Anticancer effects of melatonin via regulating lncRNA JPX-Wnt/β-catenin signalling pathway in human osteosarcoma cells

Osteosarcoma (OS) is a type of malignant primary bone cancer, which is highly aggressive and occurs more commonly in children and adolescents. Thus, novel potential drugs and therapeutic methods are urgently needed. In the present study, we aimed to elucidate the effects and mechanism of melatonin on OS cells to provide a potential treatment strategy for OS. The cell survival rate, cell viability, proliferation, migration, invasion and metastasis were examined by trypan blue assay, MTT, colony formation, wound healing, transwell invasion and attachment/detachment assay, respectively. The expression of relevant lncRNAs in OS cells was determined by real-time qPCR analysis. The functional roles of lncRNA JPX in OS cells were further examined by gain and loss of function assays. The protein expression was measured by western blot assay. Melatonin inhibited the cell viability, proliferation, migration, invasion and metastasis of OS cells (Saos-2, MG63 and U2OS) in a dose-dependent manner. Melatonin treatment significantly downregulated the expression of lncRNA JPX in Saos-2, MG63 and U2OS cells. Overexpression of lncRNA JPX into OS cell lines elevated the cell viability and proliferation, which was accompanied by the increased metastasis. We also found that melatonin inhibited the OS progression by suppressing the expression of lncRNA JPX via regulating the Wnt/β-catenin pathway. Our results suggested that melatonin inhibited the biological functions of OS cells by repressing the expression of lncRNA JPX through regulating the Wnt/β-catenin signalling pathway, which indicated that melatonin might be applied as a potentially useful and effective natural agent in the treatment of OS.     

LncRNA MEG3 inhibits the progression of prostate cancer by modulating miR‐9‐5p/QKI‐5 axis

This study was designed to detecting the influences of lncRNA MEG3 in prostate cancer. Aberrant lncRNAs expression profiles of prostate cancer were screened by microarray analysis. The qRT‐PCR and Western blot were employed to investigating the expression levels of lncRNA MEG3, miR‐9‐5p and QKI‐5. The luciferase reporter assay was utilized to testifying the interactions relationship among these molecules. Applying CCK‐8 assay, wound healing assay, transwell assay and flow cytometry in turn, the cell proliferation, migration and invasion abilities as well as apoptosis were measured respectively. LncRNA MEG3 was a down‐regulated lncRNA in prostate cancer tissues and cells and could inhibit the expression of miR‐9‐5p, whereas miR‐9‐5p down‐regulated QKI‐5 expression. Overexpressed MEG3 and QKI‐5 could decrease the abilities of proliferation, migration and invasion in prostate cancer cells effectively and increased the apoptosis rate. On the contrary, miR‐9‐5p mimics presented an opposite tendency in prostate cancer cells. Furthermore, MEG3 inhibited tumour growth and up‐regulated expression of QKI‐5 in vivo. LncRNA MEG3 was a down‐regulated lncRNA in prostate cancer and impacted the abilities of cell proliferation, migration and invasion, and cell apoptosis rate, this regulation relied on regulating miR‐9‐5p and its targeting gene QKI‐5.     

Downregulation of lncRNA HCP5 has inhibitory effects on gastric cancer cells by regulating DDX21 expression

LncRNA HCP5 has been confirmed to play crucial roles in many types of cancers. However, the role of lncRNA HCP5 in regulating the occurrence and development of gastric cancer (GC) remains unknown. In the current study, we aimed to investigate the precise effects of lncRNA HCP5 on cell proliferation, migration and invasion and molecular mechanisms in gastric cancer. Using RT-qPCR analysis, we found that lncRNA HCP5 was differentially expressed in GC cell lines. CCK-8, wound healing and transwell assay indicated that the proliferation, migration and invasion of gastric cancer cells were inhibited by downregulation of lncRNA HCP5 and lncRNA HCP5 overexpression exhibited the opposite effects in gastric cancer cells. Mechanistically, RNA binding protein immunoprecipitation and dual luciferase reporter assay confirmed the interaction between lncRNA HCP5 and DDX21. The effects of lncRNA HCP5 overexpression the proliferation, migration and invasion of GC cells were partly rescued by DDX21 silencing. Taken together, downregulation of lncRNA HCP5 exerted inhibitory effects on GC cell proliferation, migration and invasion through modulation of DDX21 expression, demonstrating the function of lncRNA HCP5 and DDX21 in GC progression.     

