrp1 3’ UTR promoted IL-6 protein production during TLR-induced inflammatory responses both in vitro and in vivo. To further investigate the pathophysiological significance of these observations, we analysed the role of Rasgrp1 3’ UTR in the development of DSS-induced colitis. After subjecting mice to DSS treatment, we found that the Il6^+/+ mice but not the Il6^-/- mice that had been intravenously injected with cholesterol-conjugated let-7a, exhibited significantly less weight loss (Fig. 5a, b). However, the rate of body weight loss was significantly higher in the Il6^+/+ mice intravenously injected with cholesterol-conjugated let-7a-sponging RNA (Fig. 5a and Supplementary Fig. 11a). Moreover, the mice intravenously injected with cholesterol-conjugated let-7a mimic showed less severe rectal bleeding and colon shortening (Fig. 5c, d), lower serum IL-6 levels (Fig. 5e, g, h, j, k), less bowel wall destruction and greater bowel wall tissue proliferation, and less inflammatory cell infiltration than the control mice (Fig. 5f, i and Supplementary Fig. 11b). In contrast, the mice intravenously injected with cholesterol-conjugated let-7a-sponging RNA showed more severe rectal bleeding and greater colon shortening (Fig. 5c, d), higher serum IL-6 levels (Fig. 5e, g, j, k), greater bowel wall destruction and higher inflammatory cell infiltration than the control mice (Fig. 5f, i). When the cholesterol-conjugated let-7a mimic and let-7a-sponging RNA were simultaneously injected intravenously, the mice acquired a phenotype that was similar to that of the control mice (Fig. 5). Collectively, our data suggested that the Rasgrp1 3’ UTR aggravates the development of colitis by promoting IL-6 protein production.

**Fig. 5: The 3’ UTR of *Rasgrp1* aggravates dextran sulphate sodium (DSS)-induced colitis in *Il6*^+/+ mice.**

RasGRP1 inhibits the growth of inflammation-associated cancer cells

A previous study showed that IL-6 signalling contributes to the malignant progression of liver cancer progenitors³⁹. Therefore, we induced HCC progenitor cell-like spheroid formation by growing Huh7 cells on low-attachment plates and found that IL-6 significantly promoted the malignant progression of liver cancer progenitors (Supplementary Fig. 12). An acute inflammatory response usually leads to high IL-6 production, but it rarely leads to tumorigenesis. How is the tumour-promoting effect of IL-6 counteracted during the acute inflammatory response? In our experiments, we observed that RasGRP1 expression was significantly upregulated, but the function of the RasGRP1 protein in the acute inflammatory response is largely unknown. To ascertain the role of RasGRP1, we used lentivirus to overexpress RasGRP1 in Huh 7 cells and found that RasGRP1 overexpression significantly inhibited the tumour-promoting effect of IL-6 (Fig. 6a, b and Supplementary Fig. 13). A recent study has reported that upregulation of RasGRP1 expression is associated with a better prognosis in colorectal cancer patients because it limits proliferative EGFR-SOS1-Ras-ERK signals⁴⁰. Therefore, we speculated that RasGRP1 may inhibit the development of liver cancer by inhibiting EGFR-SOS1-Ras-ERK signalling pathway activation to suppress the tumour-promoting effect of IL-6 during the acute inflammatory response. To test our hypothesis, we evaluated the expression levels of RasGRP1, SOS1, SOS2, EGF and EGFR in Huh7 and HepG2 cells and found that EGF and EGFR exhibited higher expression in Huh7 cells than in HepG2 cells (Fig. 6c). Then, we explored the role of RasGRP1 in Huh7 cells treated with 10 ng/ml EGF and found that RasGRP1 inhibited Huh7 cell proliferation (Fig. 6d–f), mainly by limiting EGFR-SOS1-Ras-AKT signalling (Fig. 6g, h). Next, we evaluated the specificity of the anti-RasGRP1 antibodies (Fig. 7a and Supplementary Fig. 14) and performed IHC staining for RasGRP1 in a human liver cancer tissue microarray. Representative images of the liver cancer tissue samples with high, moderate or weak (according to the IHC score in which weak≤1; 1<moderate≤2; and 2<high≤3) RasGRP1 expression are shown in Fig. 7b. Interestingly, only 22.2% of the liver cancer tissue samples exhibited high RasGRP1 expression, with a greater percentage of samples showing moderate and low RasGRP1 expression (40% and 37.8%, respectively) (Fig. 7c). However, 50% of the liver cancer tissue samples showed upregulated RasGRP1 expression compared to that in matched adjacent liver tissues (Fig. 7d). The RasGRP1 IHC score for the tumour samples with upregulated RasGRP1 expression was higher than that in tumour samples with downregulated RasGRP1 expression (Fig. 7e). The liver cancer patients with upregulated RasGRP1 expression exhibited significantly smaller tumour sizes (Fig. 7f), lower γ-glutamyl transferase levels (Fig. 7g) and longer total survival (Fig. 7h) than the liver cancer patients with downregulated RasGRP1 expression. Taken together, our results indicated that the RasGRP1 protein can inhibit liver cancer by limiting EGFR-SOS1-Ras-AKT signalling.

