The Functional Analyses Of The Different Splice Variants Of Osteopontin And Its Polymorphisms In Metastasis Of Human Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is one of the most common and aggressive malignancies worldwide.It has been ranked the third in terms of cancer mortality worldwide and the 2nd cancer killer in China since 1990s.Despite clinical advances in surgical resection,the prognosis of HCC still remains dismal.Metastatic recurrence after HCC resection is one of the major obstacles to prolonging survival.Therefore, there is a tremendous interest and urgency to search for molecules related to HCC metastasis that would provide new predictors for HCC metastasis as well as new targets for intervention.Osteopontin(OPN),a secreted phosphoglycoprotein,is a promoting factor for tumorigenicity and metastasis of cancer cells in several types of tumors through a variety of mechanisms.OPN binds with several integrins and CD44 variants in an RGD sequence-dependent and-independent manner.In our previous work,we collaborated with the National Cancer Institute(NCI)of USA to compare the difference in gene expression profiles between HCCs without and with accompanying intra-hepatic metastases using a cDNA microarray of 9180 cDNA clones.We found that OPN was one of the leading genes that over-expressed in the HCCs with metastasis.An OPN-neutralizing antibody efficiently blocked in vitro invasion and in vivo pulmonary metastasis of HCC cells.Moreover,we also found that preoperative plasma OPN level can be used as a predictive marker for tumor recurrence and prognosis of patients with HCC after operation.These suggest that OPN is associated with metastasis of HCC.However,the mechanism by which OPN mediates progression and metastasis of HCC remains unknown.Based on the previous study,we tried to find out what happened with the nonmetastatic HCC cell line(Hep G2)if different osteopontin splice variants were upregulated respectively.In addition,we sequenced the OPN gene across the promoter and detected all the SNPs of OPN gene,evaluated the associations of SNP genotypes and haplotypes with metastasis of HCC,postoperative overall survival(OS) and disease-free survival(DFS).And then we explored the possible mechanisms of different splice variants,SNP genotypes and haplotypes of OPN in HCC progression.PARTⅠEffects of the different splice variants of osteopontin on the tumor growth and metastasis of human hepatocellular carcinoma1.The expression levels of OPN splice variants in HCCObjective:Analysis of osteopontin in HCC.Methods:The different expression levels of OPN and its different splice variants were examined by quantitive real-time PCR using specific primers.Results:A significantly increased expression of OPN in primary HCC tumors and their secondary metastatic lesions compared with surrounding non-cancerous tissue. The expression levels of OPN-1 and OPN-2 were significantly higher than that of OPN-3 in tumor tissue.Conclusions:The results suggested that alternative RNA splicing of OPN existed with three OPN cDNAs identified in HCC.Different expression levels of three splicers of OPN were detected in HCC.2.The function and mechanism of different splice variants of osteopontin in HCC(1)Construction of vectors expressing splice variants and transfection into HCC cells.Objective:To obtain the stable cell line expressing the special splice variant.Methods:The three specific fusion expression vectors of GFP-splicer were constructed and transfected into HepG2 cell line.All the stable cell lines which expressing the particular splice variant and empty GFP were screened by G418.Results:The sequences of three different constructs were completely right.The expression of three special splice variants in the stable cell lines were upregulated which confirmed by Western blot and fluorescence microscope.Conclusions:We successfully obtained the stable cell lines which respectively expressing the three splice variants.(2) The effect of OPN on the proliferative and invasive abilities of human HCC cells in vitroObjective:To investigate the effects of the different splicers of OPN on adhesion, migration,invasion and cell cycle distribution of human HCC cells,and evaluate the influence of OPN splice variants on MMP2,uPA and the related signal pathway of HCC in vitro.Methods:The results of the three upregulated splicers on cell adhesion,cell migration,invasive abilities and cell cycle distribution were determined by CyQUANT assay,Matrigel assay,flow cytometry,respectively.The expression levels of MMP2,uPA and proteins related with the signal pathway induced by the three splice variants were measured with ELISA and Western blot.Results:1)The effects of OPN overexpression induced by transfection