Berberine Modulates Macrophage Activation by Inducing Glycolysis.

Classical activation of macrophage and monocyte differentiation induced by ß-glucan is accompanied with metabolic change in glucose. However, the role of the metabolic rewiring in monocyte/macrophage activation remains elusive. In this study, we show that berberine induces aerobic glycolysis by blocking the tricarboxylic acid cycle and modulates cytokine responses in bone marrow-derived macrophages (BMDMs) from mice and human PBMC. 13-Methyberberine had activities on glucose metabolism and BMDM activation similar to those of berberine, whereas other tested derivatives lost both activities. Glucose transporter (GLUT)1 expression and total cellular hexokinase activity increased gradually in BMDMs in the presence of berberine. In the contrast, LPS upregulated GLUT1 and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) levels in 6 h. Extracellular glucose levels and replacing glucose with galactose in culture medium affected the cytokine secretion of BMDMs. Berberine alleviated enteritis of Salmonella typhimurium infection and protected mice against endotoxic shock. In mice i.p. injected with LPS, the increase of serum TNF-α and the drop of blood glucose were attenuated by berberine treatment. These data together demonstrated that macrophage activation was closely related with glucose metabolism.

CircSRSF4 Enhances Proliferation, Invasion, and Migration to Promote the Progression of Osteosarcoma via Rac1.

(1) Background: At present, cancer cell metastasis is the main cause of death in patients with malignant tumors, and up to 23% of osteosarcoma patients have died due to lung and lymph node metastasis. Therefore, finding new molecules involved in tumor development can provide new strategies for the diagnosis and treatment of osteosarcoma patients. Circular RNAs (circRNAs) are a type of RNA molecule that are connected head-to-tail to form a closed ring. There is increasing evidence that circRNAs are RNA molecules with many biological functions in various diseases. However, the role and mechanism of circRNAs in osteosarcoma have rarely been reported. (2) Methods: The expression of circSRSF4 in osteosarcoma tissues and cell lines was detected by quantitative real-time PCR (RT-qPCR), and the result of high-throughput sequencing was verified. In order to explore the effect of circSRSF4 on tumor proliferation, invasion, and migration, a dual-luciferase reporter assay, RNA binding protein immunoprecipitation assay, cell counting kit-8 (CCK-8), transwell assay, scratch wound healing assay, Western blot analysis, and other experiments were carried out in vitro. Rescue experiments and a xenograft model confirmed that circSRSF4 directly acted on miR-224 to regulate Rac1 expression. (3) Results: The expression of circSRSF4 was significantly higher in osteosarcoma tissues and cell lines. Down-regulating the expression of circSRSF4 in vitro significantly inhibited the proliferation, invasion, and migration of cells, and also reduced the expression of Rac1, while the overexpression of Rac1 and miR-224 inhibition could reverse these effects. The inhibition of circSRSF4 expression in vivo also attenuated tumor growth. A mechanistic study showed that circSRSF4 can be used as an miR-224 sponge to up-regulate the expression of Rac1, thereby promoting the development of osteosarcoma. (4) Conclusions: CircSRSF4 acting as a ceRNA promotes the malignant behavior of osteosarcoma through the circSRSF4/miR-224/Rac1 axis, which provides a new theoretical basis for the clinical prevention and treatment of osteosarcoma and the study of related markers and intervention targets.

Long noncoding RNA APTR contributes to osteosarcoma progression through repression of miR-132-3p and upregulation of yes-associated protein 1.

