| BackgroundHepatocellular carcinoma(HCC)is one of the most common malignant tumors in China.Compared with other malignant tumors,the mortality rate of HCC is very high,accounting for the second highest mortality rate among all malignant tumors in China.It is estimated that the global annual increase of HCC has exceeded 1 million[1l.Since HCC cells have strong proliferative and metastatic capabilities,most HCC patients are already in the middle to advanced stage at the time of the diagnosis.Therefore,these patients have often a poor treatment outcome and prognosis.Current studies found that the 5-year overall survival(OS)of HCC patients is less than 5%[2],which indicates that early detection,diagnosis and treatment are necessary to improve the long-term survival of these patients.However,the epidemiological characteristics of HCC must first be understood in order to achieve these goals.Previous studies reported various risk factors that induce HCC,and chronic liver disease was identified to be the primary one[3].In fact,risk factors-inducing HCC vary across different continents and/or countries around the world and this aspect may be closely associated with the differences in economic level,dietary habits and lifestyle.For example,chronic hepatitis B and aflatoxin are the main inducers of HCC in East Asia and Africa[4],while hepatitis C virms and alcoholic liver damage are the major causes of HCC in North America,Europe and Japan[5].Compared with other countries,China is a hepatitis endemic country with a high incidence rate of hepatitis B.In fact,most hepatitis patients ultimately develop HCC as the disease progresses,A study showed that the risk of HCC is more than 100-fold higher in hepatitis B patients than in non-hepatitSs s virus(HBV)carriers[S· Therefore,Lo avoid HCC development,an effective antiviral treatment must be administeredto patients with HBV infection to prevent the progression of acute hepatitis to chronic hepatitis or hepatic fibrosis.This is important especially because once hepatic fibrosis occurs,the development of HCC will become inevitable even if the patients receive effective antiviral treatments.Many methods are available for the clinical diagnosis of HCC.Imaging techniques currently represent the primary methodfor HCC diagnosis,including ultrasound,four-phase multilayer spiral CT or dynamic contrast-enhanced MRI.Liver biopsy can also be performed if the imaging features of the liverare unclear.For patients suspected of early HCC,relevant guidelines also recommend the use of immunological methods for the determination of primary tumor cell characteristics or the evaluation of angiogenesis[8].However,many molecular tools are currently still inadequate for individualized diagnosis due to limitations in the availability of tissue samples[9].As regard the diagnostic method,disease stage must be determined once HCC is definitively diagnosed because it not only dictates the selection of treatment regimens,but also helps the evaluation of a disease prognosis.The clinical staging systems that arecommonly used for HCC include those from the American Joint Committee on Cancer(TNM system),Barcelona-Clinic Liver Cancer Group(BCLC),Cancer of the Liver Italian Program(CLIP),Chinese Society of Liver Cancer(CUPI system),and Japan Integrated Staging(JIS)[10].The corresponding treatCent regimen should then be selected base on the cancer stage.There are many clinical treatment options for HCC patients.The common clinical treatments for early and non-metastatic HCC patients are surgical resection and liver transplant,but these treatments are often restricted by the preoperative liver functions of the patients and the lack of organdonors,respectively[8,11].Furthermore,studies demonstrated that local therapy such as radiofrequency ablation(RFA)and transcatheter arterial chemoembolization(TACE)can prolong the median survival time of HCC patients,but these methods are not applicable to patients with hematogenous metastasis or extrahepatic dissemination.In short,treatment optionsareextremely limited for metastatic HCC patients.The rise of research in the field of molecular biology in recent years has opened up new directions for the study of HCC etiology and treatment and created the opportunity for targeted treatment.However,HCC has a complex pathogenesis,which involves a large number of signaling molecules.These molecules can affect tumor development and suppression by inducing the generation of isomeric profile and the expansion and deletion of chromosomal fragments.HCC formation is a complex process involving hepatic cell injury,inflammation,genetic instability and potentially other processes such