GRIM-19Suppresses Hepatocellular Carcinoma Proliferation And Metastasis In Vitro And In Vivo

Background and Objective:The morbidity and mortality of hepatocellular carcinoma (HCC) account for theforefront of malignant tumors in the whole world. Early diagnosis and effective clinicaltreatment are severely limited by its insidious onset, rapid progression, early metastasisand postoperative recurrence. The clinical application of molecular targeted anti-cancerdrugs has brought new hope for the HCC patients, especially for traditional treatmentfailure patients, but there is still clinical shortcomings such as the development of drugresistance. Drug resistance is mainly due to the genetic complexity of the occurrence anddevelopment of HCC,which involves a progression of changes in multiple genes andsignaling pathways change. Therefore, the search for effective molecularly targetedtherapy remains a problem that is dependent upon the elucidation of the pathogenesis ofHCC.The occurrence of malignant carcinoma results from a series of genetic mutations thatactivate oncogenes, inactivate tumor suppressor genes, and persistently activate certainsignal transduction pathways. Gene associated with retinoid-interferon inducedmortality-19(GRIM-19) is a newly discovered tumor suppressor gene. GRIM-19hasimportant roles in cell cycle regulation, apoptosis, innate immune and energy metabolism.Recent studies have shown that the abnormal expression of GRIM-19(i.e. mutationsresulting in low expression or null expression) exist in many malignant carcinomas suchas breast cancer, prostate cancer, stomach cancer and glioma.Although reduced GRIM-19expression is involved in the occurrence and development of these varied tumors, itsmechanism may vary, involving abnormal control of specific signaling pathways,whichthen upregulate cell cycling, reduce apoptosis, promote metastasis, and increase cellmetabolism. However, the role of GRIM-19on the occurrence, development, andprognosis of human liver cancer has not been elucidated. Therefore, this study is directed at discerning the effect of GRIM-19expression on the occurrence and development ofHCC. More specifically, can HCC be inhibited, by increasing GRIM-19expression,through regulation of signal transduction pathways controlling cell cycling, apoptosis andmetastasis? Can exogenous GRIM-19be used to treat HCC?To address these questions, we first analyzed GRIM-19protein expression in HCCpatient tissues vs. normal tissues, and analyzed the correlation between GRIM-19expression level and clinicopathological indicators. We used human hepatocellularcarcinoma SMMC-7721cells, with low GRIM-19expression, as our cellular model. Next,we established stable transfection of wildtype GRIM-19in the SMMC-7721cell lineusing a recombinant lentivirus pLVX-Puro-GRIM-19-Myc plasmid. Thirdly, weestablished an in situ liver cancer transplantation tumor model in mice, and then employedtargeted GRIM-19therapy of the HCC-mice utilizing attenuated Salmonella to deliverGRIM-19to tumors. Specifcally, we observed:(1) the effect of GRIM-19on theoccurrence, development and prognosis of HCC in vivo and in vitro;(2) the effect ofGRIM-19on the HCC-related signal transduction pathways including the PI3K/Akt andJAK/Stat pathway. These studies are aimed at revealing if GRIM-19is a potentialtherapeutic target for effective HCC treatment.Methods:(1) HCC tissues and adjacent non-tumor liver tissues were collected from54in-patientsfrom First Hospital of Jilin University during June2012to June2013; GRIM-19protein expression in the HCC tissues and adjacent non-tumor liver tissues wereanalyzed by immunohistochemical (IHC) and the mean optical density of GRIM-19expression was analyzed utilizing NIH Image J software.(2) In vitro analyses:①Astable cell line expressing wild-type GRIM-19was established by transfectingrecombinant lentivirus pLvx-Puro-GRIM-19-Myc to SMMC-7721cells.②GRIM-19mRNAand protein expression in the stable SMMC-7721cells wereanalyzed by semi-quantitative RT-PCR and Western blot (WB) and compared tonontransfected SMMC-7721cells.③The MTT assay was used to evaluate cell proliferation. Cell cycle was detected by Flow Cytometry (FCM) analysis with PI staining. Mitochondrial membranepotential was monitored by JC-1. Cell apoptosis was detected by FCM analysiswith an Annexin-V-FITC apoptosis kit.