Inhibition of Hypoxia Inducible Factor 1α by siRNA‐Induced Apoptosis in Human Retinoblastoma Cells

Hypoxia, which activates the hypoxia inducible factor 1α (HIF‐1α), is an essential feature of retinoblastoma (RB) and contributes to poor prognosis and resistance to conventional therapy. In this study, the effect of HIF‐1α knockdown by small interfering RNA (siRNA) on cell proliferation, apoptosis, and apoptotic pathways of human Y‐79 RB cells was first investigated. Exposure to hypoxia induced the increased expression of HIF‐1α both in mRNA and protein levels. Then, knockdown of HIF‐1α by siRNA_HIF‐1α resulted in inhibition of cell proliferation and induced cell apoptosis in human Y‐79 RB cells under both normoxic and hypoxic conditions, with hypoxic conditions being more sensitive. Furthermore, knockdown of HIF‐1α could enhance hypoxia‐induced slight increase of Bax/Bcl‐2 ratio and activate caspase‐9 and caspase‐3. These results together indicated that suppression of HIF‐1α expression may be a promising strategy for the treatment of human RB in the future.     

LncRNA HOTAIR/miR‐613/c‐met axis modulated epithelial‐mesenchymal transition of retinoblastoma cells

Since lncRNAs could modulate neoplastic development by modulating downstream miRNAs and genes, this study was carried out to figure out the synthetic contribution of HOTAIR, miR‐613 and c‐met to viability, apoptosis and proliferation of retinoblastoma cells. Totally 276 retinoblastoma tissues and tumour‐adjacent tissues were collected, and human retinoblastoma cell lines (ie, Y79, HXO‐Rb44, SO‐Rb50 and WERI‐RB1) were also gathered. Moreover, transfections of pcDNA3.1‐HOTAIR, si‐HOTAIR, miR‐613 mimic, miR‐613 inhibitor, pcDNA3.1/c‐met were performed to evaluate the influence of HOTAIR, miR‐613 and c‐met on viability, apoptosis and epithelial‐mesenchymal transition (EMT) of retinoblastoma cells. Dual‐luciferase reporter gene assay was also arranged to confirm the targeted relationship between HOTAIR and miR‐613, as well as between miR‐613 and c‐met. Consequently, up‐regulated HOTAIR and down‐regulated miR‐613 expressions displayed associations with poor survival status of retinoblastoma patients (P < 0.05). Besides, inhibited HOTAIR and promoted miR‐613 elevated E‐cadherin expression, yet decreased Snail and Vimentin expressions (P < 0.05). Simultaneously, cell proliferation and cell viability were also less‐motivated (P < 0.05). Nonetheless, c‐met prohibited the functioning of miR‐613, resulting in promoted cell proliferation and viability, along with inhibited cell apoptosis (P < 0.05). Finally, HOTAIR was verified to directly target miR‐613, and c‐met was the direct target gene of miR‐613 (P < 0.05). In conclusion, the role of lncRNA HOTAIR/miR‐613/c‐met signalling axis in modulating retinoblastoma cells’ viability, apoptosis and expressions of EMT‐specific proteins might provide evidences for developing appropriate diagnostic and treatment strategies for retinoblastoma.     

Rho‐kinase inhibitor Y‐27632 facilitates the proliferation,migration and pluripotency of human periodontal ligament stem cells