as multiple vascular tumors and angiogenesis.VEGF,PDGFs,FGF,TGF-? and TGF-? are proangiogenic growth factors that can induce angiogenic signaling and thereby activate key pathways such as RAF/MEK/ERK and PI3K/AKT/mTOR to promote the development of HCC[12].Therefore,according to these evidences,the search of new therapeutic targets and the improvement of HCC patient prognosis are the majoraimsof the current research.In recent years,researchers worldwide performed studies on numerous signaling pathways that may lead to HCC growth and metastasiswiththe hope of improving HCC patient survival rate and identifying new therapeutic drug targets.Some of these studies were focused on members of the protease-activated receptor(PAR)family.PARs are seven-transmembrane G-protein-coupled receptors that include PAR1,PAR2,PAR3 and PAR4 subtypes,and they are the targets for various proteolytic enzyme signaling.PARs have a unique activation mechanism that involves the degradation of their tethered ligands.These receptors are not only involved in the regulation of various physiological processes,such as vascular regulation,pain reception and inflammation[13-16],but also in the pathogenesis of many diseases including tissue fibrosis[17]and cancer[18-25].Due totheir role in diseases of the cardiovascular,skeletomuscular,gastrointestinal,respiratory and central nervous systems,PARs have now become the focus of new treatment development.Since mid-60s,scientists were already aware that pepsin and chymotrypsin can induce hormone-like effects in target tissues,such as the promotion of glycogen formation in rat diaphragm.Moreover,the mitotic effects of thrombin and trypsin on cell membrane have been recognized since early 70s.However,receptors ofthese enzymes,namelyG-protein-coupled receptors(GPCRs),were only recently identified.Since PARs play an extensive role in normal and pathological tissue functions,these receptors became potential therapeutic targets for various diseases such as arthritis,colitis,asthma,neurodegenerative diseases,tumor invasion and cardiovascular diseases.Different members of the PAR family exert different functions during different pathological processes.In particular,the role of PAR2 in tumor cell development,tumor growth and tumor metastasis has gained increasing attention byresearchers.In addition,tumor development and progression may also be associated with several molecular pathways,such as that of tissue factor(TF).TF is a transmembrane glycoprotein that can form a complex with factor ?/?a(F?/F?a),and this complex is the primary activator of the coagulation system in cancer patients.Under an inflammatory microenvironment,TF forms a complex with FVIIa on the surface of tumor cells and extracellular vesicles released by immune cells.This complex can significantly exacerbate coagulation disorders in cancer patients and thereby have harmful effects onthese patients.Each PAR subtype is activated by a unique,overlapping tyrosine protease.PAR2 is primarily activated by trypsin but can also be activated by the TF/F?/?a/?a to induce tumor cell migration.Furthermore,synthesized activating peptides(PAR-APs)can specifically activate PARsin vitro without receptor protein degradation,making them a useful tool for the study of PARs.PAR2 participates in the fibrotic transformation of hepatic and pancreatic tumor tissues[26].It can also promote the proliferation of connective tissues in pancreatic cancer.These tumor microenvironments,including the extracellular matrix(ECM),are essential for cancer growth and metastasis.Tumor-associatedECM is composedof myofibroblasts/stellate cells,tumor-associated fibroblasts and various immune cellstypes.The interactions between tumor cells and stromal cells can promote primary tumor growth and metastasis.Although PAR2 is considered a tumor suppressor factor in skin cancer[27],most members of the PAR family are thought to be associated with cancer development and progression,since they can regulate the growth,invasion and metastasis of various cancers including HCC.PAR2 expression and activation induce pancreatic cancer cell proliferation,migration and invasion.In colorectal cancer,PAR2(and PARI)expression is upregulated in tumor-associated ECM compared with normal ECM.Furthermore,PAR2 expression is closely associated with the depth of tumor invasion in the stomach wall,lymphatic and venous infiltration,and the presence and absence of liver metastasis in gastric cancer.Some studies examined the role of PAR2 secreted by hepatic stellate cells(HSCs)on HCC growth.In vivo studies revealed that the HSC cell line LX-2 can promote tumor growth and stimulate angiogenesis in mice bearingHCC