④CDK4, PCNA, CyclinD1, Bax, Bcl-2, Cytochrome C, Cleaved Caspase-3,Cleaved Caspase-9, VEGF, MMP2, MMP9, Akt1, p-Akt1, State3and p-Stat3were detected by WB and Immunofluorescence cytochemistry (IFC).(3) In vivo anaylses:①We established an HCC in situ transplantation tumor model in C57BL/6miceutilizing H22cells (a mouse hepatoma cell line). A GRIM-19plasmid carriedby attenuated S. Typhimurium was administered to HCC in situ transplantationtumor mice by gavage.②Changes in tumor volume in situ were detected by animal ultrasound imagingsystem.③Tumor cell cycling was detected by FCM analysis with PI staining. Tumor cellapoptosis was detected by FCM with an Annexin-V-FITC apoptosis kit andTUNEL analysis.④Protein expression levels of GRIM-19,CDK4,PCNA,CyclinD1,Bax, Bcl-2,Cleaved Caspase-3, Cleaved Caspase-9, VEGF, MMP2, MMP9, Akt1, p-Akt1,Stat3and p-Stat3were analyzed by WB and IHC.Results:(1) GRIM-19protein expression was significantly decreased in HCC comparing toadjacent non-tumor liver tissues. The positive rate of the GRIM-19proteinexpression was55.6%(30/50) in HCC tissues, and the expression intensity wasconsidered weakly positive. However, in adjacent tissues, the positive rate was100%(54/54),prioritized as moderate or strong positive.(2) There was no large differences among the level of GRIM-19protein in humanhepatocellular carcinoma tissues and the age, sex, serum AFP level, and tumor sizefor HCC patients. However, there was a negative correlation between GRIM-19protein and tumor tissue pathologic classification (P=0.015), AJCC TNM staging(P=0.024),and metastasis (P=0.022). (3) In vitro experiment, we successfully built a stable SMMC-7721cell lineover-expressing GRIM-19. Compared with SMMC-7721group (Mock) or theempty plasmid group (EV), GRIM-19mRNA and protein expression were increasedsignificantly in transfection GRIM-19group (GRIM-19)；GRIM-19over-expressionsignificantly induced liver cancer cell cycle arrest in G0-G1phase, and itsmechanism may be associated with down-regulation CDK4, Cyclin D1, and PCNAprotein expression. Simultaneously,GRIM-19over-expression lessened mitochondr-ial membrane potential.Hepatocellular carcinoma cell proliferation were significant-ly suppressed, and cell apoptosis was greatly induced by GRIM-19over-expression,which may be related to up-regulation of Bax, cleaved caspase-9, and cleavedcaspase-3expression, plus down-regulation of Bcl-2expression. In addition,GRIM-19over-expression significantly reduced VEGF, MMP2,MMP9, Akt1、p-Akt1、Stat3and p-Stat3protein expression.(4) In vivo experiment, the orthotopic transplantation tumor model of liver cancer mice(HCC-mice) was successfully established. These HCC-mice were then treared withdifferent recombinant plasmid carried by attenuated Salmonella. Treatment of HCC-mice with Salmonella expressing GRIM-19showed significantly reduced averagetumor weights and abdomen water volumes, and induced tumor cell cycle arrest inG0-G1phase, increased tumor cell apoptosis, and reduced the abdominal cavitymetastasis rate compared with other groups. In addition, the changes observed incell cycle arrest related-proteins, cell apoptosis related-proteins,cell metastasisrelated-proteins, and key proteins in signaling pathways were consistent with thechanges observed in vitro experiment.Conclusions:(1) Compared to paired adjacent non-tumor liver tissues, GRIM-19protein expressionwas markedly reduced in human HCC tissues, and the lever of GRIM-19proteinexpression was negatively correlated with increased tumor tissue pathologicclassification, AJCC TNM staging, and metastasis.(2) Using gene recombination technology, we successfully established over-expressionGRIM-19. In vitro and in vivo over-expression of the GRIM-19plasmid had a significant inhibitory effect on HCC, reduced abdominal cavity metastasis rate, andimproved the survival rate.(3) Both in vitro and in vivo studies suggest that GRIM-19might play a crucial role inhepatocarcinogenesis, development and prognosis, through negatively regulatingthe PI3K/Akt and JAK/Stat signaling pathways and then affecting downstreamtarget genes associated with apoptosis, cell cycling and metastasis.