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@#[Abstract] Objective: To investigate the expression of long non-coding RNA (lncRNA) HULC in bladder cancer tissues and its relationship with the clinicopathological features of patients, as well as the effect of silencing HULC on the proliferation, apoptosis, migration and invasion of bladder cancer 5637 cells. Methods: A total of 102 pairs of cancer tissue and adjacent normal tissue samples from bladder cancer patients who underwent surgical resection in Zhengzhou People’s Hospital from June 2014 to December 2017 were selected, as well as bladder cancer 5637 cell line and human normal bladder epithelial SV-HUC-1 cell line. The expression of HULC in bladder cancer tissues and cells was detected by qPCR, and the correlation between HULC and clinicopathological features of bladder cancer patients was analyzed. The effect of HULC on prognosis was evaluated by Kaplan-Meier survival curve. si-HUL and si-NC plasmids were transfected into 5637 cells by siRNA interference technology, and the effects of silencing HULC on proliferation, apoptosis, migration and invasion of 5637 cells were determined by CCK-8, Flow cytometry, Wound-healing assay and Transwell method, respectively. Results: The expression of HULC in bladder cancer tissues was significantly higher than that in normal tissues (P<0.05), and its expression level was correlated with tumor grade, tumor stage and lymph node metastasis (P<0.05). The OS and PFS of patients with high HULC expression were significantly lower than those with low expression (all P<0.05). The expression level of HULC in 5637 cells was significantly higher than that in SV-HUC-1 cells (P<0.01). After silencing HULC, the proliferation, migration and invasion of 5637 cells were significantly decreased (P<0.01), and the apoptosis rate was significantly increased (P<0.01). Conclusion: lncRNA HULC is highly expressed in bladder cancer tissues and 5637 cells. Silencing HULC expression can inhibit the proliferation, migration and invasion but promote apoptosis of bladder cancer cells.
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@#【Objective】To investigate the mechanism of action of long non-coding RNA highly up-regulated in liver cancer（LncRNA HULC）on the growth of glioblastoma U87 cells in vitro and in vivo.【Methods】The cultured glioblastoma U87 cells were divided into four groups：overexpression group（HULC-over）and its vector control group（VEC），silent expression group（HULC- siRNA）and its negative control group（NC）.Quantitative real- time polymerase chain reaction PCR（qRT-PCR）was used to verify the expression levels of HULC. CCK8 proliferation assay and colony formation assay were adopted to monitor the proliferation of glioblastoma U87 cells. Flow cytometry was utilized to detect the apoptosis of glioblastoma U87 cells. By injecting U87 cells，we divided the orthotopic xenograft mouse model into HULC- over group（n=10），VEC group（n=10），HULC-siRNA group（n=10）and NC group（n=10）accordingly. The survival of the mice in each group was observed. The expression of Ki67 was analyzed by immunohistochemistry. 【Results】 The expression level of HULC was significantly higher in HULC-over group than that in VEC group and significantly lower in HULC-siR NA group than that in NC group（P < 0.01）. The cell proliferation ability was significantly increased in HULC-over group compared with that in VEC group and significantly decreased in HULC- siRNA group compared with that in NC group（P < 0.01 on days2，3and4）. The colony formation rates in VEC group，HULC-over group，NC group and HULC-siRNA group were，respectively，（34.47 ± 1.56）% ，（95.4 ± 2.74）% ，（23.83 ± 0.92）% and （10.23 ± 0.61）% ，which revealed that overexpression of HULC elevated the colony formation rate and silencing expression of HULC reduced the colony formation rate（P < 0.01）. The early apoptosis rates in VEC group，HULC- over group，NC group and HULC- siRNA group were，respectively，（3.55±0.56）% ，（0.09±0.01）% ，（2.89±0.67）% ，and（7.13±0.14）% ，which showed that overexpression of HULC elevated the early apoptosis rate and silencing expression of HULC reduced the early apoptosis rate （P <0.01）. The survival curve of nude mouse indicated shorter survival time in HULC-over group than that in VEC group and longer survival time in HULC-siRNA group than that in NC group（P < 0.05）. Ki67 protein expression was up-regulated in the HULC-over group compared with that in VEC group and down-regulated in the HULC-siRNA group compared with that in NC group（P < 0.05）.【Conclusion】LncRNA HULC can enhance the growth of glioblastoma U87 cells in vitro and in vivo.
