TGF-ß1-triggered BMI1 and SMAD2 cooperatively regulate miR-191 to modulate bone formation.

Transforming growth factor ß 1 (TGF-ß1), as the most abundant signaling molecule in bone matrix, is essential for bone homeostasis. However, the signaling transduction of TGF-ß1 in the bone-forming microenvironment remains unknown. Here, we showed that microRNA-191 (miR-191) was downregulated during osteogenesis and further decreased by osteo-favoring TGF-ß1 in bone marrow mesenchymal stem cells (BMSCs). MiR-191 was lower in bone tissues from children than in those from middle-aged individuals and it was negatively correlated with collagen type I alpha 1 chain (COL1A1). MiR-191 depletion significantly increased osteogenesis and bone formation in vivo. Hydrogels embedded with miR-191-low BMSCs displayed a powerful bone repair effect. Mechanistically, transcription factors BMI1 and SMAD2 coordinately controlled miR-191 level. In detail, BMI1 and pSMAD2 were both upregulated by TGF-ß1 under osteogenic condition. SMAD2 activated miR-191 transcription, while BMI1 competed with SMAD2 for binding to miR-191 promoter region, thus disturbing the activation of SMAD2 on miR-191 and reducing miR-191 level. Altogether, our findings reveal that miR-191 regulated by TGF-ß1-induced BMI1 and SMAD2 negatively modulated bone formation and regeneration, and inhibition of miR-191 might be therapeutically useful to enhance bone repair in clinic.

MIR503HG silencing promotes endometrial stromal cell progression and metastasis and suppresses apoptosis in adenomyosis by activating the Wnt/ß­catenin pathway via targeting miR­191.

MIR503HG is a 786 bp long lncRNA located on chromosome Xq26.3, and it can regulate diverse cellular processes. The pathogenesis of adenomyosis (AD) is associated with endometrial stromal cells (ESCs). The present study investigated the specific role of MIR503HG in AD pathogenesis and progression using ESCs derived from the endometrium of patients with AD as a model. Expression of MIR503HG and microRNA (miR)-191 were assessed using reverse transcription-quantitative PCR. An immunocytochemistry assay was used to detect cytokeratin- or vimentin-positive ESCs. Transfections of ESCs with MIR503HG overexpression plasmid, short hairpin-MIR503HG and miR-191 inhibitor were performed. ESC viability, migration, invasion and apoptosis were evaluated using Cell Counting Kit-8, Transwell and flow cytometry assays. The association between MIR503HG and miR-191 was predicted by StarBase and confirmed using a dual-luciferase reporter assay. Expression of epithelial-mesenchymal transition-related markers (E-cadherin and N-cadherin) and Wnt/ß-catenin pathway-related molecules (ß-catenin) in ESCs were analyzed by western blotting. The isolated ESCs were vimentin-positive and cytokeratin-negative. MIR503HG was lowly expressed in the endometrial tissues derived from patients with AD. MIR503HG overexpression hindered ESC viability, migration and invasion while enhancing the apoptosis and downregulating miR-191 expression. MIR503HG knockdown induced the opposite effects, accompanied by downregulation of the E-cadherin expression and upregulation of N-cadherin and ß-catenin levels. MIR503HG directly targeted miR-191 that was highly expressed in endometrial tissues derived from patients with AD. In ESCs, downregulation of miR-191 inhibited the viability, migration and invasion and the expression of N-cadherin and ß-catenin levels while enhancing the apoptosis and E-cadherin expression in ESCs. Moreover, downregulation of miR-191 partially reversed the effect of MIR503HG knockdown. Collectively, overexpressed MIR503HG impeded the proliferation and migration of ESCs derived from endometrium of patients with AD, while promoting apoptosis via inhibition of the Wnt/ß-catenin pathway via targeting miR-191.

[Correlation between miR-21, miR-191 and Clinical Stage of Patients with Diffuse Large B-Cell Lymphoma].

