Early growth response factor 1 promotes HCC progression by activating the MAPK/ERK pathway through transcriptional upregulation of PAR1

Early growth response factor 1 promotes HCC progression by activating the MAPK/ERK pathway through transcriptional upregulation of PAR1 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Early growth response factor 1 promotes HCC progression by activating the MAPK/ERK pathway through transcriptional upregulation of PAR1 Jian-gang Bi, Qi Li, Yu-sheng Guo, Hong-gui Tang, Ping Xu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4744749/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. The prognosis of HCC patients who undergo surgical resection is still poor. Therefore, it is urgent to clarify the potential mechanism of HCC progression. This article reports the important role of the transcription factor early growth response 1 (Egr1) in promoting HCC progression. First, Egr1 expression was abnormally elevated in clinical HCC samples and enhanced the proliferation, invasion and migration of cancer cells. Moreover, we found that the mRNA expression levels of protease-activated receptor 1 (PAR1) and Egr1 in clinical specimens were positively correlated. Dual luciferase reporter gene assays verified that Egr1 is an upstream transcriptional regulator of PAR1, that enhances the proliferation, invasion and migration of cancer cells by upregulating PAR1. Mechanistically, we found that Egr1 activates the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway through PAR1. Finally, we demonstrated that thrombin does not affect the regulatory effect of Egr1 on PAR1 in HCC cells. In conclusion, Egr1 promotes HCC progression by upregulating PAR1 to activate the MAPK/ERK pathway. early growth response factor 1 protease-activated receptor 1 transcription mitogen-activated protein kinase hepatocellular carcinoma Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Liver cancer is undoubtedly a global public health challenge, with the incidence expected to exceed 1 million cases per year by 2025 (Sung et al., 2021 ). Primary liver cancer is the sixth most common cancer worldwide and the third leading cause of death (Sung et al., 2021 ). Hepatocellular carcinoma (HCC) accounts for the majority of liver cancer cases at diagnosis and mortality (Singal et al., 2020 ). In addition to surgical resection, some progress has been made in the clinical treatment of HCC in recent years, such as in transplantation, ablation and systemic therapy, but the treatment efficacy is not satisfactory. The mechanisms that drive the malignant transformation and progression of HCC have not been fully elucidated (Bruix et al., 2019 ; Bruix et al., 2015 ); therefore, identifying key molecules and gaining a deeper understanding of their mechanisms of action are crucial for developing new and effective treatments to improve the prognosis of HCC patients. Early growth response factor 1 (Egr1), an important transcriptional regulator, regulates the transcription of downstream target genes by recognizing and binding the specific DNA sequence 5’-CGCCCCCGC-3’ (Egr1 binding site) in the gene promoter (Khachigian et al., 1998). When cells are subjected to injury stress, the expression of Egr1 is rapidly upregulated to regulate growth and proliferation (Gashler et al., 1995). Egr1 regulates tumors in a bidirectional manner, promoting some cancers (Stamatakis et al., 2015 ; Parra et al., 2014 ; Hansson et al., 2012 ; Lee et al., 2012 ) and suppressing others (Sun et al., 2017 ; Yang et al., 2016 ). However, there are few studies on the role of Egr1 in HCC, and there is still controversy. Some studies have shown that Egr1 inhibits the development of HCC (Wang et al., 2016 ), but more studies have supported the view that Egr1 promotes the progression of HCC (Lee et al., 2009; Archer et al., 2009 ; Archer et al., 2016 ). Our previous studies revealed that Egr1 expression is significantly upregulated in HCC and promotes cell proliferation (Bi et al., 2019 ), but the downstream molecular mechanism involved needs further study. Protease-activated receptor 1 (PAR1) is a G protein-coupled receptor (GPCR) with seven transmembrane units, that is widely present in the digestive system and regulates intracellular signal changes. Many studies have confirmed that PAR1 plays an important role in promoting HCC progression (Kaufmann et al., 2007 ; Xiao et al., 2018 ; Mußbach et al., 2015 ). In addition, thrombin is known to regulate PAR1 at the posttranscriptional level by recognizing PAR1 at specific sites in the extracellular N-terminal region (O’Brien et al., 2001 ), but does not regulate PAR1 expression at the transcriptional level. There are few studies on the upstream transcriptional regulatory mechanism of PAR1 in HCC. Some studies have shown that Egr1 upregulates PAR1 at the transcriptional level in prostate cancer (Salah et al., 2007 ). Therefore, we speculated that Egr1 promotes the progression of HCC by regulating PAR1 expression. 2. Materials and Methods 2.1 Patients and clinical specimens This study was approved by the Ethics Committee of the Second Affiliated Hospital of Jinan University, and written informed consent was obtained from each patient. Eighteen patients with a histological diagnosis of HCC between 2018 and 2020 were recruited. Tumor tissues and paired adjacent nontumor samples at least 5 cm from the tumor edge were collected. Clinical specimens were immediately frozen in liquid nitrogen and stored at -80°C. Patients who received radiotherapy or chemotherapy before surgery were excluded. The clinical pathological parameters are shown in Table 1 . Table 1 Clinical characteristics of HCC patients (n = 18) Characteristics Number of cases Age (years) ≤ 65 11 > 65 7 Gender Male 14 Female 4 Type of hepatitis HBV 17 HCV 0 HBV + HCV 1 Tumor size (cm) ≤ 3 15 3–5 2 > 5 1 Tumor number Solitary 15 Multiple 3 Liver cirrhosis with 16 without 2 TNM stage Ⅰ 8 Ⅱ 5 Ⅲ 4 Ⅳ 1 2.2 Cell culture and transfection The immortalized liver cell line THLE-2, and HCC cell lines (PLC/PRF-5, HCCLM3, MHCC97H, MHCC97L, SMMC-7721 and Hep3B) were purchased from the Cell Bank of Type Culture Collection (Chinese Academy of Sciences, Shanghai, China). All cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco) at 37°C in 5% CO2. The medium was changed every 48 hours. pENTER-Egr1 and pENTER-PAR1 plasmids were purchased from WZ Biosciences (Shandong, China) for overexpression of Egr1 or PAR1. Cells were cultured in 12-well plates, and when the cell confluence reached approximately 50–60%, the cells were transfected with 2 µg of plasmid or empty vector according to the product instructions. Small interfering RNAs targeting Egr1 (siEgr1) and PAR1 (siPAR1) and the corresponding negative controls were purchased from Ribobio (Guangzhou, China) and used to inhibit the expression of Egr1 or PAR1. The cells were grouped and cultured in 12-well plates, and when the cell confluence was approximately 50–60%, transfection was performed at a concentration of 100 nmol/L according to the product instructions. LipofectamineTM3000 (Invitrogen, USA) was used for transfection. Recombinant hirudin (R-hirudin) (Sigma-Aldrich) was added to the medium according to the manufacturer’s instructions to inhibit the effect of thrombin on PAR1 at a concentration of 100 µg/ml. The cells were harvested 72 hours after transfection for subsequent experiments. 2.3 Quantitative real-time polymerase chain reaction (qRT‒PCR). Total RNA was extracted from specimens and cells using TRIzol reagent (Thermo Fisher Scientific, MA, USA). Complementary DNA (cDNA) synthesis was conducted using M-MLV Reverse Transcriptase (Invitrogen, MA, USA). qRT‒PCR analysis was performed using the SYBR Green qPCR Kit (Takara, Dalian, China) on a Roche LightCycler 480 II system (Roche, Basle, Switzerland). Relative gene expression was calculated using the 2 −ΔΔCt method. GAPDH was used as an endogenous control to normalize gene expression. The primers used are listed in Table 2 . Table 2 Sequences of primers for qRT-PCR in this study Genes Sequences Egr1 forward: 5’-CACCTGACCGCAGAGTCTTT-3’ reverse: 5’-CTGACCAAGCTGAAGAGGGG-3’ PAR1 forward: 5’TGTGAACTGATCATGTTTATG-3’ reverse: 5’TTCGTAAGATAAGAGATATGT-3’ GAPDH forward: 5’GGGAGCCAAAAGGGTCATCATCT-3’ reverse: 5’GACGCCTGC TTCACCACCTTCTTG-3’ 2.4 Western blot analysis Total proteins from tissues or cells were extracted using RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) mixed with protease and phosphatase inhibitors. The protein concentration was determined using a BCA Protein Assay Kit (Abcam). Identical quantities of protein were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, followed by transfer to polyvinylidene difluoride (PVDF) membranes (Merck Millipore, MA, USA). After blocking with 5% skim milk for 1 h at room temperature, the membranes were incubated with primary antibodies against Egr1 (ab133695; 1:1000; rabbit; Abcam), PAR1 (ab32611; 1:1000; rabbit; Abcam), ERK1/2 (ab184699; 1:2000; rabbit; Abcam), p-ERK1/2 (ab32538; 1:1000; rabbit; Abcam), and GAPDH (ab181602; 1:1000; rabbit; Abcam) at 4°C overnight. Next, the membranes were incubated with peroxidase-conjugated rabbit secondary antibodies for 1 h at room temperature, detected using an Odyssey infrared imaging system (LI-COR, Lincoln, NE) and quantified using ImageJ software. 