Dual Regulatory Mechanisms of Expression and Mutation Involving Metabolism-Related Genes FDFT1 and UQCR5 during CLM.

Colorectal cancer (CRC) is the third most common cancer worldwide, and liver metastasis presents a major cause of CRC-associated death. Extensive genomic analysis has provided valuable insight into the pathogenesis and progression of CRC; however, a comprehensive proteogenomic characterization of CRC liver metastasis (CLM) has yet to be reported. Here, we analyzed the proteomes of 44 paired normal colorectal tissues and CRC tissues with or without liver metastasis, as well as analyzed genomics of CRC characterized previously by The Cancer Genome Atlas (TCGA) to conduct integrated proteogenomic analyses. We identified a total of 2,170 significantly deregulated proteins associated with CLM, 14.88% of which were involved in metabolic pathways. The mutated peptide number was found to have potential prognosis value, and somatic variants revealed two metabolism-related genes UQCR5 and FDFT1 that frequently mutated only in the liver metastatic cohort and displayed dysregulated protein abundance with biological function and clinical significance in CLM. Proteogenomic characterization and integrative and comparative genomic analysis provides functional context and prognostic value to annotate genomic abnormalities and affords a new paradigm for understanding human colon and rectal cancer liver metastasis.

Inhibiting survivin expression enhances TRAIL-induced tumoricidal activity in human hepatocellular carcinoma via cell cycle arrest.

Human Hepatocellular carcinoma (HCC) cell types exhibit a major resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell death, and the key determinants of mechanisms accounting for TRAIL susceptibility, still remain controversial. Our previous studies showed that overexpression of survivin reduced sensitivity of HCC cells to TRAIL. The aim of this study is to investigate how tumor cells escape TRAIL-mediated surveillance through survivin expression and how to reverse the resistance of TRAIL-inducing apoptosis. Seven tumor cell lines were treated with or without TRAIL protein and antisense oligodeoxynucleotides (ODNs) against survivin in culture. HepG(2) and SMMC7721 cells were treated with mimosine, thymidine or nocodazole to synchronize their cell cycle phases and then used to test their sensitivity to TRAIL. In vivo effects of TRAIL plasmid alone or in combination with survivin antisense ODNs on tumor growth were evaluated in a nude mouse hepatoma model of HepG(2) cell grafts. Varied levels of survivin mRNA in various cell lines were evaluated and negatively correlated to TRAIL-induced apoptosis. Hepatoma HepG(2) and SMMC7721 cells in G (1) or S phase are more sensitive to TRAIL than those in G(2) phase. Treatment with survivin antisense ODNscaused S phase arrest and significantly enhanced TRAIL-induced apoptosis. TRAIL protein caused G(2)/M arrest and resulted in an increase of survivin in HepG(2) cells. Combined TRAIL plasmid and survivin antisense ODNs significantly supressed the growth of tumor xenografts as compared to TRAIL plamid or antisense ODNs alone during four weeks of observation. The findings indicate that survivin may play a role in tumor cell resistance to TRAIL-induced apoptosis, at least in part, through cell cycle regulation. Manipulation of survivin expression levels may sensitizes tumor cells to TRAIL-induced apoptosis.

[Exploration of the alternative splicing variants of rat phospholipase C-gamma 1 pre-mRNA].

OBJECTIVE: To explore the expression of phospholipase C-gamma 1 (PLC-gamma1) alternative splicing variants in rats. METHODS: According to the sequence of human PLCG1 splicing variant, specific primers for rat PLC-gamma1 were designed and synthesized. The rat RNA was reverse transcribed into cDNA, which was amplified using the specific primers, and the PCR products were sequenced and analyzed using BLAST and bioinformatics methods. Totally 21 rat tissue samples were examined, including the heart, liver, lung, kidney, eyeball, and brain obtained in 3 different embryonic stages, 7 different early postnatal stages, and in adulthood. RESULTS: The result did not show that rat PLC-gamma1 had the same splicing variant (PLC-gamma1a, NM_002660) as human does. CONCLUSIONS: The same splicing variant of PLC-gamma1 detectable in human may not exist in rats, and the pre-mRNA may undergo splicing resulting predominantly in PLC-gamma1b mRNA. Very likely, the alternative splicing site of rat PLC-gamma1 is not identical to that of human.

