Role Of G Protein-coupled Receptor Kinase2in The Invasion And Metastasis Of Hepatocellular Carcinoma And Its Possible Mechanism

Hepatocellular carcinoma (HCC) is one of the deadliest forms of human livercancer and does not respond well to conventional therapies. Novel effective treatmentsare urgently in need. G protein-coupled receptor kinase2(GRK2) is uniqueserine/threonine kinase that involves in many signaling pathways and regulates variousessential cellular processes. Altered levels of GRK2have been linked with severalhuman diseases including cancer. However, the role of GRK2in regulating HCC cellmigration, invasion and its possible mechanisms remain unclear. Prostaglandin receptorEP2belongs to the G protein-coupled receptors (GPCRs) family. It has been reportedthat EP2is involved in the angiogenesis, invasion and metastasis of various tumors. Ourprevious study found that selective EP2agonist Butaprost could significantly stimulatethe proliferation and invasion of HCC cells. The present experiment was designed toinvestigate the role of GRKs in the invasion and metastasis of HCC and its possiblemechanism. Human para-tumorous tissues and tumor tissues from HCC patients wereused to assess the expression of GRK2and GRK3. To further characterize the role ofGRK2in the development of HCC, diethylnitrosamine (DEN) was intraperitoneallyinjected into14-day-old mice, the dynamic expression of GRK2, GRK3and EP2was observed. Moreover, the change of GRK2expression and the cytoplasm/membranedistribution of EP2were analyzed in HCC cell lines with stepwise metastasis. The smallinterfering RNA (siRNA) technique was used to explore the role of GRK2in themigration of HCC cells and EP2-induced Akt activation.OBJECTIVE Clinical HCC specimens from patients and animal model of DEN-induced liver cancer were used to observe and analyze the expression of GRK2andGRK3. The expression and distribution of GRK2, GRK3and EP2in HCC cell lineswith different metastatic potentials was analyzed in vitro. The siRNA technique wasapplied to explore the role of GRK2in the invasion and metastasis of HCC and theassociated mechanism.METHODS The expression of GRK2and GRK3in HCC patients and intrahepaticbiliary lithiasis patients were determined by immunohistochemistry. Meanwhile, qPCRand Western blot analysis were used to detect changes of GRK2and GRK3in livertissues of18self-paired HCC patients. C57BL/6J mice of14-day-old were injectedintraperitoneally with DEN to establish the liver cancer model. The mice were dividedinto normal control group (0wk) and liver cancer model group (8,16,24,32and40wk)randomly. Western blot analysis was used to detect the dynamic expression of GRK2,GRK3and EP2in mice liver. In addtion, HCC cell lines with stepwise metastasis(HepG2, SMMC-7721, MHCC97L, MHCC97H and HCCLM3) and human liver cellline L02were applied in vitro. Change of GRK2and the cytoplasm/membranedistribution of EP2were analyzed by Western blot and laser confocal microscopy.Furthermore, qPCR and Western blot analysis were used to detect the expression ofGRK2after transfected with GRK2siRNA in HCC cell lines. Wound healing andtranswell assay were applied to measure the migration and invasion of HCC cells aftertransfection. Meanwhile, the activation of ERK and Akt, which were the EP2downstream signaling pathways, were determined by Western blot． RESULTS1. The expression of GRK2and GRK3in HCC patientsThe results of immunohistochemistry showed that the expression of GRK2inintrahepatic biliary lithiasis patients was significantly higher than that in HCC patients.In addition, the expression of GRK2in poorly differentiated HCC patients was almostnegative, and significantly lower than that in well differentiated HCC patients, whilethere was no significant change in the expression of GRK3. The qPCR and Western-blotresults showed that the expression of GRK2in tumor tissues was lower than that in thenon-tumor tissues, while the expression of GRK3was not significantly different.2. The expression of GRK2and GRK3in DEN-induced liver cancer miceTo further explore the role of GRK2in HCC, animal model was established byintraperitoneal injection of DEN. Western blot results showed that GRK2expression inmice liver was gradually decreased with time passing, while there was no significantdifference in the expression of GRK3.3. Expression and distribution of GRK2, GRK3and EP2in human HCC cell linesThe results showed that expression of GRK2gradually decreased with increasingmetastatic potential of HCC cell lines, while there was no significant difference in theexpression of GRK3in different cell lines. Western blot and immunofluorescenceanalysis revealed a differential expression between the GRK2and EP2membraneproteins, HCC cell lines with high metastatic potential exhibit low expression of GRK2and elevated EP2levels compared with HCC cell lines with low metastatic potentialafter stimulation with selective EP2receptor agonist Butaprost.4. Effects of siGRK2on the migration and invasion of human SMMC-7721cellsThe change of GRK2in HCC cell lines with stepwise metastatic potentialsuggested a role of GRK2in metastasis. To further investigate the role of GRK2in themigration and invasion of HCC cells, siRNA targeting GRK2was used to silence themRNA expression of GRK2in SMMC-7721cells. Results of wound healing and transwell assay showed a significant increase of migration and invasion by transfectingGRK2siRNA in SMMC-7721cells.5. Effects of GRK2siRNA on activation of ERK1/2and Akt in SMMC-7721cellsIn order to investigate the relevant mechanism, siRNA targeting GRK2wastransfected in SMMC-7721cells. The results indicated that transfection GRK2siRNAsignificantly increased p-Akt level, while p-ERK1/2level had no obvious change. Itsuggested that low expression of GRK2may contribute to activation of Akt, which wasthe downstream signaling molecule of EP2.CONCLUSIONS1. In HCC tissues, GRK2had low expression in poorly differentiated HCC patients,while GRK3was not significantly changed among the different differentiated HCCpatients. In DEN-induced liver cancer mice, the expression of GRK2graduallydecreased with time passing, while the expression of GRK3had no remarkable change.The results above indicated that GRK2may play an important role in inhibiting thedevelopment of HCC.2. In vitro, the expression of GRK2was decreased gradually with increasing metastaticpotential of HCC cell lines. Membrane protein of cells with high metastatic potentialshowed low level of GRK2and high level of EP2compared with HCC cell lines withlow metastatic potential, which indicated that GRK2might be involved in the invasionand metastasis by regulating EP2signaling pathways of HCC cells.3. Our data showed that silenced endogenous GRK2by siRNA promoted the metastasisand invasion of SMMC-7721cells, with the activation of Akt. The results suggested thatGRK2may suppress HCC invasion and metastasis through regulating the PI3K/Aktsignaling pathway.