ABSTRACT H1 histamine receptor (H1HR) belongs to the family of rhodopsin-like G-protein-coupled receptors. Recent studies have shown that H1HR expression is increased in several types of

cancer. However, its functional roles in tumor progression remain largely unknown, especially in hepatocellular carcinoma (HCC). We found that H1HR is frequently unregulated in HCC, which is

significantly associated with both recurrence-free survival and overall survival in HCC patients. Functional experiments revealed that H1HR promoted both the growth and metastasis of HCC

cells by inducing cell cycle progression, formation of lamellipodia, production of matrix metalloproteinase 2, and suppression of cell apoptosis. Activation of cyclic adenosine

demonstrate that H1HR plays a critical role in the growth and metastasis of HCC cells, which provides experimental evidence supporting H1HR as a potential drug target for the treatment of

HCC. SIMILAR CONTENT BEING VIEWED BY OTHERS INHIBITION OF HISTAMINE RECEPTOR H3 SUPPRESSES THE GROWTH AND METASTASIS OF HUMAN NON-SMALL CELL LUNG CANCER CELLS VIA INHIBITING PI3K/AKT/MTOR

AND MEK/ERK SIGNALING PATHWAYS AND BLOCKING EMT Article 06 November 2020 CYCLIC INCREASE IN THE HISTAMINE RECEPTOR H1-ADAM9-SNAIL/SLUG AXIS AS A POTENTIAL THERAPEUTIC TARGET FOR EMT-MEDIATED

PROGRESSION OF ORAL SQUAMOUS CELL CARCINOMA Article Open access 20 March 2025 EPHA2–YES1–ANXA2 PATHWAY PROMOTES GASTRIC CANCER PROGRESSION AND METASTASIS Article Open access 03 May 2021

INTRODUCTION Histamine is a ubiquitous messenger molecule with multiple physiological activities that released from neurons, mast cells, and enterochromaffin-like cells [1]. It exerts

various biological effects through its four cognate G protein-coupled histamine receptors: H1, H2, H3, and H4 (H1HR, H2HR, H3HR, and H4HR, respectively) [2]. Previous studies have shown that

H1 histamine receptor (H1HR) is extensively involved in a wide array of pathological processes of allergy, such as atopic dermatitis, allergic rhinitis, and asthma [3, 4]. Recently, it was

observed that H1HR promotes cell proliferation in several cancer types, including astrocytoma and breast cancer [5, 6], suggesting that H1HR is a possible oncoprotein and potential target in

cancer therapy. However, the functional roles of H1HR in tumor growth and metastasis remain largely unknown, especially in hepatocellular carcinoma (HCC). The human H1HR gene encodes an

integral membrane protein consisting of 487 amino acids, which predominantly couples to Gαq/11 proteins [7], resulting in the activation of phospholipase C and subsequent release of the

secondary response elements inositol trisphosphate (IP3) and diacylglycerol (DAG), followed by the activation of protein kinase C (PKC). In addition, H1HR activation also stimulates adenylyl

cyclase (AC) to generate cyclic adenosine monophosphate (cAMP), which in turn activates protein kinase A (PKA) [8]. Activated PKC and PKA phosphorylate a number of substrates involved in

cell signal transduction. However, activation of the H1HR signaling pathway is poorly understood in cancer cells. In this study, we systematically investigated the expression and functional

roles of H1HR in HCC as well as its clinical implication. Our study facilitates an understanding of the pathological roles of H1HR and provides experimental evidence for H1HR as a potential

therapeutic target in HCC. RESULTS H1HR IS UPREGULATED IN HCC TISSUES AND CONTRIBUTES TO TUMOR PROGRESSION AND A POOR PROGNOSIS We first evaluated the mRNA and protein expression levels of