Angiotensin II type 2 receptor gene polymorphisms and cardioprotective role in essential hypertension.

Angiotensin II type 2 receptor (AT2R) has been proved to be involved in a cardioprotective role, but only a few studies have addressed the association of AT2R-genotype with this role. Whether the AT2R genotype is associated with hypertension is controversial. The aim of the study was to explore the information of single-nucleotide polymorphisms (SNPs) of the AT2R gene in Cantonese, an essential subpopulation of Chinese, and study the association of SNPs in the AT2R gene with hypertension, and to detect the genotypes that indicate a cardioprotective role. Two hundred and sixty-two patients with essential hypertension and 75 normotensive subjects were enrolled for a case-control study. All of the subjects were Cantonese. Sixteen individuals were chosen to sequence the AT2R gene and 16 SNPs were acquired. G/T rs5193 and G/A rs5194 were two SNPs in the 3' untranslated region which were focused on the association of the AT2R-genotype and phonotype. Polymerase chain reaction was performed to amplify the fragment spanning the two SNPs. Genotype and haplotype were identified by dot blot hybridization. Four haplotypes in males (G-G, G-A, T-A, T-G) and eight haplotype combinations in females (G-G/G-G, G-A/G-A, G-G/G-A, G-G/T-A, G-G/T-G, T-A/T-A, T-G/T-G, and T-A/T-G) were detected. G-G and G-A haplotype were predominant, while T-A and T-G were rare in Cantonese. None of these was associated with hypertension. T-A carriers with essential hypertension indicated lower levels of left ventricular mass (LVM) and left ventricular hypertrophy index (LVHI). The levels of LVM and LVHI were still significantly lower in T-A carriers with hypertension adjusted for age or body mass index for men and women separately. No episodes of coronary heart disease and heart failure were detected in T-A carriers with hypertension. Haplotypes of G/T rs5193-G/A rs5194 are not associated with essential hypertension. Among these haplotypes, T-A may be implicated in a cardioprotective role to protect hypertense subjects from left ventricular hypertrophy.

[Analysis of ERG11 gene mutation in Candida albicans].

OBJECTIVE: To study the relationship between fluconazole (FCZ)-resistance of Candida albicans and the mutation of ERG11 gene encoding FCZ-targeted enzyme. METHOD: Three strains of FCZ-susceptible and 10 FCZ-resistent C. albicans were isolated from the urethra, vagina, oropharynx, respiratory tract, prostate secretion and blood samples. ERG11 gene was amplified by PCR using C.albicans genomic DNA extracts as the templates and the DNA sequences of the PCR products were determined and compared using BLAST and Clustal-W softwares. RESULTS: The comparison of ERG11 gene sequences identified mutations at 21 sites in 13 strains, including 17 same-sense and 4 missense mutations. Base substitutions at the sites of 348 bp and 383 bp resulting in D116E and K128T conversion may take place in both drug-resistant and drug-susceptible strains. The point mutation at the site of 1309 bp of FCZ-resistant strain may cause V437I change, and the base inversion at 1320 bp may give rise to A/C heterozygosity mutant of ERG11 gene, probably resulting in N440K conversion. CONCLUSION: FCZ-resistance of C. albicans may be associated with point mutations of V437I and N440K in ERG11 gene, but not with the point mutations of D166E and K128T.

[p53 gene codon 72 polymorphism and susceptibility to keloid].

