Effect of sirtuin 6 on proliferation of hepatocellular carcinoma cells

AIM: To illuminate the effect of sirtuin 6 (SIRT6) on the proliferation of hepatocellular carcinoma (HCC) cells. METHODS: The mRNA expression of SIRT6 in the peripheral blood from 200 cases of HCC patients and 50 cases of healthy people was detected by real-time quantitative PCR (RT-qPCR). The mRNA expression levels of SIRT6 in the peripheral blood from 200 cases of HCC patients were combined with multiple clinicopathologic parameters for statistical analysis. The protein expression of SIRT6 in primary hepatocytes, immortalized hepatocytes and 4 hepatoma cell lines were determined by Western blotting. The silencing of SIRT6 was conducted by transfection of vector expressing short hairpin RNA targeting on SIRT6, and the protein level of SIRT6 was measured by Western blotting. The viability of HCC cells was tested by MTS assay. DNA synthesis was analyzed by Cell-Light^TM EdU Apollo® 488 In Vitro Imaging Kit. The abilities of colony formation and anchorage-dependent growth were measured by colony formation assay and soft agar assay, respectively. RESULTS: The mRNA expression of SIRT6 in the peripheral blood of HCC patients was significantly higher than that in the healthy people, and its expression was highly associated with tumor size, tumor grade and vascular invasion. SIRT6 expression in 4 hepatoma cell lines was significantly higher than that in the others. SIRT6 silencing led to a significant decrease in the cell viability as tested by MTS assay. EdU staining revealed that SIRT6 silencing reduced DNA synthesis. SIRT6 silencing reduced the ability of colony formation and anchorage-dependent growth as determined by colony formation assay and soft agar assay, respectively. CONCLUSION: Sirtuin 6 promotes the proliferation and malignant transformation of HCC cells.     

Effect of TRIM44 silencing on proliferation of hepatocellular carcinoma cells and its molecular mechanism

ATM: To investigate the effect of tripartite motif-containing protein 44 (TRIM44) on the proliferation of hepatocellular carcinoma (HCC) cells and to study the molecular mechanism. METHODS: The expression of TRIM44 at mRNA and protein levels in normal liver tissues, HCC tissues, adjacent nontumor liver tissues, immortalized hepatocytes and hepatoma cell lines was determined by RT-qPCR and Western blot, respectively. The silencing of TRIM44 was conducted by transfection of vector expressing shRNA targeting TRIM44 (shTRIM44) in the HCC cells, and the protein level of TRIM44 was measured by Western blot. The viability of the HCC cells was analyzed by MTS assay. The DNA synthesis of HCC cells was detected by Click-iT EdU Imaging Kit. The ability of anchorage-independent growth was determined by the method of colony formation on the soft agar. The effects of TRIM44 on the total protein and phosphorylation of mammalian target of rapamycin (mTOR) levels were measured by Western blot. The HCC cells were transfected with shTRIM44 and treated with mTOR agonist MHY1485, and the cell viability was analyzed by MTS assay. RESULTS: The mRNA and protein levels of TRIM44 in the HCC tissues were significantly higher than those in the adjacent nontumor liver tissues and normal liver tissues. In addition, the mRNA and protein levels of TRIM44 in the hepatoma cell lines were significantly higher than those in the immortalized hepatocytes. TRIM44 silencing significantly inhibited the viability of HCC cells and reduced the abilities of DNA synthesis and anchorage-independent growth of the HCC cells. TRIM44 silencing decreased the phosphorylation level of mTOR protein. MHY1485 significantly antagonized the inhibitory effect of TRIM44 silence to the viability of HCC cells. CONCLUSION: TRIM44 silencing inhibits the proliferation of HCC cells possibly through down-regulating the activity of mTOR.     