H1HR in 30 paired tumor and peritumor tissues from HCC patients. Both qRT-PCR and western blot analyses showed that H1HR was significantly upregulated in HCC tissues compared with peritumor

tissues (Fig. 1a–c). Correlation analysis indicated a significant positive correlation between the mRNA and protein levels for individual patients (Fig. 1d). To further confirm these

results, we evaluated the mRNA expression of H1HR using publicly accessible data from The Cancer Genome Atlas (TCGA) database (http://cancergenome.nih.gov). In TCGA cohort of 50 paired HCC

tissues, H1HR mRNA expression levels were significantly higher in tumor tissues than in their adjacent normal tissues (Fig. 1e). The expression of H1HR was further evaluated by IHC analysis

in another 217-paired tumor and peritumor tissues. As shown in Fig. 1f, the IHC score of H1HR was markedly increased in HCC tissues compared with paired peritumor tissues. In addition, the

primary tumor tissues from 36 HCC patients exhibited lower H1HR expression compared with unpaired metastatic tissues from another 36 HCC patients (Fig. 1g). We further evaluated the

clinicopathological and prognostic significance of H1HR in HCC patients based on IHC data from 217 tumor tissues. As shown in Supplementary Table 1 H1HR expression was significantly

associated with tumor size (_P_ < 0.001) and a more advanced tumor–node–metastasis stage (_P_ = 0.031). Kaplan–Meier survival analysis showed that HCC patients with high H1HR expression

had significantly shorter overall survival (OS) and recurrence-free survival (RFS) than those with low H1HR expression (Fig. 1h, I). Considering that infiltration of myeloid cells has been

well-known as a poor prognostic factor in several tumor types [9, 10], multiple staining of H1HR, GPC3 [11,12,13] (marker of HCC), and CD68 [14, 15] (marker of myeloid cell) by IHC analysis

was applied to explore whether H1HR is also expressed in myeloid cells and thus plays a role in the progression of HCC. We found that cells with positive GPC3 and CD68 staining also

exhibited H1HR positive staining, indicating that H1HR was expressed in both HCC cells and infiltration myeloid cells (Supplementary Fig. S1A). In addition, bioinformatic analysis based on

TCGA database (http://cancergenome.nih.gov) indicated a significant positive correlation between the mRNA expression levels of H1HR and CD68 (_r_ = 0.314, _P_ = 0.026) (Supplementary Fig.

S1B), implying that also myeloid cells present in the tumor microenvironment express H1HR and may contribute to the observed negative impact of H1HR expression on prognosis. Furthermore,

TCGA-based survival analysis indicated that overexpressed H1HR predicted a poor survival only in HCC patients with high CD68 expression levels, but not in HCC patients with low CD68

expression levels (Supplementary Fig. S1C, D), which further suggest that increased infiltration of myeloid may aggravate the oncogenic effects exerted by H1HR in HCC. Taken together, these

data indicate that H1HR overexpression contributes to the progression of HCC and poor prognosis. H1HR PROMOTES HCC CELL GROWTH BOTH IN VITRO AND IN VIVO To assess the potential effects of

H1HR on cell growth, a series of in vitro biological experiments were performed with gain-of-function or loss-of-function of H1HR. SNU-368 cells with relatively high H1HR expression and HLE

cells with relatively low H1HR expression (Supplementary Fig. S2A, B) were selected to establish cell models with knocked down or overexpression of H1HR (Supplementary Fig. S2C, D). The MTS

cell viability assay showed that SNU-368 cells with H1HR knockdown had a much slower growth rate than control cells, whereas HLE cells with H1HR overexpression grew faster than control cells

(Fig. 2a). Accordingly, the number of colonies and 5-ethynyl-2′-deoxyuridine (EdU) incorporation were markedly decreased in SNU-368 cells with H1HR knockdown, but were significantly

increased in HLE cells with H1HR overexpression compared with controls. (Fig. 2b, c). To further investigate the in vivo tumourigenic ability of H1HR, we constructed a xenograft nude mice

model using SNU-368 and HLE cell lines with stable H1HR knockdown or overexpression (Supplementary Fig. S2E, F). Xenograft tumors developed from SNU-368 cells with stable H1HR knockdown

exhibited a significant decrease in growth capacity and less net weight compared with control tumors. In contrast, the growth capacity and net weight of xenografts developed from HLE cells

with stable H1HR overexpression were much higher than controls (Fig. 2d, e). Moreover, when compared with controls, those xenografts developed from SNU-368 cells with stable H1HR knockdown

exhibited a considerable decrease of positive Ki-67 staining. However, overexpression of H1HR significantly increased Ki-67-positive staining in xenografts developed from HLE cells (Fig.

