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Objective To investigate the effect and underlying mechanism of micro ribonucleic acid (miR)-101 on the epithelial-mesenchymal transition in human hepatocellular carcinoma MHCC97H cells. Methods MHCC97H cell lines were transfected with miR-101 mimics (M101 group) and negative control mimic (NCM group), and the cell lines without transfection were used as the control group. The expression levels of miR-101 in cells of 3 groups were quantitatively measured using reverse transcription polymerase chain reaction (RT-PCR). Transwell assay was performed to evaluate the cell migration and invasion ability of 3 groups. The expression levels of vimentin, α-catenin, E-cadherin and USP22 proteins in cells of 3 groups were measured by Western blot. The relationship between miR-101 and USP22 was analyzed by dual-luciferase assay. Results In the M101 group, the expression level of miR-101 was significantly up-regulated, which was approximately 761 times of that in the control group (P<0.05). In the M101 group, the quantity of migrating cells was 15.7±1.6, significantly lower than 94.1± 1.8 in the control group (P<0.05). In the M101 group, the quantity of invasive cells was 9.1±0.4, significantly lower compared with 51.6±0.9 in the control group (P<0.05). In the M101 group, the expression levels of vimentin and USP22 protein were significantly down-regulated, whereas the expression levels of α-catenin and E-cadherin protein were significantly up-regulated. Dual-luciferase assay revealed that USP22 was the target gene of the downstream miR-101 signaling pathway. Conclusions miR-101 regulates the expression of epithelial-mesenchymal transition-related proteins and suppresses the epithelial-mesenchymal transition of hepatocellular carcinoma MHCC97H cells probably through down-regulating the expression level of USP22.
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[Objective]To explore the correlation of the expression of alpha-fetoprotein(AFP)mRNA in the peripheral blood and postoperative survival and metastasis of patients with liver cancer.[Methods] A total of 66 patients with liver cancer who received radical resection surgery from January 2005to December 2006 was enrolled in this study.The cell total RNA was extracted from peripheral blood and the expression of AFP mRNA was detected by nested PCR.All the patients were followed up for 60 months after surgery.[Results]The expression rate of AFP mRNA in the peripheral blood was 40.91％(27/66).The expression of AFP mRNA in the peripheral blood in patients with liver cancer was significantly related to microvascular invasion and metastasis(P ＜ 0.05 or ＜ 0.01),but the expression had no relationship with sex,age,HBV infection,cirrhosis,AFP concentration,tumour size and number,and Edmondson grading(P＞0.05).The overall 1,2,and ＞ 3 years survival rates of patients with positive AFP mRNA after surgery were 66.7％(18/27),38.9％(7/18),28.6％(2/7),respectively.The overall 1,2,and ≥3 years survival rates of patients with negative AFP mRNA after surgery were 84.6％(33/39),60.6％(20/33),45.0％(9/20).There was statistical significance between the survival rates of AFP mRNA-negative patients and AFP mRNApositive patients(P ＜ 0.01).[Conclusions] The detection of AFP mRNA in the peripheral blood may provide clue for early microscopic metastasis.It can be a prediction index for postoperative recurrence.
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Objective To design and prepare an RNA interfering vector for effectively inhibiting the cellular expression of zinc finger protein A20 and observe the effect of A20 gene silence on cellular inflammatory response. Methods Specific RNA interfering oligonucleotide fragments (ASRF) were designed and synthesized artificially and the A20 RNA interfering vector pSUPER-EGFP-A20 siRNA constructed. Human monocyte cell line THP1 was used to infect the pSUPER-EGFP-A20 siRNA by means of genetic transfection technique; then, silence rate of cellular A20 was analyzed by real-time polymerase chain reaction (PCR). In the meantime, the activity of nuclear transcription factor nuclear factor-κB (NF-κB) and the level of tumor necrosis factor-α (TNF-α) in culture supernatant were measured by ELISA. Results Of two specific inhibitory oligonueleotide fragments of A20, the fragment M59465-385R/F had a higher inhibition to A20 expression, with rate of A20 gene silence of 83.86%. Preliminary application showed that after A20 gene silence, the activity of NF-κB was increased by 78.13% and the level of TNF-α in cell culture supernatant was increased by 49.30%. Conclusions Vector of A20gene silence with a high efficiency is obtained successfully. Preliminary application indicates that the expression of A20 can down-regulate the degree of cellular inflammatory responses.
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Objective: To investigate androgen receptor (AR) mRNA expression in human peripheral leukocytes. Methods: Total RNA was isolated from peripheral leukocytes of healthy men and women, AR mRNA was determined by RT PCR and Northern blot. Results: An AR cDNA fragment of the expected size of 390 bp was determined by RT PCR. 9.4 kb AR mRNA was detected by Northern blot, which was consistent with AR mRNA in human prostate reported by Lubahn et al . Conclusion:AR mRNA expresses in human peripheral leukocytes, this provides basis for studying the effect of androgen on the function of the human peripheral leukocytes and the changes of AR in pathological stages. [
