Diosgenin exerts its tumor suppressive function via inhibition of Cdc20 in osteosarcoma cells

Osteosarcoma (OS) is one of the aggressive malignancies for young adults. Cdc20 (cell division cycle 20 homologue) has been reported to exhibit an oncogenic role in OS, suggesting that inhibition of Cdc20 could be a novel strategy for the treatment of OS. Since Cdc20 inhibitors have side effects, it is important to discover the new CDC20 inhibitors with non-toxic nature. In the present study, we determine whether natural agent diosgenin is an inhibitor of Cdc20 in OS cells. We performed MTT, FACS, Wound healing assay, Transwell, Western blotting, transfection assays in our study. We found diosgenin inhibited cell growth and induced apoptosis. Moreover, diosgenin exposure led to inhibition of cell migration and invasion. Notably, diosgenin inhibited the expression of Cdc20 in OS cells. Overexpression of Cdc20 abrogated the inhibition of cell growth and invasion induced by diosgenin. Our data reveal that inhibition of Cdc20 by diosgenin could be helpful for the treatment of patients with OS.     

The tumor suppressive role of eIF3f and its function in translation inhibition and rRNA degradation

Deregulated translation plays an important role in human cancer. We previously reported decreased eukaryotic initiation factor 3 subunit f (eIF3f) expression in pancreatic cancer. Whether decreased eIF3f expression can transform normal epithelial cells is not known. In our current study, we found evidence that stable knockdown of eIF3f in normal human pancreatic ductal epithelial cells increased cell size, nuclear pleomorphism, cytokinesis defects, cell proliferation, clonogenicity, apoptotic resistance, migration, and formation of 3-dimensional irregular masses. Our findings support the tumor suppressive role of eIF3f in pancreatic cancer. Mechanistically, we found that eIF3f inhibited both cap-dependent and cap-independent translation. An increase in the ribosomal RNA (rRNA) level was suggested to promote the generation of cancer. The regulatory mechanism of rRNA degradation in mammals is not well understood. We demonstrated here that eIF3f promotes rRNA degradation through direct interaction with heterogeneous nuclear ribonucleoprotein (hnRNP) K. We showed that hnRNP K is required for maintaining rRNA stability: under stress conditions, eIF3f dissociates hnRNP K from rRNA, thereby preventing it from protecting rRNA from degradation. We also demonstrated that rRNA degradation occurred in non-P body, non-stress granule cytoplasmic foci that contain eIF3f. Our findings established a new mechanism of rRNA decay regulation mediated by hnRNP K/eIF3f and suggest that the tumor suppressive function of eIF3f may link to impaired rRNA degradation and translation.     

Induced expression of miR-1250-5p exerts tumor suppressive role in triple-negative breast cancer cells

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, and it has a prevalence rate of 15%–20% among all breast cancer cases in younger women. Still, the underlying molecular mechanisms of its pathogenesis are not entirely understood. In the previous study, we identified that microRNA (miR)-1250-5p is significantly down-expressed in TNBC cells. Thus, in the present study, we explore the functional anticancer role of miR‑1250‑5p in the transient mimic transfected TNBC cells. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to examine the effect of miR-1250-5p on cell viability of TNBC (MDA-MB-231 and MDA-MB-453) cells. The confocal microscopy, quantitative real-time polymerase chain reaction, and western blot analysis techniques were used to assess the effect of miR-1250-5p on cancer hallmarks in test cells. Induced miR‑1250-5p expression in MDA-MB-231 and MDA-MB-453 cells decreased cell viability in a time-dependent manner. Increased miR‑1250-5p expression levels significantly decreased cell cycle G1/S phase transition markers (Cyclin D1 and CDK4) at messenger RNA (mRNA) and protein levels in TNBC cells compared to scrambled sequence transfected cells. Transient transfection of TNBC cells with miR-1250-5p mimic increased apoptosis in TNBC cells by increasing the level of active caspase (Caspase 8 and Caspase 3) of the intrinsic pathway. Apoptosis-related morphological changes were also observed in the test cells. Further, the induced expression of miR-1250-5p significantly decreased epithelial-mesenchymal transition (EMT) by altering the mRNA and protein levels of E-cadherin and Vimentin. Moreover, results of confocal microscopy revealed increased reactive oxygen species generation, and decreased mitochondria membrane potential in miR-1250-5p mimic transient transfected TNBC cells. In conclusion, miR‑1250-5p acts as tumor suppressor in TNBC cells and its induction by therapeutics might be a novel strategy for the disease treatment.     