OBJECTIVE: To analyze the relationship between microRNA (miR)-21, miR-191 and clinical stage of patients with diffuse large B-cell lymphoma (DLBCL). METHODS: 100 patients with DLBCL treated in Shanxi Fenyang Hospital from January 2019 to January 2021 were selected as the research subjects. All patients was divided into stage I, stage II, stage III and stage IV according to Ann-Arbor (Cotswolds) staging system at admission. The baseline data of patients at different clinical stages were counted and compared in detail. The relationship between the levels of miR-21 and miR-191 and the clinical stage of DLBCL patients was mainly analyzed. RESULTS: Among the 100 patients with DLBCL, there were 15 patients at stage I, 25 patients at stage II, 37 patients at stage III and 23 patients at stage IV. The levels of miR-21 and miR-191 in patients at stage Ⅰ, Ⅱ, Ⅲ and Ⅳ were increased gradually, which showed statistically significant differences (P<0.05). According to Kendall's tau-b correlation analysis, it was found that the levels of miR-21 and miR-191 were positively correlated with the clinical stage of DLBCL patients (r=0.566, 0.636). Multiple logistic regression analysis showed that the overexpression of serum miR-21 and miR-191 was a risk factor for high clinical stage in patients with DLBCL (OR>1, P<0.05). Bivariate Pearson correlation analysis showed that there was a positive correlation between miR-21 and miR-191 levels in patients with DLBCL (r=0.339). CONCLUSION: The overexpression of miR-21 and miR-191 in patients with DLBCL is related to high clinical stage.

Correlation between miR-21, miR-191 and Clinical Stage of Patients with Diffuse Large B-Cell Lymphoma / 中国实验血液学杂志

OBJECTIVE@#To analyze the relationship between microRNA (miR)-21, miR-191 and clinical stage of patients with diffuse large B-cell lymphoma (DLBCL).@*METHODS@#100 patients with DLBCL treated in Shanxi Fenyang Hospital from January 2019 to January 2021 were selected as the research subjects. All patients was divided into stage I, stage II, stage III and stage IV according to Ann-Arbor (Cotswolds) staging system at admission. The baseline data of patients at different clinical stages were counted and compared in detail. The relationship between the levels of miR-21 and miR-191 and the clinical stage of DLBCL patients was mainly analyzed.@*RESULTS@#Among the 100 patients with DLBCL, there were 15 patients at stage I, 25 patients at stage II, 37 patients at stage III and 23 patients at stage IV. The levels of miR-21 and miR-191 in patients at stage Ⅰ, Ⅱ, Ⅲ and Ⅳ were increased gradually, which showed statistically significant differences (P<0.05). According to Kendall's tau-b correlation analysis, it was found that the levels of miR-21 and miR-191 were positively correlated with the clinical stage of DLBCL patients (r=0.566, 0.636). Multiple logistic regression analysis showed that the overexpression of serum miR-21 and miR-191 was a risk factor for high clinical stage in patients with DLBCL (OR>1, P<0.05). Bivariate Pearson correlation analysis showed that there was a positive correlation between miR-21 and miR-191 levels in patients with DLBCL (r=0.339).@*CONCLUSION@#The overexpression of miR-21 and miR-191 in patients with DLBCL is related to high clinical stage.

Correlation between the expression levels of plasma microRNA-191,microRNA-23a and microRNA-145 and the prognosis of patients with prostate cancer / 新乡医学院学报