A growing amount of evidence has shown that long noncoding RNAs (lncRNAs) play crucial roles in osteosarcoma (OS). However, little knowledge is available about the functional roles and molecular mechanisms of lncRNA Alu-mediated p21 transcriptional regulator (APTR) in OS. Herein, APTR expression was demonstrated to be significantly upregulated in OS tumor tissues and four OS cell lines (including MG63, 143B, Saos-2, and HOS) compared with the adjacent tissues and human osteoblast cell line hFOB1.19, respectively. We confirmed miR-132-3p to be a target for APTR, and its expression was demonstrated to be inhibited by APTR. In functional terms, knockdown of APTR and overexpression of miR-132-3p both, remarkably repressed human OS cell proliferation, invasion and migration, and induced apoptosis. Also, Yes-associated protein 1 (YAP1) was determined as an inhibitory target of miR-132-3p. Moreover, our findings demonstrated that the repression of YAP1 protein expression and the suppression of Ki-67, MMP9, and Bcl2 expression induced by APTR knockdown required increased miR-132-3p. Thus, APTR contributed to OS progression through repression of miR-132-3p and upregulation of YAP1 expression. Therefore, we have uncovered a novel regulatory mechanism by which the APTR/miR-132-3p/YAP1 axis can regulate OS progression.

miR-17-5p promotes proliferation and epithelial-mesenchymal transition in human osteosarcoma cells by targeting SRC kinase signaling inhibitor 1.

MicroRNA-17-5p (miR-17-5p) and epithelial-mesenchymal transition (EMT) have been reported to participate in the development and progression of multiple cancers. However, the relationship between the miR-17-5p and EMT in osteosarcoma (OS) is still poorly understood. This study was to investigate the effects of the miR-17-5p and its potential mechanism in regulating proliferation, apoptosis, and EMT of human OS. Quantitative real-time PCR was used to detect the miR-17-5p and SRC kinase signaling inhibitor 1 (SRCIN1) messenger RNA expression in OS specimens and cell lines. After transfection with miR-17-5p inhibitors, proliferation, apoptosis, migration, and invasion of OS cells were assessed by using the Cell Counting Kit-8, the annexin V-FITC apoptosis, wound-healing, and transwell assays. The SRCIN1 was validated as a target of the miR-17-5p through bioinformatics algorithms and luciferase reporter assay. Moreover, the expression of EMT markers, E-cadherin, N-cadherin, and Snail was identified by the Western blot analysis. MiR-17-5p was significantly upregulated in OS tumor samples and cell lines. It inhibited proliferation and EMT, and promoted apoptosis in OS. The SRCIN1 was identified as a direct target of the miR-17-5p. Silenced miR-17-5p could change the expression of EMT markers, such as upregulating the expression of E-cadherin, and downregulating the expression of N-cadherin and Snail through targeting the antioncogenic SRCIN1. These findings suggest that the miR-17-5p promotes cell proliferation, and EMT in human OS by directly targeting the SRCIN1, and reveal a branch of the miR-17-5p/SRCIN1/EMT signaling pathway involved in the progression of OS.

Long non-coding RNA TCONS_00041960 enhances osteogenesis and inhibits adipogenesis of rat bone marrow mesenchymal stem cell by targeting miR-204-5p and miR-125a-3p.

A growing number of long non-coding RNAs (lncRNAs) have been found to be involved in diverse biological processes such as cell cycle regulation, embryonic development, and cell differentiation. However, limited knowledge is available concerning the underlying mechanisms of lncRNA functions. In this study, we found down-regulation of TCONS_00041960 during adipogenic and osteogenic differentiation of glucocorticoid-treated bone marrow mesenchymal stem cells (BMSCs). Furthermore, up-regulation of TCONS_00041960 promoted expression of osteogenic genes Runx2, osterix, and osteocalcin, and anti-adipogenic gene glucocorticoid-induced leucine zipper (GILZ). Conversely, expression of adipocyte-specific markers was decreased in the presence of over-expressed TCONS_00041960. Mechanistically, we determined that TCONS_00041960 as a competing endogenous RNA interacted with miR-204-5p and miR-125a-3p to regulate Runx2 and GILZ, respectively. Overall, we identified a new TCONS_00041960-miR-204-5p/miR-125a-3p-Runx2/GILZ axis involved in regulation of adipogenic and osteogenic differentiation of glucocorticoid-treated BMSCs.

