Expression Levels Of Livin Research Articles

MiR-20a lowers chemosensitivity of liver cancer Huh-7 cells via regulating NF-кB expression.

The aim of this study was to explore the regulatory effect of micro ribonucleic acid (miR)-20a on nuclear factor-κB (NF-кB) in liver cancer Huh-7 cells, and to elucidate its influence on the chemosensitivity of Huh-7 cells. Huh-7 cells with overexpression of miR-20a or knockout of miR-20a were first constructed. Quantitative polymerase chain reaction (qPCR) was adopted to detect the expression level of miR-20a in each group of cells. The sensitivity of cells to cisplatin and doxorubicin in each group was measured using methyl thiazolyl tetrazolium (MTT) assay, and the 50% inhibitory concentration (IC50) was calculated. Hoechst 33258 staining was performed to detect the apoptosis of cells in each group. Furthermore, the expression levels of apoptosis-associated proteins and the NF-кB signaling pathway-related proteins in each group of cells were determined via Western blotting. The expression level of miR-20a in blank control group was considerably higher than that in knockout group (p<0.01). Meanwhile, cells in overexpression group exhibited a notably higher expression level of miR-20a than blank control group (p<0.01). Cells in knockout group had dramatically enhanced sensitivity to doxorubicin and cisplatin (p<0.01), with a prominently decreased IC50 value (p<0.01). However, cells in overexpression group exhibited remarkably weakened sensitivity (p<0.01) and increased IC50 value (p<0.01). After treatment with doxorubicin and cisplatin, the apoptosis level of cells rose substantially in knockout group (p<0.01), whereas declined significantly in overexpression group (p<0.01). Moreover, knockout group exhibited a notably elevated expression level of Caspase-3 (p<0.01), and a considerably decreased ratio of B-cell lymphoma 2 (Bcl-2)/Bcl-2 associated X protein (Bax) (p<0.01). The expression level of Caspase-3 declined remarkably (p<0.01), however, the ratio of Bcl2/Bax increased substantially (p<0.01) in overexpression group. The expression level of NF-кB inhibitor beta (NF-кBIB) was markedly up-regulated (p<0.01), while the expression levels of Livin and Survivin declined remarkably (p<0.01) in knockout group. Furthermore, overexpression group had a considerably decreased expression level of NF-кBIB (p<0.01), but notably increased expression levels of Livin and Survivin (p<0.01). MiR-20a up-regulates the expressions of the downstream proteins Livin and Survivin, decreases the expressions of apoptosis-associated proteins, weakens the sensitivity of cells to chemotherapy drugs and lowers the apoptosis level of cells by activating the NF-кB signaling pathway in liver cancer Huh-7 cells.

A novel role for Livin in the response to ultravioletB radiation and pterygium development.

A pterygium is an inflammatory, invasive and proliferative lesion on the ocular surface, which can decrease visual acuity, damage the ocular surface and affect the appearance of the eye. However, the underlying molecular mechanisms of the pathogenesis remain unclear. In the present study, the role of apoptosis-associated protein Livin in the occurrence and development of pterygium was investigated. Primary samples from quiescent or advanced clinical stages of pterygium and normal human conjunctival tissues were used to assess mRNA and protein expression levels of Livin using reverse transcription-quantitative PCR and immunohistochemistry, respectively. Livin was knocked down in pterygium epithelial cells (PECs) using small interfering RNA (siRNA), to investigate the role of Livin in PEC viability, migration, invasion ability and apoptosis. The cell viability, invasion ability and apoptosis of PECs following ultraviolet B (UVB) radiation alone or in combination with Livin silencing were also analyzed. Expression levels of Livin increased in the pterygium tissues compared with those in the normal conjunctiva at both the mRNA and protein levels. Livin expression levels in advanced pterygium were significantly higher compared with those in quiescent pterygium samples. Knockdown of Livin expression levels significantly reduced cell migration, invasion ability and cell viability, and induced apoptosis of PECs. Inhibition of Livin expression in PECs increased the expression levels of caspase-7, caspase-3 and E-cadherin, whereas expression levels of Snail were downregulated. Cell viability and invasion ability in PECs was enhanced following UVB radiation and Livin expression upregulated. UVB irradiation induced cell invasion ability of PECs and this was attenuated by Livin-silencing. Transfection with Livin siRNA also partially recovered the apoptosis rate of PECs, which was reduced by UVB irradiation. In conclusion, Livin was upregulated in pterygium, and UVB radiation functions in the development of pterygium by inducing Livin expression.