The selective in vitro expansion and differentiation of multipotent stem cells are critical steps in cell‐based regenerative therapies, while technical challenges have limited cell yield and thus affected the success of these potential treatments. The Rho GTPases and downstream Rho kinases are central regulators of cytoskeletal dynamics during cell cycle and determine the balance between stem cells self‐renewal, lineage commitment and apoptosis. Trans‐4‐[(1R)‐aminoethyl]‐N‐(4‐pyridinyl)cylohexanecarboxamidedihydrochloride (Y‐27632), Rho‐associated kinase (ROCK) inhibitor, involves various cellular functions that include actin cytoskeleton organization, cell adhesion, cell motility and anti‐apoptosis. Here, human periodontal ligament stem cells (PDLSCs) were isolated by limiting dilution method. Cell counting kit‐8 (CCK8), 5‐ethynyl‐2′‐deoxyuridine (EdU) labelling assay, cell apoptosis assay, cell migration assay, wound‐healing assay, alkaline phosphatase (ALP) activity assay, Alizarin Red S staining, Oil Red O staining, quantitative real‐time polymerase chain reaction (qRT‐PCR) were used to determine the effects of Y‐27632 on the proliferation, apoptosis, migration, stemness, osteogenic and adipogenic differentiation of PDLSCs. Afterwards, Western blot analysis was performed to elucidate the mechanism of cell proliferation. The results indicated that Y‐27632 significantly promoted cell proliferation, chemotaxis, wound healing, fat droplets formation and pluripotency, while inhibited ALP activity and mineral deposition. Furthermore, Y‐27632 induced PDLSCs proliferation through extracellular‐signal‐regulated kinase (ERK) signalling cascade. Therefore, control of Rho‐kinase activity may enhance the efficiency of stem cell‐based treatments for periodontal diseases and the strategy may have the potential to promote periodontal tissue regeneration by facilitating the chemotaxis of PDLSCs to the injured site, and then enhancing the proliferation of these cells and maintaining their pluripotency.     

KCNQ1OT1 regulates the retinoblastoma cell proliferation,migration and SIRT1/JNK signaling pathway by targeting miR-124/SP1 axis

Objective: Long non-coding RNA (lncRNA) KCNQ1OT1 was reported to be tightly associated with tumorigenesis and progression of multiple cancers. However, the expression and biological functions of KCNQ1OT1 in retinoblastoma (RB) are still unknown. We aim to elucidate the potential function and underlying mechanism of KCNQ1OT1 in regulating the progression of RB. Methods: The levels of KCNQ1OT1 were assayed by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) analysis. The cell proliferation of RB cells (Y79 and WERI-Rb-1) were evaluated through Cell Counting Kit 8 (CCK-8) assay. Meanwhile, Y79 and WERI-Rb-1 cell apoptosis and cell cycle were assessed by Flow Cytometry analysis. Dual luciferase reporter assay were performed to illustrate the interaction between KCNQ1OT1, miR-124, and SP1. Results: We found that KCNQ1OT1 was up-regulated and miR-124 was down-regulated in RB tissues and cells. Moreover, knockdown of KCNQ1OT1 reduced the proliferation, migration, and cell cycle, as well as promoted cell apoptosis of Y79 and WERI-Rb-1 cells. Western blot analysis consistently proved cell cycle and apoptosis related protein expression levels. More importantly, KCNQ1OT1 was a sponge of microRNA (miR)-124. MiR-124 inhibition strongly reversed the effect on cell proliferation, cycle arrest, and apoptosis by KCNQ1OT1 knockdown mediation. In addition, KCNQ1OT1 regulated expression of SP1, a direct target of miR-124 in RB. On the other hand, miR-124 inhibitor abrogated the active effect of KCNQ1OT1 silencing on silent information regulator 1 (SIRT1)/c-Jun N-terminal kinase (JNK) signaling pathway. The function of KCNQ1OT1 was verified in vivo. Conclusions: These findings implied that KCNQ1OT1 silencing inhibited RB progression and activated SIRT1/JNK signaling pathway partially by modulating the miR-124/SP1 axis.     

MAPK‐mediated upregulation of fibrinogen‐like protein 2 promotes proliferation,migration, and invasion of colorectal cancer cells