Upregulation of miR-192 inhibits cell growth and invasion and induces cell apoptosis by targeting TCF7 in human osteosarcoma.

MicroRNAs (miRNAs) can function as oncogenes or tumor suppressor genes and are involved in multiple processes in cancer development and progression. For example, miR-192 is dysregulated in multiple human cancers, including osteosarcoma (OS). However, the pathophysiological role of miR-192 and its relevance to OS cell growth and invasion has not yet been clarified. This study aimed to investigate the expression of miR-192 in OS and elucidate the molecular mechanisms by which miR-192 acts as a tumor suppressor in this disease. The qRT-PCR data identified significant down-regulation of miR-192 in 20 OS tissue samples and two OS cell lines when compared with adjacent normal tissues and a human osteoblast cell line, respectively. Furthermore, Western blot analysis revealed overexpression of T cell-specific transcription factor (TCF) 7 protein in tumor tissues compared with matched adjacent normal tissues. Further in vitro studies demonstrated that enforced expression of miR-192 inhibited U2OS and MG63 cell proliferation, invasion, and migration and induced apoptosis. Finally, Western blot and Luciferase assays identified TCF7 as a target of miR-192. Collectively, these findings suggest an important role for miR-192 in regulating the proliferation, migration, invasion, and apoptosis of OS cells through the regulation of TCF7.

Downregulation of PPARγ by miR-548d-5p suppresses the adipogenic differentiation of human bone marrow mesenchymal stem cells and enhances their osteogenic potential.

BACKGROUND: Human bone marrow mesenchymal stem cells (hBMSCs) are multipotent cells that can differentiate into a variety of cell types. Elevated expression of peroxisome proliferator-activated receptor-γ (PPARγ) promotes the adipogenic differentiation of hBMSCs, and reduces their osteogenic differentiation. MicroRNAs (miRNAs) have been shown to play important roles in the regulation of hBMSCs differentiation. Because bioinformatic analysis has indicated that PPARγ is a candidate target of miR-548d-5p, the aim of this study was to assess the impact of miR-548d-5p on the dexamethasone-induced adipogenic differentiation of hBMSCs. METHODS: A quantitative RT-PCR (qRT-PCR) assay was used to compare miR-548d-5p expression levels in dexamethasone-induced hBMSCs and uninduced control cells. Oil red O staining, cellular triglyceride (TG) content, and the mRNA and protein levels of PPARγ and CCAAT/enhancer binding protein α (C/EBPα) were used to evaluate the adipogenic differentiation of hBMSCs. Alkaline phosphatase (ALP) activity and levels of osteocalcin (OCN) and Runx2 were used to evaluate the osteogenic potential of hBMSCs. RESULTS: Compared with untreated cells, miR-548d-5p expression levels were downregulated during dexamethasone-induced adipogenic differentiation of hBMSCs. In contrast to the profuse Oil Red O staining in the cytoplasm of dexamethasone + scrambled miRNA-treated cells, there was limited staining in the cytoplasm of dexamethasone + miR-548d-5p-treated cells, indicating the absence of adipocytes. Moreover, compared with scrambled miRNA-treated cells, treatment with miR-548d-5p suppressed cellular levels of PPARγ and C/EBPα mRNA and protein, and cell TG content (P < 0.05). In contrast, compared with scrambled miRNA-treated cells, cellular levels of OCN and Runx2 mRNA and protein, as well as ALP activity, were significantly higher in miR-548d-5p-treated cells (P < 0.05). Western blot and luciferase reporter assays confirmed that miR-548d-5p directly targeted the 3'-untranslated region of PPARγ. CONCLUSIONS: miR-548d-5p is downregulated during dexamethasone-induced adipogenic differentiation of hBMSCs. By directly targeting and downregulating PPARγ, miR-548d-5p suppresses the dexamethasone-induced adipogenic differentiation of hBMSCs and enhances their osteogenic potential. Our findings suggest that miR-548d-5p has potential in the treatment of corticosteroid-induced osteonecrosis of the femoral head.

