Construction of a metastasis-associated gene subtracted cDNA library of human colorectal carcinoma by suppression subtraction hybridization

AIM: To construct a differentially-expressed gene subtracted cDNA library from two colorectal carcinoma (CRC) cell lines with different metastatic phenotypes by suppression subtractive hybridization. METHODS: Two cell lines of human CRC from the same patient were used. SW620 cell line showing highly metastatic potential was regarded as tester in the forward subtractive hybridization, while SW480 cell line with lowly metastatic potential was treated as tester in the reverse hybridization. Suppression subtractive hybridization (SSH) was employed to obtain cDNA fragments of differentially expressed genes for the metastasis of CRC. These fragments were ligated with T vectors, screened through the blue-white screening system to establish cDNA library. RESULTS: After the blue-white screening, 235 white clones were picked out from the positive-going hybridization and 232 from the reverse. PCR results showed that 200-700 bp inserts were seen in 98% and 91% clones from the forward and reverse hybridizations, respectively. CONCLUSIONS: A subtractive cDNA library of differentially expressed genes specific for metastasis of CRC can be constructed with SSH and T/A cloning techniques.     

Profiling of differentially expressed genes in human Gastric carcinoma by cDNA expression array

AIM: To investigate the expression of cancer relatedgenes in gastric carcinoma (GC) through the use of AtlasHuman Cancer Array membranes with 588 well-characterized human genes related to cancer and tumorbiology.METHODS: Hybridization of cDNA blotting membranewas performed with 32P-labeled cDNA probessynthesized from RNA isolated from gastric carcinomaand adjacent noncancerous gastric epithelial tissue.AtlasImage, which is a software specific to array, wasused to analyze the result.RESULTS: The differentially expression cell cycle/growth regulator in GC showed a stronger tendencytoward cell proliferation with 2.7-fold up-regulation ofCK1. The promoter genes of apoptosis were down-regulated, including caspase-8 precursor, caspase-9and caspase-10. Among the oncogene/tumorsuppressor genes, ABL2 was down-regulated. Inaddition, some genes were up-regulated, includingmatrix metalloproteinse 2(MMP-2), MMP-16(MT3-MMP), SKY, CD9 and semaphorin V. A number of geneswere down-regulated, including neuroendocrine-dlg(NE-dig), retinoic acid receptor gamma and tumorsuppressor DCC colorectal. In general, The expressionof the cancer progression genes were up-regulated,while the expression of anti-cancer progression geneswero down-regulated.CONCLUSION: Investigation of these genes should helpto disclose the molecular mechanism of the onset,progression and prognosis of GC. Several genes arereported herein to be altered in GC for the first time.The quick and high-throughout method of profiling geneexpression by cDNA array provides us with an overviewof key factors that may involved in GC, and may aid thestudy of GC carcinogenesis and provide moleculartargets for diagnosis and therapy. The preciserelationship between the altered genes and gastriccarcinogenesis is a matter for further investigation.     

Differential gene expression in human hepatocellular carcinoma Hep3B cells induced by apoptosis-related gene BNIPL-2

AIM:Bd-2/adenovirus E1B 19 ku interacting protein 2-like(BNIPL-2) is a novel protein recently identified in ourlaboratory.BNIPL-2 is homologous to human BNIP-2,apotentially proapoptotic protein,and can interact with Bcl-2 and Cdc42GAP and promote apoptosis in BEL-7402 cells.Here we report the gene-expression profile regulated byBNIPL-2 in human hepatocarcinoma Hep3B cells and theanalysis of its potential roles in cell apoptosis.METHODS:BNIPL-2 was overexpressed in Hep3B cellsusing tetracycline inducible or Tet-on system.Screened byWestern blot,the cells with low background and highinduction fold of BNIPL-2 were obtained.We performedAtlas human cDNA expression array hybridization on thesecells and analyzed the data with Quantarray~(?) software toidentify BNIPL-2-regulated genes and their expressionprofile.RT-PCR was used to confirm the altered expressionlevel of part of genes identified by the Atlas array hybridization.RESULTS:Fifteen of 588 genes spotted on the Atlasmembrane showed altered expression levels in BNIPL-2-transfected Hep3B-Tet-on cells,in which 8 genes involvedin cell apoptosis or growth inhibition were up-regulatedand 7 genes involved in cellular proliferation were down-regulated following overexpression of BNIPL-2.CONCLUSION:cDNA array is a powerful tool to exploregene expression profiles under inducible conditions.Thedata obtained using the cDNA expression microarraytechnology indicates that BNIPL-2 may play its roles inapoptosis through regulating the expression of genesassociated with cell apoptosis,growth inhibition and cellproliferation.     