Expression of Livin and PlGF in human osteosarcoma is associated with tumor progression and clinical outcome.

Baculoviral IAP repeat containing 7 (BIRC7/Livin/ML-IAP/KIAP; referred to as Livin throughout the present study) and placental growth factor (PlGF) are not detectable in the majority of normal differentiated tissues, but are present in a number of types of cancer, including hepatocellular carcinoma, ovarian cancer and renal cell carcinoma. The aim of the present study was to assess the expression levels of Livin and PlGF in human osteosarcoma specimens and cell lines, and to analyze the functions of Livin and PIGF in the prognosis of osteosarcoma. The expression levels of Livin and PlGF in 48 osteosarcoma specimens and three osteosarcoma cells were determined using immunohistochemistry and reverse transcription-quantitative polymerase chain reaction. The positivity rates of Livin and PlGF in osteosarcoma specimens were 58.3 and 60.4%, respectively, but were 0% in normal bone tissues. The expression levels of Livin and PlGF were increased in MG-63 cells, compared with those in the other cell lines evaluated in the present study. In addition, the expression levels of Livin and PlGF were significantly associated with tumor diameter and Enneking staging, but were independent of tumor site, age and sex of patients. The expression level of Livin was not associated with PlGF. Furthermore, the 5-year overall survival rate was decreased in the Livin or PlGF expression group, compared with that in the non-expression group (P=0.034 and P=0.012, respectively). The expression levels of Livin and PlGF were independent prognostic factors for patients with osteosarcoma. The results of the present study demonstrated that Livin and PlGF may participate in the pathogenesis of osteosarcoma. Therefore, pharmacological inhibition of Livin or PlGF may provide a novel strategy for osteosarcoma treatment.

Expression of Apoptosis-related Protein Livin and Caspase-3 in Diffuse Large B Cell Lymphoma and Its Clinical Significance

To detect the expression levels of Livin and Caspase-3 proteins in diffuse large B cell lymphoma (DLBCL), and to explore its correlation with clinicopathological characteristics, so as to provide a reference for clinical treatment and evaluation prognosis of DLBCL. Immunohisto-chemical staining was performed to detect the expression of Livin and Caspase-3 in the samples from 51 DLBCL patients and 20 patients with reactive hyperplasia of lymph node (RHL). The relation between Livin and Caspase-3 with the factors influencing the prognosis of DLBCL was analyzed. Livin+ was not expressed in the tissues of RHL, but its expression rates reached to 58.82%(30/51) in 51 cases of DLBCL(P<0.05). The expression rats of Caspase-3 in DLBCL and RHL were 25.49%(13/51) and 85.0%(17/20) respectively, and the difference was statistically significant(P<0.05). The expression level of Livin in DLBCL tissues was related with clinical stage and pathological type(P<0.01, P<0.01). The expression of Livin was not obviously related with sex, age and extranodal involvement. The expression level of Caspase-3 in DLBCL tissues was related with clinical stage (P<0.05). The expression of Caspase-3 was not obviously related with sex, age, pathological type and extranodal involvement. There was a negative correlation between Livin and Caspase-3 in DLBCL(γs=-0.333,P=0.017). Kaplan-meier survival analysis revealed that the expressions of Livin and Caspase-3 were related stalistically significantly with the patients prognosis (P<0.01, P<0.05). Over-expression of Livin in DLBCL and DLBCL of non-GCB subtypes, and low-expression of Caspase-3 in DLBCL may play a significant role in clinical prognosis of DLBCL.

Transfection with Livin and Survivin shRNA inhibits the growth and proliferation of non‑small cell lung cancer cells.