The effect of combined regulation of the expression of peroxisome proliferator-activated receptor-γ and calcitonin gene-related peptide on alcohol-induced adipogenic differentiation of bone marrow mesenchymal stem cells.

Studies have shown that alcohol can upregulate the expression of peroxisome proliferator-activated receptor-γ (PPARγ) gene in bone marrow mesenchymal stem cells (BMSCs). High expression of PPARγ can promote adipogenic differentiation of BMSCs, and reduce their osteogenic differentiation. Abnormal proliferation of adipocytes and fatty accumulation in osteocytes can result in high intraosseous pressure and disturbance of blood circulation in the femoral head, which induces osteonecrosis of the femoral head (ONFH). Downregulation of PPARγ is efficient in inhibiting adipogenesis and maintaining osteogenesis of BMSCs, which might potentially reduce the incidence of ONFH. Calcitonin gene-related peptide (CGRP) is a neuropeptide gene which has been closely associated with bone regeneration. In this study, we aimed to observe the effect of combined regulation of the expression of PPARγ and CGRP genes on alcohol-induced adipogenic differentiation of BMSCs. Our results demonstrated that simultaneous downregulation of PPARγ and upregulation of CGRP was efficient in suppressing adipogenic differentiation of BMSCs and promoting their osteogenic differentiation. These findings might enlighten a novel approach for the prevention of ONFH.

Preventive effects of siRNA targeting PPARγ gene on steroid-induced osteonecrosis in rabbits.

UNLABELLED: PURPOSES/AIM: Glucocorticoid steroids can induce expression of PPARγ gene and enhance adipogenesis by bone marrow mesenchymal stem cells (BMSCs), which may result in osteonecrosis of the femoral head. Currently, there are no medications available to prevent steroid-induced osteonecrosis. We hypothesized that siRNA targeting PPARγ gene may prevent steroid-induced adipogenesis and osteonecrosis in rabbit. The purpose of this study was to evaluate the preventive effects of siRNA targeting PPARγ gene on steroid-induced adipogenesis and osteonecrosis. METHODS: Forty-eight healthy New Zealand rabbits were randomized into four groups with Group M treated with dexamethasone only, Group S with dexamethasone and a recombinant adenovirus shuttle vector carrying siRNA targeting PPARγ gene, Group Con with dexamethasone and a vector carrying irrelative sequence, and Group N with no treatment serving as control. Expressions of the PPARγ, osteocalcin and Runx2 genes, as well as histopathologic changes were evaluated. RESULTS: The levels of PPARγ gene expression were decreased while the levels of osteocalcin and Runx2 gene expression were increased in rabbits treated with dexamethasone and recombinant adenovirus shuttle vector carrying siRNA targeting PPARγ gene (Group S), compared to rabbits treated either with dexamethasone alone (Group M) or with both dexamethasone and a vector carrying irrelative sequence (Group Con). The marrow necrosis, adipocyte hypertrophy and proliferation, diminished hematopoiesis, thinner and sparse trabeculae, and increased empty osteocyte lacunae in the femoral head were observed in Group M and Group Con rabbits. However, no such changes were seen in Group S rabbits that were treated with dexamethasone and a recombinant adenovirus shuttle vector carrying siRNA targeting PPARγ gene. CONCLUSION: siRNA targeting PPARγ gene can inhibit adipogenic differentiation of BMSCs and prevent steroid-induced osteonecrosis in rabbit. The inhibition of bone-marrow adipogenesis and concomitant enhancement of osteogenesis with RNAi may provide a novel approach to the prevention of steroid-induced osteonecrosis.

DNA polymerase ß promoter mutations and transcriptional activity in esophageal squamous cell carcinoma.