Loss of heterozygosity on hromosome 1 in sporadic colorectal carcinoma

AIM: Loss of heterozygosity (LOH) on tumor suppressor genes is believed to play a key role in carcinogenesis of colorectal cancer. When it occurs at a tumor suppressor gene locus with abnormal allele, neoplastic transformation happens. In this study, we analyzed the LOH at 21 loci on chromosome 1 in sporadic colorectal cancer to identify additional loci involved in colorectal tumorigenesis. METHODS: Twenty-one polymorphic micro-satellite DNA markers were analyzed with PCR both in 83 cases of colorectal cancer and in normal tissues. PCR products were eletrophoresed on an ABI 377 DNA sequencer. Genescan 3.1 and Genotype 2.1 software were used for LOH scanning and analysis. χ² test was used to compare LOH frequency with clinicopathological data. P < 0.05 was considered as statistically significant. RESULTS: The average LOH frequency of chromosome 1, short arm and long arm was 19.83%, 18.00% and 21.66%, respectively. The 2 highest LOH loci with a frequency of 36.54% and 32.50% were identified on D1S468 (1p36.33-p36.31) and D1S413 (1q31.3), respectively. On D1S2726 locus, LOH frequency of rectal cancer was 28.57% (6/21), which was higher than that of colon cancer (0.00%, 0/33) (P = 0.002), suggesting that the mechanism of carcinogenisis was different in both groups. CONCLUSION: Putative tumor suppressor genes on chromosome 1 may relate to sporadic colorectal carcinomas. Tumor-suppressor-genes might locate on 1p36.33-36.31 and/or 1q31.3.     

Clinical significance of vascular endothelial growth factor expression and neovascularization in colorectal carcinoma

AIM: To clarify the association of vascular endothelial growth factor (VEGF) and microvascular density (MVD)expression with the angiogenesis and prognosis of colorectal cancer.METHODS: A total of 97 cases of colorectal carcinomas were examined by immunohistochemical staining (SP method), using anti-VEGF and anti-factor CD34+ monoclonal antibodies. RESULTS: VEGF positive staining was obtained in 68 out of 97 cases (70.1%), and observed mainly in the cytoplasm of tumor cells, and also frequently in stromal cells. VEGF expression was more intense in poorly differentiated adenocarcinoma in comparison with others, but there was no significant correlation between VEGF expression and age,sex and stage. A significant correlation was found between the MVD and grades, and there was no significant relationship between the MVD and age, sex, and stage. The MVD in the VEGF positive group (68 cases) was higher than that in the negative group. Upon multivariate analysis, the significant variables were stage, tumor grade and MVD; VEGF expression was not an independent prognostic factor. CONCLUSION: The expression of VEGF has a significant correlation with MVD; MVD expression has prognostic value but VEGF has not in colon cancer.     

The point mutation of p53 gene exon7 in hepatocellular carcinoma from Anhui Province, a non HCC prevalent area in China

AIM: In hepatocellular carcinoma (HCC) prevalent areas ofChina, the point mutation of p53 exon7 is highly correlatedwith Hepatitis B virus(HBV) infection and aflatoxin B intake.While in non-HCC-prevalent areas of China, these factorsare not so important in the etiology of HCC. Therefore, thepoint mutation of p.53 exon7 may also be different than thatin HCC-prevalent areas of China. The aim of this study is toinvestigate the status and carcinogenic role of the pointmutation of p53 gene exon7 in hepatocellular carcinoma fromAnhui Province, a non-HCC-prevalent area in China.METHODS: PCR,PCR-SSCP and PCR-RFLP were applied toanalyze the homozygous deletion and point mutation of p53exon7 in HCC samples from Anhui, which were confirmedby DNA sequencing and Genbank comparison.RESULTS: In the 38 samples of hepatocellular carcinoma, nohomozygous deletion of p53 exon7 was detected and pointmutations of p53 exon7 were found in 4 cases, which werefound to be heterozygous mutation of codon 249 With amutation rate of 10.53 %(4/38). The third base mutstion(G→T) of p53 codon 249 was found by DNA sequencing andGenbank comparison.CONCLUSION: The incidence of point mutation of p53 codon249 is lower in hepatocellular carcinoma and theheterozygous mutation of p53 exon7 found in these patientsonly indicate that they have genetic susceptibility to HCC.p53 codon 249 is a hotspot of p53 exon7 point mutation,suggesting that the point mutation of p53 exon 7 may notplay a major role in the carcinogenesis of HCC in AnhuiProvince, a non-HCC-prsvalent area in China.     