Fibrinogen‐like protein 2 (FGL2) has been reported to play a key role in the development of human cancers. However, it is still unmasked whether FGL2 plays a potential role in colorectal carcinogenesis. In this study, the messenger RNA and protein expression levels were measured by quantitative real‐time polymerase chain reaction and western blot. Cell counting kit‐8 assay, transwell migration, and invasion assay were carried out to evaluate the proliferation, migration, and invasion of LOVO and SW620 cells. FGL2 was upregulated in colorectal cancer (CRC) tissues, as well as cell lines. Mitogen‐activated protein kinase (MAPK) signaling was activated in CRC tissues and cell lines. FGL2 was confirmed to be downregulated by MAPK signaling inhibitor U0126. Further, we determined that knockdown of FGL2 caused a reduction of proliferation, migration, and invasion in LOVO and SW620 cells. Consistently, treatment of LOVO and SW620 cells with U0126 led to a decrease in cell proliferation, migration, and invasion. However, these changes initiated by U0126 were abolished by FGL2 overexpression. To conclude, MAPK‐mediated upregulation of FGL2 promotes the proliferation, migration, and invasion of CRC cells.     

Shikonin suppresses progression and epithelial–mesenchymal transition in hepatocellular carcinoma (HCC) cells by modulating miR‐106b/SMAD7/TGF‐β signaling pathway

Shikonin is a natural naphthoquinone component with antioxidant and anti‐tumor function and has been used for hepatocellular carcinoma (HCC) treatment. According to the previous study, many herbs can regulate cancer cell progression by targeting specific microRNA (miRNA) (Liu, 2016). However, the underlying pathological mechanism of shikonin in HCC therapy is still unclear. The detection of cell growth and death rate were performed by hemacytometry and trypan blue staining, respectively. The expression of miR‐106b and SMAD7 messenger RNA (mRNA) in HCC cells was evaluated by quantitative real‐time polymerase chain reaction. Cell proliferation, apoptosis, and migration ability were measured by cell counting kit‐8 (CCK‐8), flow cytometry, and transwell assay. The expression of proteins E‐cadherin, N‐cadherin, vimentin, SMAD7, TGF‐β1, p‐SMAD3, SMAD3, and GAPDH was examined by western blot. The interaction between SMAD7 and miR‐106b was assessed by luciferase reporter system. Shikonin inhibited Huh7 and HepG2 cell growth in a dose‐dependent manner while induced cell death in a time‐dependent manner. In addition, the expression of miR‐106b was reduced after shikonin treatment. Moreover, miR‐106b attenuated the suppressive effects of shikonin on HCC cell migration and epithelial–mesenchymal transition (EMT). SMAD7 was predicted as a target of miR‐106b and the prediction was confirmed by luciferase reporter system. Additionally, we observed that SMAD7 reversed the promotive effects of miR‐106b on HCC cell progression and EMT. The subsequent western blot assay revealed that shikonin could modulate SMAD7/TGF‐β signaling pathway by targeting miR‐106b. In conclusion, Shikonin suppresses cell progression and EMT and accelerates cell death of HCC cells via modulating miR‐106b/SMAD7/TGF‐β signaling pathway, suggesting shikonin could be an effective agent for HCC treatment.     

MircoRNA‐1275 promotes proliferation,invasion and migration of glioma cells via SERPINE1

This study was designed to explore the relationship between miR‐1275 and SERPINE1 and its effects on glioma cell proliferation, migration, invasion and apoptosis. Differentially expressed miRNAs and mRNAs in glioma tissues were screened out by bioinformatic analysis. Dual‐luciferase reporter gene assay was used to validate the targeted relationship between miR‐1275 and SERPINE1. qRT‐PCR was used to detect the expression of miR‐1275 and SERPINE1 in glioma tissues. The expressions of SERPINE1 and p53 pathway‐related proteins in glioma cells were detected by western blot. Glioma cell proliferation, apoptosis, migration and invasion were respectively detected by CCK‐8 assay, flow cytometry, wound healing assay and transwell assay. Tumour xenograft model was developed to study the influence of miR‐1275 and SERPINE1 on glioma growth in vivo. The results of microarray analysis, qRT‐PCR and western blot showed that miR‐1275 was low‐expressed while SERPINE1 was high‐expressed in glioma. Dual‐luciferase assay showed that miR‐1275 could bind to SERPINE1. Overexpression of miR‐1275 could promote the p53 pathway‐related proteins’ expression. Highly expressed miR‐1275 could repress the migration, proliferation and invasion of glioma cells while highly expressed SERPINE1 had inverse effects. Tumour xenograft showed that up‐regulated miR‐1275 or down‐regulated SERPINE1 could repress glioma growth in vivo. Up‐regulation of miR‐1275 activated p53 signalling pathway via regulating SERPINE1 and therefore suppressed glioma cell proliferation, invasion and migration, whereas promoted cell apoptosis.     