Lung cancer is reported to be a major public health issue worldwide and the overall prognosis of patients remains poor. The expression levels of Livin and Survivin, of the inhibitors of apoptosis (IAP) family, are associated with prognostic significance in the majority of solid tumors. Therefore, in the presents study, short hairpin (sh)RNA expression vectors inhibiting the Livin and Survivin genes were constructed to examine the effects of the transfection of Livin shRNA and/or Survivin shRNA on the biological functions of tumor cells. The transfection efficiency was measured using fluorescence reverse transcription‑quantitative polymerase chain reaction and western blot analyses. The cell growth inhibition ratio was measured using a CCK assay. Cell apoptosis following transfection and in tumor tissues were measured using a TUNEL assay, and a cancer xenograft model was used to investigate the effect of Livin shRNA and/or Survivin shRNA on tumor growth. The results indicated that the mRNA and protein expression levels were suppressed following the transfection of Livin and Survivin shRNA into tumor cells (P<0.05, compared with control group). The growth of tumor cells invivo and invitro was significantly inhibited following transfection with Livin and Survivin shRNA, compared with that in the other groups (P<0.05). Taken together, the transfection of cells with Livin and Survivin inhibited tumor growth invivo and invitro, with the co‑transfection of Livin and Survivin shRNA showing increased efficiency, compared with transfection of either the Livin vector or Survivin vector alone. The combined inhibition of Livin and Survivin may be a promising multitargeted gene therapeutic strategy in cancer treatment.

Silencing Livin induces apoptotic and autophagic cell death, increasing chemotherapeutic sensitivity to cisplatin of renal carcinoma cells.

Renal cell carcinoma (RCC) accounts for 3% of all adult malignancies and is the most lethal urological cancer. Livin is a member of the inhibitor of apoptosis protein (IAP) family, which is associated with tumor resistance to radiotherapy and chemotherapy. Clinical data also showed that patients with high tumor grades and stages have higher expression levels of Livin in RCC cells. Autophagy is a survival mechanism activated in response to nutrient deprivation. A possible role of Livin in the autophagy of RCC cells has not been investigated; therefore, this pioneer study was carried out. Livin was silenced in RCC cells (slow virus infection [SVI]-shLivin cells) by lentiviral transfection. Then, mRNA and protein expression levels in the transfected cells were assessed by quantitative fluorescence PCR and Western blotting, respectively. In addition, acridine orange staining and electron microscopy were used to assess autophagy in SVI-shLivin cells. The cisplatin IC50 values for RCC cells were measured by the CCK8 assay. Potent antitumor activities were observed in xenograft mouse models generated with Livin-silenced RCC cells in terms of delayed tumor onset and suppressed tumor growth. These results suggested that Livin silencing could increase the chemotherapeutic sensitivity of RCC cells to cisplatin and induce autophagic cell death. A possible mechanism of Bcl-2 and Akt pathway involvement was discussed specifically in this study. Overall, Livin silencing induces apoptotic and autophagic cell death and increases chemotherapeutic sensitivity of RCC cells to cisplatin.

Effects of monomethoxypolyethylene glycol-chitosan nanoparticle-mediated dual silencing of livin and survivin genes in prostate cancer PC-3M cells.

Monomethoxypolyethylene glycol-chitosan (mPEG-CS) nanoparticles were used as interfering RNA carriers to transfect human prostate cancer PC-3M cells to evaluate the effects of livin and survivin gene silencing on the proliferation and apoptosis. mPEG-CS nanoparticles with sizes of approximately 60 nm were first synthesized by ionic crosslinking. Through electrostatic adsorption, mPEG-CS-livin short hairpin RNA (shRNA), mPEG-CS-survivin shRNA, and mPEG-CS-(livin shRNA + survivin shRNA) nanoparticles were then prepared to transfect PC-3M cells. The mRNA and protein expression levels of livin and survivin were measured by reverse transcription-PCR and western blotting, respectively. The inhibitory effects of down-regulated livin and survivin gene expression on the cell proliferation were evaluated by MTT assay. Cell apoptosis was assessed visually using Hoechst staining. Livin and survivin expression levels in all shRNA interference groups were effectively down-regulated at both the mRNA and protein levels. Dual silencing of livin and survivin genes markedly inhibited cell proliferation and facilitated apoptosis, with better outcomes than those of individual shRNA treatments. mPEG-CS nanoparticle-mediated dual shRNA interference of livin and survivin genes significantly reduced the expression levels in PC-3M cells, inhibited proliferation, and promoted apoptosis. As these effects were superior to single interference, this method may have synergistic effects.