Effect of NANOG silencing on cyclin D1 expression and proliferation in human hepatoma HepG2 cells

AIM:To investigate the effect of NANOG silencing on cyclin D1 expression and proliferation in human hepatoma HepG2 cells. METHODS:Transient transfection of NANOG targeting siRNA into HepG2 cells was performed. The expression of NANOG and cyclin D1 at mRNA and protein levels was detected by real-time PCR and Western blotting. Cell proliferation was examined by CCK-8 assay and colony formation assay, and cell cycle was tested by flow cytometry. RESULTS:After transfection with NANOG-targeting siRNA, the inhibition of NANOG expression was observed. Compared with mock group, the mRNA and protein expression levels of NANOG and cyclin D1 were decreased (P<005). In addition, knockdown of NANOG expression inhibited the cell proliferation and increased the proportion of G 0/G 1-phase cells (P<0.05). CONCLUSION:Silencing of NANOG expression in HepG2 cells causes down-regulation of cyclin D1 expression and decreases the cell proliferation ability.     

Gene silencing of indoleamine 2,3-dioxygenase 2 influences proliferation,migration and invasion of B16-BL6 melanoma cells

AIM: To investigate the effects of indoleamine 2, 3-dioxygenase 2 (IDO2) silencing on proliferation, migration and invasion of B16-BL6 melanoma cells.METHODS: IDO2-siRNA was transfected into the B16-BL6 melanoma cells in vitro. The expression of IDO2 or IDO1 at mRNA and protein levels was detected by real-time PCR and Western blot. Colony formation assay was performed to analyze the proliferation of IDO2-silencing tumor cells. The migration ability of B16-BL6 cells after silencing of IDO2 was measured by wound healing assay and Transwell cell migration assay. The invasion ability of the tumor cells was detected by Transwell cell invasion assay.RESULTS: IDO2-siRNA signi-ficantly down-regulated IDO2 expression in B16-BL6 melanoma cells, and did not affect IDO1 expression. Compared with control group, the colony formation ability, the migratory distance measured by wound healing assay, and the migration and the invasion cell numbers detected by Transwell assay all remarkably decreased in the IDO2-silencing cells.CONCLUSION: IDO2 silencing affects the proliferation, migration and invasion abilities of the B16-BL6 melanoma cells.     

ZNF580 is up-regulated in S1P-induced migration and proliferation of endothelial cells

AIM: To investigate whether a novel human C2H2-type zinc finger protein ZNF580 is involved in the proliferation and migration of endothelial cells induced by sphingosine 1-phosphate (S1P). METHODS: The cDNA of EA.hy926 cells was analyzed by RT-PCR to determine the S1P receptor expression profile. The cells were incubated with S1P at different concentrations and for different time intervals. Total RNA and protein in treated EA.hy926 cells were analyzed by RT-PCR and Western blotting. SB203580, a chemical inhibitor of p38 MAPK, was used to determine whether p38 MAPK pathway had any effect on the up-regulation of ZNF580 expression by S1P. The plasmid pEGFP-ZNF580 or the synthetic ZNF580-siRNA was transfected into EA.hy926 cells with Lipofectamine 2000 for 48 h. Cell migration assay and MTT colorimetric assay were used to investigate the effects of ZNF580 on the motility and growth of endothelial cells. RESULTS: EA.hy926 endothelial cells expressed S1P1, S1P3 and S1P5 receptors. Furthermore, S1P up-regulated ZNF580 at mRNA and protein levels in a concentration- and time-dependent manner. The p38 MAPK pathway specific inhibitor SB203580 blocked the S1P-induced up-regulation of ZNF580 expression. Moreover, overexpression/silencing of ZNF580 in EA.hy926 cells led to enhancement/decrease of the migration and proliferation of the cells. CONCLUSION: S1P-induced migration and proliferation of endothelial cells are critical for angiogenesis. ZNF proteins usually play an essential role in altering gene expression and regulating the angiogenesis.     