Inhibitory effects of c9, t11-conjugated linoleic acid on invasion of human gastric carcinoma cell line SGC-7901

AIM: To investigate the effect of c9, t11-conjugated linoleic acid (c9, t11-CLA) on the invasion of human gastric carcinoma cell line and its possible mechanism of preventing metastasis.METHODS: Using reconstituted basement membrane invasion, chemotaxis, adhesion, PAGE substrate zymography and RT-PCR assays, we analyzed the abilities of invasion,direct migration, adhesion of intracellular matrix, as well as the activity of type IV collagenase and expression of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 mRNA in SGC-7901 cells which were treated with gradually increased concentrations (25, 50, 100 and 200 μmol/L) of c9, t11-CLA for 24 h.RESULTS: At the concentrations of 200 μmol/L, 100 μmol/L and 50 μmol/L, cg, t11-CLA suppressed the invasion of SGC7901 cells into the reconstituted basement membrane by 53.7%, 40.9% and 29.3%, respectively, in comparison with the negative control. Only in the 200 μmol/L c9,t11CLA group, the chemotaxis of SGC-7901 cells was inhibited by 16.0% in comparision with the negative control. cg, t11CLA also could inhibit the adhesion of SGC-7901 cells to laminin, fibronectin and Matrigel, increase the expression of TIMP-1 and TIMP-2 mRNA, and reduce type IV collagenase activities in the serum-free medium supernatant of SGC7901 cells.CONCLUSION: c9, t11-CLA can inhibit the invasion of SGC7901 cells at multiple procedures in tumor metastasis cascade, which may be associated with the induction of TIMP-1 and TIMP-2 mRNA expression.     

Morphological and functional changes of mitochondria in apoptotic esophageal carcinoma cells induced by arsenic trioxide

AIM: To demonstrate that mitochondrial morphological andfunctional changes are an important intermediate link in thecourse of apoptosis in esophageal carcinoma cells inducedby As2O3.METHODS: The esophageal carcinoma cell line SHEEC1,established in our laboratory, was cultured in 199 growthmedium, supplemented with 100mL@ L-1 calf serum and3 mol@L-1 As2O3( the same below). After 2, 4, 6, 12, 24 hof drug adding, the SHEEC1 cells were collected for light-and electron-microscopic examination. The mitochondriawere labeled by Rhodamine fluorescence probe and thefluorescence intensity of the mitochondria was measured byflow cytometer and cytofluorimetric analysis. Further, themitochondrial transmembrane potential ( MTP, ΔΨm )change was also calculated.RESULTS: The mitochondrial morphological change afteradding As2O3 could be divided into three stages. In theearly-stage (2-6h) after adding As2O3, an adaptiveproliferation of mitochondria appeared; in the mid-stage (6-12 h ) e degenerative change was observed; and in the late-stage (12-24 h ) the mitochondria swelled with outermembrana broken down and then calls death with apoptoticchanges of nucleus. The functional change of themitochondria indicated by fluorescent intensity, whichreflected the MTP status of mitochondria, was in accordancewith morphological change of the mitochondria. Thefluorescent intensity increased at early-stage, declined inmid-stage and decreased to the lowest in the late@ stage. 24h after As2O3 adding, the cell nucleus showed typicalapoptotic changes.CONCLUSION: Under the inducement of As2O3, the earlyapoptotic changes of SHEEC1 cells were the apparentmorphological and functional changes of mitochondria,afterwards the nucleus changes followed. lt is consideredthat changes of mitochondria are an important intermediatelink in the course of apoptosis of esophageal carcinomacalls induced by As2O3.     