OBJECTIVE: To investigate the relationship between p53 codon 72 polymorphism and susceptibility to keloid in a southern Chinese population. METHODS: The p53 genotypes were determined by polymerase chain reaction-reverse dot blot (PCR-RDB) and DNA direct sequencing in 45 patients with keloid and 60 unrelated healthy controls. RESULTS: The frequency of the p53 Pro allele among keloid patients was significantly higher than that among healthy controls (chi2 = 4.485, P = 0.034). The Pro/Arg and Arg/Arg genotype distribution among keloid patients was not significantly different from that among healthy controls (chi2 = 0.949, 1.346; P = 0.330, 0.246, respectively). However, the Pro/Pro genotype frequency among keloid patients was significantly higher than that among healthy controls (chi2 = 4.375, P = 0.036). The p53 Pro/Pro genotype significantly increased the risk for developing keloid, compared to the combination of Pro/Arg and Arg/Arg genotypes,with the odds ratio (OR) of 2.400 (95%CI: 1.048-5.498). CONCLUSIONS: Determination of the p53 codon 72 genotype may be used as a stratification marker to predicate high-risk individuals for keloid.

[Phospholipase C-gamma1 dynamic mRNA expression patterns in rats during early postnatal period].

OBJECTIVE: To investigate the expression of phospholipase C-gamma1 (PLCG1) mRNA in rats during early postnatal period. METHODS: Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of PLCG1 mRNA in 28 samples extracted from the liver, lung, kidney and brain of rats aged 1, 3, 5, 7 days, and 2, 3, 5 weeks. Specific PLCG1 product and GAPDH product as the internal control were both amplified by RT-PCR, and the ratio of their integral optical density was calculated to estimate the relative mRNA expression of PLCG1. RESULTS: PLCG1 was expressed in rat liver, lung, kidney and brain at the 7 postnatal time points, and the expression varied significantly with time and between the different organs (P<0.01), virtually undetectable in the liver on postnatal day 1 and reaching the highest level in the brain tissues on postnatal day 7. CONCLUSION: The differences in PLCG1 expression in various organs and development periods suggest that PLCG1 is involved in cell proliferation and differentiation during the early development of rats.

[Analysis of T cell receptor V gamma I-J gamma gene rearrangement in elderly patients with acute leukemia].

OBJECTIVE: To obtain insight into the molecular mechanism of acute leukemia in elderly patients in relation to the clinical features of the disease. METHODS: DNA specimens were obtained from 34 elderly patients with acute nonlymphoblastic leukemia (ANLL), 9 elderly patients with acute lymphoblastic leukemia (ALL) and 10 healthy blood donors. Polymerase chain reaction (PCR) coupled with single-strand conformation polymorphism (SSCP) was performed to detect T cell receptor (TCR) V gamma I-J gamma gene rearrangement. RESULTS: In the 34 elderly ANLL patients, 47.1% (16/34) were found with TCRV gamma I-J gamma gene rearrangement, showing a higher rate of the rearrangement than in younger ANLL patients (P<0.025). TCRV gamma I-J gamma gene rearrangement was identified in 7 (77.8%) of the 9 elderly patients with ALL, and 3 of the identified cases had oligoclonal/ subclonal rearrangement, suggesting a higher rate of oligoclonal/subclonal rearrangement in elderly ALL patients than in younger ALL patients (P<0.01). None of the blood donors was found with TCRV gamma I-J gamma gene rearrangement. The complete remission rate was lower in the elderly ANLL patient with TCRV gamma I-J gamma gene rearrangement than in those without (P<0.05). CONCLUSION: The detection of TCRV gamma I-J gamma gene rearrangement can be of value in assessing the therapeutic effect and making prognostic prediction in elderly patients with acute leukemia.

[Fluconazole resistance in Candida albicans assayed by PCR fingerprinting with M13 prime].

OBJECTIVE: To understand the molecular and genetic mechanism underlying fluconazole resistance in Candida albicans by PCR fingerprinting with M13 primer. METHODS: Paper disc diffusion method was employed for assay of fluconazole resistance in 41 clinical isolates of Candida albicans, followed by PCR fingerprinting with M13 primer to study the gel patterns with cluster analysis using neighbor joining (NJ) method performed with RAPD200 software. RESULTS: Of the 41 clinical isolates, 11 strains (26.8%) were fluconazole-sensitive, 8 (19.5%) fluconazole-dependent and 22 (53.7%) fluconazole-resistance. Two to twelve bands could be observed among these strains, and the gel patterns revealed by cluster analysis were associated with the reactions of the strains against fluconazole and the location of infection. CONCLUSION: There is high prevalence of fluconazole resistance in clinical Candida albicans isolates, and PCR fingerprinting with M13 primer is convenient for assay of fluconazole resistance and molecular epidemiological study of Candida albicans.

