Knockdown of GCF2/LRRFIP1 by RNAi causes cell growth inhibition and increased apoptosis in human hepatoma HepG2 cells.

BACKGROUND: GC-binding factor 2 (GCF2) is a transcriptional regulator that represses transcriptional activity of the epidermal growth factor receptor (EGFR) by binding to a specific GC-rich sequence in the EGFR gene promoter. In addition to this function, GCF2 has also been identified as a tumor-associated antigen and regarded as a potentially valuable serum biomarker for early human hepatocellular carcinoma (HCC) diagnosis. GCF2 is high expressed in most HCC tissues and cell lines including HepG2. This study focused on the influence of GCF2 on cell proliferation and apoptosis in HepG2 cells. MATERIALS AND METHODS: GCF2 expression at both mRNA and protein levels in HepG2 cells was detected with reverse transcription (RT) PCR and Western blotting, respectively. RNA interference (RNAi) technology was used to knock down GCF2 mRNA and protein expression. Afterwards, cell viability was analyzed with a Cell Counting Kit-8 (CCK-8), and cell apoptosis and caspase 3 activity by flow cytometry and with a Caspase 3 Activity Kit, respectively. RESULTS: Specific down-regulation of GCF2 expression caused cell growth inhibition, and increased apoptosis and caspase 3 activity in HepG2 cells. CONCLUSIONS: These primary results suggest that GCF2 may influence cell proliferation and apoptosis in HepG2 cells, and also provides a molecular basis for further investigation into the possible mechanism at proliferation and apoptosis in HCC.

The prognostic role of BRAF mutation in metastatic colorectal cancer receiving anti-EGFR monoclonal antibodies: a meta-analysis.

BACKGROUND: BRAF mutation has been investigated as a prognostic factor in metastatic colorectal cancer (mCRC) undergoing anti-EGFR monoclonal antibodies (moAbs), but current results are still inconclusive. The aim of this meta-analysis was to evaluate the relationship between BRAF mutation status and the prognosis of mCRC patients treated with moAbs. METHODS: Eligible studies were identified by systematically searching Pubmed, the Cochrane Library, Web of Knowledge, and OVID. Risk ratio (RR) for overall response rate (ORR), Hazard ratios (HRs) for Progression free survival (PFS) and Overall survival (OS) were extracted or calculated. Prespecified subgroup analyses were conducted in KRAS wild-type and in different study types. The source of between-trial variation was explored by sensitivity analyses. Quality assessment was conducted by the Hayden's criteria. RESULTS: A total of twenty one trials including 5229 patients were identified for the meta-analysis. 343 patients displayed BRAF mutations of 4616 (7.4%) patients with known BRAF status. Patients with BRAF wild-type (WT) showed decreased risks of progression and death with an improved PFS(HR 0.38, 95% confidence intervals 0.29-0.51) and an improved OS (HR 0.35 [0.29-0.42]), compared to BRAF mutant. In KRAS WT population, there were even larger PFS benefit (HR 0.29[0.19,0.43]) and larger OS benefit (HR 0.26 [0.20,0.35]) in BRAF WT. A response benefit for BRAF WT was observed (RR 0.31[0.18,0.53]) in KRAS WT patients, but not observed in unselected patients (RR 0.76 [0.43-1.33]). The results were consistent in the subgroup analysis of different study types. Heterogeneity between trials decreased in the subgroup and explained by sensitivity analysis. No publication bias of ORR, PFS and OS were detected. CONCLUSIONS: The results indicate that BRAF mutant is a predictive biomarker for poor prognosis in mCRC patients undergoing anti-EGFR MoAbs therapy, especially in KRAS WT patients. Additional large prospective trials are required to confirm the predictive role of BRAF status.

Screening for differentially methylated genes among human colorectal cancer tissues and normal mucosa by microarray chip.