**Fig. 6: RasGRP1 affects the growth of liver cancer cells.**

**Fig. 7: RasGRP1 affects the prognosis of liver cancer patients.**

To investigate whether the RasGRP1 protein exerts an inhibitory effect on other cancers, we examined the effect of RasGRP1 gene expression on the survival of cancer patients with one of the multiple types of cancer. Specifically, we accessed the Kaplan–Meier Plotter database (http://kmplot.com/analysis/), and our analysis of the database samples indicated that high RasGRP1 expression was positively correlated (Fig. 8a–l) or not significantly correlated (Supplementary Fig. 15a–k) with higher overall survival for cancer patients with a specific type of cancer. Notably, high RasGRP1 expression was positively correlated with lower overall survival only for patients with kidney renal papillary cell carcinoma (Supplementary Fig. 15l). These results suggested that the RasGRP1 protein may inhibit the growth of multiple kinds of cancer cells.

**Fig. 8: RasGRP1 expression correlates with the overall survival of cancer patients.**

Discussion

In this study, we showed that RasGRP1 has an important function in regulating the acute inflammatory response and cancer. An acute inflammatory response is crucial for the efficient clearance of pathogens but needs to be strictly regulated to prevent deleterious side effects, such as the development of tumours and autoimmune diseases^9,41. Our study demonstrated that RasGRP1 is a key regulator that promotes production of the proinflammtory cytokine IL-6 and decreases the probability of inflammation-associated cancer developing during acute inflammation (Fig. 8m).

Let-7 was one of the first miRNAs identified, and to uncover its biological role, extensive research has focused on let-7 target prediction⁴². Let-7a family members are highly conserved across animal species, particularly with respect to seed sequences, and have been reported to be important regulators of tumours and immunity, with specific roles in repressing the production of IL-6, a key proinflammatory cytokine in acute inflammatory responses⁸. We found that macrophages preferentially express let-7a to inhibit IL-6 production during the acute inflammatory response. A key question regarding the acute inflammatory response remains: How do macrophages rapidly release the inhibitory effect of let-7 on IL-6 during pathogenic infection. Our analysis of the gene expression profiles of mouse peritoneal macrophages stimulated with LPS showed that Rasgrp1 mRNA may be the most important ceRNA for Il6 binding because it sponges sufficient let-7a molecules to derepress the inhibitory effect of let-7 on IL-6 production. To determine whether the other known targets of let-7, such as Edn1, Socs1, Olr1 and Tnfaip3^{43,44,45,46,47}, can synergistically regulate IL-6 expression with Rasgrp1 during an acute inflammatory response, further investigation is needed. The reason why Rasgrp1 mRNA did not affect Il6 mRNA levels via let-7a was not discerned, which is a limitation of this study; further investigation is needed. Upon phosphorylation, Ago2 controls the activation of macrophages by reversing miRNA-mediated repression during the initial transient activation of macrophages⁴⁸. Whether Rasgrp1 and Ago2 exert a synergistic effect on IL-6 expression during macrophage activation also requires further investigation.