lncRNA CASC11 promotes cancer cell proliferation in bladder cancer through miRNA-150

Long noncoding RNAs (lncRNAs) CASC11 is an oncogenic lncRNA in gastric cancer and colorectal cancer. Our study aimed to investigate the role of lncRNA CASC11 in bladder cancer. In this study we showed that plasma lncRNA CASC11 was upregulated, while plasma miRNA-150 was downregulated in patients with early-stage bladder cancer than in healthy controls. Altered expression of plasma lncRNA CASC11 and miRNA-150 separated patients with bladder cancer from healthy controls. lncRNA CASC11 expression was inversely correlated with miRNA-150 expression in patients with bladder cance but not in healthy controls. Overexpression of lncRNA CASC11 mediated the inhibition of miRNA-150 expression in cancer cells, while miRNA-150 overexpression did not significantly alter lncRNA CASC11 expression. lncRNA CASC11 overexpression promoted, while miRNA-150 overexpression inhibited cancer cell proliferation. miRNA-150 also attenuated the enhancing effects of lncRNA CASC11 overexpression on cancer cell proliferation. However, overexpression of lncRNA CASC11 showed no significant effects on cancer cell migration and invasion. Therefore, lncRNA CASC11 may promote cancer cell proliferation in bladder cancer, and the actions of lncRNA CASC11 are likely through miRNA-150.     

Gastrin mediates resistance to hypoxia-induced cell death in xenografts of the human colorectal cancer cell line LoVo

Aim: Gastrins act as growth factors for the normal and neoplastic colorectal mucosa. The aim of this study was to determine the role of gastrins in the response of human colorectal cancer (CRC) cells to hypoxia in vitro and in vivo. Methods: Expression of the gastrin gene in the human CRC cell line LoVo was examined under normoxia and hypoxia by quantitative PCR and by radioimmunoassay. Gastrin expression was knocked down with shRNA, and the effect on cell proliferation was measured by cell counting, on cell apoptosis by annexin V staining, and on cell migration by Boyden chamber assay. The effect of gastrin knockdown on tumourigenesis in mouse xenografts was analysed by measurement of tumour volumes and weights, and by immunohistochemistry. Results: Gastrin gene expression in LoVo cells was stimulated by hypoxia via binding of hypoxia-inducible factor-1α to the gastrin promoter. The viability of gastrin knockdown cells exposed to hypoxia (1% O₂) in vitro was diminished because of loss of resistance against hypoxia-induced apoptosis, and the effect was partly reversed by treatment with non-amidated, but not amidated, gastrin. Conditioned medium from control LoVo cells under hypoxia simulated proliferation but not migration, and the effect was blocked by an inhibitor of non-amidated gastrins, but not by an inhibitor of amidated gastrins. In xenografts in mice exposed to hypoxia (10% O₂) for 21 days, tumour necrosis was significantly increased by knocking down gastrin expression. Conclusion: These results provide evidence that non-amidated gastrins are involved in the adaptation of CRCs to hypoxic microenvironments through increasing resistance to apoptosis.     

PTH-related protein enhances LoVo colon cancer cell proliferation, adhesion, and integrin expression

Parathyroid hormone-related protein (PTHrP) has been localized in human colon cancer tissue and cell lines. Tumor cell adhesion to extracellular matrix (ECM) proteins plays a major role in the invasion and metastasis of tumor cells, and is mediated via integrin subunits. The LoVo human colon cancer cell line was used as a model system to study the effects of PTHrP on cell proliferation and adhesion to ECM proteins found in normal liver. Clones of LoVo cells engineered to overexpress PTHrP by stable transfection with a PTHrP cDNA showed enhanced cell proliferation vs. control (empty vector-transfected) cells. PTHrP-overexpressing cells also showed significantly higher adhesion to collagen type I, fibronectin, and laminin, and enhanced expression of the [symbol: see text] integrin subunits. These results indicate that PTHrP may play a role in colon cancer invasion and metastasis by increasing cell proliferation and adhesion to the ECM via upregulation of proinvasive integrin expression.     