Intronic polyadenylation in the human glycinamide ribonucleotide formyltransferase gene.

The mouse glycinamide ribonucleotide formyltransferase (GART) locus is known to produce two functional proteins, one by recognition and use of an intronic polyadenylation site and the other by downstream splicing. We now report a similar intronic polyadenylation mechanism for the human GART locus. The human GART gene has two potential polyadenylation signals within the identically located intron as that involved in intronic polyadenylation in the mouse gene. Each of the potential polyadenylation signals in the human gene was followed by an extensive polyT rich tract, but only the downstream signal was preceded by a GT tract. Only the downstream signal was utilized. The polyT rich tract which followed the functional polyadenylation site in the human GART gene was virtually identical in sequence to a similarly placed region in the mouse gene. An exact inverted complement to the polyT rich stretch following the active polyadenylation signal was found in the upstream intron of the human gene, suggesting that a hairpin loop may be involved in this intronic polyadenylation.     

Intronic gene conversion in the evolution of human X-linked color vision genes

Human red and green visual pigment genes are X-linked duplicate genes. To study their evolutionary history, introns 2 and 4 (1,987 and 1,552 bp, respectively) of human red and green pigment genes were sequenced. Surprisingly, we found that intron 4 sequences of these two genes are identical and that the intron 2 sequences differ by only 0.3%. The low divergences are unexpected because the duplication event producing the two genes is believed to have occurred before the separation of the human and Old World monkey (OWM) lineages. Indeed, the divergences in the two introns are significantly lower than both the synonymous divergence (3.2% +/- 1.1%) and the nonsynonymous divergence (2.0% +/- 0.5%) in the coding sequences (exons 1-6). A comparison of partial sequences of exons 4 and 5 of human and OWM red and green pigment genes supports the hypothesis that the gene duplication occurred before the human-OWM split. In conclusion, the high similarities in the two intron sequences might be due to very recent gene conversion, probably during evolution of the human lineage.      

miR-216 and miR-217 expression is reduced in transgenic mouse models of pancreatic adenocarcinoma,knockout of miR-216/miR-217 host gene is embryonic lethal

Mice harboring a G12D activating Kras mutation are among the most heavily studied models in the field of pancreatic adenocarcinoma (PDAC) research. miRNAs are differentially expressed in PDAC from patients and mouse models of PDAC. To better understand the relationship that Kras activation has on miRNA expression, we profiled the expression of 629 miRNAs in RNA isolated from the pancreas of control, young, and old P48^+/Cre;LSL-KRAS^G12D as well as PDX-1-Cre;LSL-KRAS^G12D mice. One hundred of the differentially expressed miRNAs had increased expression in the advanced disease (old) P48^+/Cre;LSL-KRAS^G12D compared to wild-type mice. Interestingly, the expression of three miRNAs, miR-216a, miR-216b, and miR-217, located within a ～30-kbp region on 11qA3.3, decreased with age (and phenotype severity) in these mice. miR-216/-217 expression was also evaluated in another acinar-specific ELa-Kras^G12D mouse model and was downregulated as well. As miR-216/-217 are acinar enriched, reduced in human PDAC and target KRAS, we hypothesized that they may maintain acinar differentiation or represent tumor suppressive miRNAs. To test this hypothesis, we deleted a 27.9-kbp region of 11qA3.3 containing the miR-216/-217 host gene in the mouse’s germ line. We report that germ line deletion of this cluster is embryonic lethal in the mouse. We estimate that lethality occurs shortly after E9.5. qPCR analysis of the miR-216b and miR-217 expression in the heterozygous animals showed no difference in expression, suggesting haplosufficiency by some type of compensatory mechanism. We present the differential miRNA expression in Kras^G12D transgenic mice and report lethality from deletion of the miR-216/-217 host gene in the mouse’s germ line.     

