摘要：
The role of microRNA-195 in developing acquired drug resistance in hepatocellular carcinoma cells was investigated. Expression pro fi ling of miRNAs revealed a limited set of miRNAs with altered expression in drug resistant hepatocellular carcinoma cell line BEL-7402/5-FU compared to its parental BEL-7402 cell line. Real-time PCR confirmed down-regulation of miRNA-195 in BEL-7402/5-FU cells. Overexpression of miRNA-195 sensitized BEL-7402/5-FU cells to anticancer drugs. Consistent with these findings, miR-195 antisense oligonucleotide induced drug resistance in BEL-7402/5-FU cells. Also, the basal levels of the anti-apoptotic protein Bcl-w were high in BEL-7402/5-FU cells and miR-195 overexpression repressed Bcl-w protein level and inhibited the luciferase activity of a Bcl-w 3' untranslated region-based reporter construct in both BEL-7402/5-FU and BEL-7402 cells. These results indicate that miR-195 could improve the drug sensitivity at least in part by targeting Bcl-w to increase cell apoptosis in hepatocellular carcinoma cells.
作者机构：
[Tang, Huifang; Yang, Xiaoyan; Yin, Jie; Xiang, Qiong; Lei, Xiaoyong; Yu, Jia] Univ S China Hengyang, Affiliated Hosp 1, Inst Pharm & Pharmacol, Hengyang, Peoples R China.;[Yang, Xiaoyan] Univ S China, Coll Pharm & Life Sci, 28 Changsheng Rd, Hengyang 421001, Hunan, Peoples R China.
通讯机构：
[Yang, Xiaoyan] U;Univ S China, Coll Pharm & Life Sci, 28 Changsheng Rd, Hengyang 421001, Hunan, Peoples R China.
摘要：
Recently, we have reported tissue- and stage-specific expression of miR-195 in human hepatocellular carcinoma cells and so far, not many reports discuss the function of this microRNA (miRNA). Expression profiling of miRNAs revealed a limited set of miRNAs with altered expression in drug resistant hepatocellular carcinoma cell line BEL-7402/5-FU compared to its parental BEL-7402 cell line. Real-time PCR confirmed down-regulation of miR-195 in BEL-7402/5-FU cells. Western blots were performed to determine protein levels of LATS2, P53 and CDK2. MTT analysed the cell proliferation activity. Flow cytometry were performed to determine apoptosis rate. Up-regulation of miR-195 increased expression of LATS2 and increased apoptosis of HCC cells, while Anti-miR-195 treatment inhibited expression of LATS2. miR-195 over-expression inhibited the luciferase activity of a LATS2 3' untranslated region-based reporter construct in BEL-7402/5-FU cells. These results indicate that miR-195 could increase cell apoptosis by targeting LATS2 in hepatocellular carcinoma cells.
作者机构：
[Yin, Jie; Yang, Xiao Yang; Lei, Xiao Yong; Xiang, Qiong; Hu, Nan; Yu, Jia] Univ S China, Inst Pharm & Pharmacol, Hengyang, Hunan, Peoples R China.;[Tang, Hui Fang] Univ S China, Affiliated Hosp 1, Hengyang, Hunan, Peoples R China.;[Lei, Xiao Yong] Univ S China, Inst Pharm & Pharmacol, 28 Changsheng Rd, Hengyang, Hunan, Peoples R China.
通讯机构：
[Lei, Xiao Yong] U;Univ S China, Inst Pharm & Pharmacol, 28 Changsheng Rd, Hengyang, Hunan, Peoples R China.
摘要：
To investigate the changes in drug sensitivity of miR-122 transfected BEL-7402/5-FU cells. MiR-122 and negative miRNA expression vectors were constructed and stably transfected into BEL-7402/5-FU cells. Real-time RT-PCR was used to detect the level of miR-122, Bcl-XL, Bcl-2 and P53 mRNA. Western Blotting was used to detect Bcl-2, Bcl-XL and P53 protein expression. Drug sensitivity of the cells to 5-fluorouracil (5- FU) was analyzed with MTT and flow cytometry. Compared with negative miRNA transfectants or untreated cells, mRNA and protein expression level of Bcl-2, Bcl-XL in stable miR-122 transfectants were decreased. Accordingly, P53 protein expression showed a significant up-regulation; MTT results showed that after incubation with 5-FU, miR-122 transfectants had higher cell inhibitory rates than negative miRNA or untreated cells; flow cytometry results demonstrated that apoptosis rate increased in miR-122 transfected cells, compared with negative miRNA or untreated cells. After addition of 5-FU (10 and 100 μmol/l), miR-122 transfected cells showed higher apoptosis rate than negative miRNA or untreated cells. MiR-122 can specifically down-regulate the expression of Bcl-2 and Bcl-XL, and increase P53 activity in BEL-7402/5-FU cells, which increased cells spontaneous apoptosis and sensitize cells to 5-FU. Therefore, MiR-122 can be used as a potential therapy agent against human hepatoblastoma.
摘要：
AIM: To investigate the effects of macrophage migration inhibitory factor (MIF) on proliferation of human gastric cancer MGC-803 cells and expression of cyclin D1 and p27~(Kip1) in them, and further determine whether the effects are related to the PI3K/Akt signal transduction pathway. METHODS: Gastric cancer MGC-803 cells were cultured and then treated with 50 μg/L recombinant human MIF (rhMIF) with and without a PI3K inhibitor, LY294002 (25 μmol/L). MTT assay was used to detect the proliferation of MGC-803 cells. Cell cycle was detected by flow cytometry. Expression of cyclin D1 and p27~(Kip1) mRNA was by reverse transcription-polymerase chain reaction. Protein expression of phosphorylated Akt (p-Akt), Akt, cyclin D1 and p27~(Kip1) was examined by immunocytochemistry and Western blotting. RESULTS: rhMIF significantly stimulated the proliferation of MGC-803 cells and cell cycle progression from G1 phase to S phase in a concentration- and time-dependent manner. After the MGC-803 cells were treated with rhMIF for 24 h, the expression of cyclin D1 was significantly up-regulated compared with the cells not treated with rhMIF at both mRNA and protein levels (0.97± 0.02 vs 0.74±0.01,P = 0.002;0.98±0.05 vs 0.69±0.04,P =0.003). The p27~(Kip1) was downregulated but only statistically significant at the protein level. rhMIF significantly increased the expression of p-Akt, which reached the peak at 30 min, but did not affect the expression of Akt. However, LY294002 inhibited all the effects of rhMIF. CONCLUSION: Macrophage MIF increases the proliferation of gastric cancer cells, induces the expression of cyclin D1 at the transcriptional level and inhibits the expression of p27~(Kip1) at the post-transcriptional level via the PI3K/Akt pathway.