Long noncoding RNA ENST00000455974 plays an oncogenic role through up-regulating JAG2 in human DNA mismatch repair-proficient colon cancer

DNA mismatch repair-proficient colon cancer is the most common type of colon cancer, but its initiation and progression are still unknown. Our previous study has revealed that a long noncoding RNA (lncRNA) ENST00000455974 was significantly associated with TNM stage and distant metastasis in patients with DNA mismatch repair-proficient (pMMR) colon cancer (CC). Here, firstly, we observed that ENST00000455974 was gradual increased across colon normal-adenoma-carcinoma-metastasis sequence by quantitative real-time PCR. Secondly, ENST00000455974 showed a better sensitivity and specificity than CEA and CA19-9 in the diagnosis of pMMR CC by drawing the receiver operating characteristic (ROC) curve. Thirdly, a higher level of ENST00000455974 was associated with a poorer patient survival. Furthermore, Knockdown of ENST00000455974 led to reduced proliferation and migration of colon cancer cells. Mechanistically, ENST00000455974 was mainly located in the nucleus of colon cancer cells and it promoted the growth and metastasis of pMMR CC cells through up-regulating JAG2.     

lncRNA CEBPA-AS1靶向miR-455-3p调控胃癌细胞生物学行为的分子机制研究

摘要 目的：探讨lncRNA CEBPA-AS1对胃癌细胞生物学行为的影响及其可能作用机制。方法：qRT-PCR法检测胃癌组织、癌旁组织与正常人胃上皮GES1细胞和人胃癌SNU-1、AGS、HS-746T细胞系中lncRNA CEBPA-AS1、miR-455-3p的表达量。si-NC、si-lncRNA CEBPA-AS1、miR-NC、miR-455-3p mimics、si-lncRNA CEBPA-AS1与anti-miR-NC、si-lncRNA CEBPA-AS1与anti-miR-455-3p分别转染至SNU-1细胞（分别命名为si-NC组、si-lncRNA CEBPA-AS1组、miR-NC组、miR-455-3p组、si-lncRNA CEBPA-AS1+anti-miR-NC组和si-lncRNA CEBPA-AS1+anti-miR-455-3p组）后,MTT实验与平板克隆形成实验分别检测细胞增殖及克隆形成能力,Transwell小室实验检测细胞迁移及侵袭能力,双荧光素酶报告基因实验与qRT-PCR实验验证lncRNA CEBPA-AS1与miR-455-3p的靶向调控关系,Western blot法检测MMP2、MMP9蛋白表达情况。结果：与癌旁组织比较,胃癌组织中lncRNA CEBPA-AS1的表达量显著升高,miR-455-3p的表达量显著降低,差异均有统计学意义（均P＜0.05）。与GES1细胞比较,SNU-1、AGS、HS-746T细胞中lncRNA CEBPA-AS1的表达量显著升高,miR-455-3p的表达量显著降低,其中SNU-1细胞的lncRNA CEBPA-AS1表达量最高,差异均有统计学意义（均P＜0.05）。与si-NC组比较,si-lncRNA CEBPA-AS1组细胞活力降低,细胞克隆形成数、迁移及侵袭细胞数减少,MMP2、MMP9蛋白表达水平降低,差异均有统计学意义（P＜0.05）。与miR-NC组比较,miR-455-3p组细胞活力降低,细胞克隆形成数、迁移及侵袭细胞数减少,MMP2、MMP9蛋白表达水平降低,差异均有统计学意义（P＜0.05）。lncRNA CEBPA-AS1可靶向结合miR-455-3p,并可负调控miR-455-3p的表达。与si-lncRNA CEBPA-AS1+anti-miR-NC组比较,si-lncRNA CEBPA-AS1+anti-miR-455-3p组细胞活力升高,细胞克隆形成数、迁移及侵袭细胞数增多,MMP2、MMP9蛋白表达水平升高,差异均有统计学意义（P＜0.05）。结论：干扰lncRNA CEBPA-AS1表达可通过靶向调控miR-455-3p而抑制胃癌细胞增殖、克隆形成、迁移及侵袭。     