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Objective@#To explore the effects of lncRNA HULC on hepatocellular carcinoma (HCC) growth by down-regulating miR-29.@*Methods@#The expression levels of HULC and miR-29 in HCC tissues and cells were detected by real-time quantitative PCR (RT-qPCR), and the correlation analysis was performed. After HCC cells were transfected with HULC overexpressed plasmid or siRNA, the expressions of miR-29 and its target gene SETDB1 were determinate by RT-qPCR. According to the bioinformatic prediction of the miR-29 binding site in the HULC sequence, the report gene plasmids were constructed. The HCC cells were co-transfected with miR-29 mimics or miR-29 inhibitor, and the HULC targeted regulation of miR-29 was verified by dual luciferase reporter assay. The effect of miR-29 on the HULC-mediated proliferation in HCC cells was detected by cell count kit 8 (CCK-8) experiment. Expression of tumor proliferation antigen Ki-67 was detected by RT-qPCR.The Hep3B cells were inoculated in mice and miR-29 mimics and miR-29 negative control (NC) further injected into the lesions. The tumor volume was observed, and the expressions of tumor proliferation antigen ki-67 in tumor tissues were detected by immunohistochemical staining.@*Results@#The expression of HULC was significantly up-regulated while the expression of miR-29 was significantly down-regulated in HCC tissues and cells (P<0.01). The level of HULC was negatively correlated with miR-29 in tumor tissues (r=-0.754, P<0.01) and HCC cells (r=-0.865, P<0.05). The in vitro experiments showed that, compared with the blank control group, the expression of miR-29 in HULC overexpressed Huh7 cells was significantly reduced, while the mRNA level of miR-29 target gene SETDB1 was increased (P<0.01). The expression of miR-29 was significantly increased in HULC deleted Hep3B cells, while the mRNA expression of SETDB1 was decreased (P<0.01). Double luciferase reporter gene assay showed that miR-29 mimics significantly inhibited the luciferase activity of Hep3B cells transfected with HULC wide type (psi-HULC-WT) plasmid but had no effect on Hep3B cells transfected with mutant plasmid (psi-HULC-Mut). However, the miR-29 inhibitor antagonized the inhibitory effect of miR-29 mimics on luciferase activity of psi-HULC-WT (P<0.01). Cell proliferation experiments showed that, compared with the control group, the proliferation ability of miR-29 mimics overexpressed Huh7 cells was significantly reduced.After 24, 48 and 72 hours of treatment, the proliferation rates of Huh7 cells in the HULC overexpressed group were (43.87±3.82)%, (83.45±7.46)% and (123.34±8.67)%, respectively, significantly higher than (13.45±1.77)%, (23.54±1.37)% and (38.21±2.09)% of control group (P<0.05). After treatment for 48 and 72 hours, the proliferation rates of miR-29 mimics transfected Huh7 cells were (57.10±1.94)% and (73.76±3.46)%, respectively, significantly lower than (83.45±7.46)% and (123.34±8.67)% of control group (P<0.05). After treatment for 48 and 72 hours, the proliferation rates of Huh7 cells transfected with miR-29 mimics and miR-29 inhibitor group were (76.45±3.24)% and (89.37±4.37)%, respectively, significant higher than (57.10%±1.94)% and (73.76±3.46)% of the control group (P<0.05). After 48 h transfection, the expression of Ki-67 in Huh7 transfected with miR-29 mimics was significantly inhibited compared with the control group (P<0.01). However, the expression of Ki-67 mRNA was increased in Huh7 cells transfected with miR-29 inhibitor (P<0.01). The results of in vivo experiments showed that the tumor volumes of the control group, miR-29 mimics group and miR-29 mimics + miR-29 inhibitors group were (504.0±19.6) mm3, (310.0±24.3) mm3 and (483.7±21.2) mm3, respectively. Injection of miR-29 mimics reduced while miR-29 inhibitor promoted tumorigenesis ability of Huh7 in nude mice (P<0.01). The immunohistochemical staining showed that the average optical density values of Ki-67 protein in tumor tissues of the control group, miR-29 mimics group and miR-29 analogue+ miR-29 inhibitor group were 0.65±0.08, 0.36±0.07 and 0.56±0.06, respectively. The expression level of Ki-67 protein in miR-29 mimics group was significantly reduced (P<0.01) while increased in the miR-29 mimics+ miR-29 inhibitor group (P<0.01).@*Conclusion@#LncRNA HULC promotes HCC growth by down-regulating miR-29.