Effects of HIF-1α silencing on proliferation of rat hepatoma cells

AIM：To study the effect of hypoxia-inducible factor 1α (HIF-1α) silencing on the proliferation of hepatoma cells under hypoxia. METHODS：Rat hepatoma cell line CBRH-7919 was used in this study. Hypoxia model was established by treating the cells with cobalt chloride (CoCl2). The expression of HIF-1α was silenced by small interfe-rence RNA. Real-time RT-PCR and Western blotting were used to detect the mRNA and/or protein expression of HIF-1α, vascular endothelial growth factor (VEGF), p21 and cyclin D1 in CBRH-7919 cells under hypoxia. The proliferation of CBRH-7919 cells was measured by the technique of 5-bromo-2’-deoxyuridine (BrdU) incorporation. RESULTS：The expression of HIF-1α and VEGF at mRNA and protein levels was significantly increased under hypoxia (P<0.05). Silencing of HIF-1α significantly inhibited the expression of HIF-1α, VEGF and cyclin D1 at mRNA and/or protein levels, while increased the protein expression of p21 (P<0.05). The BrdU-positive cells in HIF-1α siRNA transfection group were significantly less than those in control group. CONCLUSION：HIF-1α silencing significantly inhibits the proliferation of hepatoma cells under hypoxia.     

Effects of HIF-1α silencing on expression of p27 and Ki67 in rat hepatoma cell line CBRH-7919

AIM: To study the effect of hypoxia-inducible factor 1α(HIF-1α) silencing on the expression of p27 and Ki67 in rat hepatoma cell line CBRH-7919 under hypoxia. METHODS: Hypoxic condition was induced by CoCl₂ and the expression of HIF-1α was silenced by small interference RNA. HIF-1α-specific RNAi lentiviral vector was constructed. Real-time RT-PCR and Western blotting were used to detect the expression of HIF-1α at mRNA and protein levels in CBRH-7919 cells under hypoxia. The expression of p27 and Ki67 was observed by Western blotting after HIF-1α silencing was performed. The cell cycle of hepatoma cells was detected by flow cytometry. RESULTS: Under hypoxic condition, the expression of HIF-1α at mRNA and protein levels in CBRH-7919 cells increased significantly (P<0.05). HIF-1α silencing significantly reduced the expression of Ki67 but increased the expression of p27 (P<0.05) in CBRH-7919 cells. In transfected cells, the number of cells in G₀/G₁ phase was much higher and that in S phase was much lower than those in the control cells. CONCLUSION: Hypoxia induces the expression of HIF-1α. HIF-1α silencing can regulate the proliferation of hepatoma cells through reducing the expression of Ki67 and increasing the expression of p27.     

Effects of RNA interference targeting CDC25agene on proliferation of human liver cancer HepG2 cells

AIM: To investigate the effect of silencing cell division cycle 25a (CDC25a) gene on the proliferation of human hepatoma HepG2 cells. METHODS: CDC25agene in human hepatoma HepG2 cells was silenced by RNA interference. Real-time PCR was applied to detect the expression of CDC25a, cyclin E and CDK2 at mRNA levels in the HepG2 cells. Western blotting was applied to detect the expression of CDC25a at protein level. In addition, MTT assay, Giemsa staining and flow cytometry were used to measure the proliferation of human hepatoma HepG2 cells. RESULTS: The expression of CDC25a at mRNA and protein levels in RNA silence group was lower than those in negative control group and normal control group (P＜0.05). The mRNA expression of cyclin E and CDK2 in silence group was lower than that in negative control group and normal control group (P＜0.05). The cell proliferation in silence group was lower than that in negative control group and normal control group (P＜0.05). The results of flow cytometry revealed that the cells in silence group were blocked in G1 phase. CONCLUSION: Infection of LV-CDC25a-RNAi recombinant to the HepG2 cells effectively inhibits the CDC25agene expression and the proliferation of human hepatoma cells, and arrests the cells in G1 phase, suggesting that CDC25agene may be a key target for the treatment of liver cancer.     

