Effects of GATA6 silencing mediated by lentivirus on apoptosis of human liver cancer Huh-7 cells

AIM: To explore the effect of GATA6 gene silencing on apoptosis of hepatocellular carcinoma Huh-7 cells. METHODS: RNA interference vectors of the target gene GATA6 mediated by lentivirus were constructed in vitro to transfect the hepatocellular carcinoma cell line Huh-7. The apoptotic rate of transfected cells was measured by flow cytometry. The protein expression of GATA6, NF-κB and Bcl-2 in transfected cells was determined by Western blotting. RESULTS: The transfection efficiency was 57.4%. The mRNA and protein expression of GATA6 reduced significantly after the carcinoma cell line Huh-7 being transfected by RNA interference vectors mediated by lentivirus. The apoptotic rate of the carcinoma cells with silent GATA6 gene was significantly increased (P<0.05). The protein expression levels of NF-κB and Bcl-2 were also significantly decreased. CONCLUSION: Lentiviral vector-mediated RNA interference of GATA6 has an inhibitory effect on the expression of the gene itself, and promotes the apoptosis of hepatocellular carcinoma cells. Regulation of the apoptosis-related protein expression by the NF-κB signaling to influence the proliferation and apoptosis of hepatocellular carcinoma cells might be one of the possible mechanisms.     

The c-Myc expression and apoptosis of mouse thymocytes treated with dexamethasone

AIM: To study c-Myc expression and its relationship with caspase-3 in a dexamethasone (DEX)- induced mouse thymocyte apoptosis model, and discuss the role of c-Myc in cell apoptosis. METHODS: Mouse thymocyte apoptosis was induced by 1 μmol/L DEX, the apoptotic and necrosis cells were measured by Annexin V-FITC/PI double staining flowcytometry at 30 min, 3 h, 6 h and 9 h . Electron microscopy observation was carried out at 6 h, and c-Myc and caspase-3 contents were tested by Western blot at 0, 30, 60, 180 min. RESULTS: By 1μmol/L DEX treatment, the apoptosis rates of thymocytes at 30 min, 3 h, 6 h, 9 h were (5.70±0.46)%, (35.79±1.13)%, (50.61±2.15)% and (35.52±1.66)%,respectively; in control group, they were (5.97±0.25)%, (10.20±0.71)%, (12.10±0.66)% and (15.45±0.51)% (P0.01). At same time intervals, the necrosis rates of thymocytes in DEX group were (4.58±0.51)%, (4.66±0.67)%, (25.36±1.64)% and (46.99±2.67)%; in control group, they were (4.38±0.39)%, (4.19±0.73)%, (9.63±1.25)% and (13.38±0.72)%. Typical apoptotic cells were observed at 6 h in DEX group by electron microscopy. Obvious expression of c-Myc was detected at 0 min in control group, then c-Myc content increased at 30 min and reduced at 1 h and 3 h, in DEX group, c-Myc expression was higher than that in control group, and got a peak expression at 30 min, then significantly reduced at 1h and 3 h. Caspase-3 increased following culture time lapse in control group, while its content was more in DEX group at 0 min, peak content was detected at 30 min, and significant reduction at 1 h and 3 h. CONCLUSION: These results implied a c-Myc mediated cell apoptosis pattern in the DEX- induced mouse thymocyte apoptosis model. c-Myc and caspase-3 signals may have feedback inhibitory regulation in this process.     

Effects of MAGEA3 inhibition by shRNA on apoptosis in human hepatocellular cancer cells

AIM:To investigate the inhibitory effect of vector-based RNA interference ( RNAi) on the expression of melanoma associated antigen A3 (MAGEA3) protein in hepatocellular carcinoma cells and on apotposis of hepatocellular carcinoma cells. METHODS:A vector for transcribing specific small hairpin RNA ( shRNA) targeting MAGEA3 gene was constructed ,introduced into hepatocellular carcinoma MEL-ED1 cells by Lipofectamine 2000. The MAGEA3 protein and mRNA expression levels of MEL-ED1 cells were detected by Western blotting and RT-PCR, respectively. The cell apoptosis was studied by DNA fragmentation, electron microscopy ，TUNEL assay, and annexin V/PI staining. RESULTS:The vector of RNA interference was successfully constructed and MAGEA3 expression was descreased significantly in MEL-ED1 cells. After the shRNA expression vector was transfected into the MEL-ED1 cells, the expression of MAGEA3 gene was inhibited significantly ( by 90% ). DNA fragmentation，electron microscopy and TUNEL assay showed classic apoptosis characters in the MEL-ED1 cells transfected with pSilencer-MAGEA3 plasmid with an apoptosis rate of 21.41% ±1.98%, significantly higher than those in the negative control group transfected with pSilencer-neo and in the non-transfected group (both P<0.01). CONCLUSION:The specific small hairpin RNA targeting MAGEA3 mRNA can inhibit the expression of MAGEA3 and cause apoptosis of hepatocellular carcinoma cells , which suggests inhibitory effect of MAGEA3 on apoptosis in cancer and provides an experimental basis for treating human tumors with RNAi.     