RasGRP1 is a Ras guanine nucleotide-releasing factor that activates Ras and its downstream ERK and AKT signalling pathways^49,50. However, the function of RasGRP1 in tumorigenesis has been debated⁵¹. The EGFR signalling-driven ERK and AKT signalling cascades are critical for the development and tumorigenesis of HCC, lung cancer, breast cancer and other cancers^52,53,54. A recent study reported that upregulation of RasGRP1 expression is associated with a better prognosis in colorectal cancer patients because it inhibits proliferative EGFR-SOS1-Ras-ERK signalling⁴⁰. We found that overexpression of RasGRP1 suppressed liver cancer cell growth by inhibiting proliferative EGFR-SOS1-Ras-AKT signalling. However, further investigation may be required to understand whether RasGRP1-mediated EGFR-SOS1-Ras-ERK or EGFR-SOS1-Ras-AKT signalling inhibition is a general mechanism that affects other inflammation-associated cancers.

In summary, our study demonstrated that RasGRP1 is an essential regulator that promotes the inflammatory response by increasing IL-6 production and suppressing liver cancer cell growth via inhibition of EGFR-SOS1-Ras-AKT signalling pathway activation. Under physiological conditions, especially during an acute inflammatory response, RasGRP1 may be important in regulating the inflammatory response and decreasing the probability that inflammation-associated cancer will develop.

Methods

Ethics statement

All animal experiments were conducted in accordance with the principles of the Declaration of Helsinki and were approved by the Animal Care and Use Committee of the School of Medicine, Zhejiang University. Human tissue specimens were obtained with informed consent and approved by the institutional review boards of Hunan Cancer Hospital, and the experiments were conducted in accordance with the principles of the Declaration of Helsinki.

Mice, cells and reagents

Wild-type C57BL/6 mice (male, 6–8 weeks of age) were purchased from Joint Ventures Sipper BK Experimental Animals (Shanghai, China). Il6^−/− mice (C57BL/6, 6–8 weeks of age) were purchased from the Jackson Laboratory. The mice were bred under pathogen-free conditions and housed in groups of three to five per cage on a 12 h light/dark cycle at 23 °C and 50% relative humidity. All animal experiments were conducted in accordance with the principles of the Declaration of Helsinki and were approved by the Medical Ethics Committee of Zhejiang University School of Medicine. HEK293 cells, HEK293T cells, Huh7 cells and RAW264.7 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultured according to ATCC-recommended protocols. Mouse peritoneal macrophages and human peripheral blood MDMs were prepared and cultured as described previously³⁰. LPS (0111:B4) and poly (I:C) were purchased from Sigma (St. Louis, MO, USA). CpG ODN was obtained from Invitrogen (San Diego, California). Antibodies against RasGRP1 (ab37927, 1:1000 for WB, 1:100 for IHC) and Cy3 (ab52060, 1:100) and GAPDH (ab128915, 1:1000) were purchased from Abcam, Inc. (Cambridge, MA). Antibodies against IL-6 (12912, 1:100), F4/80 (30325, 1:200), Ki-67 (9129, 1:200), JNK1/2 (9252, 1:1000), phosphorylated (p)-JNK1/2 (Thr183/Tyr185) (4668, 1:1000), ERK1/2 (9101, 1:1000), p-ERK1/2 (Thr202/Tyr204) (4695, 1:1000), p38(9215, 1:1000), p-p38 (Thr180/Tyr182) (9212, 1:1000), AKT (9272, 1:1000), p-AKT (Ser473) (4060, 1:1000), IkBα (4814, 1:1000), p-IkBα (Ser32/36) (9246, 1:1000) and p-IKKα/β (2697, 1:1000) were purchased from Cell Signalling Technology (Beverly, MA). An antibody against β-actin (A1978, 1:2000) was purchased from Sigma–Aldrich.