2.5 Cell Counting Kit-8 (CCK-8) assay HCC cells were cultured in 96-well plates containing complete medium. Cell proliferation was measured using a CCK-8 (Dojindo, Kumamoto, Japan) according to the manufacturer’s protocols. Optical density values at 450 nm were recorded at 0, 24, 48 and 72 hours using a microplate reader (Dynex Technologies, UK). Proliferation curves were drawn using the absorbance at each time point. 2.6 Transwell assay After HCC cells were suspended in 200 µl of serum-free medium, approximately 1×10 5 HCC cells were seeded in the upper chamber of a transwell chamber (Millipore, USA) coated with Matrigel (BD Biosciences, USA), and 600 µl of DMEM containing 10% FBS was added to the lower chamber. After incubation for 48 hours, the cells that invaded the lower chamber were first fixed with 4% paraformaldehyde and then stained with crystal violet (Sigma). Finally, images were acquired using a Nikon microscope. 2.7 Dual luciferase reporter assay The PAR1 promoter (-1 Kb) sequence containing the predicted wild-type Egr1 binding site or the mutant Egr1 binding site was constructed using the luciferase reporter gene pLG3 promoter vector (Promega, Madison, WI, USA). PLC/PRF/5 cells or HLLCM3 cells were seeded in 96-well plates and cultured for 24 hours. Then the pLG3 promoter vector, pRL-TK (Renilla luciferase control reporter vector) (Promega, Madison, WI) and pENTER-Egr1 plasmid were cotransfected into PLC/PRF/5 cells according to the group. A mixture of the pLG3 promoter vector, pRL-TK and siEgr1 was cotransfected into HCCLM3 cells according to the group. Transfection was performed using LipofectamineTM3000 reagent (Invitrogen, USA) according to the product instructions. Cells were harvested 48 hours after transfection and detected using a dual luciferase reporter system (Promega, Madison, WI, USA). 2.8 Statistical analysis All experiments were conducted in triplicate. The data are presented as the mean ± standard deviation (SD) and were analyzed using Student’s t - test or ANOVA. Correlation analysis was performed using the Pearson method. Statistical analysis was performed using SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). P values < 0.05 were considered to indicate statistical significance. 3. Results 3.1 Egr1 and PAR1 were overexpressed in HCC tissues We first detected the expression of Egr1 and PAR1 in 18 pairs of HCC specimens and adjacent normal tissues by immunohistochemical staining, and found that the expression of both genes was significantly increased in tumor specimens ( Fig. 1 A ) . Then we detected the expression of Egr1 and PAR1 in 18 pairs of samples by qPCR and Western blot analysis, and found that the mRNA and protein levels of the two genes were indeed increased in HCC tissues ( Fig. 1 B, C and D) . These results indicate that the expression levels of both Egr1 and PAR1 are abnormally increased in HCC. 3.2 Overexpressed Egr1 enhanced proliferation, invasion, migration, and PAR1 expression in HCC cells To further clarify the role and relationship between Egr1 and PAR1, we detected the mRNA and protein expression of these two genes in different HCC cell lines and normal cell lines. The results showed that the expression of Egr1 and PAR1 in PLC/PRF/5 cells was significantly lower than that in other HCC cell lines, while the expression in HCCLM3 cells was significantly increased and the highest among HCC cell lines ( Fig. 2 A, B ) . Therefore, we selected the PLC/PRF/5 and HCCLM3 cell lines for subsequent in vitro experiments. The pENTER-Egr1 plasmid was used to increase the expression of Egr1 in PLC/PRF/5 cells, and siEgr1 was used to reduce the expression of Egr1 in HCCLM3 cells. We found that Egr1 overexpression upregulated PAR1 expression ( Fig. 2 C, D ) and promoted the proliferation of PLC/PRF/5 cells ( Fig. 2 E ) ; and that Egr1 inhibition downregulated PAR1 expression ( Fig. 2 F, G ) and inhibited the proliferation of HCCLM3 cells ( Fig. 2 H ) . In addition, Egr1 upregulation promoted the invasion and migration of HCC cells, while Egr1 downregulation reduced the invasion and migration of HCC cells ( Fig. 2 I, J ) . These results suggest that Egr1 has a regulatory effect on the proliferation, invasion, migration and expression of PAR1 in HCC cells. 3.3 Egr1 increased the transcription of PAR1 in HCC cells We performed a correlation analysis on the mRNA expression levels of Egr1 and PAR1 in 18 HCC specimens and found that the two were positively correlated ( Fig. 3 A ) . To further investigate whether Egr1 regulates PAR1 expression, we performed a dual luciferase reporter assay. There is a potential Egr1 binding site between − 354 bp and − 335 bp in the h PAR1 promoter gene sequence (Salah et al., 2007 ). Therefore, we constructed dual-luciferase reporter genes containing wild-type or mutant Egr1 binding sites in the PAR1 promoter sequence ( Fig. 3 B ) . These reporters were cotransfected with the pENTER-Egr1 plasmid into PLC/PRF/5 cells and cotransfected with siEgr1 into HCCLM3 cells. The results showed that overexpression of Egr1 in PLC/PRF/5 cells significantly increased the luciferase activity of the reporter gene containing the wild-type Egr1 binding site, but had no effect on the reporter gene containing the mutant binding site ( Fig. 3 C ) . In contrast, knockdown of Egr1 expression in HCCLM3 cells significantly reduced the luciferase activity of a reporter gene containing the wild-type Egr1 binding site, but did not alter the luciferase activity of a reporter gene containing the mutant binding site ( Fig. 3 D ) . These data suggest that Egr1 is a transcriptional regulator of PAR1 in HCC cells. 3.4 Egr1 promoted the proliferation, invasion and migration of HCC cells by activating the MAPK/ERK pathway through PAR1 Aberrant activation of the MAPK/ERK pathway plays an important role in promoting HCC progression (Moon et al., 2021). PAR1, a GCPR, can activate MAPK/ERK signaling during tumorigenesis (Rabinovitch et al., 2021 ; Alturkistani et al., 2019 ). Therefore, we hypothesized that Egr1 activates the MAPK/ERK pathway through PAR1 and promotes HCC development. Western blot data showed that Egr1 overexpression resulted in increased levels of PAR1 and p-ERK1/2 in PLC/PRF/5 cells, while Egr1 inhibition led to decreased levels of PAR1 and p-ERK1/2 in HCCLM3 cells, while total ERK1/2 remained unchanged ( Fig. 4 A ) . To further clarify whether PAR1 in HCC cells mediates the regulatory effect of Egr1 on the MAPK/ERK signaling pathway, we transfected PCL/PRF-5 cells with the pENTER-Egr1 plasmid and siPAR1 or cotransfected the cells with the pENTER-Egr1 plasmid and siPAR1. Moreover, we treated HCCLM3 cells with siEgr1 and pENTER-PAR1 plasmids separately, or cotransfected them with siEgr1 and pENTER-PAR1 plasmids. The results showed that in PLC/PRF/5 cells, inhibition of PAR1 alleviated the promoting effect of Egr1 overexpression on phosphorylated ERK1/2 levels and cell proliferation, invasion and migration ( Fig. 4 B, C, D and E) . In contrast, in HCCLM3 cells, upregulation of PAR1 restored the inhibitory effect of silencing Egr1 expression on phosphorylated ERK1/2 levels, and cell proliferation, invasion and migration ( Fig. 4 F, G, H and I) . These findings suggest that abnormal Egr1 overexpression can promote proliferation, invasion and migration through PAR1 to activate the MAPK/ERK pathway. 