Bcl-2 antisense oligodeoxynucleotide increases the sensitivity of HL60 and K562 cells to daunorubicin

AIM:To investigate whether the bcl-2 antisense oligonucleotide increases the sensitivity of HL60 and K562 cell lines to daunorubicin.METHODS:IC50 for HL60 and K562 was determined with MTT method, the expression levels of Bcl-2 protein were assayed by immunofluorescence using fluoresce isothiocyanate labeling. In addition, apoptosis was detected by morphological observation and flow cytometric analysis of DNA fragmentation.RESULTS:It was found that the two oligonucleotides directed against the coding region and the translation initiation of bcl-2 mRNA, combined respectively with daunorubicin, inhibited expression of bcl-2 protein, increased apoptosis in HL60 and K562 cells, and decreased IC50 of daunorubicin significantly (P＜0.05). Compared to the antisense oligonucleotide directed against the translation initiation of bcl-2 mRNA, the antisense oligonucleotide directed against the coding region showed stronger effects in the aspects of increasing the sensitivity of HL60 cells to daunorubicin (P＜0.05).CONCLUSIONS:These two antisense sequences in the translation initiation and the coding region of bcl-2 mRNA increased the sensitivity of HL60 and K562 cell lines to daunorubicin in a sequence-specific manner.     

Effects of baicalin on CA46 cell proliferation inhibition and apoptosis induction

AIM: To investigate the effects of baicalin on proliferation inhibition and apoptosis induction in human Burkitt lymphoma cell line CA46 and to explore its underlying mechanisms. METHODS: CA46 cells were exposed to baicalin at different dosages and its proliferation inhibition was detected by MTT assay. The ability of baicalin to induce CA46 cell apoptosis was examined by Annexin V-FITC/PI double staining analysis, TUNEL labeling method and DNA fragmentation. The mRNA expressions of c-myc and bcl-2 were detected by RT-PCR, and the protein expressions of c-Myc, Bcl-2, caspase-3 precursor (procaspase-3) and poly ADP-ribose polymerase (PARP) were detected by Western blotting. RESULTS: Baicalin remarkably inhibited the CA46 cell proliferation, with an IC50 value of 10 μmol/L. Apoptosis was remarkably induced by baicalin in a dose-dependent manner, and its earlier and later stages were detected by annexin V-FITC/PI double staining analysis, TUNEL labeling method and DNA fragmentation, respectively. Furthermore, RT-PCR showed that the mRNA expressions of c-myc and bcl-2 in treated CA46 cells decreased in a time-dependent manner. Western blotting showed that the protein expressions of c-Myc, Bcl-2, procaspase-3 and PARP (116 kD) in baicalin treated CA46 cells were down-regulated in a time-dependent manner, while the expression of PARP(85 kD) was up-regulated. CONCLUSION: Baicalin efficiently induces proliferation inhibition and apoptosis in CA46 cells, which may be related with the down-regulation of c-Myc and Bcl-2 expressions, as well as the up-regulation of caspase-3 activity.     