2f). H1HR PROMOTES HCC CELL GROWTH BY INDUCING CELL CYCLE PROGRESSION AND SUPPRESSING CELL APOPTOSIS To characterize the oncogenic mechanism of H1HR, we further investigated the functional

roles of H1HR in cell cycle progression and cell apoptosis. We found that knockdown of H1HR significantly increased the number of SNU-368 cells in the G1 phase, but decreased the number in

the S phase compared with the control. Conversely, ectopic expression of H1HR in HLE cells promoted the G1–S cell cycle transition (Fig. 3a). As expected, western blot analysis showed that

the protein expression of two major G1–S checkpoint regulators cyclin D1 and cyclin-dependent kinase was decreased in SNU-368 cells with H1HR knockdown, while overexpression of H1HR in HLE

cells had opposite effect (Fig. 3b), confirming role of H1HR in promoting cell cycle progression in HCC cells. Next, the effect of H1HR on cell apoptosis was assessed by flow cytometry. We

found that knockdown of H1HR significantly enhanced the apoptosis of SNU-368 cells, whereas overexpression of H1HR in HLE cells showed an opposite effect (Fig. 3c). Consistently, cytochrome

c release and the cleavage of caspase 9 and 3 were significantly induced by H1HR knockdown, while were significantly inhibited by H1HR overexpression upon CCCP treatment (Fig. 3d, e). We

further confirmed the protective role of H1HR against apoptosis in xenograft tumor models. When compared with the control, xenografts developed from SNU-368 cells with stable knockdown of

H1HR exhibited a considerable increase in positive TUNEL staining. In contrast, overexpression of H1HR significantly decreased TUNEL-positive staining in xenografts developed from HLE cells

(Fig. 3f). Collectively, these data showed that H1HR promoted HCC growth by inducing cell cycle progression and suppressing cell apoptosis. H1HR PROMOTES THE MIGRATION AND INVASION OF HCC

CELLS BOTH IN VITRO AND IN VIVO We also investigated the effects of H1HR on the metastasis of HCC cells using both in vitro and in vivo metastasis assays. The scratch wound healing and

transwell migration assays showed that knockdown of H1HR significantly inhibited the migratory ability of SNU-368 cells. By contrast, the migratory ability of HLE cells with H1HR

overexpression was significantly greater than that of control cells (Fig. 4a, b). The transwell matrigel invasion assay also showed that knockdown of H1HR significantly impaired the invasion

of SNU-368 cells, whereas overexpression of in HLE cells had the opposite effect (Fig. 4c). These results indicate that H1HR increases the in vitro migration and invasion abilities of HCC

cells. To determine whether H1HR promotes tumor metastasis in vivo, we performed a cancer metastatic assay by injecting SNU-368 and HLE cells with stable H1HR knockdown or overexpression,

respectively, into the lateral tail vein of nude mice. We found that mice injected with SNU-368 cells with H1HR knockdown exhibited a dramatic decrease in the incidence of lung metastases,

whereas overexpression of H1HR in HLE cells significantly increased the incidence of lung metastases in the metastasis mouse model (Fig. 4d). H1HR PROMOTES THE MIGRATION AND INVASION OF HCC