UNLABELLED: High density DNA methylation microarrays were used to study the differences of gene methylation level in six pairs of colorectal cancer (CRC) and adjacent normal mucosa. We analyzed the profile of methylated genes by NimbleGen Microarray and the biologic functions by NIH-NAVID. In addition, preliminary validation studies were done in six pairs of samples by MSP (methylation-specific PCR). A total of 4,644 genes had a difference in methylation levels. Among them 2,296 were hypermethylated (log2ratio > 1), 2,348 genes were hypomethylated (log2ratio < -1), in which 293 hypermethylated and 313 hypomethylated genes were unmapped according to the NIH-NAVID. All these genes were randomly distributed on all the chromosomes. However, chromosome 1 contained the most of the hypermethylated genes (232 genes), followed by chromosome 19 (149 genes), chromosome 11 (144 genes), chromosome 2 (141 genes), chromosomes 3 (127 genes). Through the analysis of the statistics, There were 2 hypermethylated/3 hypomethylated genes involved in six pairs of samples simultaneously, followed by 10/14 in five samples, 34/37 in four samples, 101/113 in three samples, 341/377 in two samples, 1,808/1,804 in one sample. According to gene ontology analysis, some physiological processes play important roles in the cell division and the development of tumor, such as apoptosis, DNA repair, immune, cell cycle, cell cycle checkpoint, cell adhesion and invasion etc. Through Preliminary validation, there were two genes (St3gal6, Opcml) in thirty top-ranking genes shown hypermethylated in six pairs of CRC and adjacent normal mucosa. CONCLUSIONS: High density DNA methylation microarrays is an effective method for screening aberrantly methylated genes in CRC. The methylated genes should be further studied for diagnostic or prognostic markers for CRC.

Clinicopathologic characteristics and prognoses for multicentric occurrence and intrahepatic metastasis in synchronous multinodular hepatocellular carcinoma patients.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and the outcomes for patients are still poor. It is important to determine the original type of synchronous multinodular HCC for preoperative assessment and the choice of treatment therapy as well as for the prediction of prognosis after treatment. AIMS: To analyze clinicopathologic characteristics and prognoses in patients with multicentric occurrence (MO) and intrahepatic metastasis (IM) of synchronous multinodular hepatocellular carcinoma (HCC). METHODS: The study group comprised 42 multinodular HCC patients with a total of 112 nodules. The control group comprised 20 HCC patients with 16 single nodular HCC cases and 4 HCC cases with a portal vein tumor emboli. The mitochondrial DNA (mtDNA) D-loop region was sequenced, and the patients of the study group were categorized as MO or IM based on the sequence variations. Univariate and multivariate analyses were used to determine the important clinicopathologic characteristics in the two groups. RESULTS: In the study group, 20 cases were categorized as MO, and 22 as IM, whereas all 20 cases in the control group were characterized as IM. Several factors significantly differed between the IM and MO patients, including hepatitis B e antigen (HBeAg), cumulative tumor size, tumor nodule location, cirrhosis, portal vein and/or microvascular tumor embolus and the histological grade of the primary nodule. Multivariate analysis further demonstrated that cirrhosis and portal vein and/or microvascular tumor thrombus were independent factors differentiating between IM and MO patients. The tumor-free survival time of the MO subjects was significantly longer than that of the IM subjects (25.7 ∓ 4.8 months vs. 8.9 ∓ 3.1 months, p=0.017). Similarly, the overall survival time of the MO subjects was longer (31.6 ∓ 5.3 months vs. 15.4 ∓ 3.4 months, p=0.024). The multivariate analysis further demonstrated that the original type (p=0.035) and Child-Pugh grade (p<0.001) were independent predictors of tumor-free survival time. Cirrhosis (p=0.011), original type (p=0.034) and Child-Pugh grade (p<0.001) were independent predictors of overall survival time. CONCLUSIONS: HBeAg, cumulative tumor size, tumor nodule location, cirrhosis, portal vein and/or microvascular tumor embolus and histological grade of the primary nodule are important factors for differentiating IM and MO. MO HCC patients might have a favorable outcome compared with IM patients.