Effect of extract of Oratosquilla oratoria on telomerase activity in human nasopharyngeal carcinoma cell line

AIM: To study the inhibitory effect and its mechanisms of the extract of Oratosquilla oratoria (EOS) on the activity of telomerase in human nasopharyngeal carcinoma cell line CNE-2Z. METHODS: MTT assay was used to determine the effect of different doses of EOS on the proliferation of CNE-2Z cells. The activity of telomerase was analyzed by TRAP-ELISA. The mRNA expression of hTERT was determined by RT-PCR, and the protein expression of c-Myc was detected by Western blotting. RESULTS: EOS inhibited the proliferation of CNE-2Z cells in a dose-dependent manner (P<0.01). Telomerase activity was decreased, the mRNA expression of hTERT and c-Myc in CNE-2Z cells was also decreased (P<0.01) by the treatment of EOS. The correlation between the down-regulatory expression of hTERT mRNA and inhibitory expression of c-Myc protein (P<0.05) under the condition of EOS exposure was observed. CONCLUSION: EOS inhibits the proliferation of CNE-2Z cells by reducing the activity of telomerase, which is related with the inhibitory expression of hTERT mRNA caused by the decrease in c-Myc production.     

Inhibition of miR-9 expression suppresses proliferation,invasion and migration of nasopharyngeal carcinoma cells

AIM: To investigate the effects of down-regulated miR-9 expression on the proliferation, invasion and migration of nasopharyngeal carcinoma (NPC) cells. METHODS: Human NPC CNE1 and CNE2 cells were transfected with the inhibitor of miR-9 by Lipofectamine to down-regulate the expression of miR-9, and the cells transfected with an inhibitor control were also set up. The cell proliferation and cell cycle were evaluated by CCK-8 assay and flow cytometry. The cell invasion and migration abilities were detected by Transwell invasion and wound-healing assays. Immunoblotting was applied to analyze the levels of the proteins. RESULTS: Compared with control group, inhibition of miR-9 expression in the NPC cells by transfection of the miR-9 inhibitor significantly decreased the proliferation ability (P<0.05). The percentages of the cells in G0/G1 phase ［CNE2: (57.96±1.39)% vs (47.93±1.76)%, P<0.05; CNE1: (51.24±0.88)% vs (48.29±0.39)%, P<0.05］ were significantly increased. The migration distances ［CNE2: (186.50±7.94)μm vs (247.56±15.56)μm, P<0.05; CNE1: (139.06±16.73)μm vs (230.66±14.27)μm, P<0.01］ and the invasion ability of the CNE2 cells (43.00±3.17 vs 65.80±5.20, P<0.01) were also significantly inhibited. Moreover, the tumor cells transfected with the inhibitors produced lower β-catenin. CONCLUSION: Inhibition of miR-9 expression suppresses the proliferation, invasion and migration of nasopharyngeal carcinoma cells.     

Effects of lentivirus-mediated caspase-3 silencing on proliferation and apoptosis of rat bone marrow mesenchymal stem cells

AIM：To investigate the effects of caspase-3 gene silencing on proliferation, cell cycle and apoptosis of rat bone marrow mesenchymal stem cells (MSCs). METHODS：A lentiviral vector expressing caspase-3 shRNA was constructed and transfected into rat bone marrow MSCs．The expression of caspase-3 at mRNA and protein levels was detected by real-time PCR and Western blotting, respectively. Cell proliferation and cell cycle were evaluated by MTS assay and flow cytometry, respectively. The expression of bcl-2 and bax mRNA was detected by real-time PCR. The apoptosis of the cells was evaluated by Hoechst 33258 staining. RESULTS：Recombinant lentivirus was successfully transfected into MSCs. The proliferation of the MSCs transfected with caspase-3 shRNA was significantly promoted (P<0.05) and the proportion of the cells in S phase was increased to (52.66±0.30) %. Compared with control groups, caspase-3 silencing up-regulated the mRNA level of bcl-2 and down-regulated the mRNA level of bax, and the ratio of bcl-2 to bax increased (P<0.05). The apoptotic rate in MSCs-shRNA group was (15.01±1.73) %, which was significantly lower than those in MSCs and MSCs-vector group ［(23.67±1.16) % and (25.67±3.05) %, respectively; P<0.05］. CONCLUSION: Caspase-3 silencing regulates cell cycle, promotes the proliferation and attenuates the apoptosis of rat bone marrow MSCs.     