Let‐7b‐5p inhibits proliferation and motility in squamous cell carcinoma cells through negative modulation of KIAA1377

KIAA1377 has been found to be linked with lymph node metastasis in esophageal squamous cell carcinoma (SCC) in our previous study; however, the regulation of KIAA1377 remains far from understood. Herein, to understand the regulation of KIAA1377 from the angle of microRNA (miRNA)–messenger RNA (mRNA) modulation in the setting of SCC cells, the basal level of KIAA1377 was determined by quantitative real‐time polymerase chain reaction (qRT‐PCR) and western blot analysis in KYSE‐150 and HeLa cells; biological roles of KIAA1377 contributing in the proliferation, migration, and invasion were evaluated using 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT), wound‐healing and Transwell assays, respectively, after KIAA1377 was knocked out mediated by the CRISPR‐Cas9 system. Bioinformatic prediction revealed that let‐7b‐5p was a putative miRNA regulating KIAA1377, which was ensuingly validated by the luciferase reporter assay; after which, variation of KIAA1377 expression was further verified by qRT‐PCR and western blot analysis. Moreover, the biological roles of let‐7b‐5p in proliferation, migration, and invasion of KYSE‐150 and HeLa cells were also evaluated. It was exhibited that KIAA1377 was able to promote the proliferation and motility of both KYSE‐150 and HeLa cells, which can be reverted by re‐expression of let‐7b‐5p. The luciferase reporter assay verified that let‐7b‐5p can diametrically target KIAA1377. Collectively, our data demonstrated that let‐7b‐5p can directly but negatively regulate KIAA1377 in SCC cell lines, Ecal109, and HeLa cells.     

Recoverin in Cultured Human Retinoblastoma Cells: Enhanced Expression During Morphological Differentiation

Abstract: Recoverin is a calcium-binding protein expressed in retinal photoreceptors. It appears to delay the termination of the phototransduction cascade by blocking the phosphorylation of photoexcited rhodopsin. The goal of this study was to determine if recoverin mRNA and protein are expressed in cultured human Y79 retinoblastoma cells, so that this cell line could be used as a model to study the mechanism of recoverin gene expression in the retina. A cDNA encoding human recoverin was PCR cloned and used for prokaryotic expression of recoverin protein. Polyclonal antibodies raised against pure recombinant recoverin were used for western blotting and immunocytochemistry of Y79 cells grown as attachment cultures in the presence of the differentiating agents dibutyryl cyclic AMP (dbcAMP) or butyrate. Northern blot analysis was performed on mRNA extracted from Y79 cells that were also treated with the differentiating agents. In Y79 cell monolayer cultures, recoverin was immunolocalized to the cell cytoplasm, and immunoreactivity was increased dramatically by the addition of 2 m M butyrate to the culture medium. Butyrate treatment also caused an increase in the development of neurite-like cellular processes. Addition of 4 m M dbcAMP resulted in a moderate increase in both recoverin immunoreactivity and number of cellular processes. Western and northern blots of butyrate and dbcAMP-treated Y79 cell cultures demonstrated an increase in recoverin protein and RNA expression, respectively, comparable with that observed with immunocytochemistry. These data suggest that, under the influence of the differentiating agent butyrate, Y79 cells exhibit an increase in expression of the photoreceptor protein recoverin and a concomitant morphological differentiation toward a neuronal phenotype.     