The present study analyzed the correlation of DNA polymerase ß (DNA polß) promoter mutations and activity in esophageal squamous cell carcinoma (ESCC). The DNA polß promoter was amplified from 108 ESCC samples and adjacent paracancerous samples by PCR and cloned into the pGL3-enhancer luciferase vector. The recombined vectors were transfected into esophageal carcinoma cells (EC9706, Eca109, and KYSE30), and luciferase activity was detected using dual luciferase reporter gene technology. Eleven polß promoter mutations were identified and submitted to GenBank. The mutation rate of the DNA polß promoter was higher in ESCC tissues (36/108, 33.3 %) than in the paired paracancerous tissues (21/108, 19.4 %) (P = 0.021). The C → A mutation at locus -37 was the hotspot mutation in cancerous tissues, and its frequency was higher in ESCC tissues (26/108) than in paracancerous tissues (7/108) (P = 0.00). The highest relative luciferase activity (RLA) was observed in the DNA polß promoter, with a C → A mutation at -37. Significant differences in RLA were observed between mutant DNA polß promoters (except for C detected at -19, T → C at -194, C → A at -37, and T → C at 30) and the wild-type DNA polß promoter (P = 0.000), and RLA was significantly higher in ESCC tissues than in paracancerous tissues (P = 0.003). Our findings suggest that the upregulation of transcriptional activity induced by mutations in the DNA polß promoter in ESCC tissues may be one of the molecular mechanisms mediating abnormal overexpression of DNA polß in ESCC.

DNA polymerase beta promoter mutations affect gene transcription, translation and the sensitivity of esophageal cancer cells to cisplatin treatment.

The ability of a promoter to initiate transcription is important for the control of gene expression. Mutations in the DNA polymerase beta (po1ß) promoter may affect the transcription of this gene; however, the relationship between these mutations and the upregulation of the expression of po1ß remains unclear. Therefore, in the present study, three po1ß promoter mutants (M1, -37 C→A; M2, -114 G→A, -37 C→A; M3, -194 T→C) were generated to examine the effect of promoter mutations on polß gene expression and sensitivity to cisplatin. We found that the M1 and M2 mutant polß promoter constructs showed higher RLA than the wild-type polß promoter (P < 0.01), whereas the activity of the M3 polß promoter did not differ significantly from that of the wild-type polß promoter (P > 0.05). The expression levels of polß mRNA and protein were significantly higher (P < 0.01) and the sensitivity to cisplatin was significantly lower (P < 0.05) in Eca9706(-/-)-M1 and Eca9706(-/-)-M2 cells than in Eca9706(-/-)-W. The expression levels of polß mRNA and protein and the sensitivity to cisplatin were not significantly different between Eca9706(-/-)-M3 and Eca9706(-/-)-W cells (P > 0.05).These results revealed that specific mutations of the polymerase beta gene promoter significantly enhanced the gene's transcriptional activity. These mutations correspondingly increased the gene's mRNA and protein product, at the same time reduced the esophageal cancer cells' sensitivity to cisplatin.

Erratum: MicroRNA-183 suppresses the vitality, invasion and migration of human osteosarcoma cells by targeting metastasis-associated protein 1.

[This corrects the article DOI: 10.3892/etm.2018.6068.].

Nucleolar small molecule RNA SNORA75 promotes endometrial receptivity by regulating the function of miR-146a-3p and ZNF23.

Endometrial receptivity enables the embryo to attach, invade and develop, forming a new individual and species continuity. Small nucleolar RNAs (SnoRNAs) are a class of non-coding RNAs comprising two classes: C/D box snoRNAs and H/ACA box snoRNAs. Aberrant expression of SNORNAs has been reported in tumorigenesis. However, the role of SNORNAs in maintaining endometrial receptivity has not been reported. First, we detected SNORNA expression in endometrial tissues during proliferative and secretory endometrial periods using RNA sequencing. SNORA75 expression was higher in the secretory endometrium, and its overexpression significantly promoted the proliferation, migration and invasion of endometrial cells. The results of analysis with bioinformatics software and RNA pulldown experiments showed that miR-146a-3p interacted with SNORA75. Western blotting showed that miR-146a-3p regulated the expression of ZNF23, whose overexpression significantly promoted the proliferation, migration and invasion of endometrial cells. SNORA75 modulates endometrial receptivity through the miR-146a/ZNF23 signaling pathway.