MiRNA mimics, sponges and inhibitors

Let-7a mimics (cholesterol-modified oligonucleotides labelled with FAM), let-7a sponges #1, #2, and #3 (cholesterol modified oligonucleotides labelled with Cy3) and let-7a inhibitors (dsRNA oligonucleotides) obtained from GenePharma (Shanghai, China) were used for the overexpression and inhibition of let-7a. Macrophages were transfected with miRNAs to establish a final concentration of 20 nM.

Plasmid construction and transfection

The CDSs and 3’ UTRs of Rasgrp1 and Il6 were obtained from mouse macrophage cDNA, and each was cloned into a pcDNA3.1 vector. The CDS of Tnf and the 3’ UTR of Il6 were obtained and cloned into a pcDNA3.1 vector. The wild-type Il6 3’UTR or a mutant Il6 3’ UTR was cloned into the pMIR-Report vector (Ambion, Austin, TX). The wild-type Rasgrp1 3’UTR or a mutant Rasgrp1 3’ UTR was cloned into the pcDNA3.1 vector or pMIR-Report vector (Ambion, Austin, TX). Each constructed plasmid was confirmed by sequencing. The corresponding primers are listed in Supplementary information Table S1. JetPEI transfection reagents (Polyplus Transfection, Illkirch, France. Cat: 409-10) were used for the cotransfection of plasmids and miRNAs into HEK293T cells. Gene-specific primer pairs for plasmid construction are listed in Supplementary Table 1.

RNA interference

siRNA duplexes³⁰ were synthesised for knocking down mouse Rasgrp1 and human RasGRP1 expression (Shanghai GenePharma Co., Shanghai, China). The siRNA duplexes of 5’-GCUCCAUCUAUUCCAAGCUTT-3’ (sense) and 5’-AGCUUGGAAUAGAUGGAGCTT-3’ (antisense) were used for mouse Rasgrp1 expression knockdown, whereas siRNA duplexes of 5’-GCGGGAUGAACUGUCACAATT-3’ (sense) and 5’-UUGUGACAGUUCAUCCCGCTT-3’ (antisense), were used for human RasGRP1 expression knockdown. The siRNA duplexes of 5’-UUCUCCGAACGUGUCACGUTT-3’ (sense) and 5’-ACGUGACACGUUCGGAGAATT-3’ (antisense) were synthesised as RNA interference controls (Ctrls). The siRNA duplexes were transfected into macrophages using INTERFERin according to the manufacturer’s protocol (Polyplus-Transfection, Illkirch, France).

Quantitative PCR (qRCR)

Total cellular RNA was extracted using TRIzol reagent, and complementary DNA was synthesised using a reverse transcription kit. Quantitative real-time RT‒PCR was performed to analyse the levels of the indicated molecules by using a SYBR RT‒PCR kit. Data obtained for each molecule were obtained by evaluating the threshold cycle (Ct) of the target molecule after normalisation against the Ct value of β-actin. Gene-specific primer pairs for qPCR are listed in Supplementary Table 2.

Enzyme-linked immunosorbent assays (ELISAs) for cytokines

ELISA kits for human and mouse TNF (human, Cat: DTA00D; mouse, Cat: MTA00B), IL-1β (mouse, Cat: MLB00C) and IL-6 (human, Cat: D6050; mouse, Cat: SM6000B) were obtained from R&D Systems. The concentrations of TNF, IL-1β and IL-6 in the culture supernatants or serum were measured by ELISA. Briefly, 50 μl of diluted culture supernatant and 50 μl of assay diluent were added to each well and incubated for 2 h at room temperature, and the wells were washed three times with washing buffer. Then, 100 μl of anti-IL-6, anti-IL-1β or anti-TNF antibody was added to each well, and the plate was incubated for 2 h at room temperature and washed three times with washing buffer. After adding 100 μl per well of horseradish peroxidase-conjugated secondary antibody and incubating the plate in the dark for 1 h at room temperature, 50 μl of substrate per well was added to the extensively washed plate and incubated in the dark for 30 min at room temperature. Then, 50 μl of stopping buffer was added to each well to stop the reaction. Finally, the optical intensity of each sample was measured at 450 nm. The concentration of cytokines was calculated with a standard curve of standard samples.