BY INDUCING LAMELLIPODIA FORMATION AND UPREGULATING MATRIX METALLOPROTEINASE 2 The epithelial–mesenchymal transition (EMT) plays a crucial role in tumor metastasis by reducing cell–cell

contacts and increasing cell motility. We clarified whether EMT was involved in H1HR-mediated HCC invasion and migration. Quantitative RT-PCR and western blot analysis showed no changes in

the expression of epithelial (E-cadherin and zonula occludens-1) or mesenchymal (N-cadherin and vimentin) markers when H1HR was knocked down or overexpressed (Fig. 5a, b), suggesting that

H1HR promotes the migration and invasion of HCC not through regulating the EMT. Lamellipodia is a dynamic surface extension of the cell, which plays a pivotal role in cell migration. To

explore the mechanisms by which H1HR promotes HCC cell migration, the formation of lamellipodia was examined in SNU-368 and HLE cells with H1HR knockdown or overexpression. F-actin staining

revealed that lamellipodia formation was markedly decreased in SNU-368 cells after H1HR was knocked down. In contrast, overexpression of H1HR in HLE cells significantly increased

lamellipodia formation (Fig. 5c), suggesting that H1HR may promote the migration of HCC cells by modulating actin rearrangements. To determine how H1HR facilitates the invasion of HCC cells,

the expression of four matrix metalloproteinases (MMPs), which are involved in degradation of the basement membrane, were analyzed by qRT-PCR in SNU-368 and HLE cells with H1HR knockdown

and overexpression, respectively. Our data showed that only MMP-2 was dramatically downregulated in SNU-368 cells with H1HR knockdown and upregulated in HLE cells with H1HR overexpression

(Fig. 5d), which was further confirmed by western blot analysis (Fig. 5e). In addition, we found a significant positive correlation between the IHC scores of H1HR and MMP2 (_r_ = 0.357, _P_ 

< 0.001) in 217 HCC tissue samples (Fig. 5f). To determine whether H1HR promotes cell invasion through MMP-2 production, RNA interference was used to knockdown MMP-2 expression in HLE

cells with H1HR overexpression (Supplementary Fig. S3). As shown in Fig. 5g, knockdown of MMP-2 was sufficient to inhibit the increase of cell invasion ability induced by H1HR

overexpression, suggesting that H1HR may facilitate the invasion of HCC cells by upregulating MMP-2. ACTIVATION OF PKA PATHWAYS IS INVOLVED IN H1HR-MEDIATED GROWTH AND METASTASIS OF HCC

CELLS It is well established that PKC and PKA signaling is commonly triggered by the activation of histamine receptors [16,17,18,19,20]. To test whether PKC and PKA were activated by H1HR in

HCC cells, the activities of PKC and PKA were detected in SNU-368 and HLE cells with H1HR knockdown or overexpression. As shown in Fig. 6a, b, the activity of PKA was significantly

decreased in SNU-368 cells with H1HR knockdown and significantly increased in HLE cells with H1HR overexpression, while PKC activity remained unchanged, suggesting that the PKA pathway is

activated by H1HR in HCC cells. To confirm this observation, we examined the levels of phosphorylated cAMP response element-binding protein (p-CREB, an indicator of PKA activation) in 217

HCC tissue samples, and found a significant positive correlation between the IHC scores of H1HR and p-CREB (_r_ = 0.417, _P_ < 0.001) (Fig. 6c). Because PKA activation plays a critical

role in tumor growth and metastasis, we hypothesized that the oncogenic phenotype induced by H1HR may be associated with activation of the PKA pathway. To test this possibility, we treated

HLE cells or SNU-368 cells with the specific PKA inhibitor H89 or PKA activator 8-bromo-cAMP. As shown in Fig. 6d–g, H89 treatment significantly decreased the growth and metastasis of HLE

cells induced by H1HR overexpression, whereas 8-bromo-cAMP treatment significantly increased the growth and metastasis of SNU-368 cells suppressed by H1HR knockdown. Collectively, these

results indicate that H1HR promotes the growth and metastasis of HCC cells mainly by activating the PKA signaling pathway. H1HR OVEREXPRESSION IS MAINLY MEDIATED BY DOWNREGULATION OF MIR-940

MicroRNA plays an important role in the gene-expression regulation network. To identify potential microRNAs involved in the overexpression of H1HR in HCC, we used microRNA Data Integration

Portal-based target prediction programs [21]. Among the top ten predicted miRNAs targeting H1HR (Supplementary Table 2), only miR-940 repressed H1HR expression in SNU-368 cells (Fig. 7a).