Effects of RNA interference-mediated knockdown of livin and survivin using monomethoxypolyethylene glycol-chitosan nanoparticles in MG-63 osteosarcoma cells

MG-63 human osteosarcoma cells were transfected with short hairpin RNA (shRNA) against livin and survivin using monomethoxypolyethylene glycol‑chitosan (mPEG‑CS) nanoparticles (NPs) as carriers, with the aim of evaluating the effect on cell proliferation and apoptosis. mPEG‑CS NPs sized ~100 nm were prepared by ionic crosslinking. mPEG‑CS‑livin shRNA, mPEG‑CS‑survivin shRNA and mPEG‑CS‑(livin shRNA + survivin shRNA) NPs were constructed by electrostatic adsorption at NP suspension/gene solution ratios of 3:1 to transfect MG‑63 cells. The expression levels of livin and survivin mRNA and protein were measured by reverse transcription‑polymerase chain reaction and western blotting, respectively. The inhibitory effects of downregulated livin and survivin expression on cell proliferation were measured using an MTT assay. The apoptosis‑inducing effects of livin and surivin knockdown were investigated using a Hoechst staining kit. All shRNA groups resulted in reduced expression of livin and survivin mRNA and protein in MG‑63 cells. The MTT assay and Hoechst staining indicated that simultaneous knockdown of livin and survivin genes inhibited the proliferation of MG‑63 cells and promoted their apoptosis, to a greater extent than knocking down either gene individually. The simultaneous interference mediated by mPEG‑CS NPs significantly reduced livin and survivin expression in MG‑63 cells, suppressed proliferation and facilitated apoptosis, to a greater extent than knockdown of either livin or survivin alone were. Thus the results indicate a synergistic effect of livin and survivin.

Effect of siRNA-Livin on drug resistance to chemotherapy in glioma U251 cells and CD133+ stem cells.

The aim of the present study was to observe the effect of siRNA-Livin on the expression of multidrug resistance-associated protein (MRP) genes in a U251 cell line and U251 stem cells. CD133+ cancer stem cells were identified and isolated from the U251 glioblastoma cells, and morphological observations were used to detect the cell survival conditions. In addition, quantitative polymerase chain reaction was used to detect the mRNA expression levels of Livin, MRP1 and MRP3. Following transfection with the lentivirus containing the siRNA-Livin, the expression of Livin was significantly inhibited in the U251 cells and stem cells (P<0.01). Following temozolomide intervention, the proliferation of the U251 cells and U251 stem cells was restrained, with a lot of cell debris present and the structure of the cell spheres destroyed. The inhibitory effect was more significant following transfection with siRNA-Livin. Prior to siRNA-Livin transfection, the expression of MRP1 presented an increasing trend in the U251 cells and U251 stem cells with increasing drug concentrations and intervention times (P<0.05). Following siRNA-Livin transfection, the expression of MRP1 decreased in the U251 cells and U251 stem cells under the same drug concentration and intervention time (P<0.05), while the expression of MRP3 increased in the U251 stem cells under the same intervention concentration and time (P<0.05). Therefore, siRNA-Livin was shown to decrease the expression of MRP1 in U251 cells and U251 stem cells, increase the expression of MRP3 in U251 stem cells and decrease the proliferation of U251 cells and U251 stem cells. Thus, Livin may be associated with the high expression of MRP1, and siRNA-Livin may be used to lower the expression of MRP1 in order to reduce the drug resistance to chemotherapy in cases of glioblastoma.