3.5 Thrombin did not affect the Egr1/PAR1/MAPK pathway in HCC cells Thrombin is an upstream regulator of PAR1 that stimulates PAR1 expression at the posttranscriptional level (Ramachandran et al, 2012 ). To clarify whether thrombin is involved in the regulatory effect of Egr1 on PAR1 expression in HCC cells, we cultured PLC/PRF/5 cells transfected with the pENTER-Egr1 plasmid in DMEM supplemented with or without the thrombin inhibitor R-hirudin, and then performed subsequent experiments. Western blot analysis revealed that R-hirudin had no significant effect on the Egr1 overexpression-induced upregulation of PAR1 and p-ERK1/2 expression ( Fig. 5 A, B and C) . In addition, the enhanced cell proliferation, invasion and migration caused by Egr1 upregulation were not affected by R-hirudin ( Fig. 5 D, E and F) . These results indicate that thrombin is not related to the activation of the PAR1/MAPK/ERK pathway by Egr1 in HCC cells. 4. Discussion HCC is a very aggressive tumor with frequent intrahepatic and distant metastasis, which is also the main reason for the high recurrence and low survival rate after surgical resection of HCC (Yang et al., 2019 ). However, the exact mechanism of HCC progression is not yet completely clear, and an increasing number of studies have attempted to clarify the detailed mechanism of HCC progression. In this study, we found that the expression levels of Egr1 and PAR1 were significantly increased in HCC clinical samples, and their mRNA levels were positively correlated. Using PLC/PRF/5 cells and HCCLM3 cells, we further found that Egr1 regulates the expression of PAR1 and cell proliferation, invasion and migration. Furthermore, we elucidated that Egr1 can transcriptionally regulate the expression of PAR1 and activate the MAPK/ERK signaling pathway through PAR1 to promote the proliferation, invasion and migration of HCC cells. Finally, we demonstrated that the regulation of PAR1 by Egr1 was not affected by the PAR1 inhibitor thrombin, and clarified the important role of the Egr1/PAR1/MAPK pathway in the progression of HCC. The transcription factor Egr1 plays an important regulatory role in the occurrence, metastasis and angiogenesis of various malignant tumors (De Mestre et al., 2005 ). However, its role in HCC remains controversial. Some studies have reported that Egr1 can inhibit the progression of HCC (Wang et al., 2016 ; Hao et al., 2002 ), while more studies believe that Egr1 promotes HCC (Lee et al., 2009; Archer et al., 2009 ; Archer et al. 2016 ; Bi et al., 2019 ). We found that Egr1 expression was significantly increased in clinical HCC specimens and could stimulate the proliferation, invasion and migration of cancer cells. The results of this study showed that Egr1 expression was upregulated in HCC and promoted tumor progression, which is consistent with the findings that Egr1 is an oncogene in HCC. PAR1 is a GPCR with 7 transmembrane units. Abnormal expression of PAR1 can promote the growth and metastasis of various types of tumors (Kaufmann et al., 2007 ; Ramachandran et al., 2012 ). Abnormally high expression of PAR1 in HCC promotes the proliferation and invasion of cancer cells (Kaufmann et al., 2007 ; Xiao et al., 2018 ; Mußbach et al., 2015 ). However, the upstream regulatory mechanism of PAR1 in HCC is unclear. Previous studies have shown that in prostate cancer, Egr1 can directly bind to the PAR1 promoter region and regulate the transcription of PAR1 (Salah et al., 2007 ). In this study, the mRNA expression levels of the Egr1 and PAR1 genes in 18 HCC clinical specimens were positively correlated. Therefore, does Egr1 promote the progression of HCC by regulating the expression of PAR1? In HCC, is Egr1 an upstream transcriptional regulator of PAR1? To answer these questions, we performed dual luciferase reporter assays and in vitro gain-of-function and loss-of-function experiments. The results showed that Egr1 regulates the transcription of PAR1 by directly binding to the promoter region of PAR1, thereby promoting the proliferation, invasion and migration of cancer cells. The MAPK/ERK signaling pathway is activated by signal transduction from cell surface receptors such as receptor tyrosine kinases (RTKs) or GPCRs (Delire et al., 2015). The MAPK/ERK pathway plays a key role in regulating cell survival and proliferation, and its abnormal activation is closely related to cell transformation and carcinogenesis (Guo et al., 2020 ). MAPK/ERK signaling is considered to be activated in approximately 50% of early HCC patients and almost all advanced patients (Neuzillet et al., 2014 ). Previous studies have shown that PAR1, a GPCR, can activate the MAPK/ERK pathway to promote tumor progression (Rabinovitch et al. 2021 ; Darmoul et al., 2003 ). Therefore, does Egr1 promote HCC progression by activating the MAPK/ERK pathway through PAR1? We found that in PLC/PRF/5 cells, silencing PAR1 restored the upregulation of ERK1/2 phosphorylation caused by Egr1 overexpression; in HCCLM3 cells, overexpression of PAR1 restored the downregulation of ERK1/2 phosphorylation caused by Egr1 downregulation. This finding suggested that Egr1 promotes HCC progression by regulating PAR1 to activate the MAPK/ERK pathway. Thrombin is a clear regulator of PAR1 that promotes the expression of PAR1 at the posttranscriptional level by recognizing a specific site in the extracellular N-terminus of PAR1 (Ramachandran et al., 2012 ). Therefore, in HCC, is thrombin involved in the regulation of PAR1 by Egr1? We used the thrombin inhibitor R-hirudin (Wakui et al., 2019 ) for in vitro experiments and found that R-hirudin had no significant effect on the activation of the MAPK/ERK pathway caused by Egr1 overexpression through the upregulation of PAR1 transcription. 5. CONCLUSION In summary, Egr1 plays a role as an oncogene in HCC. The abnormally high expression of Egr1 upregulates PAR1 expression through transcription, thereby activating the MAPK/ERK pathway and promoting the proliferation, invasion and migration of cancer cells. Abbreviations CCK-8 Cell Counting Kit-8 DMEM Dulbecco’s modified Eagle’s medium Egr1 early growth response factor 1 FBS fetal bovine serum GPCR G-protein coupled receptor HCC hepatocellular carcinoma PAR1 protease-activated receptor 1 MAPK Mitogen-activated protein kinase ERK extracellular signal-regulated kinase qRT‒PCR quantitative real-time polymerase chain reaction siEgr1 small interfering RNA targeting Egr1 siPAR1 small interfering RNA targeting PAR1 R-hirudin recombinant hirudin Declarations Author Contributions Jian-gang Bi and Ping Xu designed the experiments and wrote the manuscript. Qi Li, Yu-sheng Guo and Hong-gui Tang performed all the experiments. Jian-gang Bi analyzed the data. Qi Li searched the literature. All authors reviewed the results and agreed to the final version of the manuscript. Conflict of interest The authors declare no conflicts of interest with respect to the authorship, research, or publication of this article. Data availability statement All the data generated or analyzed during this study are included in this manuscript. Funding information This study was supported by the Guangdong Medical Science and Technology Research Foundation (A2021275, A2020559). The funders had no role in the design of the study, the execution of the experiment, the collection and analysis of the data, the decision to publish, or the preparation of the manuscript. References Alturkistani, A., Ghonem, N., Power-Charnitsky, V.A., Pino-Figueroa, A., Miglioreet, M.M., 2019. Inhibition of PAR-1 Receptor Signaling by Enoxaparin Reduces Cell Proliferation and Migration in A549 Cells. Anticancer Res 39(10), 5297-5310. Archer, K.J., Mas, V.R., David, K., Maluf, D.G., Bornstein, K., Fisher, R.A., 2009. 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A global view of hepatocellular carcinoma: trends, risk, prevention and management. Nat Rev Gastroenterol Hepatol 16, 589-604. Yang, M., Teng, W., Qu, Y., 2016. Sulforaphene inhibits triple negative breast cancer through activating tumor suppressor Egr1. Breast Cancer Res Treat 158(2), 277-286. