Inhibiting effect and its mechanism of BDH2 gene on the proliferation of liver cancer cells
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Abstract:
Objective
To investigate the inhibiting effect and its mechanism of β-hydroxybutyrate dehydrogenase 2 (BDH2) gene on the proliferation of liver cancer cells.
Methods
The lentiviral vectors expressing BDH2 were constructed. The liver cancer cell line HepG2-BDH2 stably expressing BDH2 (experimental group) and the control cell line HepG2-Vector (control group) were established. The expression levels of BDH2 mRNA in two groups were detected by RT-PCR. The proliferation of liver cancer cells in two groups was detected by CCK-8 assay. The colony formation ability of cells was observed in two groups by colony formation assay. The expression of BDH2 protein and apoptosis-associated protein Bcl-2 was detected by Western blot. The experimental data were compared using t test.
Results
In experimental group, the expression level of BDH2 mRNA was (2.20±0.10)×10-3, which was significantly higher than (0.20±0.01)×10-3 in control group (t=34.95, P<0.05). In experimental group, the A450 values at 3, 4, 5, 6 and 7 d after cell culture were 0.55±0.20, 0.73±0.02, 1.26±0.12, 1.62±0.14 and 2.19±0.12, which were significantly lower than 0.70±0.06, 1.13±0.08, 1.77±0.15, 2.45±0.12 and 3.02±0.15 in control group (t=-5.19, -11.34, -5.96, -10.35, -9.54; P<0.05), respectively. The cell proliferation curve showed that the proliferation of cells in experimental group was significantly weaker than that in control group. Colony formation assay indicated that the number of cell clones in experimental group was 184±7, which was significantly less than 429±15 in control group (t=-25.84, P<0.05). Compared with control group, the expression levels of BDH2 and cleaved caspase-3 protein in experimental group were up-regulated significantly, whereas the expression level of Bcl-2 protein was down-regulated significantly.
Conclusions
BDH2 gene can inhibit the proliferation of liver cancer cells probably through promoting the apoptosis of liver cancer cells via Bcl-2 signaling pathway.
Key words:
Carcinoma, hepatocellular; Cell proliferation; Apoptosis; BDH2 gene; Bcl-2Keywords:
Liver Cancer
To explore the influence of micro ribonucleic acid (miR)-101 on breast cancer cell proliferation and apoptosis via nuclear factor (erythroid-derived 2)-like 2 (Nrf2) signaling pathway.All MCF-7 cells were divided into 3 groups, namely control group, miR-101 mimic group (the cells were treated with 50 nmol/L miR-101 mimic), and miR-101 inhibitor group (the cells were treated with 50 nmol/L miR-101 inhibitor). The impact of miR-101 expression level on MCF-7 cell proliferation was evaluated via cell counting kit-8 (CCK-8) and colony formation assays. After the MCF-7 cells in the three groups were treated with 100 nM H2O2 for 12 h, the change in the apoptosis rate was detected via flow cytometry. Moreover, the influence of miR-101 expression level on the Nrf2 signaling pathway was detected via reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting.According to the CCK-8 assay results, compared with that in control group, the proliferation rate of cells notably declined at 48, 72, and 96 h in miR-101 mimic group, and the difference was statistically significant (p<0.01), while it was substantially raised in miR-101 inhibitor group, showing a statistically significant difference (p<0.01). Compared that in control group, the cell colony formation rate was remarkably lowered in miR-101 mimic group, and the difference was statistically significant (p<0.01), while it was substantially raised in miR-101 inhibitor group (p<0.01). According to the flow cytometry assay results, compared with that in control group, the apoptosis of MCF-7 cells was markedly enhanced in miR-101 mimic group, showing a statistically significant difference (p<0.01), while it was weakened in miR-101 inhibitor group, with a statistically significant difference (p<0.01). The influence of miR-101 on the expression level of Nrf2 was detected via RT-PCR, and it was found that the messenger RNA (mRNA) expression level of Nrf2 was notably lower in miR-101 mimic group than that in control group (p<0.01), while it was raised in miR-101 inhibitor group. Western blotting results showed that compared with control group, miR-101 mimic group had a substantially lowered protein expression level of Nrf2 in the cell nucleus, with a statistically significant difference (p<0.01), while it was notably raised in miR-101 inhibitor group and the difference was statistically significant (p<0.01), indicating that miR-101 can remarkably lower the nucleoprotein expression level of Nrf2.The results of this study imply that miR-101 can inhibit the expression of Nrf2 to suppress the proliferation of breast cancer cells and enhance their sensitivity to oxidative stress, which provides a theoretical basis for reversal of tumor resistance.