3’ UTR luciferase reporter

The wild-type and mutated Il6 or Rasgrp1 3’UTR luciferase reporter construct was prepared by amplifying the mouse wild-type and mutated Il6 or Rasgrp1 mRNA 3’UTR and cloning into the pGL3-promoter vector (Promega). Eighty nanograms of luciferase reporter plasmid, 10 ng of pRL-TK-Renilla-luciferase plasmid and 50 nM let-7a mimics or matched control were cotransfected into HEK293 cells. After 24 h, luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega). Data were normalized by dividing the firefly luciferase activity by the Renilla luciferase activity. Primer pairs for 3’UTR luciferase reporter vector construction are listed in Supplementary Table 3.

Western blotting

Total cell lysates were prepared in cell lysis buffer (Cell Signaling Technology) containing phosphatase inhibitor cocktail (Sigma) as described previously³⁰. Cell extracts were subjected to SDS–PAGE, and the proteins were transferred onto nitrocellulose membranes and subjected to immunoblot analysis. The primary antibodies and secondary antibodies were used at a 1:1000 dilution. Full-sized scans of western blots are provided in the Source Data file.

Gene microarray analysis

RNA was extracted from macrophages or LPS-stimulated macrophages using an RNA extraction kit (Fastagen Biotech Co., Shanghai, China. Cat: 220011). Gene expression microarray analysis was performed using a Mouse OneArray Plus® Microarray (Hsinchu, Taiwan) according to the manufacturer’s instructions. The changes in the let-7a target genes predicted by TargetScan (http://www.targetscan.org/vert_72) are presented in a heatmap on the basis of the microarray results.

RIP analysis

Biotin-tagged let-7a and streptomycin tagged magnetic beads were used to pull down Il6 mRNA for RIP analysis as previously described⁵⁵. Briefly, HEK293 cells were cotransfected with Il6 mRNA plasmids and/or Rasgrp1 3’UTR plasmids for 24 h. Total cellular RNA was extracted using TRIzol reagent, and the target mRNAs of let-7a were captured by biotin-tagged let-7a and streptomycin-tagged magnetic beads, washed, and extracted using TRIzol reagent again. The RIP RNAs were reverse transcribed and detected by qPCR assay. The primers used are listed in Supplementary Table 3. Gene-specific primer pairs for RIP analysis are listed in Supplementary Table 4.

H&E staining

Mouse colons and lung tissues were embedded in paraffin, followed by slicing and dewaxing. The slices were then stained with haematoxylin (BOSTER, Wuhan, China) for 5 min and counterstained with eosin (BOSTER) for 1 min. Images were captured using an inverted microscope.

Immunofluorescence

Mouse colons and lung tissues were embedded in paraffin, followed by slicing and deparaffinization. After antigen retrieval by boiling in antigen retrieval buffer (sodium citrate, pH 6.0), the tissue sections were blocked with goat serum for 1 h at room temperature. The slides were rinsed with PBS and then incubated with anti-F4/80 and anti-IL-6 or anti-Cy3 antibodies overnight at 4 °C, followed by incubation with fluorescent dye-conjugated secondary antibodies at room temperature for 1 h. Nuclei were counterstained with DAPI for 5 min. Images were captured using a confocal microscope.