Hepatitis B virus infection of transplanted human hepatocytes causes a biochemical and histological hepatitis in immunocompetent rats

AIM: To characterize the host response to hepatitis B virus (HBV) infection in human hepatocytes transplanted into immunocompetent rodent rats tolerized by, and transplanted with primary human hepatocytes.METHODS: One week after the transplantation, rats were inoculated with HBV, and viral gene expression, replication,and host response was monitored.RESULTS: HBV DNA was detectable in serum for at least 60 days. HBsAg levels rose steadily for 3 weeks postinoculation and then plateaued at a level of about 0.6 pg/mi. HBV RNA was also found in liver at levels that remained constant through the time course. Immunofluorescence revealed clusters of hepatocytes that stained positive for HBcAg. The presence of HBV covalently closed circular DNA (cccDNA) in liver was demonstrated using nuclease digestion of single-stranded DNA followed by PCR. Serum ALT levels rose and reached a peak level of 180 IU/L on day 18, but remained elevated for 60 days. Histology revealed a progressive predominantly mononuclear lobular hepatitis.CONCLUSION: These data indicate that human hepatocytestransplanted into rats rendered tolerant to these cells, when infected by HBV, results in biochemical as well as histological evidence of hepatitis that accompanies viral gene expression,and DNA replication.     

Tumor type M2 pyruvate kinase expression in gastric cancer,colorectal cancer and controls

AIM: Tumor formation is generally linked to an expansionof glycolytic phosphometabolite pools and aerobic glycolyticflux rates.To achieve this,tumor cells generally overexpressa special glycolytic isoenzyme,termed pyruvate kinase typeM_2.The present study was designed to evaluate the useof a new tumor marker,tumor M_2-PK,in discriminatinggastrointestinal cancer patients from healthy controls,andto compare with the reference tumor markers CEA andCA72-4.METHODS: The concentration of tumor M2-PK in body fluidscould be quantitatively determined by a commerciallyavailable enzyme-linked immunosorbent assay (ELISA)-kit(ScheBo(?) Tech,Giessen,Germany).By using this kit,thetumor M_2-PK concentration was measured in EDTA-plasmaof 108 patients.For the healthy blood donors a cut-offvalue of 15 U/mL was evaluated,which corresponded to90% specificity.Overall 108 patients were included in thisstudy,54 patients had a histological confirmed gastriccancer,54 patients colorectal cancer,and 20 healthyvolunteers served as controls.RESULTS: The cut-off value to discriminate patients fromcontrols was established at 15 U/mL for tumor M_2-PK.Themean tumor M_2-PK concentration of gastric cancer was26.937 U/mL.According to the TNM stage system,the meantumor M_2-PK concentration of stage Ⅰ was 16.324 U/mL,ofstage Ⅱ 15.290 U/mL,of stage Ⅲ 30.289 U/mL,of stage Ⅳ127.31 U/mL,of non-metastasis 12.854 U/mL and of metastasis35.711 U/mL.The mean Tumor M_2-PK concentration ofcolorectal cancer was 30.588 U/mL.According to the Dukesstage system,the mean tumor M_2-PK concentration ofDukes A was 16.638 U/mL,of Dukes B 22.070 U/mL,andof Dukes C 48.024 U/mL,of non-metastasis 19.501 U/mL,ofmetastasis 49.437 U/mE The mean tumor M_2-PK concentrationallowed a significant discrimination of colorectal cancers(30.588 U/mL) from controls (10.965 U/mL) (P<0.01),andgastric cancer (26.937 U/mL) from controls (10.965 U/mL)(P<0.05).The overall sensitivity of tumor M_2-PK for colorectalcancer was 68.52%,while that of CEA was 43.12%.Ingastric cancer,tumor M_2-PK showed a high sensitivity of50.47%,while CA72-4 showed a sensitivity of 35.37%. CONCLUSION: Tumor M_2-PK has a higher sensitivity thanmarkers CEA and CA72-4,and is a valuable tumor markerfor the detection of gastrointestinal cancer.     