Cell counting
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Objective To study the expression difference of family with sequence similarity 96,member B (FAM96B) in human hepatocellular carcinoma (HCC) and its impact on the proliferation and apoptosis of HepG2 cell line.Methods FAM96B mRNA and protein expression was examined respectively by real-time fluorogenic quantitative polymerase chain reacton (FQ-PCR) and Western blotting in tumor tissues and adjacent tissues from 42 patients with HCC.The FAM96B mRNA and protein expression was detected in HepG2 and L-02 cells.Moreover the eukaryotic vector of human FAM96B was constructed and transfected into HepG2 cells.The proliferative rate and apoptosis rate of HepG2 cells were examined by the methyl thiazol tetrazolium (MTT) assay and fluorescence-activated cell sorting (FACS).Results In 42 patients with HCC,the global mRNA expression of FAM96B was 0.304 ± 0.094 times in tumor tissues than in adjacent tissues.And its mRNA expression was 0.406 ±0.115 times in HepG2 cell than in L-02 cell.While the Western blotting analysis confirmed that FAM96B protein expression in tumor tissues was 0.288±0.132 times lower than in the adjacent tissues (P ＜ 0.05).And FAM96B protein expression in HepG2 cell was 0.359 ± 0.143 times lower than in L-02 cell.The MTT assay showed that the A value inFAM96B group was lower than in control group,and overexpression of FAM96B significantly inhibited proliferation of HepG2 cells (P ＜0.05).FACS analysis indicated that there was significant difference in cell apoptosis rate between FAM96B-expressing clones group and the control group (vector-transfected cells group) (P ＜ 0.05).Conclusion FAM96B expression showed statistically significant difference between tumor tissues and adjacent tissues in 42 cases of HCC.Moreover FAM96B expression in HepG2 cells was significantly lower than in L-02 cells (P ＜ 0.05).Furthermore,overexpression of FAM96B can induce apoptosis of HepG2 cells and inhibit proliferation of HepG2 cells.The data suggest that FAM96B probably plays an important role in carcinogenesis of HCC.
Key words:
Carcinoma, hepatocellular; Family with sequence similarity 96, member B; Proliferation
Cell Sorting
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Objective
To investigate the activation effect of microRNA-1280 (miR-1280) on the expression of p21 gene in bladder cancer cell line BIU-87 and its effect on cell cycle and proliferation of bladder cancer cell line.
Methods
Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the expressions of miR-1280 in bladder cancer cell lines T24, 5637, J82, BIU-87 and normal bladder epithelial cells SV-HUC-1. miR-1280 mimics (experimental group) and miR-NC (control group) were transfected into the bladder cancer cells with the lowest expression of miR-1280. The expressions of miR-1280 and p21 mRNA were detected by qRT-PCR. Chromatin immunoprecipitation (ChIP) was used to verify the targeting effect of miR-1280 and p21 gene promoter. Western blotting was used to detect the expressions of p21, cell cycle-dependent kinase 1 (CDK1), Cyclin A2 mRNA and protein in the two groups. Cell cycle was detected by flow cytometry, and cell proliferation was detected by methyl thiazolyl tetrazclium (MTT) assay.
Results
The results of qRT-PCR indicated that the expression levels of miR-1280 in bladder cancer cell lines T24, 5637, J82 and BIU-87 and normal urothelium cell line SV-HUC-1 were 0.503±0.094, 0.611±0.054, 0.567±0.077, 0.257±0.032 and 1.014±0.090 respectively, with a significant difference (F=1.880, P<0.001). Compared with bladder cancer cell lines T24, 5637 and J82 cells, the expression of miR-1280 in BIU-87 cell was the lowest (P=0.026, P=0.003, P=0.008). Compared with the control group, the expre-ssion of miR-1280 in BIU-87 cell was significantly increased (1 041.000±157.500 vs. 1.023±0.118, t=6.606, P<0.001), and the expression of p21 mRNA was also significantly increased (5.280±0.660 vs. 1.007±0.070, t=6.440, P<0.001). Western blotting showed that p21 protein expression was up-regulated, CDK1 and Cyclin A2 protein expressions were down-regulated. ChIP experiments showed that compared with the miR-NC transfection group, the concentration of biotin modified miR-1280 in the p21 gene promoter region was significantly increased (1.246±0.171 vs. 0.519±0.087, t=3.787, P=0.009). The proportion of G0-G1 cells in the experimental group BIU-87 cells was significantly higher than that in the control group (68.360%±3.064% vs. 46.970%±3.971%, t=4.263, P=0.005). The results of MTT showed that compared with the control group, the cell proliferation ability of BIU-87 cells after being transfected miR-1280 was significantly decreased starting from day 3 (0.826±0.099 vs. 1.224±0.057, t=3.505, P=0.013).