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4744749","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":331200958,"identity":"f7805e36-90f5-4f57-be4c-446aaaea06e5","order_by":0,"name":"Jian-gang Bi","email":"","orcid":"","institution":"Shenzhen People's Hospital (The Second Clinical Medical College of Jinan University, Southern University of Science and Technology)","correspondingAuthor":false,"prefix":"","firstName":"Jian-gang","middleName":"","lastName":"Bi","suffix":""},{"id":331200959,"identity":"01e90091-348e-4ba5-8e7f-3b2525ae1114","order_by":1,"name":"Qi Li","email":"","orcid":"","institution":"Shenzhen People's Hospital (The Second Clinical Medical College of Jinan University, Southern University of Science and Technology)","correspondingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Li","suffix":""},{"id":331200960,"identity":"99d14048-0aea-49d5-ae06-1aeb256dce2b","order_by":2,"name":"Yu-sheng Guo","email":"","orcid":"","institution":"Shenzhen People's Hospital (The Second Clinical Medical College of Jinan University, Southern University of Science and Technology)","correspondingAuthor":false,"prefix":"","firstName":"Yu-sheng","middleName":"","lastName":"Guo","suffix":""},{"id":331200961,"identity":"ae8de7cc-8a0f-4f87-8128-7f4588d53153","order_by":3,"name":"Hong-gui Tang","email":"","orcid":"","institution":"Shenzhen People's Hospital (The Second Clinical Medical College of Jinan University, Southern University of Science and Technology)","correspondingAuthor":false,"prefix":"","firstName":"Hong-gui","middleName":"","lastName":"Tang","suffix":""},{"id":331200962,"identity":"ceb124c2-00e0-476d-b3fd-c1476347dd52","order_by":4,"name":"Ping Xu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIie3QsQqCQBjA8e8QrkVxvSPIV7gQpMc5cWipNygRhGsRWmvqFWyJxkTQxZob8w1qdOskoxrSxob7wx3f8uP4DkCl+tc4wABAr0cC0PuR2E9CQPvxITd4EOgm7MzxpdzPx5vlMSmr/cg3Q0DX2+Q7oSveY26RT+PDybONghCSgkbXu+/EJBwTV2TTGEVOHwm5SwpYM1oIbsjYCnWHVpJYXaR5ZcYh0x1iSMK6CI3KBXPFYRgXul3vQrcpClt3YbmXlZXwLWtZDOWP+eYgD5PrrYUAICGvtJmD190Wlsd/JyqVSqX67A5CmEoLgjtx+gAAAABJRU5ErkJggg==","orcid":"","institution":"Shenzhen People's Hospital (The Second Clinical Medical College of Jinan University, Southern University of Science and Technology)","correspondingAuthor":true,"prefix":"","firstName":"Ping","middleName":"","lastName":"Xu","suffix":""}],"badges":[],"createdAt":"2024-07-15 18:14:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4744749/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4744749/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":62646966,"identity":"b7a1a713-d4a1-4739-a7f0-1b95c66c8fdd","added_by":"auto","created_at":"2024-08-16 21:20:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2559672,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of Egr1 and PAR1 in HCC tissues. (A)\u003c/strong\u003e Representative images of Egr1 and PAR1 expression using immunohistochemical staining in adjacent normal tissues and HCC tumor tissues.\u003cstrong\u003e (B)\u003c/strong\u003e qRT-PCR analysis of Egr1 and PAR1 expression in 18 paired nontumor and HCC tumor specimens.\u003cstrong\u003e (C)\u003c/strong\u003e Western blot and\u003cstrong\u003e (D) \u003c/strong\u003equantitative analysis of Egr1 and PAR1 protein levels in 18 HCC tissues and paired adjacent nontumor tissues. N: nontumor; T: tumor.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4744749/v1/e91bbd96977fa2aa1b55a034.png"},{"id":62647405,"identity":"2a2ba9e2-bd02-4008-9c3a-289f0384ce58","added_by":"auto","created_at":"2024-08-16 21:28:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3600050,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpressed Egr1 enhanced proliferation, invasion, migration, and PAR1 expression in HCC cells.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e qRT-PCR and \u003cstrong\u003e(B)\u003c/strong\u003e western blot with quantitative analysis of Egr1 and PAR1 expression in HCC cell lines and normal cell line.\u003cstrong\u003e (C, D)\u003c/strong\u003eExpression of Egr1 and PAR1 was detected by qRT-PCR and western blot with quantitative analysis in PLC/PRF/5 cells transfected with pENTER-Egr1 plasmids. \u003cstrong\u003e(E)\u003c/strong\u003e Effect of overexpression of Egr1 on HCC cell proliferation was evaluated by CCK-8 assay.\u003cstrong\u003e (F) \u003c/strong\u003eqRT-PCR and \u003cstrong\u003e(G)\u003c/strong\u003e western blot with quantitative analysis of Egr1 and PAR1 expression in HCCLM3 cells transfected with siEgr1. \u003cstrong\u003e(H) \u003c/strong\u003eCCK-8 assay was used to evaluate the cell viability of HCCLM3 cells with Egr1 silencing expression. \u003cstrong\u003e(I)\u003c/strong\u003e Invasive and migratory abilities of PLC/PRF/5 cells with overexpressed Egr1 and of HCCLM3 cells with silenced Egr1 detected by transwell assay. \u003cstrong\u003e(J)\u003c/strong\u003e Wound healing assay analysis of PLC/PRF/5 cells treated with pENTER-Egr1 plasmids and of HCCLM3 cells treated with siEgr1. pENTER, empty vector; pENTER-Egr1, plasmid encoding Egr1; NC, negative control; siEgr1, small interfering RNA targeting Egr1. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4744749/v1/3e6b398efdf4829e523f5783.png"},{"id":62646969,"identity":"78ae06fd-865c-4fa9-8dae-faa0686731e9","added_by":"auto","created_at":"2024-08-16 21:20:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":217443,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEgr1 increased transcription of PAR1 in HCC cells. (A) \u003c/strong\u003eCorrelation of Egr1 and PAR1 mRNA levels in 18 HCC specimens. Pearson correlation analysis was performed for ΔCt values.\u003cstrong\u003e (B)\u003c/strong\u003e The putative wild type Egr1 binding sequence and mutant Egr1 binding sites in the PAR1 promoter. \u003cstrong\u003e(C)\u003c/strong\u003e PLC/PRF/5 cells were co-transfected with pENTER-Egr1 and the wild or mutant PAR1 promoter. Dual luciferase assay was used to detect the relative luciferase activity of each group. \u003cstrong\u003e(D)\u003c/strong\u003e HCCLM3 cells were co-transfected with siEgr1 and and the wild or mutant PAR1 promoter, and the relative luciferase activity was determined of different group. wt, wild type; mt, mutant type; pENTER, empty vector; pENTER-Egr1, plasmid encoding Egr1; siNC, negative control of small interfering RNA; siPAR1, small interfering RNA targeting PAR1. ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4744749/v1/43dd86ee1824ee57c094eb6f.png"},{"id":62646967,"identity":"e78b87c2-f042-46a7-8679-8f9c5c4cd767","added_by":"auto","created_at":"2024-08-16 21:20:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1937643,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEgr1 promoted proliferation, invasion and migration of HCC cells through activating MAPK/ERK pathway via PAR1.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Western blot and quantitative analysis of PAR1, total ERK1/2 and p-ERK1/2 levels in PLC/PRF/5 cells transfected with pENTER-Egr1 plasmid and in HCCLM3 cells treated with siEgr1.\u003cstrong\u003e (B)\u003c/strong\u003e PLC/PRF/5 cells were transfected with pENTER, pENTER-Egr1, siPAR1, or pENTER-Egr1 and siPAR1, respectively. Protein expression of Egr1, PAR1, total ERK1/2 and p-ERK1/2 of each group was detected by western blot. \u003cstrong\u003e(C)\u003c/strong\u003e Proliferation ability of each group PLC/PRF/5 cells was assessed by CCK-8 assay. \u003cstrong\u003e(D)\u003c/strong\u003e For each group of cells, the representative pictures with invasion and migration ability were obtained by transwell assay and quantitative analysis was performed. \u003cstrong\u003e(E)\u003c/strong\u003eThe wound width images of different groups were detected by wound healing assay and quantitative analysis was performed. \u003cstrong\u003e(F)\u003c/strong\u003e HCCLM3 cells were treated with NC, siEgr1, pENTER-PAR1, or siEgr1 and pENTER-PAR1, respectively. Western blot analysis was performed to measure Egr1, PAR1, total ERK1/2 and p-ERK1/2 protein expression of each group.\u003cstrong\u003e (G) \u003c/strong\u003eCell viability of each group HCCLM3 cells was assessed by CCK-8 assay. \u003cstrong\u003e(H) \u003c/strong\u003eFor each group, invasion and migration were evaluated by transwell assay and quantitative analysis was performed. \u003cstrong\u003e(I)\u003c/strong\u003e Images of wound width and quantification of relative distance of wound healing were obtained by wound healing assay. pENTER, empty vector; pENTER-Egr1, plasmid encoding Egr1; siPAR1, small interfering RNA targeting PAR1; NC, negative control; siEgr1, small interfering RNA targeting Egr1; pENTER-PAR1, plasmid encoding PAR1. **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4744749/v1/1332e00bc0140b7d14965f19.png"},{"id":62647792,"identity":"08e66003-8b57-4f60-bd86-dec656ba914a","added_by":"auto","created_at":"2024-08-16 21:36:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3339462,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThrombin was not essential for Egr1/PAR1/MAPK pathway in HCC cells.