Effect of temozolomide on livin and caspase-3 in U251 glioma stem cells

The aim of the present study was to analyze the effect of temozolomide (TMZ) on the antiapoptotic gene livin and the associated gene caspase-3. Cancer stem cells were isolated from U251 glioblastoma cells using immunomagnetic beads. The glioma cells and glioma stem cells were transfected with livin or small hairpin RNA (shRNA) against livin using lentiviral vectors. Quantitative PCR, flow cytometry and a Cell Counting kit-8 assay were used to detect the expression of livin and caspase-3, analyze the cell cycle and investigate cell proliferation, respectively, following treatment with various concentrations of TMZ (0, 25, 50, 100, 200 and 400 μmol/l) for different periods of time (24, 48 and 72 h). The expression levels of livin and caspase-3 in the U251 stem cells were significantly higher than those in the U251 cells (P<0.01). At the same intervention time, the expression levels of livin decreased and those of caspase-3 increased as the concentration of TMZ increased (P<0.05). The expression levels of livin and caspase-3 in the U251 cells were lower than those in the U251 stem cells with the same intervention time and concentration of TMZ (P<0.05). The cell cycle was arrested in the G2/M phase in the U251 cells following TMZ intervention; the proportion of cells in the G2/M phase increased as the concentration of TMZ increased (P<0.05). The U251 stem cells were arrested in the S phase following treatment with TMZ; the proportion of cells in the S phase increased as the concentration of TMZ increased (P<0.05). In conclusion, the expression levels of livin and caspase-3 were effectively inhibited and increased, respectively, in all cell models following treatment with TMZ. TMZ is able to arrest the cell cycle and enhance cell apoptosis. U251 stem cells are less vulnerable than U251 cells to TMZ.

Expression and prognostic significance of livin/BIRC7 in childhood acute lymphoblastic leukemia

Livin, a member of the inhibitor of apoptosis proteins has been considered to be a poor prognostic marker in malignancies. However, little is known about the clinical relevance of livin expression in childhood acute lymphoblastic leukemia (ALL). The aim of the present study was to assess the expression of livin on leukemic blasts of de novo childhood ALL and its relevance to clinical and hematological findings, and treatment outcome. The expression of livin was analyzed in 80 patients with newly diagnosed childhood ALL using quantitative reverse transcriptase-polymerase chain reaction. The results of the study revealed that the expression levels of livin were higher in patients with favorable prognostic factors. Furthermore, livin expression was associated with a favorable early response to chemotherapy (leukemic blast <25% day 7 bone marrow response) (P = 0.001). Patients with high livin expression were associated with significantly higher CR rate (P = 0.02) and lower mortality rate (P = 0.05) than those with low livin expression. Kaplan-Meier curves demonstrated that high livin expression was associated with significantly longer DFS (P = 0.004) and overall survival (P = 0.02). Multivariate analysis demonstrated that livin expression was independent favorable prognostic factor for OS and DFS (P = 0.05 and P = 0.01, respectively). This study suggests that livin expression could be a novel prognostic marker in childhood ALL thus it could be incorporated into patient stratification and treatment protocols.

Impact of Co-transfection with Livin and Survivin shRNA Expression Vectors on Biological Behavior of HepG2 Cells

To construct short hairpin RNA (shRNA) eukaryotic expression vectors targeting Livin and Survivin genes, and to explore the impact of co-transfection of Livin and Survivin shRNA expression vectors on the biological behavior of HepG2 cells. shRNA eukaryotic expression vectors pSD11-Livin and pSD11- Survivin were designed and constructed then transfected into HepG2 cells separately or in combination. mRNA and protein expression in transfected cells was assessed by quantitative fluorescence PCR and Western blotting, respectively. Cell proliferation was measured by MTT assay and cell apoptosis by TUNEL assay. The Livin and Survivin shRNA eukaryotic expression vectors were successfully constructed and transfected into HepG2 cells. The relative mRNA expression levels of Livin and Survivin in HepG2 cells co-transfected with pSD11-Livin and pSD11-Survivin were 0.12 ± 0.02 and 0.33 ± 0.13, respectively, which was significantly lower than levels in cells transfected with either pSD11-Livin or pSD11-Survivin (P<0.05). The relative protein expression levels of Livin and Survivin in the co-transfected cells were also significantly decreased compared to single- transfection (P<0.05). The inhibition rate of cell growth in the co-transfection group was higher than that in the single-transfection groups at 48 h, 60 h, or 72 h after transfection (P<0.01). The apoptotic rate increased to the greatest extent in the co-transfection group relative to any other group (P<0.05). Co-transfection with pSD11-Livin and pSD11-Survivin was more efficient than transfection with either vector alone in reducing the mRNA and protein expression of Livin and Survivin genes in HepG2 cells. Co-transfection also inhibited the proliferation of transfected cells more than the other groups, and induced cellular apoptosis more effectively.