Epigenetic inactivation of SPINT2 is associated with tumor suppressive function in esophageal squamous cell carcinoma

Hepatocyte growth factor activator inhibitor type 2 (SPINT2), a Kunitz-type serine proteinase inhibitor, has been identified as a putative tumor suppressor gene silenced by promoter methylation. We aimed to investigate whether SPINT2 might act as an esophageal squamous cell carcinoma (ESCC) tumor suppressor gene. Four ESCC cell lines, Fifty-two ESCC tissues and twenty-nine neighboring non-cancerous tissues were included in this study. The expression of SPINT2 was monitored by real time PCR. Bisulfite genomic sequencing and methylation-specific PCR were used to analyze methylation status. The effect of SPINT2 on cell proliferation and apoptosis in EC109 and EC9706 cells was observed by CCK-8 assay and flow cytometric analysis. We found that silencing of SPINT2 was associated with promoter methylation in ESCC cell lines. The densely methylated SPINT2 promoter region was confirmed by bisulfite genomic sequencing. Ectopic expression of SPINT2 inhibited cell proliferation through inducing cell apoptosis in vitro. Furthermore, methylation-specific PCR analysis revealed that SPINT2 promoter methylation was prominent in carcinoma tissues (52.08%) compared with neighboring non-cancerous tissues (22.58%). Kaplan–Meier analysis showed that patients with SPINT2 hypermethylation had shorter survival time. The tumor suppressor gene of SPINT2 is commonly silenced by promoter hypermethylation in human ESCC and SPINT2 hypermethylation is correlated with poor overall survival, implicating SPINT2 is an underlying prognostic marker for human ESCC.     

XIAP,commonly targeted by tumor suppressive miR-3607-5p and miR-3607-3p,promotes proliferation and inhibits apoptosis in hepatocellular carcinoma

MicroRNAs (miRNAs) are frequently aberrantly expressed in hepatocellular carcinoma (HCC) and are involved in its development. However, their role and mechanism in HCC are still not fully elucidated. Differential expression analysis and survival analysis were performed to identify potential miRNAs in HCC and miR-3607 was identified as a candidate therapeutic target and prognostic biomarker. RT-qPCR confirmed the low expression of mature miR-3607-3p and miR-3607-5p in HCC. Functional experiments suggested that both miR-3607-3p and miR-3607-5p significantly inhibited HCC proliferation and induced apoptosis. Next, the detailed mechanism of miR-3607-3p and miR-3607-5p in HCC was explored by combination of bioinformatic analysis and experimental validation, and uncovered that XIAP, a common target gene of miR-3607-3p and miR-3607-5p, was involoved in their tumor suppressive effects. Finally, a XIAP-associated protein-protein interaction network, consisting of 10 positively correlated genes, was established. Collectively, we for the first time suggest that miR-3607-3p and miR-3607-5p inhibit HCC by acting one common target XIAP.     

Combined effects of radiotherapy and endostatin gene therapy in melanoma tumor model

PEgr–Endostatin–EGFP plasmid was constructed to investigate its expression properties induced by ionizing irradiation and the effect of pEgr–Endostatin–EGFP gene-radiotherapy on melanoma tumor-bearing mice. The pEgr–Endostatin–EGFP plasmid was transfected into B16 cell line with liposome. The expression property of endostatin was investigated by RT-PCR and that of EGFP was detected by flow cytometry. Tumor-bearing mice were treated by the plasmid injection and 2 Gy X-irradiation of three fractions. Tumor growth was observed for 18 days after treatment. Change of tumor capillary formation was measured with histochemistry assay at the end of the experiment. The expression of GFP in B16 melanoma cells was detected after X-irradiation with 0.05–20 Gy. Time-course studies showed that the expression of GFP in B16 cells reached its peak at 8 h after irradiation with 2 Gy. The injection of pEgr–Endostatin–EGFP recombinant plasmid into the implanted B16 melanoma in C57BL/6J mice followed by local X-irradiation could significantly inhibit tumor growth with inhibition of intratumor micro-vessel density. The inhibitory effect of pEgr–Endostatin–EGFP gene-radiotherapy on the growth of B16 melanoma is correlated with the marked decrease of intratumoral vascularization. The present data point to the potential of an anti-angiogenic approach in gene-radiotherapy of cancer.     

