HCCR, The Role Of Osteopontin In Liver Cancer And Other Malignant Tumors And Metastases

ObjectiveThe aim of this study is to prepare polyclonal antibody andmonoclonal antibody (McAb) against recombinant HCCR proteinand to identify its biological properties.MethodsThe COOH-terminal portion of HCCR cDNA encoding apolypeptide from the 167th to 360th amino acid residue which wasamplified by RT-PCR from the total RNA of human SMMC7721cells was cloned into the pMBP-C vector, and expressed in the E.coliTop10F'. Recombinant plasmid was identified by endonuclease(BamH I and Nde. I) digestion and nucleotide sequencing, andinduced by IPTG for expression. The expression product waspurified by nick-nitrilotriacetic acid (Ni-NTA) metal-affinitychromatography and was analyzed by SDS-PAGE, Western blottingand electrospmy ionization time-of-flight mass spectrometry.BALB/c mice were immunizedby intrasplenic deposition of thepurified fusion protein. The polyclonal anti-HCCR serum was testedfor its efficiency and specificity by indirect ELISA and Western blot.SP2/0 myeloma cells were fused with the splenic cells from micewith high antibody titer. Western blot and indirect ELISA were usedto identify the specific antibody secreted by fused cell lines. ResultsThe polyclonal antibody against HCCR protein produced in this studyhad both high efficiency and specificity which were tested by indirectELISA and Western blot. HCCR protein was mainly expressed on theplasma membrane and cytoplasm in hepatocellular carcinoma cell line,SMMC7721. The monoclonal antiboby against HCCR was highlyantigen specific.ConclusionsWe have successfully obtained high titer HCCR specific polyclonaland monoclonal antibodies. HCCR overexpression is associated withhepatocarcinogenesis. It could be the potential target for earlydiagnosis and treatment of HCC. ObjectiveThe aim of this study was to investigate the expression of HCCR ina wide variety of human tumors, and whether there was correlationbetween HCCR expression and clinicopathological characteristicsof different tumors.MethodsImmunofluorescence staining was used to characterize thesubcellular localization of HCCR in SMMC7721, MKN-28 andMKN-45. HCCR protein expression was detected byimmunohistochemistry in a selection of 291 human tumorsincluding malignancies from liver (116), colon (22), stomach (52),pancreas (34), esophagus (14), lung (5), kidney (7), brain (6),thyroid (4), breast (6), ovary (4), endometrium (10) and cervix(11).ResultsHigh cytoplasmic HCCR staining was observed in 83.3％of breastcancers, 81.8％of colorectal carcinomas; 80％of endometrialcancers, 80％of lung carcinomas, 79.3％of hepatocellular carcinomas, 75％of ovarian cancers, 73.5％of pancreaticcarcinomas, 72.7％of cervical cancers, 71.4％of renal carcinomas,69.2％of gastric carcinomas, 66.7％of glioblastomas, 57.1％ofesophageal carcinomas, and 50％of thyroid carcinoma. Comparedwith corresponding paraneoplastic, HCCR expression was higherin most of tumors. It was also found that the expression of HCCRin gastric carcinomas of non-antrum was significantly higher thanthat in gastric carcinomas of antrum (P＜0.05). In hepatocellularcarcinomas, the positive rate of HCCR was significantly correlatedwith minor histological grading.ConclusionThe broad expression of HCCR in the majority of human tumorssuggested its involvement in tumor formation. The significantcorrelation between the HCCR expression and histological stage inhepatocellular carcinomas suggests certain role of HCCR in tumorprogression. Objective:The aim of this study is to explore the expression of OPN indifferent kinds of human carcinomas, including hepatocellularcarcinoma, and analyze the relationship between OPN expressionand clinicopathological features.Methods:The expression of OPN was tested by immunohistochemistry in aselection of 374 carcinomas, including malignancies, fromesophagus (93), stomach (86), colon (32), pancreas (43), liver(49), lung (28), renal (17), breast (8) and neurogliocytoma (18), and147 para-tumorous tissues.Results:In esophageal carcinoma, gastric carcinoma, colonic carcinoma,pancreatic carcinoma, hepatocellular carcinoma, lung carcinoma,renal carcinoma, breast cancer and neurogliocytoma, the expressionrates of OPN were 64.5％, 65.1％, 78.1％, 65.1％, 71.4％, 71.4％,82.4％, 87.5％and 44.4％, respectively. OPN expression inmalignancy tumor tissues is higher than that in correspondingparacancious tissues (P＜0.05). There was correlation between OPNexpression and lymph nodes metastasis in esophageal carcinoma,gastric carcinoma, pancreatic