of splice variants on in vitro adhesion,migration and invasive abilities,and cell cycle distribution of Hep G2 cellsIn the transfected cells,up-regulation of OPN-2 resulted in a remarkably increased ability of cell adhesion(P＜0.01)compared with Mock.In the cell invasion assays, the migrating cell numbers of OPN-1-transfected cells(22.3±2.1)and OPN-3-transfected cells(28.5±3.8)were significantly increased compared with Mock (7.5±1.5)(P＜0.05).In contrast,there is no significant difference of migratory potential and cell cycle distribution between cells transfected with three splice variants and empty vector.2)The effects of OPN splice variants up-regulation on the expression of MMP2, uPA and the STAT3-Survivin and AKT pathways in HCCWe evaluated the protein expression such as MMP-2 and uPA relevant to tumor. ELISA assay found that the more MMP2 secreted in the supernatant of the cells transfected with the three constructs compared with Mock.Interestingly,different from the STAT3-Survivin pathway activated by OPN-1 and OPN-2,OPN-3 induced the signal pathway of AKT phosphorylation.The expressions of MMP-2 and uPA were greatly increased possibly due to different signal pathways induced by splicers.3)Influence of exogenous recombinant OPN on cell proliferation and NF-KB and AKT pathway of HCCThe cell proliferation was measured in different times when the Hep G2cell was stimulated by variant concentrations of exogenous recombinant OPN,We found that the cell proliferation reached a maximum within 24 hr at a concentration of less than 5.0μM of OPN.But the cell proliferation had a time-dependent manner with a high concentration of 5.0μM of OPN.In the OPN-untreated cells,the p65 was localized mostly in the cytoplasm compared with the nucleus.However,in the OPN-treated cells,it was translocated into the nucleus,and got the peak at 2h.To further evaluate the effect of OPN on the expression of MMP2 andμPA,10μM exogenous recombinant OPN were added in the supernatant of cells,pretreated with PDTC,LY 294002 for 30min at 37℃.Western blotting showed that OPN could increase the expression of MMP2 andμPA,and induce p65 translocation into the nucleus and activate the AKT signal pathway.Conclusions:The three splice variants had different influences on HCC cells.OPN-2 significantly increased the cell adhesion while the OPN-3 enhanced the cell invasion. In contrast,there is no remarkable difference of migratory potential and cell cycle distribution between the cells tranfected with the three OPN splice variants and the control.Different function of three splicers in HCC probably resulted from the special signal pathway induced by them.OPN-1 and OPN-2 could activate the STAT3-Survivin pathway whereas OPN-3 induced the AKT phosphorylation. Different splice variants could regulate the expression of MMP-2 and uPA possibly through activation of different signaling pathways.CyQUANT assay indicated that cell proliferation was strongly associated with the different time and concentration of exogenous recombinant OPN.Perhaps the up-regulation of MMP-2 and uPA stimulated cell proliferation through NF-KB and AKT signal pathway induced by OPN.(3)Effects of up-regulation of the three OPN variants on the in vivo tumor growth and metastasis of human HCC cellsObjective:To confirm effects of the different splice variants of OPN on the tumor growth and metastasis of human HCC by inoculated into nude mice.Methods:The stable HCC cell lines that expressing the three splice variants(5×10~6) and empty vector were subcutaneously inoculated into nude mice.Then tumor growth was monitored twice every week.At 7 weeks,the animals were killed for ethical reasons given the tumor volume,and then the tumors were excised from mice.Results:In OPN-1 and OPN-3 groups,enhanced fluorescent signal could be detected using the Luminescent and Fluorescent Image Analyzer in the subcutaneous implantation sites of Hep G2 cells.However,the enhanced GFP signal from deep small metastatic lesions was too weak to be detected.So at the 7th week,the animals were sacrificed and the metastatic lesions of liver and lung were scaned by Luminescent and Fluorescent Image Analyzer.The liver metastatic lesions were also detected in every mouse infected with the OPN-1 and OPN-3 group.However,the liver metastatic lesions could not be detected in the mice inoculated with the HCC cells transfected with empty vector and OPN-2.Conclusions:The same results both in vitro and in vivo indicated that the three OPN splice variants had different influences on HCC.The OPN-3 played a critical role in the progression of HCC among them.PARTⅡThe association of SNP genotypes and haplotypes of osteopontin promoter with clinical outcome of patients with hepatocellular