Retracted: Long noncoding RNA MEG3 is a tumor suppressor in choriocarcinoma by upregulation of microRNA-211

Tumor suppressor long noncoding RNA maternally expressed gene 3 (lncRNA MEG3) exists in various cancers. Nonetheless, the functions of lncRNA MEG3 in choriocarcinoma (CC) are still not well studied. We explored the effects of lncRNA MEG3 on human CC JEG-3 and BeWo cells. lncRNA MEG3 was overexpressed, and the effects of lncRNA MEG3 on cell viability, proliferation, apoptosis, migration, and invasion were assessed by the cell counting kit-8 assay, western blot analysis, flow cytometry (plus western blot analysis), and transwell assay (plus western blot analysis), respectively. Then, the expression level of miR-211 was detected by real-time quantitative polymerase chain reaction. After that, the effects of dysregulated microRNA-211 (miR-211) with overexpressing lncRNA MEG3 on JEG-3 cells and BeWo cells were testified. Western blot analysis was used to study the involvements of the signaling pathways in the lncRNA MEG3-associated modulation. We found that lncRNA MEG3 upregulation reduced cell viability, inhibited proliferation, migration and invasion, and promoted apoptosis. Expression of miR-211 was upregulated after lncRNA MEG3 overexpression. Effects of lncRNA MEG3 overexpression were augmented by miR-211 overexpression, while they were declined by miR-211 silencing. Phosphorylated levels of PI3K, AKT, and AMP-activated protein kinase (AMPK) were decreased by lncRNA MEG3 overexpression via regulation of miR-211. To sum up, lncRNA MEG3 could repress proliferation, migration and invasion, and promote apoptosis of JEG-3 and BeWo cells through upregulating miR-211. The PI3K/AKT and AMPK pathways were inhibited by lncRNA MEG3 overexpression via regulation of miR-211.     

慢病毒介导的靶向沉默信息调节因子1（SIRT1）的RNA干扰对肝癌细胞生长及凋亡的影响

目的探讨慢病毒介导的靶向SIRTlshRNA对肝癌细胞生长和凋亡的影响。方法Western印迹分析SIRT1在多个肝癌细胞系中的表达;通过慢病毒介导的shRNA干扰技术靶向沉默SIRT1的表达,并通过Western印迹验证SIRTl基因的沉默效果。台盼蓝排斥实验分析SIRT1基因沉默对肝癌细胞生长的影响;流式细胞术和Western印迹检测PARP蛋白的剪切物观察细胞凋亡状态。结果SIRT1在多个肝癌细胞系中表达水平明显上调;慢病毒介导的shRNA能显著抑制细胞中SIRT1的表达。流式细胞术及Western印迹结果均显示SIRT1表达沉默显著诱导了肝癌细胞的凋亡。结论慢病毒介导的靶向SIRT1shRNA显著地抑制SIRT1的表达;SIRT1基因沉默抑制肝癌细胞生长并促进了细胞凋亡。     

Retracted: PRC1 gene silencing inhibits proliferation,invasion, and angiogenesis of retinoblastoma cells through the inhibition of the Wnt/β-catenin signaling pathway

Retinoblastoma is an ocular malignancy occurring in childhood. The current study evaluates the ability of silenced PRC1 on retinoblastoma cell proliferation, and angiogenesis via the Wnt/β-catenin signaling pathway. A total of 36 cases of retinoblastoma tissues (n = 36) and normal retinal tissues (n = 10) were selected in the current study. Retinoblastoma cells presenting with the high PRC1 messenger RNA (mRNA) expression were selected among the WERI-Rb-1, HXO-RB44, Y79, SO-Rb50, and SO-Rb70 cells lines, and were transfected with siRNA-PRC1 and LiCl (the activator of the Wnt/β-catenin pathway). The expressions of PRC1, VEGF, Wnt1, β-catenin, CyclinD1, extent of β-catenin, and GSK-3β phosphorylation were evaluated. Cell proliferation, cell-cycle distribution, and cell invasion of retinoblastoma cells were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytometry, and Transwell assay. The angiogenesis of retinoblastoma cells was detected by tube formation assay. HXO-RB44 and WERI-Rb-1 cells were selected owing to the highest PRC1 mRNA expression. Meanwhile, PRC2 gene silencing presented lower expression levels of PRC1, VEGF, Wnt1, β-catenin, CyclinD1, extent of β-catenin and GSK-3β phosphorylation, decreased proliferation and invasion abilities, extended G0/G1 phase, and shortened S and G2/M phases of HXO-RB44 and WERI-Rb-1 cells, suggesting the silenced PRC2 inactivated Wnt/β-catenin pathway, so as to further restrain the retinoblastoma cell proliferation, invasion, and angiogenesis. These results support the view that PRC1 gene silencing could suppress the proliferation, and angiogenesis of retinoblastoma cells by repressing the Wnt/β-catenin pathway.     