carcinoma, hepatocellular carcinoma, lung carcinoma and neurogliocytoma (P＜0.05).Conclusion:Osteopontin was overexpressed in multiple cancer tissues,including HCC, and its, overexpression was associated with themetastasis of cancers. Osteopontin may be a marker for prognosisof multiple tumors including HCC. AimsTo explore the concentration of osteopontin (OPN) in the plasma ofpatients with liver cancer, and analyze the relationship betweenOPN expression and clinicopathological features.MethodsOPN concentration in 51 plasma samples was tested byEnzyme-Linked Immunosorbnent Assay (ELISA). And therelationship between OPN concentration and clinicopathologicfeatures was analyzed.ResultsPlasma osteopontin level in patients with liver cancer was higherthan that in normal group. OPN expression was correlated to lymphnodes metastasis, vascular encroachment and liver capsuleencroachment (P＜0.05). The sensitivity and specificity were 90.2％and 90.6％, separately.ConclusionOPN may be a potential marker for liver cancers. It may need moredata for clinical use. ObjectiveIt is unclear how osteopontin, one of metastasis-associated genes, isregulated in HepG2 cells. The aim of this study is to investigate theeffect of epidermal growth factor on the expression of osteopontin inHepG2 cells, and to explore the signal transduction pathwaymediated this expression. Another aim is to investigate the effect ofprotein kinase B (Akt) on the expression of osteopontin in HepG2cells, and to explore the relationship between Akt, a key gene inPI3K signal transduction pathway, and osteopontin expression.MethodsOsteopontin expression is detected by RNAase protection assay andwestern blot. Wortmannin, a specific inhibitor of PI3K, was used toobserve if PI3K signal transduction involved in the induction ofosteopontin gene expression. HepG2 cells were transfected withconstitutively active Akt and dominant negative Akt by lipofectmine,and transfectants were confirmed using western blot for Akt.Osteopontin expression is detected by Northern blot and westernblot.ResultsHepG2 cells constitutively express low levels of osteopontin.Treatment of epidermal growth factor increased osteopontin mRNA and protein level in a dose-and time-dependent manner. Treatmentof wortmannin caused dramatic reduction of epidermal growthfactor-induced osteopontin expression. HepG2 cells weresuccessfully transfected with Akt genes including constitutivelyactive Akt and dominant negative Akt, and overexpression ofexozegenes Akt can be detected in HepG2 cells by Western blot.Using Northern blot and Western blot, we found that Akt generegulated osteopontin expression in RNA level and protein level. Inserum-free condition, HepG2 cells constitutively express low levelsof osteopontin. Transfection of constitutively active Akt increasedosteopontin mRNA and protein expression. Transfection ofdominant negative Akt decreased osteopontin expression.ConclusionOsteopontin gene expression can be induced by treatment ofHepG2 cells with epidermal growth factor. Epidermal growth factormay regulate osteopontin gene expression through PI3K/Aktsignaling pathway. Several potential targets in the pathway can bemanipulated to block the synthesis of osteopontin and inhibit livercancer metastasis, for example, by inactivation of Akt gene. ObjectiveThe aim of this study is to investigate if osteopontin(OPN)expression was suppressed by small interfering RNA (siRNA) inHepG2 cells, and to explore the gene therapy of HCC target forOPN.MethodsThree Plasmids containing OPN-siRNA fragments wereconstructed by gene recombination with vector pGCsi andOPN-siRNA oligonucleotides. Lipofectamine was used to transfect293T and HepG2 cells. Westem blotting was used to quantify OPNprotein levels. MTT, Soft agar and wound closure assay were usedto observe the change of biologic function of the transfected cells.ResultsWe successfully constructed three plasmids of pGCsi-OPN1,pGCsi-OPN2, pGCsi-OPN3 and found that osteopontin gene wasinhibited when 293T cells were transfected with pGCsi-OPN2.After transfection of HepG2 cells with pGCsi-OPN2, we generated4 cell lines stably expressing pGCsi-OPN2 in HepG2 cells andfound OPN expression was down-regulated in these cells. Theproliferation, clony formation and migration of transfected cellswere decreased. ConclusionThis study demonstrated that RNA interference stably reducesexpression of OPN in HepG2 cells and OPN silencing can inhibitthe migration of HCC cells. With these findings in mind, OPNcould become a new target of gene therapy for HCC.