carcinoma1.The association of different genotypes and haplotypes of OPN promoter and the metastasis of hepatocellular carcinoma and their functional analysis(1)Reconstruction of the haplotypes for osteopontin promoter and their association with the metastasis of hepatocellular carcinoma.Objective:To identify the SNPs and the haplotypes in the OPN gene promoter,and evaluate their correlation with metastasis of HCC.Methods:We detected SNPs by direct sequencing of 2.3kb in OPN promoter of 30 cases of HCC,and then analyzed genotypes of the SNPs in DNA samples of 510 cases. The 510 cases were enrolled to haplotype reconstruction by PHASE software.The genotypes,haplotypes and their relation with metastasis of HCC were evaluated.Results:Four SNPs in OPN promoter were detected[-1748 A/G,-616G/T,-443C/T,-156G/-(the deletion genotype named as-)].A total of 14 haplotypes were found,three of them(GTTG,AGT-,AGC-)occurred most frequently(91.06%).Three SNPs(-1748 A/G,-616G/T and-156G/-)were found to be in a strong linkage disequilibrium(LD)(r~2=074-0.8).The other one at-443C/T had a historical recombination,and was correlated with them with a r2 range of 0.22-0.25.Association analysis revealed that carriers of alleles C at SNP-443C/T decreased the risk of metastasis and recurrence with an OR of 0.49(95%CI 0.26-0.94,P＜0.05).No significance was found between other three SNPs genotype and metastasis.The haplotype AGT-was remarkably associated with the group of higher recurrence potential(OR=1.44,95%CI 1.04-2.01,P=0.03)and the haplotype AGC-were mainly existed in the lower metastatic group.There was no different frequency of haplotype GTTG between the two groups.Conclusions:The SNP-443C/C genotype in OPN promoter was significantly associated with the lower recurrence probability while the hyplotype AGT-increased recurrence risk in these Chinese HCC patients.The functional analysis of these different genotypes and haplotypes might provide a new way in the prediction of patients' prognosis and tumor recurrence.(2)The mechanism of different genotypes and haplotypes of OPN promoter and their influence on HCC prognosisObjective:To evaluate the effect of three promoter genotypes and haplotypes on the transcriptional activity of OPN.Methods:We generated the two OPN promoter haplotype luciferase reporter vectors(pGL3-basic)spanning from-2261 to +10bp of the OPN promoter region, which transiently transfected into MHCC-97H,HepG2 and Hela cells,and detected the luciferase index.EMSA assay researched DNA-protein binding activity with the OPN promoter SNP-443 C/T genotype and Myb.Results:Compared with void pGL3-basic vectors,two recombinant promoter plasmids were observed to have obvious transcriptional activities.AGT-haplotype promoter had a higher luciferase index than AGC-haplotype promoter.EMSA assay detected the higher electrophoretic mobility shift in the T/T genotype than that in the C/C.Conclusions:The different haplotype promoters could significantly affect the transcriptional activity.Myb protein specially combined with the OPN promoter SNP-443(T/T)genotype.2.The association of OPN promoter genotype and haplotype with clinical outcome of HCC patientsObjective:To investigate the association of the genotypes and haplotypes of OPN promoter with the postoperative 5-year OS and DFS. Methods:311 cases of HCCs collected from Jan.2002 to June 2003 were enrolled for the examination of the SNPs of OPN promoter by prosequencing 96.The association of OPN genotype and haplotype with the patients' outcome was investigated.Results:Kaplan-Meier analysis showed that the SNP-443 C/C genotype and the haplotype AGC-were significantly associated with an increased postoperative OS and DFS compared with other genotypes and haplotypes(P＜0.05).We recalculated the HRs for each SNP genotypes,and for haplotype of AGC-homozygotes, heterozygotes and others/others following adjustments for clinical characteristics.This analysis showed that only the-443 C/C genotype and AGC-homozygotes were significantly associated with an increased probability of DFS(TC: OR=5.026,95%CI 1.221-22.205,P=0.026;CC:OR=5.748,95%CI 1.350-24.476,P =0.018;Ht3 heterozygotes:P=0.047,OR=6.940,95%CI 1.944-51.040,others/others: OR=9.370,95%CI 1.295-67.824,P=0.027)independently.In addition,the AGC-homozygotes could increase the overall survival time of patients after surgery.Conclusions:Our results showed that variants at OPN had different effects on HCC metastasis potential.-443 C/C genotype and AGC-homozygotes of OPN promoter were independently associated with a good clinical outcome in HCC patients after surgical resection,which indicated that-443 C/C genotype and AGC-homozygotes might have a great value in prediction of HCC metastasis and prognosis.