Objective To investigate the expression levels of microRNA(miR)-191,miR-23a,and miR-145 in plasma of patients with prostate cancer and their correlation with prognosis.Methods Sixty prostate cancer patients admitted to Zhumadian Central Hospital from December 2019 to December 2021 were selected as the observation group,and 60 healthy subjects who underwent physical examinations during the same period were selected as the control group.The expression levels of miR-191,miR-23a and miR-145 in plasma of subjects in the two groups were measured by real-time fluorescence quantita-tive polymerase chain reaction.The patients in the observation group were divided into poor prognosis group(n=11)and good prognosis group(n=49)according to their prognosis.The expression levels of miR-191,miR-23a,and miR-145 in plasma of prostate cancer patients were compared before and after treatment.The general data of patients was compared between the poor prognosis group and the good prognosis group.The receiver operator characteristic curve was drawed to analyse the predictive value of plasma miR-191,miR-23a,and miR-145 levels on prognosis.Results The relative expression levels of miR-191 and miR-23 a in plasma of patients in the observation group were significantly higher than those in the control group,while the relative expression level of miR-145 was significantly lower than that in the control group(P＜0.05).The relative expression levels of miR-191 and miR-23a of patients in the observation after treatment were significantly lower than those before treat-ment,while the relative expression level of miR-145 was significantly higher than that before treatment(P＜0.05).There was no significant difference in age,body mass index,smoking history,alcohol consumption history,and prostate volume of patients between the poor prognosis group and the good prognosis group(P＞0.05);there was significant differences in TNM staging,Gleason score,serum prostate specific antigen level and relative expressions of miR-191,miR-23a,miR-145 in plasma of patients between the two groups(P＜0.05).Plasma miR-191,miR-23a and miR-145 levels were influence factor for prognosis in patients with prostate cancer(P＜0.05).There was predictive value of plasma miR-191,miR-23a and miR-145 for prognosis of prestate cancer patients;the combined predictive value of plasma miR-191,miR-23a and miR-145 for the prognosis of prostate cancer patients was significantly higher than that of plasma miR-191,miR-23a and miR-145 alone for the prognosis of prostate cancer patients(P＜0.05).Conclusion The expression of miR-191 and miR-23a in plasma increases and the expression of miR-145 in plasma decreases in patients with prostate cancer.The combined detection of plasma miR-191,miR-23a and miR-145 has higher predictive value for the prognosis of prostate cancer.

MicroRNA-191 blocking the translocation of GLUT4 is involved in arsenite-induced hepatic insulin resistance through inhibiting the IRS1/AKT pathway.

Environmental exposure to arsenic can cause a variety of health problems. Epidemiological and experimental studies have established a diabetogenic role for arsenic, but the mechanisms responsible for arsenic-induced impairment of insulin action are unclear. MicroRNAs (miRNAs) are involved in various metabolic disorders, particularly in the development of insulin resistance. The present study investigated whether arsenite, an active form of arsenic, induces hepatic insulin resistance and the mechanisms underlying it. After male C57BL/6J mice were exposed to arsenite (0 or 20 ppm) in drinking water for 12 months, intraperitoneal glucose tolerance tests (IPGTTs) and insulin tolerance tests (ITTs) revealed an arsenite-induced glucose metabolism disorder. Hepatic glycogen levels were lower in arsenite-exposed mice. Further, for livers of mice exposed to arsenite, miR-191 levels were higher, and protein levels of insulin receptor substrate 1 (IRS1), p-IRS1, and phospho-protein kinase B (p-AKT) were lower. Further, glucose transporter 4 (GLUT4) had lower levels on the plasma membrane. For insulin-treated L-02 cells, arsenite decreased glucose consumption and glycogen levels, increased miR-191 levels, and inhibited the IRS1/AKT pathway and the translocation of GLUT4 from the cytoplasm to the plasma membrane. For insulin-treated L-02 cells, the decreases of glucose consumption, glycogen levels, GLUT4 on the plasma membrane, and p-AKT levels induced by arsenite were reversed by SC79 (agonist of AKT) and an miR-191 inhibitor; these effects caused by miR-191 inhibitor were restored by IRS1 siRNA. In insulin-treated L-02 cells, miR-191, via IRS1, was involved in the arsenite-induced decreases of glucose consumption and glycogen levels and in inhibition of the translocation of GLUT4. Thus, miR-191 blocking the translocation of GLUT4 was involved in arsenite-induced hepatic insulin resistance through inhibiting the IRS1/AKT pathway. Our study reveals a mechanism for arsenite-induced hepatic insulin resistance, which provides clues for discovering biomarkers for the development of type 2 diabetes and for prevention and treatment of arsenic poisoning.

[miRNA-191-5p represses cell growth by targeting CDK6 in gastric cancer].