\u003c/strong\u003e PLC/PRF/5 cells transfected with pENTER-Egr1 plasmid were incubated in DMEM medium with or without R-Hirudin. \u003cstrong\u003e(A, B)\u003c/strong\u003e The mRNA and protein expression of Egr1 and PAR1 of each group was detected by qRT-PCR and western blot analysis. \u003cstrong\u003e(C)\u003c/strong\u003eWestern blot was used to assess the protein levels of Egr1, PAR1, total ERK1/2 and p-ERK1/2 of different group. \u003cstrong\u003e(D)\u003c/strong\u003e CCK-8 assay was applied to evaluate the proliferation of cells. \u003cstrong\u003e(E)\u003c/strong\u003e Representative images of invasive and migrative abilities of each group were obtained via transwell assay and quantitative analysis was performed.\u003cstrong\u003e (F) \u003c/strong\u003eWound healing analysis and quantitative analysis of different group. pENTER, empty vector; pENTER-Egr1, plasmid encoding Egr1; R-Hirudin, inhibitor of thrombin. **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4744749/v1/499000c7c8761646a1eddedf.png"},{"id":78085585,"identity":"45eb64ae-0048-4a01-8dee-5ca2f16e7252","added_by":"auto","created_at":"2025-03-09 14:46:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13313393,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4744749/v1/a12a9e48-91b8-4b7d-89e0-a9d9d461eb5d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Early growth response factor 1 promotes HCC progression by activating the MAPK/ERK pathway through transcriptional upregulation of PAR1","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eLiver cancer is undoubtedly a global public health challenge, with the incidence expected to exceed 1\u0026nbsp;million cases per year by 2025 (Sung et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Primary liver cancer is the sixth most common cancer worldwide and the third leading cause of death (Sung et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Hepatocellular carcinoma (HCC) accounts for the majority of liver cancer cases at diagnosis and mortality (Singal et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In addition to surgical resection, some progress has been made in the clinical treatment of HCC in recent years, such as in transplantation, ablation and systemic therapy, but the treatment efficacy is not satisfactory. The mechanisms that drive the malignant transformation and progression of HCC have not been fully elucidated (Bruix et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Bruix et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e); therefore, identifying key molecules and gaining a deeper understanding of their mechanisms of action are crucial for developing new and effective treatments to improve the prognosis of HCC patients.\u003c/p\u003e \u003cp\u003eEarly growth response factor 1 (Egr1), an important transcriptional regulator, regulates the transcription of downstream target genes by recognizing and binding the specific DNA sequence 5\u0026rsquo;-CGCCCCCGC-3\u0026rsquo; (Egr1 binding site) in the gene promoter (Khachigian et al., 1998). When cells are subjected to injury stress, the expression of Egr1 is rapidly upregulated to regulate growth and proliferation (Gashler et al., 1995). Egr1 regulates tumors in a bidirectional manner, promoting some cancers (Stamatakis et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Parra et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Hansson et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Lee et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and suppressing others (Sun et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). However, there are few studies on the role of Egr1 in HCC, and there is still controversy. Some studies have shown that Egr1 inhibits the development of HCC (Wang et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), but more studies have supported the view that Egr1 promotes the progression of HCC (Lee et al., 2009; Archer et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Archer et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Our previous studies revealed that Egr1 expression is significantly upregulated in HCC and promotes cell proliferation (Bi et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), but the downstream molecular mechanism involved needs further study.\u003c/p\u003e \u003cp\u003eProtease-activated receptor 1 (PAR1) is a G protein-coupled receptor (GPCR) with seven transmembrane units, that is widely present in the digestive system and regulates intracellular signal changes. Many studies have confirmed that PAR1 plays an important role in promoting HCC progression (Kaufmann et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Xiao et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Mu\u0026szlig;bach et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In addition, thrombin is known to regulate PAR1 at the posttranscriptional level by recognizing PAR1 at specific sites in the extracellular N-terminal region (O\u0026rsquo;Brien et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), but does not regulate PAR1 expression at the transcriptional level. There are few studies on the upstream transcriptional regulatory mechanism of PAR1 in HCC. Some studies have shown that Egr1 upregulates PAR1 at the transcriptional level in prostate cancer (Salah et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Therefore, we speculated that Egr1 promotes the progression of HCC by regulating PAR1 expression.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Patients and clinical specimens\u003c/h2\u003e \u003cp\u003eThis study was approved by the Ethics Committee of the Second Affiliated Hospital of Jinan University, and written informed consent was obtained from each patient. Eighteen patients with a histological diagnosis of HCC between 2018 and 2020 were recruited. Tumor tissues and paired adjacent nontumor samples at least 5 cm from the tumor edge were collected. Clinical specimens were immediately frozen in liquid nitrogen and stored at -80\u0026deg;C. Patients who received radiotherapy or chemotherapy before surgery were excluded. The clinical pathological parameters are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eClinical characteristics of HCC patients (n\u0026thinsp;=\u0026thinsp;18)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNumber of cases\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (years)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026le; 65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026gt; 65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGender\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eType of hepatitis\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHBV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHCV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHBV\u0026thinsp;+\u0026thinsp;HCV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTumor size (cm)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026le; 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u0026ndash;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTumor number\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSolitary\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMultiple\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLiver cirrhosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ewith\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ewithout\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTNM stage\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eⅠ\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eⅡ\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eⅢ\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eⅣ\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Cell culture and transfection\u003c/h2\u003e \u003cp\u003eThe immortalized liver cell line THLE-2, and HCC cell lines (PLC/PRF-5, HCCLM3, MHCC97H, MHCC97L, SMMC-7721 and Hep3B) were purchased from the Cell Bank of Type Culture Collection (Chinese Academy of Sciences, Shanghai, China). All cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco) at 37\u0026deg;C in 5% CO2. The medium was changed every 48 hours. pENTER-Egr1 and pENTER-PAR1 plasmids were purchased from WZ Biosciences (Shandong, China) for overexpression of Egr1 or PAR1. Cells were cultured in 12-well plates, and when the cell confluence reached approximately 50\u0026ndash;60%, the cells were transfected with 2 \u0026micro;g of plasmid or empty vector according to the product instructions. Small interfering RNAs targeting Egr1 (siEgr1) and PAR1 (siPAR1) and the corresponding negative controls were purchased from Ribobio (Guangzhou, China) and used to inhibit the expression of Egr1 or PAR1. The cells were grouped and cultured in 12-well plates, and when the cell confluence was approximately 50\u0026ndash;60%, transfection was performed at a concentration of 100 nmol/L according to the product instructions. LipofectamineTM3000 (Invitrogen, USA) was used for transfection. Recombinant hirudin (R-hirudin) (Sigma-Aldrich) was added to the medium according to the manufacturer\u0026rsquo;s instructions to inhibit the effect of thrombin on PAR1 at a concentration of 100 \u0026micro;g/ml. The cells were harvested 72 hours after transfection for subsequent experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Quantitative real-time polymerase chain reaction (qRT‒PCR).