Effect of hPTTG1 siRNA on proliferation and apoptosis of ovarian cancer cells

AIM: To observe the proliferation and apoptosis of ovarian cancer cells by silencing the expression of human pituitary tumor-transforming gene 1 ( hPTTG1 ) using RNA interference technique.METHODS: The chemically synthesized siRNA targeting hPTTG1 was transfected into ovarian cancer cell line A2780 in vitro. The expression levels of hPTTG1 and c-myc were examined by RT-PCR and Western blotting. Cell proliferation was measured by MTT colorimetric assay and -TdR incorporation test. Cell apoptosis was detected by flow cytometry with annexin V/PI and TUNEL labeling.RESULTS: The expression of hPTTG1 at mRNA and protein levels was inhibited after transfection of hPTTG1 siRNA. The inhibitory efficiency was 70.5%±3.9% and 63.8%±4.5%, respectively. The absorbance began to decrease 24 h after transfection of hPTTG1 siRNA,and the highest inhibitory rate was 42.9%±5.2% at 48 h post-transfection. Radioactive incorporation of -TdR in hPTTG1 siRNA group was lower than that in normal and negative groups. The survival rate declined while the apoptotic rate and necrotic rate increased in hPTTG1 siRNA group. Apoptotic index in hPTTG1 siRNA group was higher than that in normal and negative groups. The expression of c-myc at mRNA and protein levels was down-regulated.CONCLUSION: Cell proliferation is inhibited and cell apoptosis is induced by hPTTG1 siRNA through down-regulating the expression of c-myc. hPTTG1 can be regarded as a candidate gene for ovarian cancer gene therapy.     

Valproate enhances imatinib-induced apoptosis and down-regulates Bcr/Abl mRNA and phosphorylated protein kinase B in chronic myeloid leukemia cell line K562

AIM：To investigate the effects of valproate and imatinib on the apoptosis of chronic myeloid leukemic cell line K562. METHODS：K562 cells were divided into 3 groups and treated with valproate, imatinib and cotreatment, respectively. Cell cycle, apoptosis, the mRNA expression of Bcr/Abl, total protein kinase B (PKB) and phosphorylated PKB (p-PKB) were analyzed. RESULTS：The apoptotic rates in valproate group, imatinib group and cotreatment group were (11.47±0.25)%, (28.43±1.70)% and (57.73±4.38)%, respectively (P<0.05). No obvious difference was observed in cell cycle between cotreatment group and monodrug group. Bcr/Abl mRNA and p-PKB in the above 3 groups were (0.00±0.00), (64.17±12.27), and (0.00±0.00) ×10 9 copies/(g total mRNA), respectively (P<005), and 0.25±0.02, 0.17±0.01 and 0.08±0.01, respectively (P<0.05). No apparent difference of PKB was found in the 3 groups. CONCLUSION：Valproate enhances imatinib-induced apoptosis and may link to the down-regulation of Bcr/Abl mRNA and p-PKB in chronic myeloid leukemic cell line K562.     

Influence on killing effect of NK cells in vitro by up-regulation of HLA-E expression in hepatocellular carcinoma Bel7402 cells

AIM: To investigate the influence on the killing effect of NK cells in vitro by up-regulation of human leukocyte antigen-E (HLA-E) expression in hepatocellular carcinoma Bel7402 cells. METHODS: The recombinant lentiviral vector (Lentivirus/CMV/GFP-HLA-E) was constructed and transfected into hepatocellular carcinoma Bel7402 cells. The HLA-E gene expression at mRNA and protein level was monitored by the methods of real-time RT-PCR and Western blotting. The influence on the killing effect of NK cells in vitro by up-regulation of HLA-E expression in hepatocellular carcinoma Bel7402 cells and by HLA-ABC antibody blocking the site on the surface of target cells was analyzed.RESULTS: Real-time RT-PCR showed that there was a significant increase in HLA-E mRNA level in hepatocellular carcinoma Bel7402 cells transfected with Lentivirus/CMV/GFP-HLA-E at 24 h (P<0.05) and 48 h,72 h, 96 h (P<0.01) as compared to blank group. There was also a significant increase in exogenous/endogenous HLA-E proteins at 12 h, 24 h, 48 h, 72 h and 96 h by Western blotting (P<0.01). Without HLA-ABC antibody blocking, there was a statistical difference for the killing effect of NK cells,comparing Bel7402 Lenti HLA-E group with Bel7402 group (P<0.05). Comparing HLA-ABC antibody blocking group with no HLA-ABC antibody blocking group, a statistical difference for the killing effect of NK cells (P<0.05) was observed. There was also a statistical difference for the killing effect of NK cells between Bel7402 with blocking group and Bel7402 Lenti HLA-E with blocking group (P<0.05). CONCLUSION: The vector of Lentivirus/CMV/GFP-HLA-E has an active up-regulation effect in HLA-E mRNA level and HLA-E protein level. While up-regulation of HLA-E in target cells, the killing effect of NK cells on target cells is obviously weakened. Blockage of the sites on the surface of target cells by HLA-ABC antibody universally enhances the killing effect of NK cells on the target cells.     