Conclusion
miR-1280 can activate the expression of p21 gene in bladder cancer cell line BIU-87 by binding the promoter region of p21 gene, blocking the progression of cell cycle and inhibiting cell proliferation, which provides a new direction for bladder cancer targeted therapy theory.
Key words:
MicroRNAs; Urinary bladder neoplasms; Proto-oncogene proteins p21 (ras); RNA activation
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Objective: To observe the phenomenon of apoptosis and the change of the expression of p53 and bcl-2 genes induced by β amyloid peptide 25-35 (Aβ 25-35 ) in PC12 cell line. Methods: After the treatment of PC12 cells with different concentrations of Aβ 25-35 (0, 5, 10, 20 μmol/L)for 24h,the MTT assay was used to evaluate the cell survival rate, the Hoechst-PI fluorescence staining and DNA Ladder-agarose gel electrophoresis were used to observe the apoptosis status, and the RT-PCR and western blot were used to detect the expression of p53 and bcl-2 genes at mRNA and protein levels, respectively. Results: In a dose-dependent manner from the low dosage to the high dosage, Aβ 25-35 reduced the survival rate of PC12 cells, induced apoptosis, increased the mRNA and protein levels of p53 gene, and decrease the mRNA and protein levels of bcl-2 gene. Conclusion: Aβ 25-35 might induce the apoptosis of PC12 cells by up-regulating the expression of p53 gene and down-regulating the expression of bcl-2 gene.
Agarose gel electrophoresis
MTT assay
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It has been reported that microRNA‑142 (miR‑142) is a tumor suppressor gene. The present study primarily investigated whether the overexpression of miR‑142 was able to inhibit the proliferation, apoptosis and expression of apoptosis‑associated proteins in osteosarcoma (OS) cells. Different concentrations of miR‑142 were transfected into the OS MG‑63 cell line using Lipofectamine 2000. The cell lines were divided into three groups: Normal group (non‑transfected group), miR‑142 transfected group, and negative group, which were transfected with random miR‑142 fragment. The proliferation of cells was detected by MTT assay. The expression of miR‑142 was detected by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). DAPI staining was performed to investigate the influence of miR‑142 on the morphology of MG‑63c ells. The apoptotic cell percentages were determined by flow cytometry with Annexin V‑fluorescein isothiocyanate/propidium iodide double staining. Expression of tumor suppressors, phosphatase and tensin homolog (PTEN) and Retinoblastoma‑associated protein (Rb), and apoptosis‑associated proteins were evaluated by western blotting. RT‑qPCR indicated a higher expression of miR‑142 in the transfected group (miR‑142 was transfected into the MG‑63 cell line) compared with that in the normal (non‑transfected group) and negative control groups. The proliferation of miR‑142 transfected cells was significantly lower compared with that in the normal and negative groups. Furthermore, an increased apoptosis rate accompanied by a statistically significant upregulation of PTEN, Rb phosphorylation, cleaved caspase‑3 and cytochrome c protein levels were detected in the transfected group, indicating an internal apoptosis pathway was involved in this process. Furthermore, no significant changes were identified between the normal and negative groups (P>0.05). The present study demonstrated that miR‑142 overexpression by liposomal transfection resulted in an inhibitory effect on MG‑63 cell proliferation. The underlying mechanisms may relate to the upregulation of tumor suppressor and activation of caspase signaling pathway, which may provide a novel horizon in short nucleotide drugs on the management of OS.