Circular RNA circMTO1 suppressed the progression of renal cell carcinoma progression by sponging miR-211 and miR-204

Despite considerable improvements in renal cell carcinoma (RCC) diagnostic and therapeutic strategy, the clinical prognosis of patients is far from satisfactory due to its recurrence and metastasis. Here, we attempted to explore the role of circMTO1 in RCC progression, and the underlying mechanism was further elucidated. We first detected the expression of circMTO1 in 90 pairs of RCC tissues and adjacent normal tissues using qRT-PCR. Besides, circMTO1, miR-211, miR-204 and KLF6 expression levels in RCC cells were also measured using qRT-PCR. MTT assay, cell migration, flow cytometry analysis, qRT-PCR and western blotting analysis were applied to evaluating the effect of circMTO1 in RCC cells. The bioinformatics analysis and the rescue experiment were devoted to the underlying mechanism. The results demonstrated CircMTO1 expression was significantly down-regulated in RCC tissues and cell lines. Besides, CircMTO1 inhibited cell proliferation, migration and invasion, induced apoptosis in RCC cells. In addition, CircMTO1 serves as a sponge for miR-211 and miR-204, KLF6 is a direct target of miR-211 and miR-204. Furthermore, circMTO1 and KLF6 overexpression rescued the suppression of miR-211/204 in RCC cell proliferation. In short, circMTO1 repressed RCC progression by regulating KLF6 via sponging miR-211 and miR-204, which may provide new idea of diagnosis and treatment in renal cell carcinoma.

     

脑胶质瘤组织miR-211、miR-374、miR-510表达水平与临床病理特征及预后的关系研究

摘要 目的：探讨脑胶质瘤组织微小RNA（miR）-211、miR-374、miR-510表达水平与临床病理特征及预后的关系。方法：选择2013年8月至2015年8月我院诊治的83例脑胶质瘤患者作为研究对象,选择同期由于脑外伤在我院行内减压术切除的正常脑组织样本31份作为对照样本。采用荧光定量PCR检测miR-211、miR-374、miR-510表达水平,生存分析采用Kaplan-Meier法,应用Cox比例风险回归模型分析预后的影响因素。结果：与正常脑组织相比,脑胶质瘤组织中miR-211、miR-374表达水平明显下降,miR-510表达水平明显升高（P<0.05）。脑胶质瘤组织miR-211、miR-374、miR-510表达均与WHO分级和卡氏功能状态量表(KPS)评分有关（P<0.05）。miR-211、miR-374低表达患者的5年总生存率明显低于高表达患者,miR-510低表达患者的5年总生存率明显高于高表达患者（P<0.05）。WHO分级、KPS评分、miR-211、miR-374和miR-510表达是脑胶质瘤患者预后的影响因素（P<0.05）。结论：脑胶质瘤组织中miR-211和miR-374表达下调,而miR-510表达上调,miR-211、miR-374和miR-510表达均与WHO分级、KPS评分和预后相关,检测miR-211、miR-374和miR-51在脑胶质瘤患者的诊断和治疗中具有一定临床意义。     

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.     

Tumour suppressive function of HUWE1 in thyroid cancer

HUWE1 (the HECT, UBA, and WWE domain-containing protein 1) is an ubiquitin E3 ligase which plays an important role in coordinating diverse cellular processes. It has been found to be dysregulated in various cancer type and its functions in tumorigenesis remain controversial. The potential tumour suppressive role of HUWE1 in thyroid cancer development was investigated by knocking down HUWE1 in three authentic thyroid cancer cell lines, WRO, FTC133 and BCPAP, followed by various functional assays, including cell proliferation, scratch wound healing and invasion assays. Xenograft experiment was performed to examine in vivo tumour suppressive properties of HUWE1. Small-interfering RNA mediated knockdown of HUWE1 promoted cell proliferation, cell migration and invasion in thyroid cancer cells. Overexpression of HUWE1 conferred partial sensitivity to chemo drugs interfering with DNA replication in these cells. Moreover, HUWE1 was found to be down-regulated in human thyroid cancer tissues compared with matched normal thyroid tissues. In addition, overexpression of HUWE1 significantly inhibited tumour growth in vivo using xenograft mouse models. Mechanistic investigation revealed that HUWE1 can regulate p53 protein level through its stabilization. HUWE1 functions as a tumour suppressor in thyroid cancer progression, which may represent a novel therapeutic target for prevention or intervention of thyroid cancer.