Effect of CD97 gene silencing by small interfering RNA on migration and invasion of gastric carcinoma cell lines

AIM: To investigate the effect of CD97 gene silencing by small interfering RNA(siRNA) on migration and invasion of gastric carcinoma cell lines. METHODS: Gastric carcinoma cell lines AGS and MGC803 were used in the study. Four pairs of siRNA were designed according to the sequence of CD97 gene and synthesized chemically. The siRNAs were transfected into the gastric carcinoma cell lines. Forty-eight hours after transfection, the total RNA was extracted and the mRNA expression of CD97 was detected by real-time RT-PCR so as to screen the most effective siRNA. The protein level of CD97 was also measured by fluorescence-activated cell sorting (FACS) 72 h after Transfection. The abilities of migration and invasion were evaluated by Transwell test. The viability of the cells was measured by MTT method. RESULTS: Real-time RT-PCR and FACS revealed that CD97-siRNA notably down-regulated CD97 expression at both mRNA and protein levels. The mRNA level decreased by (89.34±9.95)% and (95.42±1.93)% in AGS and MGC803 cells,respectively. The protein levels of CD97^EGF and CD97^stalk in AGS cells decreased by (19.29±3.45)% and (30.11±5.93)%,respectively. The protein levels of CD97^EGF and CD97^stalk in MGC803 cells decreased by (26.25±5.73)% and (16.22±3.23)%,respectively. No change of the cell viability after siRNA transfection was observed. The cell number of migration and invasion in AGS cells was decreased by (67.63±12.03)% and (68.02±15.63)%,respectively. The cell number of migration and invasion in MGC803 cells was decreased by (14.92±2.03)% and (22.09±5.43)%,respectively. CONCLUSION: The siRNA effectively inhibits CD97 expression and restrains the migration and invasion capacities of gastric carcinoma cell lines, suggesting that CD97 plays an important role in the metastasis of gastric cancer.     

Berberine promotes apoptosis of human breast cancer cells by regulating miRNA-146a

ATM: To observe the effect of berberine on apoptosis of MCF-7 cells and its potential mechanism. METHODS: The MCF-7 cells were divided into control group and the groups with 3 different doses of berberine. The cell viability was detected by MTT assay, while the cell apoptosis was measured by Hoechst 33258 staining and flow cytometry assay. The protein levels of p-P65, Bax and Bcl-2 were Western blot. The levels of microRNA-146a(miRNA-146a) in the MCF-7 cells were detected by RT-qPCR. The miRNA-146a siRNA was transfected to the MCF-7 cells after an evaluation of transfection efficacy, which was co-incubated with berberine to observe its effects on the mRNA levels of Bax and Bcl-2. RESULTS: Compared with control group, the cell viabilities were decreased significantly in medium and high doses of berberine treatment groups with a dose-dependent manner (P<0.01). The cell apoptosis was increased significantly in medium and high doses of berberine treatment groups dose-dependently (P<0.05). The protein levels of Bax were up-regulated, while those of Bcl-2 and p-P65 were down-regulated significantly by the treatment of berberine (P<0.05). In addition, the miRNA-146a levels were increased significantly in medium and high doses of berberine treatment groups (P<0.05) and showed a dose-dependent manner. The mRNA levels of Bax were decreased, while the mRNA levels of Bcl-2 were increased after transfection with miRNA-146a siRNA and co-incubated with berberine.CONCLUSION: Berberine promotes apoptosis of MCF-7 cells. The mechanism may be related to inhibit the activity of NF-κB by incresing the levels of miRNA-146a.     