Objective: To investigate the effects of miR-191-5p on cell migration, clone formation and proliferation of gastric cancer (GC) cells. Methods: The level of miR-191-5p expression was detected by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in 60 paired GC tissues and their adjacent normal tissues. miR-191-5p overexpression was achieved by transfection of construct pcDNA-miR-191-5p into GC cells. The migration, clone formation and proliferation of GC cells were detected by the scratch wound assay, clone formation assay and cell counting kit-8 (CCK-8), respectively. Low expression of miR-191-5p was achieved with miRNA-191-5p inhibitor. The binding sites of cyclin-dependent kinase 6 (CDK6) and miR-191-5p were analyzed using TargetScan software, and the interaction of CDK6 and miR-191-5p was verified using dual-fluorescence reporter gene expression. Western blot (WB) was used to detect the effect of miR-191-5p on the expression of p21 and CDK6 proteins. Results: miR-191-5p decreased in 53 cases (88%) of GC tissues compared to their controls. Furthermore, overexpression of miR-191-5p effectively inhibited the migration, clone formation and proliferation of GC cells (P<0.05). Dual-fluorescence reporter confirmed that miR-191-5p bound to 3'UTR of CDK6. WB showed that pcDNA-miR-191-5p inhibited the CDK6 expression but promoted the p21. Conclusion: Down-regulation of miR-191-5p has a correlation with the progression of GC. Overexpression of miR-191-5p can decrease the expression of CDK6 and inhibit the growth of GC cells.

IGF2BP3 and miR191-5p synergistically increase HCC cell invasiveness by altering ZO-1 expression.

Early studies have indicated that insulin-like growth factor II mRNA binding protein 3 (IGF2BP3/IMP3) may affect the progression of hepatocellular carcinoma (HCC); however, the detailed underlying mechanisms, particularly its linkage to tight junction protein-mediated cell invasion, remain unclear. The present study revealed that IGF2BP3 increased HCC cell invasiveness by suppressing zonula occludens-1 (ZO-1) expression, via direct binding to the 3' untranslated region (3'-UTR). Analysis of the molecular mechanisms demonstrated that IGF2BP3 binds to the overlapping targets of IGF2BP3-RNA cross-linkage and microRNA (miR)191-5p targeting sites, and promotes the formation of an miR191-5p-induced RNA-induced silencing complex. The knockdown of IGF2BP3 or the addition of a miR-191-5p inhibitor decreased cellular invasiveness and increased ZO-1 expression. Analysis of the human HCC database also confirmed the association between IGF2BP3 and HCC progression. Collectively, these preclinical findings suggest that IGF2BP3 increases HCC cell invasiveness by promoting the miR191-5p-induced suppression of ZO-1 signaling. This newly identified signaling effect on small molecule targeting may aid in the development of novel strategies with which to inhibit HCC progression more effectively.

miR-191 promotes radiation resistance of prostate cancer through interaction with RXRA.

Radiation therapy is a common treatment for prostate cancer, however recurrence remains a problem. MicroRNA expression is altered in prostate cancer and may promote therapy resistance. Through bioinformatic analyses of TCGA and CPC-GENE patient cohorts, we identified higher miR-191 expression in tumor versus normal tissue, and increased expression in higher Gleason scores. In vitro and in vivo experiments demonstrated that miR-191 overexpression promotes radiation survival, and contributes to a more aggressive phenotype. Retinoid X receptor alpha, RXRA, was discovered to be a novel target of miR-191, and knockdown recapitulated radioresistance. Furthermore, treatment of prostate cancer cells with the RXRA agonist 9-cis-retinoic acid restored radiosensitivity. Supporting this relationship, patients with high miR-191 and low RXRA abundance experienced quicker biochemical recurrence. Reduced RXRA translated to a higher risk of distant failure after radiotherapy. Notably, this miR-191/RXRA interaction was conserved in a novel primary cell line derived from radiorecurrent prostate cancer. Together, our findings demonstrate that miR-191 promotes prostate cancer survival after radiotherapy, and highlights retinoids as a potential option to improve radiotherapy response.

MicroRNA-191 targets CCAAT/enhanced binding protein ß and functions as an oncogenic molecule in human non-small cell lung carcinoma cells.