Tributyrin inhibits human gastric cancer SGC-7901 cell growth by inducing apoptosis and DNA synthesis arrest

AIM: To evaluate the effects of tributyrin, a pro-drug of natural butyrate and a neutral short-chain fatty acid triglyceride, on the growth inhibition of human gastric cancer SGC-7901 cell.METHODS: Human gastric cancer SGC-7901 cells were 24-72 h. MTT assay was applied to detect the cell proliferation.[3H]-TdR uptake was measured to determine DNA synthesis.Apoptotic morphology was observed by electron microscopy and Hoechst-33258 staining. Flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay were performed to detect tributyrin-triggered apoptosis. The expressions of PARP, Bcl-2 and Bax were examined by Western blot assay.RESULTS: Tributyrin could initiate growth inhibition of SGC7901 cell in a dose- and time-dependent manner. [3H]-TdR uptake by SGC-7901 cells was reduced to 33.6% after 48 h control (P＜0.05). Apoptotic morphology was detected by TUNEL assay. Flow cytometry revealed that tributyrin could induce apoptosis of SGC-7901 cells in dose-dependent manner. After 48 hours incubation with tributyrin at 2 mmol.L-1, the level of Bcl-2 protein was lowered, and the level of Bax protein was increased in SGC-7901, accompanied by PARP cleavage.CONCLUSION: Tributyrin could inhibit the growth of gastric cancer cells effectively in vitro by inhibiting DNA synthesis and inducing apoptosis, which was associated with the downregulated Bcl-2 expression and the up-regulated Bax expression. Therefore, tributyrin might be a promising chemopreventive and chemotherapeutic agent against human gastric carcinogenesis.     

Study on of bioadhesive property of carbomer934 by a gamma camera in vivo

AIM: To study the bioadhesive property of carbomer934 indog alimentary tract.METHODS: Carbomer934 and ethylcellulose wereradiolabelled with technetium-99m; and gastrointestinalemptying rate of materials was measured using thetechnique of gamma scintigraphy.RESULTS: After oral administration, the maximum intestinalradioactivity of non-bioadhesive granules and bioadhesivegranules were observed in the second hour and the sixthhour respectively. Constants of stomach emptying rate ofnonadhesive granules, bioadhesive granules Ⅰ andbioadhesive granules Ⅱ were 0.774h-1, 0.265h-1 and0.321h-1 respectively on the base of gastric residualamount. Compared to nonadhesive material(ethylcellulose), the migration rate of adhesive material(carbomer934) was remarkably slower in dog alimentarycanal.CONCLUSION: lt is concluded that, in the dog, interactionsbetween gastrointestinal mucus layer and adhesive materialor nonadhesive material were significantly different.Carbomer934 had stronger in vivo bioadhesive property thanethylcellulose.     

Loss of DPC4 expression and its correlation with clinicopathological parameters in pancreatic carcinoma

AIM: DPC4 is a tumor suppressor gene on chromosome 18q21.1 that has high mutant frequencies in pancreatic carcinogenesis. The purpose of this study was to investigate the role of DPC4 alterations in tumorigenesis and progression of pancreatic carcinomas.METHODS: We studied the immunohistochemical markers of DPC4 in 34 adenocarcinomas and 16 nonmalignant specimens from the pancreas. The 16 nonmalignant specimens from the pancreas included 8 non-neoplastic cysts and 8 normal pancreatic tissues. The relationship between DPC4 alterations and various clinicopathological parameters was evaluated by chi-square test or Fisher's exact test.Survivals were calculated using Kaplan-Meier method (by a log-rank test).RESULTS: All the 16 nonmalignant cases of the pancreas showed expression of DPC4 gene. Loss of DPC4 expression was seen in 8 of 34(23.5 %) pancreatic adenocarcinomas.The frequency of loss of DPC4 expression was higher in poorly differentiated adenocarcinoma (G3) than in well and moderately differentiated adenocarcinoma (G1 and G2)histologically (P=0.037). Loss of DPC4 expression of the patients at TNM stage Ⅳ was also higher than that of the patients at TNM stages Ⅰ, Ⅱ and Ⅲ (60.0 % at stage Ⅳ,versus14.3 % atstage Ⅰ, 18.2 % at stage Ⅱ, and 18.2 % at stage Ⅲ) (P=0.223). The mean and median survival in patients with DPC4 expression was longer than those in patients with loss of DPC4 expression. Kaplan-Meier survival analysis demonstrated patients with DPC4 expression had a higher survival rate than patients with loss of DPC4 expression, but the difference did not reach statistical significance (P =0.879).CONCLUSION: This study suggests that DPC4 is involved in the development of pancreatic carcinoma and is a late event in pancreatic carcinogenesis, DPC4 expression may be a molecular prognostic marker for pancreatic carcinoma.     