LncRNA TCONS_00023297 Regulates the Balance of Osteogenic and Adipogenic Differentiation in Bone Marrow Mesenchymal Stem Cells and the Coupling Process of Osteogenesis and Angiogenesis.

Long noncoding RNA (lncRNA) is a noncoding RNA with a length of more than 200 bases. It plays an important role in the occurrence and development of diseases. Research on lncRNAs has received increasing attention. Bone is an important organ of the human body. As the population ages, the incidence of osteoporosis gradually increases. The mechanism of action of lncRNAs in the development of osteoporosis is unclear. The imbalance between osteogenic and adipogenic differentiation in bone marrow mesenchymal stem cells (hBMSCs) and the coupling process of osteogenesis and angiogenesis plays an important role in the development of osteoporosis. Therefore, this study focused on the mechanism by which lncRNAs regulate the osteogenic differentiation of bone marrow mesenchymal stem cells and the mechanism of action of lncRNAs in bone metabolism. The expression of lncRNAs in the osteogenic differentiation of hBMSCs was detected by lncRNA microarray. Real-time quantitative PCR was used to detect the expression changes of lncRNA and osteogenic genes during hBMSC osteogenic and adipogenic differentiation. The ceRNA mechanisms were detected by RIP and luciferase reporter gene assays. The effect of lncRNAs on the osteogenesis-angiogenesis coupling process was detected by Transwell assays. TCONS_00023297 increased expression during osteogenic differentiation; TCONS_00023297 overexpression promoted osteogenic differentiation of hBMSCs; BMP2 regulated TCONS_00023297 expression in a concentration- and time-dependent manner; TCONS_00023297 regulated miR-608 via a ceRNA mechanism; TCONS_00023297 inhibited hBMSC adipogenic differentiation; and TCONS_00023297 promoted VEGF secretion by hBMSCs. TCONS_00023297 regulates osteogenic differentiation, adipogenic differentiation, and osteogenic-angiogenic coupling of hBMSCs via the TCONS_00023297/miR-608/RUNX2/SHH signaling axis.

Effect of lncRNA XLOC_005950 knockout by CRISPR/Cas9 gene editing on energy metabolism and proliferation in osteosarcoma MG63 cells mediated by hsa-miR-542-3p.

Cancer cells use glucose via glycolysis to maintain tumor cell proliferation. However, the effect of long non-coding RNAs (lncRNAs) on glycolysis in osteosarcoma (OS) cells remains unclear. The present study aimed to investigate the involvement of the lncRNA XLOC_005950/hsa-microRNA (miR)-542-3p/phosphofructokinase, muscle (PFKM) axis in the regulation of glucose metabolism, cell proliferation and apoptosis in the progression of OS. lncRNA XLOC_005950, hsa-miR-542-3p and PFKM expression in OS tissues and cells was detected via reverse transcription-quantitative PCR analysis. CRISPR/Cas9 gene editing was used to knockout lncRNA XLOC_005950 expression in MG63 cells. Cell Counting Kit-8 assay, flow cytometry, PFKM activity, and glucose and lactic acid content determination were performed to assess the effects of lncRNA XLOC_005950 knockout and overexpression of hsa-miR-542-3p on the phenotypes of OS cells. The dual-luciferase reporter assay was performed to confirm the targeting associations between lncRNA XLOC_005950, hsa-miR-542-3p and PFKM. The results demonstrated that lncRNA XLOC_005950 expression was upregulated in OS tissues and cells. Functional experiments indicated that lncRNA XLOC_005950 knockout decreased PFKM activity, the intracellular glucose and lactic acid content, and cell proliferation, while increasing apoptosis of OS cells. Furthermore, lncRNA XLOC_005950 knockout upregulated hsa-miR-542-3p expression and downregulated PFKM expression. Overexpression of hsa-miR-542-3p suppressed PFKM expression. Furthermore, lncRNA XLOC_005950, as the molecular sponge of miR-542-3p in OS, modulated the downstream target gene, PFKM. Taken together, the results of the present study suggest that lncRNA XLOC_005950 knockout may inhibit the progression of OS via hsa-miR-542-3p-mediated regulation of PFKM expression.