Real-time PCR and western blot showed that miR-940 significantly downregulated the expression of H1HR in SNU-368 and HLE cells (Fig. 7b, c). In contrast, miR-940 inhibition enhanced H1HR

expression in SNU-368 and HLE cells cells (Fig. 7d, e). In addition, a significant negative correlation (_r_ = −0.691, _p_ < 0.001) between miR-940 and H1HR was found in tumor tissues

from 30 HCC patients (Fig. 7f). Moreover, miR-940 mimics greatly attenuated the ability of H1HR to promote the growth and metastasis in HCC cells, whereas miR-940 inhibition increased the

growth and metastasis repressed by H1HR knockdown in HCC cells (Fig. 7g–k). These results suggest that miR-940 represses the expression of H1HR and its oncogenic function in HCC cells. THE

H1HR INHIBITOR TERFENADINE EXHIBITS THERAPEUTIC EFFECTS ON HCC IN VITRO AND IN VIVO To explore the therapeutic role of H1HR inhibition on HCC, we first investigated the effects of

terfenadine (TF) on HCC cell growth and found that treatment with TF significantly inhibited cell proliferation and colony formation in SNU-368 cells (Fig. 8a, b). In addition, TF treatment

also significantly decreased the migration and invasion abilities of SNU-368 cells (Fig. 8c, d). Then, we tested the in vivo effects of TF treatment on tumor growth. TF injection

significantly inhibited the growth of xenograft tumors developed from SNU-368 cells (Fig. 8e, f) and significantly inhibited the lung metastasis of SNU-368 cells (Fig. 8g). These results

indicate that inhibition of H1HR by a specific antagonist may be a promising novel therapeutic strategy for HCC. On the contrary, treatment with histamine-trifluoromethyl-toluidide

derivative (HTMT) (H1HR agonist) significantly increased the growth and metastasis abilities of HLE cells both in vitro and in vivo (Supplementary Fig. S4A–G). To further explore the

potential effect of histamine in H1HR-regulated HCC growth and metastasis, SNU-368 and HLE cells with different levels of H1HR were treated with histamine for 24 h at a concentration of 10 

μM. As shown in the Supplementary Fig. S5A, B, histamine treatment significantly increased the growth of HLE and SNU-368 cells, especially when H1HR was overexpressed in HLE cells, whereas

slightly increased the growth of SNU-368 when H1HR was knockeddown. Similar results were also obtained when scratch wound healing and transwell matrigel invasion assays were applied

(Supplementary Fig. S5C, D), showing that histamine treatment significantly increased both the migration and invasion of HLE and SNU-368 cells, especially when H1HR was overexpressed in HLE

cells, whereas slightly increased the migration and invasion of SNU-368 when H1HR was knocked down. These results collectively indicate that histamine promotes the growth and metastasis of

HCC cells by acting on H1 plasma membrane receptor, while H1HR could also play a histamine-independent role in the promotion of HCC growth and metastasis. DISCUSSION Recently, high

expression levels of histamine receptors have been reported in different human cancers including colon cancer, melanoma, and breast cancer [22]. In the present study, we found that H1HR was

frequently upregulated in HCC and contributed to tumor progression. Our results suggest that H1HR overexpression may serve as a valuable new prognostic marker for HCC patients. A previous

search for prognostic value of H1HR in the PrognoScan database (a database for meta-analysis of the prognostic value of genes) showed that H1HR is associated with the prognosis of many types

of cancers including hematological and solid cancers. In addition, we found that H1HR was expressed not only in HCC cells but also in infiltration myeloid cells. TCGA based analysis also

revealed a significantly positive correlation between the expression of H1HR and CD68, indicating that H1HR overexpression may facilitate the infiltration of myeloid cells into HCC tissue.