Effects of folic acid and vitamin B12 on human umbilical vein endothelial cells against homocysteine-induced apoptosis

AIM: To investigate the effect of folic acid and vitamin B₁₂ on homocysteine (Hcy)-induced apoptosis of human umbilical vein endothelial cells (HUVECs) through mammalian sterile 20-like kinase 1 (MST1). ME-THODS: HUVECs were cultured in the absence (control group), or presence of 100 μmol/L Hcy alone (Hcy group) or 100 μmol/L Hcy plus 30 μmol/L folic acid and vitamin B₁₂ (intervention group) for 72 h. The effect of Hcy on the apoptosis of HUVECs was analyzed by flow cytometry. The transfection efficiency of DNA methyltransferase 1 (DNMT1)-overexpressing adenovirus was observed under fluorescence inverted microscope. The mRNA and the protein levels of DNMT1 and MST1 were determined by RT-qPCR and Western blot. The DNA methylation level of MST1 promoter was detected by methylation-specific PCR. RESULTS: Compared with control group, the apoptotic rate (P<0.01) and the expression of MST1 at mRNA (P<0.01) and protein (P<0.05) levels in the HUVECs were significantly increased, while the mRNA levels of DNMT1 was decreased in Hcy group (P<0.01). In addition, folic acid and vitamin B₁₂ treatment significantly inhibited Hcy-mediated apoptosis of HUVECs (P<0.01), increase in MST1 mRNA level (P<0.01) and decrease in DNMT1 mRNA level (P<0.01). Meantime, the mRNA level of MST1 was positively correlated with the apoptotic rate of the HUVECs (r=0.943 9, P<0.001). The expression of DNMT1 at mRNA and protein levels was significantly increased after the transfection of DNMT1-overexpressing adenovirus into HUVECs (P<0.01), and a large amount of green fluorescent protein expression was observed. Meanwhile, the DNA methylation level of MST1 promoter was increased (P<0.01), while the protein level of MST1 was decreased (P<0.01).CONCLUSION: Up-regulation of MST1 promotes Hcy-induced apoptosis of HUVECs, while folic acid and vitamin B₁₂ exert an anti-apoptosis effect, which might be regulated by hypermethylation of MST1 promoter region.     

Effects of Smo gene silencing on cell activity and apoptosis of human cervical carcinoma HeLa cells

AIM: To investigate the effect of Hedgehog (Hh) signaling pathway on the viability and apoptosis of cervical carcinoma cells by shRNA technique to knock down Smoothened (Smo) gene. METHODS: Smo shRNA was used to transfect the cervical carcinoma HeLa cells. The expression of Smo and Gli1 at mRNA and protein levels in the HeLa cells was determined by RT-PCR and Western blot, respectively. The effect of Smo gene silencing on the growth of the cells was measured by MTT assay. The apoptosis and cell cycle were determined by flow cytometry. RESULTS: Compared with control group, the mRNA and protein expression of Smo and Gli1 were evenly reduced obviously after transfected with Smo shRNA for 72 h (P<0.05). The viability of HeLa cells transfected with Smo shRNA was significantly inhibited. The percentages of the cells in G₀/G₁ phase and early apoptosis rate were obviously higher in Smo shRNA transfection group than those in control group. CONCLUSION: Smo gene silencing effectively inhibits the cell growth and induces the apoptosis of human cervical carcinoma cells.     

Relationship between expression level of TIMP-3 and invasion/metastasis of hepatocellular carcinoma

AIM: To detect the levels of tissue inhibitor of metalloproteinase-3 (TIMP-3) in both plasma and the tissue of hepatocellular carcinoma (HCC), and to elucidate their association with clinical features.METHODS: Plasma protein levels of TIMP-3 in 56 HCC patients and 30 cases of controls were detected by ELISA.The mRNA and protein levels of TIMP-3 in 30 HCC tissue samples with their portal vein tumor embolus and lymphatic metastasis tissues, and in normal liver tissues from 30 controls were detected by RT-PCR and Western blotting.The relationship between mRNA and protein levels and their clinic-pathological data were analyzed.RESULTS: The plasma TIMP-3 protein levels in the extrahepatic metastasis patients were obviously lower than those in the non-extrahepatic metastasis patients (P<0.05).The mRNA levels of TIMP-3 in normal liver, carcinoma in situ, portal vein tumor embolus and lymphatic metastasis tissues were 0.78±0.09, 0.52±0.09, 0.42±0.07 and 0.40±0.08, respectively, with significant differences among them (P<0.05).The protein levels of TIMP-3 in these 4 kinds of tissues were 115.08±8.60, 77.04±8.83, 64.43±3.80 and 62.80±3.73, respectively, also with significant differences among them (P<0.05).CONCLUSION: The expression of TIMP-3 significantly decreases in the carcinoma in situ tissues of HCC patients, and decreases more obviously in the portal vein tumor embolus and lymphatic metastasis tissues, indicating that low expression of TIMP-3 may play an important role in HCC invasiveness and metastasis.     