Clinical significance of mrp gene in primary hepatocellular carcinoma.

OBJECTIVE: To study the relations among the expression of the multidrug resistance associated-protein (mrp) gene and clinicopathologic features, the influence of alpha-fetoprotein (AFP), and prognosis of patients who received adjuvant chemotherapy after resection of primary hepatocellular carcinoma (HCC). METHODS: The expression of the mrp gene encoding MRP and mRNAmrp was determined in tissues from 54 untreated patients with HCC, adjacent tissues from 24 patients with HCC and archival paraffin-embedded tissues from 12 patients with posthepatic cirrhosis. The relationship between the mrp gene expression and the change level of AFP was analyzed in the 24 postoperative HCC patients whose AFP level was measured after 2 weeks. All of the HCC patients were followed up. RESULTS: The percentage of positive expressions of MRP and mRNAmrp in the three kinds of tissues was 57.40%, 25.00%, 16.67%, and 72.22%, 37.50%, 33.33% respectively. Significant difference was noted in the untreated HCC tissue, compared to the other two tissues (P<0.05). No difference existed between the mrp gene expression and such clinicopathologic findings, as age, sex, and tumor size (P> 0.05), but the expression was related to the degree of differentiation of HCC (P<0.05). The effective rate of AFP in the mrp gene positive expression group or postoperative chemotherapeutic patients was lower than that in the negative group (P<0.05). Although no difference was seen in the 1-, 3-, 5-year survival rates of HCC patients (P>0.05), the mean survival time of postoperative HCC patients or the negative mrp gene expression group was longer than that of the positive group (P<0.05). CONCLUSIONS: Multidrug resistance (MDR) of HCC is related to mrp gene expression and initiates the intrinsic MDR. Detection of mrp gene expression is of great significance in accessing chemotherapeutic resistance of HCC, which provides evidence for reversing MDR in HCC. The mrp gene may be a useful marker in detecting prognosis of HCC patients because its expression is correlated with tumor differention and mean survival time of the patients.

[Effect of ischemic preconditioning on cytokines during lung ischemia-reperfusion injury].

OBJECTIVE: To investigate the protective role of ischemic preconditioning (IPC) during lung ischemia-reperfusion (I/R) injury and its influence on inflammatory cytokine production. METHODS: In vivo I/R injury of rabbit was induced by blocking hilum of the left lung. The wet/dry ratio of the lung, lung permeability index and neutrophils percentage in bronchoalveolar lavage fluid (BALF) were detected as indexes of the lung injury. Serum levels of tumor necrosis factor alpha (TNFalpha), interleukin-6 (IL-6) and interleukin-8 (IL-8) were also detected using enzyme-linked immunosorbent assay. The protective role of IPC and its influence on inflammatory cytokine production were observed. RESULTS: The wet/dry ratio of the lung, lung permeability index and neutrophils percentage in BALF of I/R group were 9.73 +/- 1.14, (41.62 +/- 5.77) x 10(-4) and (58.1 +/- 10.0)% respectively. The IPC group indexes were 6.23 +/- 0.69, (20.31 +/- 4.03) x 10(-4) and (23.8 +/- 5.2)% respectively. There was a significant difference between the two groups (P < 0.01). Serum levels of TNFalpha, IL-6 and IL-8 of I/R group were (0.9078 +/- 0.1062), (0.2137 +/- 0.0598) and (0.7211 +/- 0.0979) ng/ml respectively. The IPC group indexes were (0.7478 +/- 0.0843), (0.1271 +/- 0.0089) and (0.5903 +/- 0.0746) ng/ml respectively, significantly lower than that of I/R group (P < 0.01). CONCLUSIONS: Lung IPC has a marked protection effect against I/R injury. The effect was related to its inhibition of inflammatory cytokines such as TNFalpha, IL-6 and IL-8, thus reducing activation and infiltration of neutrophils.