ABSTRACT
Objective To investigate the expression and genetic alterations of KLF6 in hepatocellular carcinoma (HCC) and explore their functional mechanisms in the oncogenesis and development of HCC. Methods Real-time quantitative-PCR, direct sequencing and LOH approaches were used to detect KLF6 genetic abnormalities in HCC. The experiment had 2 groups, an experimental group and a control group. In the experimental group, the transfected plasmid pcDNA3.0 was recombined with KLF6 and tranfected into HCC HepG2 cells. MTT, flow cytometry and Western blotting were used to observe the effect of anti-oncogene wild type KLF6 on HepG2 cells by transgenic method for 48 h.Results Expression levels of KLF6 were significantly downregulated in HCCs(P＜0. 01), as detected by qRT-PCR. LOH occurred in 11 (52%) of the 21 tumors, and all the samples with LOH showed KLF6 down-regulation. The mutational frequency was 29%, and sequence changes located in activation domain of KLF6. Meanwhile, MTT assay showed a significant antiproliferative effect of the transfected wtKLF6 on HepG2 cells(42.7%, P＜0.05). Fluorescence-activated cell sorting analysis revealed that KLF6 induced apoptosis. Conclusion The deregulation of KLF6 together with genetic abnormalities of allelic imbalance and mutations may play an important role in HCC pathogenesis.
ABSTRACT
Kruppel-like factor 6 (KLF6) was reported as tumor suppressor in multiple cancers. However, loss of chromosomal locus spanning KLF6 is relatively infrequent in previous published studies. To explore the role of KLF6 in hepatocellular carcinoma (HCC), we examined the gene for expression change, loss of heterozygosity (LOH) and mutation in 26 HCC samples. The expression levels of KLF6 were significantly down-regulated in HCCs, as detected by qRT-PCR. LOH occurred in 11 (52%) of 21 tumors, and all the samples with LOH showed KLF6 down-regulation. The mutational frequency was 24%, and sequence changes located in activation domain of KLF6. Furthermore, MTT assay showed a significant antiproliferative effect of the wt KLF6 transfected in HepG2 hepatoblastoma cells. Fluorescence-activated cell sorting analysis revealed that KLF6 could induce apoptosis. These findings indicate that deregulation of KLF6, together with genetic abnormalities of allelic imbalance and mutations, may play a role in HCC pathogenesis.
ABSTRACT
To construct the specific siRNA expression vectors and investigate their effect on leptin and collagen I in HSC, which provide a new approach to the prevent and treat hepatic fibrosis. The five siRNAs against leptin gene were transcript synthesized intracellularly by expression templates of plasmid vector psiRNA-hH1neo. The recombinant leptin siRNA plasmid vectors could express in eukaryocyte , and then to evaluate them by using enzyme cutting and sequencing. The recombinant plasmids were transfected into HSCs using Lipofectamine methods respectively. The cells were selected after growing in DMEM containing 300 μg/mL G418 for about 4 weeks. Gene expression of leptin and collagen I were showed by Western blot analysis and reverse transcription polymerase chain reaction (RT-PCR). Identification by enzyme cutting and sequencing showed that the leptin siRNA expression vectors were constructed successfully, and leptin siRNA could inhibit the leptin and collagen I gene expression effectively. It was concluded that RNA interference-mediated silencing of leptin gene diminished leptin and collagen I gene expression in HSCs. Furthermore, attenuated the extracellular matrix over-deposition at the same time. Leptin gene is ideal targets of gene therapy for liver fibrosis.
ABSTRACT
To study the expression of DNA repair enzyme hMTH1 mRNA and protein in hepatocellular carcinoma (HCC) tissues, tissues adjacent to the cancers, normal liver cells and hepatoma cell lines, and to investigate their function in the progress of HCC, semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) was employed to examine the expression of hMTH1 mRNA in matched HCC tissues (HT)/surrounding tissues (HST) of HCC, normal liver cell L02 and hepatoma cell lines SMMC7721, HepG2. hMTH1 protein was detected in corresponding HT as well as their HST by immunohistochemistry. Our results showed that the expression level of hMTH1 mRNA in HT was higher than that in HST (t=2.424, P <0.05). The expression level of hMTH1 mRNA in two hepatoma cell lines was higher than that in normal liver cell line (F=6.810, P <0.01). The expression of hMTH1 mRNA in SMMC7721 was similar to that in HepG2. hMTH1 protein was 88.2 % (15 of 17) positive in HT and 82.4 % (14 of 17) in HST. The protein level of hMTH1 in HT was correspondingly higher than in their HST (t=2.618,P<0.05). It is concluded that hMTH1 mRNA and protein were over-expressed in HCC and hepatoma cell lines. It may be one of the key events during the carcinogenesis,progression of HCC and may promote the malignant growth. These results suggest that hMTH1 plays a role in HCC and may be a candidate marker for the diagnosis of HCC.