Furthermore, Our results from TCGA-based survival analysis indicated that overexpressed H1HR predicted a poor survival only in HCC patients with high CD68 expression levels, but not in HCC

patients with low CD68 expression levels, which further suggested that increased infiltration of myeloid may aggravate the oncogenic effects exerted by H1HR in HCC. However, the relationship

between the expression of H1HR and prognosis varied in different cancers, even in the same cancer from different databases [23]. This may be explained by the fact that the function of H1HR

in different tumor types may be multidimensional, and it may not simply function as just an oncogene or tumor suppressor. Histamine is a neurotransmitter that is involved in the regulation

of a variety of physiological processes including cell proliferation, differentiation, and immune responses [24]. Consistently, previous studies in breast cancer [25, 26] and

cholangiocarcinoma [27] also reported the increased levels of histamine in tumor tissues, which promoted tumor growth and angiogenesis. Unlike histamine, the functional roles of histamine

receptors are tumor type-specific. In some types of cancer, histamine receptors promote tumor growth and metastasis. For example, several studies have indicated that H2HR, H3HR, and H4HR

exert proproliferative effects on human melanoma, glioblastoma, and colon cancer cells, respectively [28,29,30]. In contrast, Meng et al. reported an antiproliferative effect of H4HR. [31]

in human cholangiocarcinoma. Our present study demonstrate that H1HR overexpression promotes HCC growth by inducing cell cycle progression and reducing cell apoptosis, which is consistent

with a previous study in human melanoma cells, showing that H1HR contributes to the reduced apoptosis of human melanoma cells [32]. Similarly, histamine receptors also play dual roles in

tumor metastasis. For example, Lin et al. observed a metastasis-promoting role for H3HR in glioblastoma [29]. In contrast, Meng et al. [31] and Cai et al. [33] showed that activation of H4HR

suppressed the metastasis of human cholangiocarcinoma and nonsmall cell lung cancer. In the present study, we showed that H1HR promoted the metastasis of HCC cells both in vitro and in

vivo. EMT is a key process in metastasis formation during malignant progression. Previous studies have shown that H3HR and H4HR affect tumor metastasis by regulating the EMT [29, 33, 34],

whereas data from our study showed that H1HR promoted migration and invasion of HCC by inducing lamellipodia formation and up-regulating MMP-2, but not by regulating the EMT. Possible

explanations for these differences include different histamine receptor and tumor origins. Remarkably, we found that histamine treatment significantly increased both the growth and

metastasis of HCC cells, especially when H1HR was overexpressed, whereas slightly increased when H1HR was knocked down. These results collectively indicate that histamine promotes the growth

and metastasis of HCC cells by acting on H1 plasma membrane receptor, while H1HR could also play a histamine-independent role in the promotion of HCC growth and metastasis. It has been

demonstrated that H1HR stimulates phospholipase to generate IP and DAG, leading to activation of PKC [16, 17]. In addition, H1 receptor activation also stimulates AC to generate cAMP, which

in turn activates PKA [20, 35]. Mechanistically, our results demonstrated that the activation of PKA but not PKC was involved in H1HR-mediated HCC growth and metastasis. However, considering

that other signaling pathways, such as the mitogen-activated protein kinases and protein kinase B, can also be activated by H1HR [36], we cannot rule out the possibility that these pathways

may be involved in this process; this theory needs further clarification. MiR-940 is a frequently downregulated tumor suppressor in several types of cancer, including HCC. For example, Xu T

et al. have demonstrated in glioma that miR-940 was downregulated in clinical samples and glioma cell lines, and suppressed tumor metastasis [37]. Rajendiran et al. have reported that