ABSTRACT
@#【Objective】 To explore the effects of long- chain non- coding RNA highly up- reglated in liver cancer（LncRNA HULC）silencing expression on proliferation and apoptosis of human glioblastoma cell line SHG44.【Methods】 Quantitative Real- time Polymerase Chain Reaction （qRT- PCR） was used to verify the expression level of HULC in HULC silent expression group （HULC- siRNA）and negative control group （NC group）. CCK8 proliferation assay and plate colony formation assay were used to detect the proliferation of glioblastoma cells. Cell cycle and apoptosis assays were used to detect the cell cycle distribution and apoptosis of glioblastoma cells. 【Results】 qRT- PCR confirmed that HULC- siRNA group had significantly lower expression of HULC than NC group （P=0.003）. CCK8 proliferation experiment showed that the proliferation rate of HULC-siRNA group was significantly lower than that of NC group on the second，third and fourth days of experiment（Day 2，P=0.003；Day 3，P=0.005；Day 4，P=0.009）. Plate colony formation assay showed the cloning rates in the NC and HULC-siRNA groups were（34.11 ± 1.24）% and（14.44 ± 0.87）%，and it showed that the cell clone formation rate was clearly decreased after silenced expression of HULC （P<0.001）. The cell cycle assay showed that the numbers of cells in NC group and HULC-siRNA group were G1 phase（36.89 ± 4.09，51.74± 0.68）and S phase（46.95 ± 2.49，36.89 ± 2.13），and it showed that the cell cycle was blocked in G1/S phase after silencing HULC expression （G1 phase，P=0.023，S phase，P=0.038）. Apoptosis experiment showed that the early apoptotic rates of NC group and HULC-siRNA group were（2.57 ± 0.22）% and（7.063 ± 0.71）%，and it showed that the early apoptotic rate of the cells was significantly increased after silencing HULC expression （P=0.004）. 【Conclusion】 Silencing of LncRNA HULC can inhibit the proliferation of glioblastoma cells and promote their apoptosis.
ABSTRACT
Prostate cancer (PCa) is the second leading cause of cancer death in men. Irradiation is one of the available options for treatment of PCa, however, approximately 10-45% of PCa are resistant to irradiation. We aimed to explore the role of long non-coding RNA highly upregulated in liver cancer (HULC) in the sensitivity of PCa cells to irradiation. Survival rate, cell apoptosis, cycle, expressions of related proteins, and caspase-3 activity were assessed to explore the effects of HULC on sensitivity of PCa cells to irradiation. Expression of HULC in DU-145, PC3, LNCaP, and RWPE-1 cells was determined and the influence of HULC on DU-145 cells was explored. Then, PC3 cells aberrantly expressing HULC were implanted into NOD-SCID mice for tumor xenograft study. Changes of autophagy after aberrant expression of HULC in vivo and in vitro were tested. Furthermore, the interacted protein of HULC and involved signaling pathway were investigated. In PC3 and LNCaP cells under irradiation, survival rate and cell cycle were decreased and apoptosis was increased by HULC knockdown. HULC knockdown arrested PC3 cells at G0/G1 phase. DU-145 was sensitive to irradiation, and resistance to irradiation of DU-145 cells was enhanced by HULC overexpression. Moreover, HULC knockdown enhanced the sensitivity of PC3 xenografts to irradiation. HULC knockdown promoted autophagy through interaction with Beclin-1 and inhibition of mTOR, resulting in increased apoptosis. HULC knockdown improved sensitivity of PCa cells to irradiation both in vivo and in vitro. HULC suppressed Beclin-1 phosphorylation, thereby reduced autophagy, involving the mTOR pathway.