\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from specimens and cells using TRIzol reagent (Thermo Fisher Scientific, MA, USA). Complementary DNA (cDNA) synthesis was conducted using M-MLV Reverse Transcriptase (Invitrogen, MA, USA). qRT‒PCR analysis was performed using the SYBR Green qPCR Kit (Takara, Dalian, China) on a Roche LightCycler 480 II system (Roche, Basle, Switzerland). Relative gene expression was calculated using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method. GAPDH was used as an endogenous control to normalize gene expression. The primers used are listed in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSequences of primers for qRT-PCR in this study\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSequences\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"1\" nameend=\"c4\" namest=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEgr1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eforward:\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-CACCTGACCGCAGAGTCTTT-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c4\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ereverse:\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-CTGACCAAGCTGAAGAGGGG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c4\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePAR1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eforward:\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;TGTGAACTGATCATGTTTATG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c4\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ereverse:\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;TTCGTAAGATAAGAGATATGT-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c4\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGAPDH\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eforward:\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;GGGAGCCAAAAGGGTCATCATCT-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c4\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ereverse:\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;GACGCCTGC TTCACCACCTTCTTG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Western blot analysis\u003c/h2\u003e \u003cp\u003eTotal proteins from tissues or cells were extracted using RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) mixed with protease and phosphatase inhibitors. The protein concentration was determined using a BCA Protein Assay Kit (Abcam). Identical quantities of protein were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, followed by transfer to polyvinylidene difluoride (PVDF) membranes (Merck Millipore, MA, USA). After blocking with 5% skim milk for 1 h at room temperature, the membranes were incubated with primary antibodies against Egr1 (ab133695; 1:1000; rabbit; Abcam), PAR1 (ab32611; 1:1000; rabbit; Abcam), ERK1/2 (ab184699; 1:2000; rabbit; Abcam), p-ERK1/2 (ab32538; 1:1000; rabbit; Abcam), and GAPDH (ab181602; 1:1000; rabbit; Abcam) at 4\u0026deg;C overnight. Next, the membranes were incubated with peroxidase-conjugated rabbit secondary antibodies for 1 h at room temperature, detected using an Odyssey infrared imaging system (LI-COR, Lincoln, NE) and quantified using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Cell Counting Kit-8 (CCK-8) assay\u003c/h2\u003e \u003cp\u003eHCC cells were cultured in 96-well plates containing complete medium. Cell proliferation was measured using a CCK-8 (Dojindo, Kumamoto, Japan) according to the manufacturer\u0026rsquo;s protocols. Optical density values at 450 nm were recorded at 0, 24, 48 and 72 hours using a microplate reader (Dynex Technologies, UK). Proliferation curves were drawn using the absorbance at each time point.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Transwell assay\u003c/h2\u003e \u003cp\u003eAfter HCC cells were suspended in 200 \u0026micro;l of serum-free medium, approximately 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e HCC cells were seeded in the upper chamber of a transwell chamber (Millipore, USA) coated with Matrigel (BD Biosciences, USA), and 600 \u0026micro;l of DMEM containing 10% FBS was added to the lower chamber. After incubation for 48 hours, the cells that invaded the lower chamber were first fixed with 4% paraformaldehyde and then stained with crystal violet (Sigma). Finally, images were acquired using a Nikon microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Dual luciferase reporter assay\u003c/h2\u003e \u003cp\u003eThe PAR1 promoter (-1 Kb) sequence containing the predicted wild-type Egr1 binding site or the mutant Egr1 binding site was constructed using the luciferase reporter gene pLG3 promoter vector (Promega, Madison, WI, USA). PLC/PRF/5 cells or HLLCM3 cells were seeded in 96-well plates and cultured for 24 hours. Then the pLG3 promoter vector, pRL-TK (Renilla luciferase control reporter vector) (Promega, Madison, WI) and pENTER-Egr1 plasmid were cotransfected into PLC/PRF/5 cells according to the group. A mixture of the pLG3 promoter vector, pRL-TK and siEgr1 was cotransfected into HCCLM3 cells according to the group. Transfection was performed using LipofectamineTM3000 reagent (Invitrogen, USA) according to the product instructions. Cells were harvested 48 hours after transfection and detected using a dual luciferase reporter system (Promega, Madison, WI, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments were conducted in triplicate. The data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) and were analyzed using Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e- test or ANOVA. Correlation analysis was performed using the Pearson method. Statistical analysis was performed using SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). \u003cem\u003eP\u003c/em\u003e values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered to indicate statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Egr1 and PAR1 were overexpressed in HCC tissues\u003c/h2\u003e \u003cp\u003eWe first detected the expression of Egr1 and PAR1 in 18 pairs of HCC specimens and adjacent normal tissues by immunohistochemical staining, and found that the expression of both genes was significantly increased in tumor specimens \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Then we detected the expression of Egr1 and PAR1 in 18 pairs of samples by qPCR and Western blot analysis, and found that the mRNA and protein levels of the two genes were indeed increased in HCC tissues \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C \u003cb\u003eand D)\u003c/b\u003e. These results indicate that the expression levels of both Egr1 and PAR1 are abnormally increased in HCC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Overexpressed Egr1 enhanced proliferation, invasion, migration, and PAR1 expression in HCC cells\u003c/h2\u003e \u003cp\u003eTo further clarify the role and relationship between Egr1 and PAR1, we detected the mRNA and protein expression of these two genes in different HCC cell lines and normal cell lines. The results showed that the expression of Egr1 and PAR1 in PLC/PRF/5 cells was significantly lower than that in other HCC cell lines, while the expression in HCCLM3 cells was significantly increased and the highest among HCC cell lines \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B\u003cb\u003e)\u003c/b\u003e. Therefore, we selected