Effects of BCL-3 gene silencing on the proliferation and drug sensitivity of human colorectal cancer cell line RKO with high expression of BCL-3

AIM：To construct lentiviral vectors for RNA interference (RNAi) of BCL-3 gene, and to detect the changes of biological behaviors and drug sensitivity of colorectal cancer cells after BCL-3 gene silencing. METHODS：The expression of BCL-3 in five human colorectal cancer cell lines was detected by RT-PCR and Western blotting. Lentiviral vectors for RNAi of BCL-3 gene were constructed and transfected into the human colorectal cancer cell line with high expression of BCL-3, and then the silencing effect was detected by Western blotting. After BCL-3 gene silencing, the change of cell proliferation was detected by MTT assay and soft agar colony formation assay, and the change of drug sensitivity was detected by MTT assay. RESULTS：BCL-3 was highly expressed in human colorectal cancer cell line RKO. Lentiviral vectors for RNAi of BCL-3 gene were successfully constructed, and Western blotting showed that BCL-3-shRNA2 could efficiently inhibit the expression of BCL-3 protein in RKO cells. After BCL-3 gene silencing, the proliferation ability and colony formation rate of RKO cells were decreased, and the median inhibitory concentration of oxaliplatin for RKO cells also decreased significantly. CONCLUSION: Inhibition of BCL-3 gene expression decreases the proliferation ability of human colorectal cell line RKO with high expression of BCL-3, and enhances the sensitivity of RKO cells to oxaliplatin.     

Effects of lentivirus-mediated c-met RNAi on biological behaviors of human papillary thyroid cancer K1 cells

AIM: To evaluate the expression of c-Met in papillary thyroid cancer (PTC) by constructing lentiviral vectors for RNA interference (RNAi) of c-met gene and detecting its silencing effect on the biological behaviors of human papillary thyroid cancer cell line K1 cells. METHODS: Immunohistochemical assay was performed to detect the expression of c-Met protein in 35 cases of PTC and 25 cases of benign thyroid disease. Lentiviral vector for RNA of c-met gene was constructed and the silencing effect was detected by RT-PCR and Western blotting. The colony-forming ability, cell cycle, migration and invasion of K1 cells were measured by colony-forming assay, flow cytometry, wound-healing observation and Transwell experiment, respectively. In vivo tumorigenicity assay was performed to analyze in vivo proliferation of K1 cells in a xenograft model. RESULTS: The expression of c-Met in PTC was significantly higher than that in benign thyroid tissues. Lentiviral RNAi vectors targeting c-met gene were successfully constructed, and they efficiently inhibited the expression of c-met at mRNA and protein levels. Transfection of c-met lentiviral RNAi vectors inhibited the colony formation, cell cycle progression, migration, invasion and tumorigenicity of K1 cells. CONCLUSION: Lentivirus-mediated c-met RNAi efficiently inhibits colony formation, cell cycle progression, migration, invasion and tumorigenicity of K1 cells.     

Effects of lentivirus-mediated RWDD3 silencing on proliferation and invasion of human glioma U251 cells

AIM: To investigate the effect of RWDD3 gene silencing on the biological characteristics of human glioma U251 cells.METHODS: A lentiviral vector expressing RWDD3 shRNA was constructed and transfeeted into the U251 cells. The expression of RWDD3 at mRNA and protein levels was detected by real-time PCR and Western blot, respectively. The cell activity was determined by MTT assay. The colony formation ability was detected by the colony formation assay. The cell proliferation ability was detected by BrdU incorporation assay. The cell invasion and migration were evaluated by Transwell assay. Flow cytometry was used to monitor the changes of cell cycle distribution and apoptosis.RESULTS: Recombinant lentivirus was successfully transfected into U251 cells. Compared with the cells transfected with the scrambled shRNA and control cells, the cell activity, colony formation ability, and the invasive and migratory activities were inhibited, the cell cycle was arrested in G₀/G₁ phase, and the apoptosis was increased in the U251 cells transfected with RWDD3 shRNA(P<0.05).CONCLUSION: RWDD3 plays a vital role in proliferation and invasion of glioma cells. It may serve as a potential target of gene therapy for glioma.     