Effects of Livin gene RNA interference on apoptosis of cervical cancer Hela cells and enhanced sensitivity to cisplatin

The recombinant plasmids pGenesil-1-BIRC71 and pGenesil-1-BIRC72 were transfected into HeLa cells and cisplatin was added with different concentrations in order to study the inhibitory effects of Livin gene, increase the apoptosis induced by cisplatin, and detect the expression of Bcl-2, Bax, caspase-3, and survivin genes. The pGenesil-1-BIRC71 and pGenesil-1-BIRC72 were transfected into HeLa cells, and the expression levels of Livin, Bcl-2, Bax, caspase-3, and survivin genes were detected by using fluorescence quantitative real-time PCR. Then cisplatin at different concentrations (3.0, 6.0 and 9.9 microg/mL) was added into the transfected HeLa cells, and 24, and 48 h later, the apoptosis rate was measured by flow cytometry. After transfection of pGenesil-1-BIRC71 and pGenesil-1-BIRC72 into HeLa cells, the expression level of Livin gene was obviously reduced, and the apoptosis rate was significantly increased in transfection group as compared with control group (P<0.05). Cisplatin could increase the apoptosis rate in a dose- and time-dependent manner. After cisplatin was added, the expression levels of Bcl-2 mRNA were reduced, and those of Bax, caspase-3, and survivin mRNA were increased in transfection group as compared with those in control group (P<0.05). It was concluded that shRNA expression vector targeting Livin gene could inhibit the expression of Livin gene in HeLa cells and enhance the apoptosis induced by cisplatin, which was related to the decreased expression of Bcl-2 and activation of Bax and caspase-3. Survivin might play an important role as an antagonist in the process of apoptosis induction.

CC Chemokine Ligand 25 Enhances Resistance to Apoptosis in CD4+ T Cells from Patients with T-Cell Lineage Acute and Chronic Lymphocytic Leukemia by Means of Livin Activation

We investigated CD4 and CD8 double-positive thymocytes, CD4(+) T cells from typical patients with T-cell lineage acute lymphocytic leukemia (T-ALL) and T cell lineage chronic lymphocytic leukemia (T-CLL), and MOLT4 T cells in terms of CC chemokine ligand 25 (CCL25) functions of induction of resistance to tumor necrosis factor alpha (TNF-alpha)-mediated apoptosis. We found that CCL25 selectively enhanced resistance to TNF-alpha-mediated apoptosis in T-ALL and T-CLL CD4(+) T cells as well as in MOLT4 T cells, but CD4 and CD8 double-positive thymocytes did not. One member protein of the inhibitor of apoptosis protein (IAP) family, Livin, was selectively expressed in the malignant cells at higher levels, particularly in T-ALL CD4(+) T cells, in comparison with the expression in CD4 and CD8 double-positive thymocytes. After stimulation with CCL25 and apoptotic induction with TNF-alpha, the expression levels of Livin in these malignant cells were significantly increased. CCL25/thymus-expressed chemokine (TECK), by means of CC chemokine receptor 9 (CCR9) ligation, selectively activated Livin to enhance resistance to TNF-alpha-mediated apoptosis in c-jun-NH(2)-kinase 1 (JNK1) kinase-dependent manner. These findings suggested differential functions of CCR9/CCL25 in distinct types of cells. CD4 and CD8 double-positive thymocytes used CCR9/CCL25 for migration, homing, development, maturation, selection, cell homeostasis, whereas malignant cells, particularly T-ALL CD4(+) T cells, used CCR9/CCL25 for infiltration, resistance to apoptosis, and inappropriate proliferation.