Cell scratching test

Huh7 cells stably transfected with RasGRP1 or not were seeded in a six-well plate at 60% confluence and cultivated at 37 °C and 5% CO₂ for one day. Then, the cells were incubated with serum-free medium for another day. A scratch was made in the cell monolayer using a 10 µl pipette tip. The cells were then cultivated in serum-free medium for 24–48 h. Images of the scratch-healing process at 24 h and 48 h were obtained using an inverted microscope.

Cultivation of HCC progenitor cell-like spheroids

Huh7 cells were cultivated on low-attachment plates in DMEM supplemented with EGF (20 ng/ml), FGF (20 ng/ml) and B27 (2%) as previously described⁵⁶. Briefly, Huh7 cells with or without overexpression of lentivirus-delivered RasGRPP1 were cultured with 20 ng/ml EGF, 20 ng/ml FGF, B27 (2%) and the indicated concentration of IL-6 on low-attachment plates and cultivated at 37 °C and 5% CO₂ for one week. Images of the HCC progenitor cell-like spheroids were captured under an inverted fluorescence microscope.

Tumour samples

All fresh tumour tissues used for immunohistochemical staining were excised from patients hospitalised at Hunan Cancer Hospital. All experiments were undertaken in accordance with the approval of the institutional review boards of Hunan Cancer Hospital and Zhejiang University School of Medicine and were performed in accordance with the Declaration of Helsinki.

Immunohistochemistry (IHC)

Colons and gastric tissues were subjected IHC staining as previously described⁵⁶. Briefly, the tissues were embedded in paraffin, followed by slicing and deparaffination. After antigen retrieval by boiling in antigen retrieval buffer (sodium citrate, PH 6.0), the tissue sections were incubated in 2% hydrogen peroxide for 30 minutes and blocked with goat serum for 30 minutes at room temperature. The slides were incubated with an RasGRP1 antibody (Abcam, ab37927) overnight at 4 °C, followed by incubation with an HRP-labelled secondary antibody (Sigma‒Aldrich, AP132P) for 50 min at room temperature and staining with DAB for 5 min. The slides were then counterstained with haematoxylin, dehydrated, and cover slipped using neutral resin.

Liver cancer tissue microarray

A liver cancer tissue microarray containing 90 carcinoma tissues and 90 matched adjacent tissues was purchased from Shanghai Outdo Biotech Co. (Shanghai, China). IHC analysis was performed using an anti-RasGRP1-specific antibody (Abcam, Inc. Cambridge, MA) according to the manufacturer’s instructions. Briefly, the tissue sections were blocked with goat serum and then incubated overnight with an anti-RasGRP1 antibody at 4 °C. The sections were stained with 3,3-diaminobenzidine and counterstained with haematoxylin after incubation with a secondary antibody. PBS was used as the negative control. The staining intensity was evaluated independently by two pathologists without knowledge of the patients’ clinical records, and the samples were categorized into negative, weakly positive, moderately positive and strongly positive groups according to the whole visual field of the tissue samples under a low-power microscope. The percentage of positively stained cells was defined as the average number of positive cells among 100 random cells in each of three visual fields with different staining intensities of each tissue sample under a high-power microscope. The IHC score (0–12) was calculated by multiplying the scores for the intensity of positive staining (negative = 0, weak = 1, moderate = 2, and strong = 3) and the percentage of positively stained cells (0–25% = 1, 26–50% = 2, 51–75% = 3, and 75–100% = 4). IHC scores≤4 indicated weak RasGRP1 expression, IHC scores > 4 but ≤8 indicated moderate RasGRP1 expression, and IHC scores > 8<but ≤12 indicated high RasGRP1 expression.

CRISPR–Cas9-mediated depletion of the Rasgrp1 3’ UTR

The Rasgrp1 3’ UTR was knocked out using a CRISPR–Cas9 system as described previously³⁰. sgRNA sequences were designed at http://crispr.mit.edu as follows: sgRNA 1, 5′-TCTGAGGTAGCGAACACTGT-3′; sgRNA 2, 5′-GGAAGGAGGCGGGCAAGTGA-3′. These sequences were subcloned into a pLentiCRISPR V2 plasmid and cotransfected into RAW264.7 cells. After induction with 2 µg/ml puromycin for 7 days, the clones propagated from single cells were selected. Depletion of the Rasgrp1 3′UTR was identified by PCR and confirmed by DNA sequencing.