the PLC/PRF/5 and HCCLM3 cell lines for subsequent in vitro experiments. The pENTER-Egr1 plasmid was used to increase the expression of Egr1 in PLC/PRF/5 cells, and siEgr1 was used to reduce the expression of Egr1 in HCCLM3 cells. We found that Egr1 overexpression upregulated PAR1 expression \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D\u003cb\u003e)\u003c/b\u003e and promoted the proliferation of PLC/PRF/5 cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e; and that Egr1 inhibition downregulated PAR1 expression \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, G\u003cb\u003e)\u003c/b\u003e and inhibited the proliferation of HCCLM3 cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH\u003cb\u003e)\u003c/b\u003e. In addition, Egr1 upregulation promoted the invasion and migration of HCC cells, while Egr1 downregulation reduced the invasion and migration of HCC cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI, J\u003cb\u003e)\u003c/b\u003e. These results suggest that Egr1 has a regulatory effect on the proliferation, invasion, migration and expression of PAR1 in HCC cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Egr1 increased the transcription of PAR1 in HCC cells\u003c/h2\u003e \u003cp\u003eWe performed a correlation analysis on the mRNA expression levels of Egr1 and PAR1 in 18 HCC specimens and found that the two were positively correlated \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. To further investigate whether Egr1 regulates PAR1 expression, we performed a dual luciferase reporter assay. There is a potential Egr1 binding site between \u0026minus;\u0026thinsp;354 bp and \u0026minus;\u0026thinsp;335 bp in the \u003cem\u003eh\u003c/em\u003ePAR1 promoter gene sequence (Salah et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Therefore, we constructed dual-luciferase reporter genes containing wild-type or mutant Egr1 binding sites in the PAR1 promoter sequence \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. These reporters were cotransfected with the pENTER-Egr1 plasmid into PLC/PRF/5 cells and cotransfected with siEgr1 into HCCLM3 cells. The results showed that overexpression of Egr1 in PLC/PRF/5 cells significantly increased the luciferase activity of the reporter gene containing the wild-type Egr1 binding site, but had no effect on the reporter gene containing the mutant binding site \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. In contrast, knockdown of Egr1 expression in HCCLM3 cells significantly reduced the luciferase activity of a reporter gene containing the wild-type Egr1 binding site, but did not alter the luciferase activity of a reporter gene containing the mutant binding site \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. These data suggest that Egr1 is a transcriptional regulator of PAR1 in HCC cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.4 Egr1 promoted the proliferation, invasion and migration of HCC cells by activating the MAPK/ERK pathway through PAR1\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAberrant activation of the MAPK/ERK pathway plays an important role in promoting HCC progression (Moon et al., 2021). PAR1, a GCPR, can activate MAPK/ERK signaling during tumorigenesis (Rabinovitch et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Alturkistani et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Therefore, we hypothesized that Egr1 activates the MAPK/ERK pathway through PAR1 and promotes HCC development. Western blot data showed that Egr1 overexpression resulted in increased levels of PAR1 and p-ERK1/2 in PLC/PRF/5 cells, while Egr1 inhibition led to decreased levels of PAR1 and p-ERK1/2 in HCCLM3 cells, while total ERK1/2 remained unchanged \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. To further clarify whether PAR1 in HCC cells mediates the regulatory effect of Egr1 on the MAPK/ERK signaling pathway, we transfected PCL/PRF-5 cells with the pENTER-Egr1 plasmid and siPAR1 or cotransfected the cells with the pENTER-Egr1 plasmid and siPAR1. Moreover, we treated HCCLM3 cells with siEgr1 and pENTER-PAR1 plasmids separately, or cotransfected them with siEgr1 and pENTER-PAR1 plasmids. The results showed that in PLC/PRF/5 cells, inhibition of PAR1 alleviated the promoting effect of Egr1 overexpression on phosphorylated ERK1/2 levels and cell proliferation, invasion and migration \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C, D \u003cb\u003eand E)\u003c/b\u003e. In contrast, in HCCLM3 cells, upregulation of PAR1 restored the inhibitory effect of silencing Egr1 expression on phosphorylated ERK1/2 levels, and cell proliferation, invasion and migration \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, G, H \u003cb\u003eand I)\u003c/b\u003e. These findings suggest that abnormal Egr1 overexpression can promote proliferation, invasion and migration through PAR1 to activate the MAPK/ERK pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Thrombin did not affect the Egr1/PAR1/MAPK pathway in HCC cells\u003c/h2\u003e \u003cp\u003eThrombin is an upstream regulator of PAR1 that stimulates PAR1 expression at the posttranscriptional level (Ramachandran et al, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). To clarify whether thrombin is involved in the regulatory effect of Egr1 on PAR1 expression in HCC cells, we cultured PLC/PRF/5 cells transfected with the pENTER-Egr1 plasmid in DMEM supplemented with or without the thrombin inhibitor R-hirudin, and then performed subsequent experiments. Western blot analysis revealed that R-hirudin had no significant effect on the Egr1 overexpression-induced upregulation of PAR1 and p-ERK1/2 expression \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B \u003cb\u003eand C)\u003c/b\u003e. In addition, the enhanced cell proliferation, invasion and migration caused by Egr1 upregulation were not affected by R-hirudin \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, E \u003cb\u003eand F)\u003c/b\u003e. These results indicate that thrombin is not related to the activation of the PAR1/MAPK/ERK pathway by Egr1 in HCC cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eHCC is a very aggressive tumor with frequent intrahepatic and distant metastasis, which is also the main reason for the high recurrence and low survival rate after surgical resection of HCC (Yang et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, the exact mechanism of HCC progression is not yet completely clear, and an increasing number of studies have attempted to clarify the detailed mechanism of HCC progression. In this study, we found that the expression levels of Egr1 and PAR1 were significantly increased in HCC clinical samples, and their mRNA levels were positively correlated. Using PLC/PRF/5 cells and HCCLM3 cells, we further found that Egr1 regulates the expression of PAR1 and cell proliferation, invasion and migration. Furthermore, we elucidated that Egr1 can transcriptionally regulate the expression of PAR1 and activate the MAPK/ERK signaling pathway through PAR1 to promote the proliferation, invasion and migration of HCC cells. Finally, we demonstrated that the regulation of PAR1 by Egr1 was not affected by the PAR1 inhibitor thrombin, and clarified the important role of the Egr1/PAR1/MAPK pathway in the progression of HCC.\u003c/p\u003e \u003cp\u003eThe transcription factor Egr1 plays an important regulatory role in the occurrence, metastasis and angiogenesis of various malignant tumors (De Mestre et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). However, its role in HCC remains controversial. Some studies have reported that Egr1 can inhibit the progression of HCC (Wang et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Hao et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), while more studies believe that Egr1 promotes HCC (Lee et al., 2009; Archer et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Archer et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Bi et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). We found that Egr1 expression was significantly increased in clinical HCC specimens and could stimulate the proliferation, invasion and migration of cancer cells. The results of this study showed that Egr1 expression was upregulated in HCC and promoted tumor progression, which is consistent with the findings that Egr1 is an oncogene in HCC.