Activated NK cells against human hepatocellular carcinoma cell lines in vitro and in vivo

AIM: To evaluate the role of natural killer(NK) cells against a variety of human hepatocellular carcinoma (HCC) cell lines in vitro and in vivo, and to investigate the expression of MHC class I chain-related protein (MIC protein) in these HCC cell lines. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from 50 mL peripheral blood donored by a healthy volunteer and cultured in the NK cell kit followed by amplification and cytokine activation. The release of lactate dehydrogenase (LDH) was detected by lysis experiment. Animal experiments were performed following vaccination with HCC cell lines (BEL7402, HepG2 and SMMC7721) in BALB/c-nu/nu mice. The diameters of the tumors were measured and the growth curves of difference cell lines were delineated. Three weeks later, these mice were sacrificed and the weight of the transplanted tumors was also detected. Furthermore, the expression of MIC protein was determined. RESULTS: The obvious differences of lysis effects of NK cells on different cell lines were observed, in which the lysis effect of NK cells on K562 cells was the strongest, the effect on BEL7402 cells was in the middle and the effect on SMMC7721 cells was the weakest. In animal experiments, NK cells also showed obvious and different restraining effects on a variety of HCC cell line-transplanted tumors in nude mice. The tumor inhibitory rates of NK cells were 43.5% in BEL-7402 cell-transplanted tumor, 40.7% in HepG2 cell-transplanted tumor and 36.0% in SMMC-7721 cell-transplanted tumor. The protein expression of MIC was 48.7%, 32.8% and 0.9% in the 3 cell lines,respectively. CONCLUSION: The lysis effects of NK cells on a variety of human HCC cell lines are different. The expression of MIC in the tumor cells may play distinct role in evaluating these differences.     

Dendritic cells transfected with tumor total RNA induce specific immune responses against hepatocellular carcinoma in vitro

AIM: To observe the ability of dendritic cells (DC) vaccine transfected with human hepatocellular carcinoma (HCC) total RNA induce specific cytotoxic T lymphocyte(CTL) response in vitro. METHODS: DCs generated from HCC patient’s peripheral blood mononuclear cells (PBMC) were incubated with recombinant human granulocyte macrophage colony-stimulationg factor (GM-CSF) and human interleukin (IL-4). Tumor total RNA was isolated from Hep G-2 cells and HCC cells. DCs transfected with tumor total RNA were used to induce specific CTL proliferation. Specific cytotoxicity was measured using MTT method. RESULTS: DC transfected with HepG-2 cell RNA and HCC RNA exhibited increased expression of CD83, CD86 and HLA-DR. The CTL from DCs transfected with HepG-2 cell RNA killed 5.84％， 14.26％， 25.19％, or 35.78％ of HepG-2 cells, and 5.26％， 11.67％， 14.68％, or 23.24％ of HCC cells, respectively, at an E/T ratio of 2.5, 5, 10, or 20. The CTL from DCs transfected with HCC cells RNA killed 4.65％， 12.23％， 15.61％, or 19.15％ of HepG-2 cells, and 7.20％， 12.83％， 27.21％, or 31.15％ of HCC cells,respectively, at an E/T ratio of 2.5, 5, 10, and 20. These CTL did not kill allogeneic malignant cells as human gastric carcinoma cells SGC-7901. CONCLUSION: DC transfected with tumor-derived total RNA could induce specific antitumor immune CTL response. These results suggest that CTL generation is applicable to adoptive immunotherapy of HCC.     