The aberrant expression of microRNAs (miRs) may be involved in tumor growth and progression in human non-small cell lung carcinoma (NSCLC). The present study aimed to investigate the potential roles of miR-191 in NSCLC. Western blotting and reverse transcription-quantitative polymerase chain reaction were performed to assess protein and/or mRNA levels. Scratch wound healing and transwell assays were performed to determine the NSCLC cell migration and invasion. A luciferase demonstrated that CCAAT/enhanced binding protein ß (C/EBPß) was a target of miR-191. Previously, miR-191 has been reported to act as an oncogenic player in multiple human cancers. C/EBPß has been identified as a target gene of miR-191; however, the roles and underlying mechanisms of miR-191 associated with the regulation of tumor invasion in NSCLC remain unknown. In the present study, it was demonstrated that miR-191 expression levels were higher in human NSCLC tumors compared with in normal adjacent tissue and elevated miR-191 expression levels were closely associated with tumor node metastasis stage in patients with NSCLC. Furthermore, transfection with miR-191 mimic inhibited C/EBPß expression at the mRNA and protein levels and promoted A549 cell migration and invasion. C/EBPß was reported to be the direct target gene of miR-191 using a dual luciferase reporter assay. Finally, C/EBPß siRNA can mimic the effects of miR-191. These findings indicated that miR-191 may function as an oncogene in NSCLC, at least partially due to its negative regulatory on C/EBPß.

Knockdown long non-coding RNA ANRIL inhibits proliferation, migration and invasion of HepG2 cells by down-regulation of miR-191.

BACKGROUND: Hepatocellular carcinoma (HCC) is a common malignant tumor with high fatality rate. Recent studies reported that up-regulation of long non-coding RNA antisense non-coding RNA in the INK4 locus (lncRNA ANRIL) was found in HCC tissues, and which could affect HCC cells biological processes. However, the potential molecular mechanism of ANRIL in HCC is still unclear. The study aimed to uncover the effect of ANRIL on HepG2 cells growth, migration and invasion. METHODS: The knockdown expression vectors of ANRIL were transfected into HepG2 cells, and qRT-PCR, CCK-8, flow cytometry, Transwell and western blot assays were performed to analyze the effect of ANRIL on cell proliferation, apoptosis, migration and invasion. The relative expression of miR-191 was then examined in ANRIL knockdown vector transfected cells. These experiments were repeated again for exploring the effect of miR-191 on HepG2 cells. NF-κB and Wnt/ß-catenin signaling pathways were examined by using western blot assay. RESULTS: Knockdown of ANRIL inhibited proliferation, induced apoptosis, meanwhile suppressed migration and invasion of HepG2 cells. Additionally, the results showed that the expression level of miR-191 was down-regulated by ANRIL knockdown in HepG2 cells. Importantly, overexpression of miR-191 reversed the anti-tumor effect of ANRIL on cell proliferation, apoptosis, migration and invasion in HepG2 cells. Besides, we found that ANRIL knockdown inactivated NF-κB and Wnt/ß-catenin pathways by regulating miR-191. CONCLUSIONS: These data demonstrated that ANRIL knockdown suppressed proliferation, migration, invasion, and promoted apoptosis in HepG2 cells by down-regulating miR-191 and inactivating NF-κB and Wnt/ß-catenin signaling pathways.

Beta-elemene increases chemosensitivity to 5-fluorouracil through down-regulating microRNA-191 expression in colorectal carcinoma cells.

Colorectal carcinoma is a common malignant tumor occurring in the alimentary system. Despite developments of modern medicine, developed resistance to 5-fluorouracil (5-FU) may lead to poor prognosis. Herein, we aimed to explore the effects of beta-elemene on colorectal carcinoma cells (HCT116 and HT29) as well as the underlying mechanisms. Beta-elemene reduced cell viability and induced apoptosis in HCT116 and HT29 cells. Increased apoptosis following beta-elemene exposure was due to enhanced sensitivity to 5-FU through down-regulating miR-191. Expression of key kinases, including Wnt3a, and ß-catenin, were down-regulated by beta-elemene through a miR-191 mechanism. Moreover, beta-elemene might improve resistance of colorectal carcinoma cells to 5-FU by down-regulating miR-191, thereby inhibiting the Wnt/ß-catenin pathway.

MicroRNA-191-5p exerts a tumor suppressive role in renal cell carcinoma.