Effects of estrogen on breast cancer development: Role of estrogen receptor independent mechanisms.

Development of breast cancer involves genetic factors as well as lifetime exposure to estrogen. The precise molecular mechanisms whereby estrogens influence breast tumor formation are poorly understood. While estrogen receptor alpha (ERalpha) is certainly involved, nonreceptor mediated effects of estradiol (E(2)) may also play an important role in facilitating breast tumor development. A "reductionist" strategy allowed us to examine the role of ERalpha independent effects of E(2) on mammary tumor development in ERalpha knockout (ERKO) mice bearing the Wnt-1 oncogene. Exogenous E(2) "clamped" at early follicular and midluteal phase levels (i.e., 80 and 240 pg/ml) accelerated tumor formation in a dose-related fashion in ERKO/Wnt-1 animals (p = 0.0002). Reduction of endogenous E(2) by oophorectomy (p < 0.001) or an aromatase inhibitor (AI) (p = 0.055) in intact ERKO/Wnt-1 animals delayed tumorigenesis as further evidence for an ER-independent effect. The effects of residual ERalpha or beta were not involved since enhancement of tumor formation could not be blocked by the antiestrogen fulvestrant. 17alpha-OH-E(2), a metabolizable but ER-impeded analogue of E(2) stimulated tumor development without measurable uterine stimulatory effects. Taken together, our results suggest that ER-independent actions of E(2) can influence breast tumor development in concert with ER dependent effects. These observations suggest 1 mechanism whereby AIs, which block E(2) synthesis, would be more effective for breast cancer prevention than use of antiestrogens, which only block ER-mediated effects.

Long non­coding RNA DLX6­AS1 is associated with malignant progression and promotes proliferation and invasion in esophageal squamous cell carcinoma.

Despite being one of the most prevalent and fatal types of cancer worldwide, the biological details of esophageal squamous cell carcinoma (ESCC) remain unknown. Recent studies have demonstrated the crucial roles of long non­coding RNAs (lncRNAs) in diverse biological processes including cancer initiation, progression and metastasis. The aim of the present study was to assess the expression profile of distal­less homeobox 6 antisense RNA 1 (DLX6­AS1) in ESCC tissues and its contributions to ESCC cell proliferation, apoptosis and invasion. The expression of DLX6­AS1 in a series of ESCC samples and paired adjacent noncancerous tissues was evaluated by reverse transcription­quantitative polymerase chain reaction. Cell proliferation, apoptosis, wound healing and Transwell invasion assays were performed to evaluate the roles of DLX6­AS1 in the ESCC cell lines EC109 and KYSE30 transfected with DLX6­AS1 small interfering RNA (siRNA). Compared with the paired adjacent noncancerous tissues, DLX6­AS1 expression was upregulated in the ESCC tissues and significantly associated with differentiation status, Tumor­Node­Metastasis stage, distant metastasis, and lymph node metastasis. Knockdown of DLX6­AS1 significantly suppressed cell proliferation, invasion and migration abilities, and enhanced the apoptotic rate in the two ESCC cell lines. Furthermore, western blot assays revealed that silencing DLX6­AS1 partly influenced the epithelial­mesenchymal transition process in ESCC cells. These results imply that the oncogenic function of DLX6­AS1 may be a novel candidate target for treating human ESCC.

MicroRNA-183 suppresses the vitality, invasion and migration of human osteosarcoma cells by targeting metastasis-associated protein 1.