Effect of WT1 silencing by siRNA on expression of Wnt/β-catenin and nephrin in mouse podocytes

AIM: To study the effect of WT1 silencing by small interfering RNA (siRNA) on podocyte vitality and expression of Wnt/β-catenin and nephrin in mouse podocytes. METHODS: Conditionally immortalized mouse podocytes were cultured at 33 ℃ in RPMI-1640 medium for proliferation and induced for differentiation at 37 ℃. The podocytes were transfected with WT1 siRNA. The cell vitality was detected by MTT assay. The expression of WT1,Wnt1,β-catenin and nephrin at mRNA and protein levels was determined by real-time qRT-PCR and Western blotting. RESULTS: WT1 siRNA induced the increase in the expression of Wnt1 at mRNA and protein levels, inhibited the phosphorylation of β-catenin, and reduced the cell vitality. Meanwhile, the expression of nephrin at mRNA and protein levels was decreased. CONCLUSION: WT1 siRNA reduces the expression of nephrin in podocytes and the vitality of the cells by activating Wnt/β-catenin signaling pathway.     

Effects of lentivirus-mediated siRNA targeting IGF-1R on migration and invasion abilities of endometrial carcinoma cells

AIM:To investigate the down-regulation of insulin-like growth factor tgpe 1 receptor(IGF-1R) on the migration and invasion abilities of human endometrial cancer cell HEC-1B. METHODS:The siRNAs targeting IGF-1R gene were synthesized, cloned into a lentivirus expression vector and transfected into endometrial cancer HEC-1B cells(HEC-1B-KD group). The control cells(without virus transfection, HEC-1B-CON group) and negative virus transfection control cells(HEC-1B-NC group) were also set up. The gene silencing effect of siRNA targeting IGF-1R was determined by real-time PCR and Western blotting at mRNA and protein levels,respectively. The proliferation rate was detected by colony formation assay. The cell migration and invasion abilities were determined by Transwell experiment. The mRNA levels of matrix metalloproteinase(MMP)-2 and MMP-9 were measured by real-time PCR. RESULTS:The mRNA and protein levels of IGF-1R in HEC-1B-KD cells were significantly reduced by 81% and 91.5%, respectively(P<0.05). In anchorage-dependent growth by colony formation assay, HEC-1B-KD cells showed much less colonies than HEC-1B-CON cells and HEC-1B-NC cells. Compared with the control cells, knockdown of IGF-1R in HEC-1B cells resulted in significant reduction of cell motility. Down-regulation of IGF-1R in HEC-1B cells also significantly reduced the invasion potential(P<0.05). Down-regulation of IGF-1R substantially reduced the expression of MMP-2 and MMP-9 compared with the control cells. CONCLUSION:Knockdown of IGF-1R reduces the migration and invasion abilities of human endometrial cancer cells in vitro accompanied with a decrease in MMP-2 and MMP-9 expression.     

Effect of long non-coding RNA-671 on proliferation and invasion of esophageal squamous-cell carcinoma cells

AIM: To detect the expression of long non-coding RNA-671 (lnc671) in esophageal squamous-cell carcinoma cell lines and to investigate the effect of lnc671 on the malignant phenotype of esophageal squamous-cell carcinoma cells. METHODS: The level of lnc671 in the esophageal squamous-cell carcinoma cells was detected by RT-qPCR. Specific lnc671 small interfering RNA (siRNA) used to explore the effects of lnc671 on proliferation, colony formation, invasion and migration abilities of esophageal squamous-cell carcinoma cells. RESULTS: The database of GEPIA analysis showed that increased expression of lnc671 was associated with shorter survival in the patients of esophageal cancer (P<0.05). Compared with normal immortalized esophageal epithelial cells, lnc671 was highly expressed in a variety of esophageal squamous-cell carcinoma cell lines. lnc671 knock-down significantly inhibited the growth, colony formation ability, migration and invasion abilities of esophageal squamous-cell carcinoma cells(P<0.01). CONCLUSION: The expression of lnc671 is increased in various esophageal squamous-cell carcinoma cell lines. Knock-down of lnc671 expression inhibits the malignant phenotype of esophageal squamous-cell carcinoma cells.