Expression of TNF-related apoptosis-inducing Ligand receptors and antitumor tumor effects of TNF-related apoptosis-inducing Ligand in human hepatocellular carcinoma.

AIM: To investigate the expression of TNF-related apoptosis -inducing Ligand (TRAIL) receptors and antitumor effects of TRAIL in hepatocellular carcinoma (HCC). METHODS: Expression of TRAIL receptors was determined in 60 HCC tissues, 20 normal liver samples and two HCC cell lines (HepG2 and SMMC-7721). The effects of TRAIL on promoting apoptosis in HCC cell lines were analyzed after the cells were exposed to the recombinant TRAIL protein, as well as transfected with TRAIL-expression construct. In vivo effects of TRAIL on tumor growth were investigated by using nude mice HCC model of hepG2. RESULTS: Both death receptors were expressed in all HCC tissues and normal hepatic samples. In contrast, 54 HCC tissues did not express DcR1 and 25 did not express DcR2. But both DcR were detectable in all of the normal liver tissues. The expression patterns of DR and DcR in HCC samples (higher DR expression level and lower DcR expression level) were quite different from those in normal tissue. DR5, DR4, and DcR2 expressed in both cell lines, while no DcR1 expression was detected. Recombinant TRAIL alone was found to have a slight activity as it killed a maximum of 15 % of HCC cells within 24 h. Transfection of the TRAIL cDNA failed to induce extensive apoptosis in HCC lines. In vivo administration of TRAIL gene could not inhibit tumor growth in nude mice HCC model. However, chemotherapeutic agents or anticancer cytokines dramatically augmented TRAIL-induced apoptosis in HCC cell lines. CONCLUSION: Loss of DcR (especially DcR1) in HCC may contribute to antitumor effects of TRAIL to HCC.HCC is insensitive towards TRAIL-mediated apoptosis, suggesting that the presence of mediators can inhibit the TRAIL cell-death-inducing pathway in HCC. TRAIL and chemotherapeutic agents or anticancer cytokines combination may be a novel strategy for the treatment of HCC.

[Antitumor effects of soluble TRAIL in human hepatocellular carcinoma].

OBJECTIVE: To investigate therapeutic potential of soluble TRAIL (sTRAIL) in hepatocellular carcinoma (HCC). METHODS: Expression of TRAILR was determined by in situ hybridization in 60 samples of resected hepatocellular carcinoma, 20 samples of normal liver tissue near the margin of benign tumor and 2 HCC cell lines of HepG2 and SMMC-7721. The clinical data of the patients were analyzed as well as cellular effects of sTRAIL in promoting apoptosis on HCC cell lines HepG2 and SMMC-7721 (p53 gene mutated) after exposure to different concentrations of recombinant protein. RESULTS: High death receptor (DR) expression and low DcR expression in HCC tissue differed from low DR expression and high DcR expression in the normal hepatic tissue with statistical significance. DR5, DR4, and DcR2 but not DcR1 were expressed in both cell lines. The expression of DR was closely correlated with HCC differentiation, with the weak expression in poor differentiation. The positive rate of DR expression in 32 cases of grade III-IV was significantly lower than that in 28 cases of grade I-II (P < 0.05). Cell apoptosis rates were 10%, 70% and 50% of HCC cells, Jurkat cells and human cholangiocarcinoma cell line QBC939 24 h after recombinant of TRAIL alone. CONCLUSION: TRAILR expression is prevalent in HCC, with different receptor types existing. HCC is resistant to TRAIL-mediated apoptosis. The treatment of TRAIL alone only has a limited effect on inducing apoptosis on HCC cell lines of HepG2 and SMMC-7721.