Overexpression of long non-coding RNA RP11-363E7.4 inhibits proliferation and invasion in gastric cancer

LncRNA RP11-363E7.4 has been shown to be downregulated in gastric cancer (GC), while the effect of lncRNA RP11-363E7.4 on GC and its potential molecular mechanisms is unclear. The purpose of this study was to explore the functional role and underlying molecular mechanisms of lncRNA RP11-363E7.4 involved in GC progress.To address the question, quantitative real-time PCR assay was performed to confirm lncRNA RP11-363E7.4 expression levels in GC tissues and cell lines. Cell proliferation, apoptosis, migration and invasion were estimated using Cell Counting Kit-8, colony formation, scratch wound healing and Transwell assays. Potential molecular mechanisms were evaluated using western blot assay. The results showed that lncRNA RP11-363E7.4 was significantly downregulated in GC cell lines and 82 paired tissues. The correlation between expression and clinicopathological features indicated that low expression of lncRNA RP11-363E7.4 was associated with T stage (P = .010). Functional experiments showed that overexpression of lncRNA RP11-363E7.4 prevented proliferation, migration, and invasion and induced apoptosis of GC cells. Western blot assay revealed that lncRNA RP11-363E7.4 functioned via the p53, Bax/Bcl-2, β-catenin pathway. In summary, this study revealed that lncRNA RP11-363E7.4 functioned as a tumour suppressor by inhibiting proliferation, migration, and invasion and inducing apoptosis of GC cells. Significance of the study :LncRNA RP11-363E7.4 has been shown to be downregulated in GC, while the effect of lncRNA RP11-363E7.4 on GC and its potential molecular mechanism is unclear. We revealed that lncRNA RP11-363E7.4 functioned as a tumour suppressor by inhibiting proliferation, migration, and invasion and inducing apoptosis of GC cells. LncRNA RP11-363E7.4 might become an attractive diagnostic and prognostic biomarker of GC and a promising target for GC treatment.     

LncRNA BDNF‐AS inhibits proliferation,migration, invasion and EMT in oesophageal cancer cells by targeting miR‐214

This study was aimed at exploring the effect of lncRNA BDNF‐AS on cell proliferation, migration, invasion and epithelial‐to‐mesenchymal transition (EMT) of oesophageal cancer (EC) cells. The expression of BDNF‐AS and miR‐214 in tissue samples and cells was measured by qRT‐PCR. The targeted relationship between BDNF‐AS and miR‐214 was analysed by dual‐luciferase reporter assay. After cell transfection, the cell proliferation activity was assessed by MTS method, while the migrating and invading abilities were evaluated by transwell assay. LncRNA BDNF‐AS was remarkably down‐regulated, while miR‐214 was up‐regulated in EC tissues and cells in comparison with normal tissues and cells. Overexpression of BDNF‐AS significantly inhibited the abilities of cell proliferation, migration and invasion as well as the EMT processes of EC cells. The bioinformatics analysis and luciferase assay indicated that BDNF‐AS could be directly bound by miR‐214. Furthermore, overexpression of miR‐214 and BDNF‐AS exerted suppressive influence on EC cell multiplication, migration, invasion and EMT processes. LncRNA BDNF‐AS restrained cell proliferation, migration, invasion and EMT processes in EC cells by targeting miR‐214.     

PI3K/Akt pathway involving into apoptosis and invasion in human colon cancer cells LoVo

In this study we determined the effects of Curcumin on human colon cancer cells line LoVo. We found that Curcumin significantly inhibited the proliferation, migration and invasion, and clone formation of LoVo cells in a dose-dependent manner. Curcumin also dose-dependently reduced the phosphorylation of proteins Akt and increased expression levels of the genes caspase-3, cytochrome-c, Bax mRNA in LoVo cells. In addition, Curcumin dose-dependently decreased gene Bcl-2 mRNA expression. Similar results were observed in LoVo cells treated with LY294002. These in vitro studies suggest that Curcumin may play its anti-cancer actions partly via suppressing PI3K/Akt signal pathway in LoVo cells.     