MiR‐9 promotes multiple myeloma progression by regulating TRIM56/NF‐κB pathway

miR‐9 has been reported to play a pivotal role in multiple human cancers by acting as an oncogene or tumor suppressor. In this study, we explored the possible role and molecular mechanism of miR‐9 in multiple myeloma (MM). The miR‐9 expression was examined by quantitative real‐time polymerase chain reaction assay. Transfection with miR‐9‐mimics, miR‐9‐inhibitor, pcDNA‐TRIM56, or si‐TRIM56 into cells was used to change the expression levels of miR‐9 and TRIM56. Western blot analysis was used to detect the expression of TRIM56, p65, p‐p65, IκBα, and p‐IκBα. The potential target of miR‐9 was confirmed by luciferase reporter assay. The 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium (MTT) assay, colony formation assay, and flow cytometry were used to assess the abilities of cell proliferation and apoptosis. miR‐9 was upregulated in MM patients and cell lines, and miR‐9 overexpression promoted proliferation and repressed apoptosis in MM cell lines. TRIM56 was confirmed as a target of miR‐9. Moreover, TRIM56 reversed miR‐9‐mediated pro‐proliferation and anti‐apoptosis effect on MM cell lines. Furthermore, nuclear factor‐κB (NF‐κB) pathway was involved in miR‐9/TRIM56‐mediated regulation on MM cell lines. miR‐9 promoted the development and progression of MM by regulating TRIM56/NF‐κB pathway, thereby providing a potential microRNA‐based target for MM therapy.     

Hsa_circ_0069094 positively regulates the expression of oncogenic ZNF217 by competitively targeting miR-758–3p to promote the development of breast cancer

To investigate the functions and potential mechanisms of hsa_circ_0069094 in this cancer. The expression of hsa_circ_0069094, zinc finger protein 217 (ZNF217) and microRNA-758–3p (miR-758–3p) was detected by quantitative polymerase chain reaction (qPCR), and the protein level of ZNF217 was detected by western blot. Cell proliferation was assessed using cell counting kit-8 (CCK-8) assay and colony formation assay. Cell cycle progression and cell apoptosis were determined using flow cytometry assay. Cell invasion and cell migration were monitored using transwell assay and wound healing assay. The protein levels of apoptosis-related proteins were quantified by western blot. The putative relationship between miR-758–3p and hsa_circ_0069094 and ZNF217 was confirmed using dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Xenograft model was constructed in mice to explore the role of hsa_circ_0069094 on solid tumor growth.Hsa_circ_0069094 and ZNF217 were highly expressed, while miR-758–3p was poorly expressed in tissues and cells of breast cancer. Hsa_circ_0069094 knockdown or ZNF217 knockdown inhibited cell proliferation, invasion and migration and induced cell apoptosis and cell cycle arrest in breast cancer cells. The inhibitory effects of hsa_circ_0069094 knockdown on cell malignant behaviors were abolished by ZNF217 overexpression. Hsa_circ_0069094 competed with ZNF217 for the binding site of miR-758–3p, and hsa_circ_0069094 positively regulated ZNF217 expression by competitively binding to miR-758–3p. Hsa_circ_0069094 knockdown also blocked solid tumor growth in mice. Collectively, Hsa_circ_0069094 played oncogenic effects in breast cancer by activating the expression of ZNF217 via competitively binding to miR-758–3p, which might be a novel strategy for breast cancer suppression.