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The functions of miR-126-mediated signal transducers and activators of the transcription 3 (STAT3) signal pathway were investigated in regulating the behavior of cells in non-small cell lung cancer (NSCLC). Cultured NSCLC A549 cells were transfected with empty, miR-126 overexpression or miR-126 knocked-down expression plasmids. After transfection efficiency verification by reverse transcription polymerase chain reaction (RT-PCR) and culture for 24 h, methyl thiazolyl tetrazolium (MTT) was applied to detect cell proliferation rate, migration distance was measured in scratch assays, cell cycle was determined through flow cytometry, the mRNA expression level of caspase-3 in cells was detected using RT-PCR and protein expression levels of STAT3 were detected using western blotting. Our results showed the cell proliferation rate was significantly higher in cells of the overexpression group than that in those of the control group (p<0.05) and the rate in the cells of the low-expression group was the lowest among the three groups (p<0.05). The migration distance of the overexpression group cells was significantly longer than that in the control group cells and the shortest migration distance was found in the low-expression group cells (p<0.05). The amount of cells in mitotic phase in the overexpression group was significantly higher than that in the control group and the same amount in the low-expression group was the lowest (p<0.05). The mRNA expression level of caspase-3 of cells in the overexpression group was significantly lower than that of cells in the control group and the highest expression level was found in the low-expression group (p<0.05). Finally, the protein expression levels of STAT3 in cells in the overexpression group were significantly lower than those in the control group and the highest expression levels were identified in the low-expression group (p<0.05). Based on our findings, the cancer-promoting miR-126 can mediate the activation of the STAT3 signal pathway to regulate the malignant biological behavior of NSCLC cells affecting their proliferation, migration, cycle and apoptosis susceptibility.
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The aim of this study was to evaluate the effects of small interfering RNA (siRNA)-inhibited expression of the Sal-like 4 (SALL4) gene on the proliferation, colony formation, and apoptosis of prostate cancer C4-2 cells.C4-2 cells were cultured and divided into a si-SALL4 group, a negative control siRNA group, and a blank control group.SALL4 mRNA levels and protein expression were detected by real-time polymerase chain reaction and western blot, respectively.Changes in the cell proliferation and colony formation capacities were observed by using the MTS colorimetric method and colony formation assay, respectively.The influence of SALL4 on apoptosis was assessed with flow cytometry, and the expression of apoptosis-related proteins B-cell lymphoma 2 (Bcl-2) and bcl-like-protein 4 (Bax) were detected by western blot.The si-SALL4 group had significantly lower mRNA and protein levels of SALL4 as well as decreased proliferation and colony formation capacities than the negative control group (P < 0.05).There were significantly more apoptotic cells in the si-SALL4 group compared to the negative control (P < 0.05), and the expression of Bcl-2 and Bax decreased and increased, respectively, after treatment with ©FUNPEC-RP www.funpecrp.com.brGenetics and Molecular Research 15 (2): gmr.15027885 si-SALL4.Silencing SALL4 expression by using siRNA technology inhibited the proliferation and colony formation of C4-2 cells, and promoted apoptosis likely mediated by Bcl-2 and Bax expression.These results provide experimental basis for further elucidating the role of SALL4 in prostate cancer cells.
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Objective
To investigate the regulatory effect of miRNA-370 (miR-370) on the expression of tumor suppressor gene p21 in renal cell carcinoma cell lines ACHN and 786-O and its effect on cell growth.
Methods
RCC cells were transfected with dsRNA known lack homology to human genes (control group) and miR-370 (experimental group) by Lipofectamine 3000 respectively. Real-time fluorescence quantitative polynucleotide chain reaction (RT-qPCR) and Western blot were used to detect the expression of p21 mRNA and protein. The cell cycle distribution was identified by flow cytometry (FCM). Cell viability and proliferation ability were measured by cell viability assay (MTS) and colony culture assay.
Results
The expression of p21 mRNA in ACTN and 786-O cells in control group was 1.04 ± 0.33, 1.04 ± 0.31, respectively. The expression of p21 mRNA in experimental group was significantly increased by 3.68 ± 0.62 (t= 7.535, P < 0.001), 3.15 ± 0.29 (t= 9.975, P < 0.001). Western blot further demonstrated that the increased expression of p21 protein in both renal cell lines was consistent with the upregulation of p21 mRNA level. FCM results showed that the cell cycle of more cells was blocked in G0-G1 phase after transfection of miR-370. MTS results showed that after transfection of miR-370, the number of colonies formed by ACHN and 786-O cells in the control group was 113 ± 30 and 106 ± 27 respectively. The number of colonies formed by experimental group was significantly reduced by 53 ± 17 (t= 2.982, P= 0.041) and 50±16 (t= 3.089, P= 0.037).
Conclusion
miR-370 can significantly up-regulate the expression of tumor suppressor gene p21 in renal cell carcinoma and inhibit the growth of renal cell carcinoma.