The aim of the present study was to investigate the effects of microRNA (miR)-183 on vitality, invasion, metastasis and apoptosis in osteosarcoma (OS) cells, mediated by its binding to metastasis-associated protein 1 (MTA1). A dual luciferase reporter assay was performed to determine whether MTA1 was a direct target of miR-183. Cell Counting Kit-8, Transwell, scratch-wound healing, fluorescence-activated cell sorting andterminal deoxynucleotidyl transferase dUTP nick end labeling assays were also performed to investigate the effects of miR-183 expression on the proliferation, invasion, migration and apoptosis of MG63 cells. It was demonstrated that that MTA1 expression levels were significantly higher in OS tissues and MG63 cells compared with corresponding adjacent noncancerous tissues and normal cells, respectively, while miR-183 expression levels were significantly lower (both P<0.05). Furthermore, miR-183 overexpression downregulated MTA1 levels and inhibited cell proliferation (P<0.05), migration (P<0.05) and invasion (P<0.01), as well as promoting apoptosis (P<0.01) by binding to the 3'-untranslated region of MTA1. These results indicate that miR-183 inhibits the vitality, invasion, migration and apoptosis of the OS cell line MG63 by targeting MTA1. These findings may contribute to the development of novel clinical therapeutic approaches for the treatment of OS.

MicroRNA-192 inhibits the proliferation, migration and invasion of osteosarcoma cells and promotes apoptosis by targeting matrix metalloproteinase-11.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression during stem cell growth, proliferation and differentiation. miRNAs are also involved in the development and progression of a number of cancer types, including osteosarcoma (OS). miR-192 is significantly downregulated in various tumors, including lung, bladder and rectal cancer. miR-192 expression is associated with the migration and invasion of OS cells. However, the expression of miR-192 and its effects on the development of OS have not been reported. In the present study, the involvement of miR-192 and its molecular mechanisms in the development of OS was investigated. The results indicate that miR-192 expression was significantly downregulated in OS tissues compared with non-tumor tissues (P<0.05). Next, a miR-192 agomir was transfected into the OS cell line MG-63 to upregulate miR-192. The effects of miR-192 overexpression were then investigated by examining cell proliferation, apoptosis, migration and invasion. Matrix metalloproteinase (MMP)-11 belongs to a family of nine or more highly homologous Zn2+-endopeptidases. It was demonstrated that the mRNA and protein expression of MMP-11 were upregulated in OS tissues compared with non-tumor tissues (P<0.05). MMP-11 was predicted by TargetScan and miRanda as a miR-192 target, which was confirmed by western blotting and dual-luciferase assays. Finally, it was demonstrated that the overexpression of miR-192 was able to downregulate MMP-11 expression and reduce proliferation, migration and invasion, and promote apoptosis in OS cells. Together, these data indicate that miR-192 may be a tumor suppressor that inhibits the progression and invasion of OS by targeting MMP-11. Therefore, miR-192 may be useful for the diagnosis and treatment of OS.

Downregulation of lncRNA ANRIL suppresses growth and metastasis in human osteosarcoma cells.

BACKGROUND: This study was designed to research the potential function of lncRNA ANRIL in osteosarcoma (OS). MATERIALS AND METHODS: Quantitative real-time PCR, cell counting kit-8, wound healing assay, Transwell assay, flow cytometric analysis, caspase activity analysis, and Western blot were carried out. RESULTS: ANRIL was remarkably upregulated in human OS tissues and cells, and knockdown of ANRIL significantly suppressed MG63 cell proliferation, migration, and invasion and promoted apoptosis. Moreover, our mechanistic research findings verified that ANRIL-influenced growth and apoptosis may be partly through regulation of caspase-3 and Bcl-2. Migration and invasion were influenced via ANRIL-mediated regulation of MTA1, TIMP-2, and E-cadherin. CONCLUSION: Our finding demonstrates that ANRIL plays vital roles in OS growth and metastasis.