\u003c/p\u003e \u003cp\u003ePAR1 is a GPCR with 7 transmembrane units. Abnormal expression of PAR1 can promote the growth and metastasis of various types of tumors (Kaufmann et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Ramachandran et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Abnormally high expression of PAR1 in HCC promotes the proliferation and invasion of cancer cells (Kaufmann et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Xiao et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Mu\u0026szlig;bach et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). However, the upstream regulatory mechanism of PAR1 in HCC is unclear. Previous studies have shown that in prostate cancer, Egr1 can directly bind to the PAR1 promoter region and regulate the transcription of PAR1 (Salah et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). In this study, the mRNA expression levels of the Egr1 and PAR1 genes in 18 HCC clinical specimens were positively correlated. Therefore, does Egr1 promote the progression of HCC by regulating the expression of PAR1? In HCC, is Egr1 an upstream transcriptional regulator of PAR1? To answer these questions, we performed dual luciferase reporter assays and in vitro gain-of-function and loss-of-function experiments. The results showed that Egr1 regulates the transcription of PAR1 by directly binding to the promoter region of PAR1, thereby promoting the proliferation, invasion and migration of cancer cells.\u003c/p\u003e \u003cp\u003eThe MAPK/ERK signaling pathway is activated by signal transduction from cell surface receptors such as receptor tyrosine kinases (RTKs) or GPCRs (Delire et al., 2015). The MAPK/ERK pathway plays a key role in regulating cell survival and proliferation, and its abnormal activation is closely related to cell transformation and carcinogenesis (Guo et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). MAPK/ERK signaling is considered to be activated in approximately 50% of early HCC patients and almost all advanced patients (Neuzillet et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Previous studies have shown that PAR1, a GPCR, can activate the MAPK/ERK pathway to promote tumor progression (Rabinovitch et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Darmoul et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Therefore, does Egr1 promote HCC progression by activating the MAPK/ERK pathway through PAR1? We found that in PLC/PRF/5 cells, silencing PAR1 restored the upregulation of ERK1/2 phosphorylation caused by Egr1 overexpression; in HCCLM3 cells, overexpression of PAR1 restored the downregulation of ERK1/2 phosphorylation caused by Egr1 downregulation. This finding suggested that Egr1 promotes HCC progression by regulating PAR1 to activate the MAPK/ERK pathway.\u003c/p\u003e \u003cp\u003eThrombin is a clear regulator of PAR1 that promotes the expression of PAR1 at the posttranscriptional level by recognizing a specific site in the extracellular N-terminus of PAR1 (Ramachandran et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Therefore, in HCC, is thrombin involved in the regulation of PAR1 by Egr1? We used the thrombin inhibitor R-hirudin (Wakui et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) for in vitro experiments and found that R-hirudin had no significant effect on the activation of the MAPK/ERK pathway caused by Egr1 overexpression through the upregulation of PAR1 transcription.\u003c/p\u003e"},{"header":"5. CONCLUSION","content":"\u003cp\u003eIn summary, Egr1 plays a role as an oncogene in HCC. The abnormally high expression of Egr1 upregulates PAR1 expression through transcription, thereby activating the MAPK/ERK pathway and promoting the proliferation, invasion and migration of cancer cells.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCCK-8 Cell Counting Kit-8\u003c/p\u003e\n\u003cp\u003eDMEM Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium \u003c/p\u003e\n\u003cp\u003eEgr1 early growth response factor 1\u003c/p\u003e\n\u003cp\u003eFBS fetal bovine serum\u003c/p\u003e\n\u003cp\u003eGPCR G-protein coupled receptor\u003c/p\u003e\n\u003cp\u003eHCC hepatocellular carcinoma \u003c/p\u003e\n\u003cp\u003ePAR1 protease-activated receptor 1\u003c/p\u003e\n\u003cp\u003eMAPK Mitogen-activated protein kinase\u003c/p\u003e\n\u003cp\u003eERK extracellular signal-regulated kinase\u003c/p\u003e\n\u003cp\u003eqRT‒PCR quantitative real-time polymerase chain reaction\u003c/p\u003e\n\u003cp\u003esiEgr1 small interfering RNA targeting Egr1\u003c/p\u003e\n\u003cp\u003esiPAR1 small interfering RNA targeting PAR1\u003c/p\u003e\n\u003cp\u003eR-hirudin recombinant hirudin\u003c/p\u003e\n\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJian-gang Bi and Ping Xu designed the experiments and wrote the manuscript. Qi Li, Yu-sheng Guo and Hong-gui Tang performed all the experiments. Jian-gang Bi analyzed the data. Qi Li searched the literature. All authors reviewed the results and agreed to the final version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest with respect to the authorship, research, or publication of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the data generated or analyzed during this study are included in this manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Guangdong Medical Science and Technology Research Foundation (A2021275, A2020559). The funders had no role in the design of the study, the execution of the experiment, the collection and analysis of the data, the decision to publish, or the preparation of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlturkistani, A., Ghonem, N., Power-Charnitsky, V.A., Pino-Figueroa, A., Miglioreet, M.M., 2019. Inhibition of PAR-1 Receptor Signaling by Enoxaparin Reduces Cell Proliferation and Migration in A549 Cells. Anticancer Res 39(10), 5297-5310.\u003c/li\u003e\n\u003cli\u003eArcher, K.J., Mas, V.R., David, K., Maluf, D.G., Bornstein, K., Fisher, R.A., 2009. Identifying genes for establishing a multigenic test for hepatocellular carcinoma surveillance in hepatitis C viruspositive cirrhotic patients. Cancer Epidemiol Biomarkers Prev 18, 2929-2932.\u003c/li\u003e\n\u003cli\u003eArcher, Y., Han, C.C., Li, Y., Wang, Y., Wei, W., 2016. 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Breast Cancer Res Treat 158(2), 277-286.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"early growth response factor 1, protease-activated receptor 1, transcription, mitogen-activated protein kinase, hepatocellular carcinoma","lastPublishedDoi":"10.21203/rs.3.rs-4744749/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4744749/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. The prognosis of HCC patients who undergo surgical resection is still poor. Therefore, it is urgent to clarify the potential mechanism of HCC progression. This article reports the important role of the transcription factor early growth response 1 (Egr1) in promoting HCC progression. First, Egr1 expression was abnormally elevated in clinical HCC samples and enhanced the proliferation, invasion and migration of cancer cells. Moreover, we found that the mRNA expression levels of protease-activated receptor 1 (PAR1) and Egr1 in clinical specimens were positively correlated. Dual luciferase reporter gene assays verified that Egr1 is an upstream transcriptional regulator of PAR1, that enhances the proliferation, invasion and migration of cancer cells by upregulating PAR1. Mechanistically, we found that Egr1 activates the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway through PAR1. Finally, we demonstrated that thrombin does not affect the regulatory effect of Egr1 on PAR1 in HCC cells. In conclusion, Egr1 promotes HCC progression by upregulating PAR1 to activate the MAPK/ERK pathway.\u003c/p\u003e","manuscriptTitle":"Early growth response factor 1 promotes HCC progression by activating the MAPK/ERK pathway through transcriptional upregulation of PAR1","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-16 21:20:54","doi":"10.21203/rs.3.rs-4744749/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"968c7f8a-e5ca-4d4d-871a-229f3e691a0c","owner":[],"postedDate":"August 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-09T14:38:32+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-16 21:20:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4744749","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4744749","identity":"rs-4744749","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}