Apoptosis of the prostate cancer cell line PC-3M induced by E2F decoy DNA

AIM：To observe the effect of E2F decoy DNA on proliferation and apoptosis of androgen-independent prostate cancer cell line PC-3M.METHODS：E2F decoy DNA,ARE decoy DNA and control decoy DNA were transfected into PC-3M cells with lipofectamine,respectively.Their effects on cell proliferation were detected by MTT assay.The changes of cell morphology were observed by inverted phase contrast microscope.The cell apoptotic rate was determined by flow cytometry (FCM) analysis and chromosome DNA ladder was detected by DNA gel electrophoresis.The expression of c-Myc mRNA and cyclin D1 mRNA was detected by RT-PCR.The protein levels of c-Myc and cyclin D1 were detected by Western blotting.RESULTS：The growth of PC-3M cells was inhibited after transfection.The transfected PC-3M cells displayed typical apoptotic morphological changes.The apoptotic rate was 26.35% and DNA ladder was observed after transfection.The expression of c-Myc and cyclin D1 were inhibited.CONCLUSION：These results indicate that E2F decoy DNA induces apoptosis of androgen-independent prostate cancer cell lines PC-3M and inhibits cell proliferation via inhibiting expression of c-Myc and cyclin D1.     

miRNA-7-mediated Bax/Bcl-2 expression promotes apoptosis of human nasopharyngeal carcinoma CNE-1 cells

AIM: To investigate the relationship of microRNA-7 (miRNA-7) over-expression and Bax/Bcl-2 expression in human nasopharyngeal carcinoma CNE-1 cells.METHODS: The CNE-1 cells were transfected with miRNA-7 mimics using Lipofectamine 2000. The expression of miRNA-7 was detected by real-time PCR. CCK-8 assay and Hoechst 33258 staining were used to detect the cell activity and apoptosis. The expression of Bax/Bcl-2 at mRNA and protein levels was determined by real-time PCR and Western blot. RESULTS: The expression of miRNA-7 was increased significantly in the CNE-1 cells compared with negative control group and mock group (P<0.01). The activity of CNE-1 cells were extremely decreased after tansfected with miRNA-7 mimics (P<0.01). The typical apoptotic nuclear morphological changes were observed in the CNE-1 cells under the fluorescence microscope with Hoechst 33258 staining. The expression of Bax at mRNA and protein levels was significantly increased compared with the other 2 groups (P<0.01), while the Bcl-2 expression at mRNA and protein levels was significantly down-regulated (P<0.01).CONCLUSION: Over-expression of miRNA-7 significantly inhibits the growth and promotes the apoptosis of nasopharyngeal carcinoma CNE-1 cells by increasing the expression of Bax and down-regulating Bcl-2.     

Effect of microRNA-100 on proliferation activity and cell cycle of hepatocarcinoma cells

AIM：To investigate the effect of microRNA-100 (miR-100) on the proliferation activity and cell cycle of hepatocarcinoma cells. METHODS：Synthetic miR-100 mimic and its negative control were transfected into human hepatocarcinoma HepG2 cells by liposome method. After transfection, the cell counting kit-8 (CCK-8) was used to measure the cell proliferation activity. The cell cycle distribution was determined by flow cytometry. The expression of Polo-like kinase 1 (Plk1) at mRNA and protein levels was detected by quantitative real-time PCR (qRT-PCR) and Western blotting. RESULTS：The transfection efficiency mediated by cationic liposome was greater than 85%. The inhibitory rates of cell proliferation in HepG2 cells were (43.5±12.2)%, (46.5±3.7)% and (52.1±0.2)% at 24 h, 48 h and 72 h after transfected with miR-100 mimic, respectively, which were significantly increased as compared with the control cells. Moreover, the cell proliferation index in experimental group (35.8 ± 1.4) was higher than that in negative control group (39.2 ± 1.0) and simple liposome group (40.7 ± 2.0) at 72 h. At the same time, the mRNA and protein expression levels of Plk1 obviously decreased in HepG2 cells transfected with miR-100 at 72 h after transfection. CONCLUSION：miR-100 suppresses the proliferation activity of hepatocarcinoma cells by down-regulating Plk1 gene expression.