Cloning and sequencing of cagA gene fragment of Helicobacter pylori with coccoid form

AIM:To clone and sequence the cagA gene fragment ofHelicobacter pylori(H pylon)with coccoid form.METHODS:Hpyloristrain NCTC11637 were transformedto coccoid form by exposure to antibiotics in subinhibitoryconcentrations.The coccoid Hpyloriwas collected,cagAgene of the coccoid Hpyloristrain was amplified by PCR.After purified,the target fragment was cloned into plasmidpMD-18T.The recombinant plasmid pMD-18T-cagA wastransformed into E.coli JM109.Positive clones were screenedand identified by PCR and digestion with restrictionendonucleases.The sequence of inserted fragment wasthen analysed.RESULTS:cagA gene of 3 444 bp was obtained from thecoccoid Hpylori genome DNA.The recombinant plasmidpMD-18T-cagA was constructed,then it was digested byBamH I Sac I,and the product of digestion was identicalwith the predicted one.Sequence analysis showed that thehomology of coccoid and the reported original sequenceH pylori was 99.7%.CONCLUSION:The recombinant plasmid containing cagAgene from coccoid H pylorihas been constructed successfully.The coccoid H pylori contain completed cagA gene,whichmay be related to pathogenicity of them.     

Expression and altered subcellular localization of the cyclin-dependent kinase inhibitor p27Kip1 in hepatocellular carcinoma

AIM:To investigate p27 expression in hepatocellularcarcinoma (HCC),adjacent nontumoral and normal livertissues,and to verify whether the subcellular localizationof p27 was altered in HCC.METHODS:The level of p27 in tumoral,nontumoral,andnormal liver tissues were assessed by immunohistochemical(IHC) analysis.Parallel immunostaining was done forproliferating cell nuclear antigen (PCNA) to evaluate cellproliferation.RESULTS:The labeling index (LI) of p27 in tumoral lesionswas significantly lower than that in adjacent nontumorallesions (t=2.444,P=0.017) and normal controls (t=2.268,P=0.029).The LI of p27 significantly decreased in patientswith massive type (t=2.227,P=0.037) and infiltration(t=2.197,P=0.036).The prognosis of patients with higherp27 LI was longer than that of patients with lower p27 LI(P=0.0247,log-rank test).The LI of PCNA was significantlyhigher in HCC than that in adjacent nontumoral lesions(t=2.092,P=0.041) and normal controls (t=3.533,P=0.002).There was no significant correlation between p27expression and cell proliferation in tumor samples.Thelevel of p27 in the cytoplasmic fraction was higher in tumoraland nontumoral liver tissues,and was associated withclinical stage (t=2.520,P=0.029) and the degree of invasion(t=2.640,P=0.019).Survival analysis showed that p27 wasan independent prognosis marker for HCC patients.CONCLUSION:These results suggest that p27 underexp-ressing in patients with HCC is closely associated withinfiltration,metastasis,and prognosis.Alterations in thesubcellular localization of p27 protein may occur earlyduring hepatocarcinogenesis.     

Pathological characteristics, PCNA labeling index and DNA index in prognostic evaluation of patients with moderately differentiated hepatocellular carcinoma

AIM: To study the relationship between prognosis and pathological characteristics, proliferating cell nuclear antigen labeling index (PCNA-LI) and DNA index (DI) in patients with moderately differentiated hepatocellular carcinoma(HCC).METHODS: 51 cases of moderately differentiated HCC were analyzed with respect to the relation between their clinical follow-up data and pathological characteristics. Meanwhile, PCNA-LI of HCC cells was detected by immunohistochemistry assay and DI was measured by Feulgen staining and automatic image analysis technique.RESULTS: Patients with a single tumor nodule, less than 5 cm in diameter, no tumor emboli, no daughter nodules and necrosis had relatively better prognosis; patients with euploidy HCC had better prognosis than those with aneuploidy; among the aneuploidy patients those with DI ＜ 1.5 had better prognosis than the cases with DI＞ 1.5; The higher the PCNA-LI, the worse would be the prognosis. The increase in DI was correlated with the increase in PCNA-LI,and both of them were correlated with the pathological changes of the tumor.CONCLUSION: A composite analysis of the pathological characteristics of tumor tissue, DI and PCNA-LI might be useful in predicting the prognosis of HCC patients.