Dextran sulfate sodium (DSS)-induced colitis and histology

To establish models of DSS-induced colitis in Il6^–/– mice and in wild-type mice, all mice (8 weeks of age) received water containing 2.5% DSS for 7 days followed by normal water. On the indicated days, the body weight of each mouse was measured. Ten days after the final DSS treatments, mouse sera were collected for use in the IL-6 ELISA, the colon length was measured, and the colons were subjected to haemoxylin & eosin (H&E) staining and analysis to evaluate the severity of inflammation³⁰.

Survival curves

The survival curves of the cancer patients were generated by Kaplan–Meier plot profiling (http://kmplot.com/analysis/).

Statistical analysis

The data were analysed using SPSS software. The results are reported as the means ± SMEs or mean ± S.D.s of at least three independent experiments. The significance of differences between two groups was assessed by Student’s t test, one-way ANOVA with the LSD test, one-way ANOVA with the Tukey test, the Mann-Whitney U-test and the Wilcoxon signed-rank test. Survival curves were generated using the Kaplan–Meier method and compared with the log-rank test. Statistical significance was defined as follow: *p value < 0.05, **p value < 0.01 or *** p value < 0.001.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The microarray data generated in this study has been deposited in the NCBI Gene Expression Omnibus database⁵⁷ and is accessible through GEO with the primary accession number GSE165800. All other data generated in this study are provided in the article and the Supplementary Information. Source data are provided with this paper.

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Acknowledgements

We thank Prof. Long fei Mao (Hunan University) for the help of statistical test. This study was supported by grants from the National Natural Science Foundation of China (81971498, 81601383, 81730064, 81902069 and 31670877), the National Science and Technology Major Project (2017ZX10202201), the China Postdoctoral Science Foundation (2015M571888), the Fundamental Research Funds for the Central Universities and the Hunan Natural Science Foundation (2018JJ3091).

Author information

These authors contributed equally: Cong Wang, Xue Li, Binbin Xue, Changping Yu, Luoling Wang.

Authors and Affiliations

Institute of Pathogen Biology and Immunology, Department of Pharmacy, College of Biology, Hunan Provincial Key Laboratory of Medical Virology, Hunan University, Changsha, 410082, China
Cong Wang, Xue Li, Binbin Xue, Changping Yu, Luoling Wang, Rilin Deng, Hui Liu, Zihao Chen, Yingdan Zhang, Hungyu Sun, Haizhen Zhu & Songqing Tang
Institute of Immunology, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, China
Suping Fan & Jianli Wang
Institute of Hematology, Zhejiang University & Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Hangzhou, 310058, China
Suping Fan & Jianli Wang
Department of Gastroduodenal and Pancreatic Surgery, Translational Medicine Research Center of Liver Cancer, Hunan Cancer Hospital, Changsha, 410013, China
Chaohui Zuo

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Contributions

S.T., J.W. and H.Z. designed the experiments and supervised the project; C.W., X.L., B.X., C.Y., L.W., R.D., H.L., Z.C., Y.Z., S.F. and C.Z. performed the experiments; S.T., H.S., J.W. and H.Z. analysed the results and wrote the manuscript.

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Correspondence to Haizhen Zhu, Jianli Wang or Songqing Tang.

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Wang, C., Li, X., Xue, B. et al. RasGRP1 promotes the acute inflammatory response and restricts inflammation-associated cancer cell growth. Nat Commun 13, 7001 (2022). https://doi.org/10.1038/s41467-022-34659-x

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Received: 23 February 2021
Accepted: 02 November 2022
Published: 16 November 2022
DOI: https://doi.org/10.1038/s41467-022-34659-x

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