miR-940 is downregulated significantly in prostate cancer and suppresses cell migration and invasion [38]. In HCC, miR-940 was also found to be significantly decreased in the HCC tissues and

cell lines, and function as a tumor suppressor through inhibition of invasion and migration of HCC cells [39, 40]. Consistently, our present study showed that miR-940 was involved in the

overexpression of H1HR in HCC cells. These results collectively suggest that miR-940 functions as an important tumor suppressor in HCC. However, we still cannot rule out the possibility that

other factors may also contribute to the elevated expression of H1HR in HCC, such as genetic and epigenetic alterations, which still needs further confirmation. TF is an antihistamine

reagent (targeting the H1 receptor) that is widely used for the treatment of allergic conditions. Several previous studies have indicated that people with allergy showed an increased risk of

certain types of cancer [41, 42], although in several other types of cancer, allergies may have the opposite effect and help protect against cancer [43, 44]. These findings suggest that the

association between allergies and cancer risk is complex, the risk of developing cancer could depend on the specific malignancy, and H1HR may be a feasible target in cancer prevention.

However, the potential use of H1HR antagonists to treat liver cancer has been unexplored until now. Our data clearly showed that TF treatment significantly inhibited the growth and

metastasis of HCC both in vitro and in vivo, indicating that inhibition of H1HR by TF may be a promising novel therapeutic strategy for treating HCC. In summary, the results of this study

indicate that upregulated H1HR promotes both the growth and metastasis of HCC by inducing cell cycle progression, lamellipodia formation, and MMP-2 production and by suppressing cell

apoptosis. These findings suggest that H1HR plays an important oncogenic role in HCC and may be a potential therapeutic target for HCC treatment in the future. MATERIALS AND METHODS REAGENTS

The PKA inhibitor H89 and activator 8-bromo-cAMP were purchased from Beyotime Biotechnology (Cat. #S1643; Beijing, China) and Sigma-Aldrich (Cat. B-5380; St. Louis, MO), respectively. The

H1HR inhibitor TF and H1HR agonist was purchased from Abcam (Cambridge, UK). Histamine was purchased from Sigma-Aldrich. The PKC kinase activity kit (ab139437) and PKA kinase activity kit

(ab139435) were purchased from Abcam (Cambridge, UK). The primary antibodies used in this study and their working concentrations were listed in Supplementary Table 3. The sequences of

primers and siRNAs used in this study were listed in Supplementary Table 4. CELL VIABILITY ASSAY Cell viability was examined by the MTS assay (G3581; Promega Corporation, Madison, WI)

according to the manufacturer’s instructions. The microplates were read in a spectrophotometer at a wavelength of 490 nm. COLONY FORMATION ASSAY The transiently transfected HCC cells

(SNU-368 and HLE) were collected and seeded (1 × 103/well) in six-well plates and cultured for 2 weeks. Colonies were counted after fixing in 70% ethanol staining with 5% crystal violet.

CELL CYCLE ANALYSIS The transiently transfected HCC cells (SNU-368 and HLE) were fixed in 70% ethanol for 24 h, and then labeled with 50 μg/ml of propidium iodide (PI; BestBio, shanghai,

China). Cell cycle profiles were analyzed on a flow cytometry (Beckman, Fullerton, CA). APOPTOSIS ASSAYS Cell apoptosis was analyzed by flow cytometry and terminal deoxynucleotidyl

transferase-mediated dUTP nick-end labeling (TUNEL) assay as previously described [45]. Nuclei with clear green staining were considered TUNEL-positive apoptotic cells. The apoptosis index

was calculated as the percentage of TUNEL-positive nuclei after counting at least 500 cells. WOUND HEALING ASSAY Cell migration was assessed using a wound healing assay. Briefly, when the

cells reached 80% confluency in six-well plates, three scratch wounds in each well were made using plastic pipette tips (100 μL pipette tips; Eppendorf). The wound closure was observed after