Key words:
Kidney neoplasms; MicroRNAs; Oncogene protein p21 (ras)
Lipofectamine
Viability assay
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The aim of the present study was to examine the effect of fatty acid binding protein-5 (FABP-5) gene on the proliferation, apoptosis and invasion of human gastric cancer SGC-7901 cells. The viability, apoptosis and cell invasion of SGC-7901 cells before and after FABP5 knockdown were taken as the study objects, design and synthesis of siRNA interference sequence were conducted according to FABP-5 mRNA coding sequences, and SGC-7901 cells were transiently transfected. The human gastric cancer SGC-7901 cells were divided into three groups: FABP-5 siRNA group, negative control group and blank control group. FABP-5 gene mRNA and protein expression levels were detected by RT-PCR and western blot analysis. The CCK-8 assay was used to detect in vitro cell proliferation, flow cytometry (FCM) was used to detect changes in cell cycle and apoptosis in each group, TUNEL staining was used to detect apoptosis in each group, and the cell invasion chamber assay was used to detect cell invasiveness in each group. Each test was repeated three times. The results of the RT-PCR and western blot analysis showed that, expression of FABP-5 mRNA and protein in the FABP-5 siRNA group was significantly decreased compared with the negative and blank control groups. The cell growth rate in the FABP-5 siRNA group was significantly retarded, cell cycle was arrested in G0/G1 phase, the number of cells in S phase was reduced, and compared with the negative and blank control groups, the apoptotic rate in the FABP-5 siRNA group was significantly increased (P<0.01), while proliferation and invasiveness were significantly decreased (P<0.05). In conclusion, specific FABP-5 gene silencing may reduce the invasiveness of gastric cancer cells, inhibit cell proliferation, and arrest cell cycle in G0/G1 phase, resulting in a significant increase in apoptosis.
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Objective
To investigate the expression of prostate cancer-associated transcript 6 (PCAT6) in the serum of hepatocarcinoma patients and its effect on the proliferation, migration, invasion and apoptosis of Hep3B cells.
Methods
The qPCR method was used to detect the level of long non-coding RNA (lncRNA) PCAT6 in serum and THLE-3, Hep3B and HepG2 cells. The Hep3B cells were randomly divided into blank control group (NC group), negative empty vector transfection group (si-con group) and lncRNA PCAT6 silencing group (si-PCAT6 group). Western blotting was used to detect the expression of matrix metalloproteinase (MMP)-2, MMP-9, cleaved cysteinyl aspartate-specific protease (Caspase)-3, cleaved Caspase-9, phosphatidylinositol 3 kinase (PI3K), phosphorylated-PI3K (p-PI3K), protein kinase B (Akt), and phosphorylated Akt (p-Akt) proteins. The cell proliferation was measured by methyl thiazol tetrazolium (MTT) assay. The apoptosis was examined by flow cytometry, and the migration and invasion was tested detected by Transwell chamber method. The SPSS 21.0 software was used for statistical analysis, and the measurement data were expressed as mean±standard deviation (SD).
Results
The expression level of lncRNA PCAT6 in patients with moderate or high differentiation (60.94%) was significantly higher than that in patients with low differentiation (6.25%) (χ2=22.968, P<0.01). The percentage of patients with high expression of lncRNA PCAT6 in T-stage Ⅱ-Ⅳ (57.81%) was significantly higher than that in stage Ⅰ (9.38%) (χ2=8.529, P<0.01). The expression of lncRNA PCAT6 in Hep3B cells (3.72±0.67) and HepG2 cells (3.38±0.53) was significantly higher than that in THLE-3 cells (1.03±0.14) (t=9.322 and 8.144, P<0.05). The expression of lncRNA PCAT6 (0.21±0.11) in si-PCAT6 group was significantly lower than that in si-CON group (0.96±0.15) (t=9.915, P<0.05), and the cell viability was significantly reduced at 24 h, 48 h and 72 h (t=3.280, 6.144 and 6.373, P<0.05). Western blotting showed that the expression of cleaved Caspase-3, cleaved Caspase-9, p-pi3k and p-Akt increased significantly (t=11.408, 14.628, 8.683 and 9.585, P<0.01), and the expression of MMP-2 and MMP-9 decreased significantly in si-PCAT6 group (t=10.568 and 10.814, P<0.01). The apoptosis rate of Hep3B cells increased significantly (t=21.075, P<0.01), and the migration and invasion of Transwell cells decreased significantly (t=12.816 and 12.707, P<0.01).
Conclusion
The serum lncRNA PCAT6 is highly expressed in HCC patients, which is related to the degree of HCC differentiation and T stage. Silencing lncRNA PCAT6 can inhibit the biological behaviors of HCC probably by inhibiting the Akt signaling pathway activation.
Key words:
Long non-coding RNA; Prostate cancer-associated transcript 6; Liver cancer; Biological function
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