xenograft.However,downregulation of PAR2 expression by RNAi-mediated silencing of various signaling pathways can significantly reduce tumor cell growth and metastasis.Therefore,these findings demonstrate that PAR2 expression plays a critical role in the promotion of HCC growth and can serve as a new target for the treatment of HCC.Objectives1.To evaluate PAR2 mRNA and protein expression in liver tumor of HCC patients to confirm high PAR2 expression in HCC tissues.2.To determine the effect of PAR2 expression in the proliferation,invasion and metastasis of various HCC cell linesin vitro.3.To determine the effects of PAR2 expression in the proliferation and metastasis of various HCC cell lines in vivo.4.To elucidate PAR2 downstream mechanisms regulating the promotion of HCC cell migration.Methods1.PAR2 expression in the liver tumor of HCC patients was measured by immunohistochemistry.Tumor progression,tumor volume,capillary invasion rate,disease-free survival(DFS)and OS were compared between high PAR2 expression group and low PAR2 expression group.2.HepG2 and SMMC-7721 cells were transduced with shRNAlentiviral vector to stably knockdown PAR2 expression(shPAR2).In addition,HepG2 and SMMC-7721 cells were transduced with pcDNA3.1-PAR2 to achieve a transient PAR2 overexpression.3.The induction of HCC cell proliferation by PAR2 was determined using Cell Count Kit 8(CCK8)and clonal colony-forming assay,and tumor cell invasion and migration were measured by the Transwell assay.4.The HCC xenograft model.was established by subcutaneous(s.c.)injection ofstable PAR2-knockdown HepG2 and SMMC-7721 cells into nude mice toevaluate the proliferative ability of these cells in vivo.The liver metastasis model was established by intrasplenic(i.s.)injection of stable PAR2-knockdown Hep2 and SMCC-7721 cells into nude mice to evaluate the migratory ability of these cells in vivo.5.Intracellular MAPK and EMT expression was determined by Western blot.Results1.Tumor progression,tumor size and capillary invasion were significantly different between the high PAR2 expression group and low PAR2 expression group(P=0.001,0.032 and 0.037,respectively).OS and DFS were significantly lower in the high PAR2 expression group than in the low PAR2 expression group(P=0.03 and 0.02,respectively).2.Stable PAR2-knockdown cell lines(shPAR2)were established by transducing shRNAlentivirus into HepG2 and SMMC-7721 cells.PAR2 mRNA and protein expression wassignificantly downregulated in the shPAR2 group than in the negative control(shNC)group(P<0.01).In contrast,PAR2 mRNA and protein expression wassignificantly upregulated in pcDN A3.1-P AR2-transduced HepG2 and SMMC-7721 cells than in the blank pcDNA3.1-transduced cells(P<0.01).3.Compared with the control cells,PAR2-knockdown HepG2 and SMMC-7721 cells showed asignificantly reduced cell count and clonal formation(P<0.01 and P<0.001,respectively).In addition,a decreased number of PAR2-knockdown cells was observed in the lower chamber of the transwell insert with or without matrigel coating(P<0.05 and P<0.01× respectively).The migration rate was significantly lower in the PAR2-knockdown group than in the control group at 24h and 48h post-scratching.On the other hand,transient PAR2 overexpression by pcDNA3.1-PAR2 transduction significantly increased cell count and clonal formation inboth cell lines(P<0.01 compared to???)and significantly increased the number of cells in the lower chamber of the transwellinsertwith or without matrigel coating(both P<0.01 compare to???).Furthermore,these cells showed asignificantly higher rate of migration than control cells at 24h and 48h post-scratching.4.Stable PAR2-knockdown HepG2 and SMMC-7721 cells were s.c.injected into nude mice.The tumor volume and tumor weight of these mice at 4 weeks post-engraftment were significantly different than those in control mice(P<0.01 and P<0.001,respectively).Stable PAR2-knockdown HepG2 and SMMC-7721 cells were i.s.injected into nude mice.Liver tumor count and percentage of liver metastasis in these mice at 4 weeks post-engraftment were significantly different than those in control mice(both P<0.01).5.PAR2 gene knockdown induced epithelial-like morphological changes in HepG2 and SMMC-7721 cells.PAR2 gene knockdown upregulated E-cadherin and downregulated N-cadherin protein expression,while transient PAR2 overexpression downregulated E-cadherin but upregulated N-cadherin protein expression in these cells.Conclusions1.PAR2 promotes HCC cell proliferation,invasion and migration.2.PAR2 activates ERK to induce epithelial-mesenchymal transition and thereby promote HCC cell proliferation. |
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