48 h and photographs were taken at 0 and 48 h. PKC AND PKA ACTIVITY ASSAY The activities of PKC and PKA were examined using a PKC and PKA enzyme activity kit, respectively. Briefly, cells

were washed twice in ice-cold phosphate-buffered saline and lysed on ice for 10 min. Then, the lysates were centrifuged at 13,000 rpm for 15 min and the clear supernatant extract was

analyzed for kinase activity according to the manufacturer’s protocol. Finally, the microplates were read in a spectrophotometer at a wavelength of 450 nm. STATISTICAL ANALYSIS Experiments

were repeated three times when appropriate. Data were represented as the mean ± standard error of the mean. SPSS 17.0 software (SPSS, Chicago, IL) was used for all statistical analyses and

_P_ values < 0.05 were considered statistically significant. Paired or unpaired _t_ tests were used for comparisons between two groups where appropriate. Correlations between measured

variables were tested by Pearson or Spearman rank correlation analyses. OS and RFS in relation to expression were evaluated by the Kaplan–Meier survival curve and the log-rank test.

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through the ROS-modulated coordinated regulation of the NFKB and TP53 pathways. Autophagy. 2016;12:1–16. PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS This

work was supported by the State Key Laboratory of Cancer Biology Project (CBSKL2017Z01), National Natural Science Foundation of China (grants 81472298, 81502085 and U1604167) and Natural

Science Basic Research Plan in Shaanxi Province of China (Program no. 2018JM7018). AUTHOR INFORMATION Author notes * These authors contributes equally: Jing Zhao, Yiran Hou AUTHORS AND

AFFILIATIONS * Center for Molecular Pathology, First Affiliated Hospital, Gannan Medical University, Ganzhou, Jiangxi, 341000, China Jing Zhao & Shaogui Wan * State Key Laboratory of

Cancer Biology and Experimental Teaching Center of Basic Medicine, Fourth Military Medical University, Xi’an, 710032, China Jing Zhao, Chun Yin, Jing Hu, Xiaojun Huang, Jinliang Xing & 

Jibin Li * Medical College of Yan’an University, Yan’an, Shaanxi, 716000, China Yiran Hou * Department of Gynecology and Obstetrics, Xijing Hospital, Fourth Military Medical University,

Xi’an, 710032, China Tian Gao & Xiaohong Zhang * Department of Hepatobiliary Surgery, Xijing Hospital, Fourth Military Medical University, Xi’an, 710032, China Jiaze An Authors * Jing

Zhao View author publications You can also search for this author inPubMed Google Scholar * Yiran Hou View author publications You can also search for this author inPubMed Google Scholar *

Chun Yin View author publications You can also search for this author inPubMed Google Scholar * Jing Hu View author publications You can also search for this author inPubMed Google Scholar *

Tian Gao View author publications You can also search for this author inPubMed Google Scholar * Xiaojun Huang View author publications You can also search for this author inPubMed Google

Scholar * Xiaohong Zhang View author publications You can also search for this author inPubMed Google Scholar * Jinliang Xing View author publications You can also search for this author

inPubMed Google Scholar * Jiaze An View author publications You can also search for this author inPubMed Google Scholar * Shaogui Wan View author publications You can also search for this

author inPubMed Google Scholar * Jibin Li View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHORS Correspondence to Shaogui Wan or Jibin

Li. ETHICS DECLARATIONS CONFLICT OF INTEREST The authors declare that they have no conflict of interest. ETHICAL APPROVAL The study was approved by the Ethics Committee of the Fourth

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receptor H1 promotes tumor progression and contributes to poor prognosis in hepatocellular carcinoma. _Oncogene_ 39, 1724–1738 (2020). https://doi.org/10.1038/s41388-019-1093-y Download

citation * Received: 23 May 2019 * Revised: 25 October 2019 * Accepted: 29 October 2019 * Published: 18 November 2019 * Issue Date: 20 February 2020 * DOI:

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