Ligustrazine inhibits the proliferation and migration of ovarian cancer cells via regulating miR-211

Ovarian cancer (OC) is a commonly diagnosed female cancer. Ligustrazine (LSZ), a natural compound, has been reported to exert anti-cancer activity, although the mechanisms underlying the anti-cancer effects are not clear. The present study investigated the impact of LSZ on cell proliferation and migration by regulating microRNA-211 (miR-211) expression using the human ovarian cancer SK-OV-3 and OVCAR-3 cell lines. OC cells were treated with 0, 0.5, 1, and 2 mM LSZ, and quantitative real-time PCR was utilized to measure miR-211 levels in SK-OV-3 and OVCAR-3 cells with different treatment. Moreover, to further confirm the roles of miR-211 in LSZ induced anti-tumor effects, miR-211 expression was inhibited by transfection of miR-211 inhibitors in SK-OV-3 cells. Cell proliferation of transfected cells was evaluated using the CCK-8 and colony formation assay. The scratch assay was employed to assess cell migration and transwell assay was performed for evaluating the cell invasion. Protein levels of epithelial–mesenchymal transition (EMT) markers were determined by Western blotting. We found that LSZ inhibited the viability, proliferation, migration and invasion ability of SK-OV-3 and OVCAR-3 cells in a dose-dependent manner; moreover, LSZ could significantly increase the expression of miR-211 in both SK-OV-3 and OVCAR-3, and knockdown of miR-211 in SK-OV-3 cells partially abrogated the anti-tumor behavior of LSZ by promoting the viability, proliferation, migration, invasion and EMT of SK-OV-3 cells. Thus, we found that LSZ can inhibit the proliferation and migration of OC cells via regulating miR-211. Our study suggests that LSZ might be a potential and effective treatment for OC.     

NF-kappaB and Hsp70 are involved in the phospholipase Cgamma1 signaling pathway in colorectal cancer cells

The majority of deaths from colorectal cancer are due to tumor invasion and metastasis. Induced migration of tumor cell is generally considered to be one critical step in cancer progression to the invasive and metastatic stage. Phospholipase Cgamma1 (PLCgamma1) is a key molecular switch in the process. But, the mechanism and function of PLCgamma1 in colorectal cancer motility are unclear. We showed first in this report that epidermal growth factor (EGF) stimulated the phosphorylation of PLCgamma1 in human colorectal cancer cell line LoVo. Inhibition of PLCgamma1 with the pharmacologic agent U73122 decreased the migration of LoVo cells in a dose-dependent manner while EGF treatment reversed it partially. PLCgamma1 signaling pathway also upregulated the activity of NF-kappaB. Furthermore, expression of Hsp70 was increased by treatment with U73122 or pyrrolidine dithiocarbamate (PDTC), a NF-kappaB inhibitor. These data indicated that PLCgamma1 played a pivotal role in the migration of human colorectal cancer cell and first demonstrated that upregulation of NF-kappaB binding activity and downregulation of Hsp70 expression were PLCgamma1-dependent in LoVo cells.     

DLC-1基因表达对结肠癌细胞侵袭转移能力的影响

目的:研究DLC-1基因对结肠癌细胞侵袭迁移能力的影响.方法:将DLC-1 shRNA(短发夹状RNA,short hairpin RNA)序列克隆到质粒pGCsi-U6/Neo载体,采用脂质体介导的转染方法将构建的DLC-1 shRNA表达质粒转入结肠癌细胞系LoVo细胞.采用RT-PCR技术和Western Blot技术分别检测LoVo细胞中DLC-1mRNA和蛋白表达水平的变化.Transwell小室人工重组基底膜侵袭转移实验观察LoVo细胞侵袭迁移能力的改变.结果:结肠癌细胞系LoVo细胞表达DLC-1分子.所构建质粒表达载体能有效地干扰LoVo细胞DLC-1 mRNA和蛋白质表达水平;Transwell小室人工重组基底膜侵袭转移实验结果显示,转染后LoVo细胞侵袭转移能力明显增强(p＜0.05).结论:结肠癌细胞系LoVo细胞表达DLC-1基因,应用RNAi技术可特异性降低其表达.DLC-1的表达水平与结肠癌细胞侵袭转移相关.     

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.