Renal cell carcinoma (RCC) is a common tumor of the urinary system. Previously, miR-191-5p has been reported to be associated with various types of cancer; however, its specific functions in RCC have not been investigated to date. In the present study, the expression of miR-191-5p in the 786-O and ACHN cell lines was detected in vitro by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The results of RT-qPCR revealed that miR-191-5p was significantly downregulated in the two cell lines compared with the 293T cell line. miR-191-5p was also significantly downregulated in RCC tissue compared with paired normal tissue. In addition, the effects of miR-191-5p on cell proliferation, migration, invasion and apoptosis were examined by CCK-8, MTT, wound scratch, Transwell and flow cytometry assays. Downregulation of miR-191-5p was observed to promote cell proliferation, migration and invasion, as well as to repress the cell apoptosis of 786-O and ACHN cells. Therefore, the current study suggests that miR-191-5p functions as a tumor suppressor in RCC. Further studies are required to uncover the underlying signaling pathway of miR-191-5p and its potential role as a biomarker for early detection and prognosis prediction, and as a therapeutic target of RCC.

Exosomal microRNA-191 derived from arsenite-transformed cells promotes the proliferation of human liver cells / 中华地方病学杂志

Objective To investigate the role of exosomal microRNA(miR)-191 derived from NaAsO2-transformed cells in proliferation of human liver L-02 cells.Methods The normal wild-type L-02 cells (recipient L-02 cells)were treated with media or exosome derived from 2 μmol/L NaAsO2-transformed L-02 cells.Anti-miR-191 and anti-miR-NC were transfected into NaAsO2-transformed L-02 (T-L-02) cells by lipofectamineTM 2000,respectively,while untreated group was set as control.The expression of miR-191 was detected by qRT-PCR.Cell proliferation was evaluated by CCK-8 assay.Results The proliferation [(207 ± 24)％ vs (105 ± 21)％,t =5.462,P ＜ 0.01] and the expression of miR-191 [(206 ± 25)％ vs (105 ± 20)％,t =4.116,P ＜ 0.05] of recipient L-02 cells were significantly increased in the transformed L-02 cells media (T-CM) treated group compared with in the normal L-02 cells media (CM) group.Several concentrations of exosomes derived from CM did not change the proliferation and miR-191 expression of recipient L-02 cells (F =2.213,2.213,all P ＞ 0.05).Several concentrations of exosomes derived from T-CM increased the proliferation and miR-191 expression of recipient L-02 cells in a dose-response manner (F =10.910,4.553,P ＜ 0.01 or ＜ 0.05).The proliferation [(160 ± 32)％ vs (102 ± 8)％,(203 ± 7)％ vs (111 ± 5)％,t =2.999,18.750,P ＜ 0.05 or ＜ 0.01] of recipient L-02 cells treated with 20 or 50 mg/L exosomes derived from T-CM was higher than that treated with the same concentration of exosomes derived from CM.The expression of miR-191 [(166 ± 13)％ vs (113 ±9)％,(211 ± 55)％ vs (102 ± 8)％,(206 ± 31)％ vs (105 ± 6)％,t =5.611,3.357,5.509,P ＜ 0.05 or ＜ 0.01] of recipient L-02 cells treated with 10,20 or 50 mg/L exosomes derived from T-CM was higher than that treated with the same concentration of exosomes derived from CM.The miR-191 levels of T-L-02 cells [(39 ± 10)％ vs (100 ± 0)％ or (106 ±17)％,all P ＜ 0.01] or exosomes [(30 ± 19)％ vs (100 ± 0)％ or (104 ± 17)％,all P ＜ 0.01] in the anti-miR-191 treated group were significantly lower than that in the untreated group or anti-miR-NC treated group.The exosomes derived from untreated group promoted the proliferation [(395 ± 31)％ vs (100 ± 0)％,t =16.290,P ＜ 0.01] and miR-191 expression [(208 ± 47)％ vs (100 ± 0)％,t =4.015,P ＜ 0.05] of recipient L-02 cells.The proliferation [(157 ± 19)％ vs (395 ± 31)％ or (411 ± 55)％,P ＜ 0.05] and miR-191 expression of [(103 ± 44)％ vs (208 ± 47)％ or (197 ± 37)％,P＜ 0.05 or ＜ 0.01] of recipient L-02 cells treated with exosomes derived from anti-miR-191 treated group were lower than those treated with exosomes derived from untreated group or anti-miR-NC treated group.Conclusion The exosomal miR-191 secreted by NaAsO2-transformed L-02 cells promotes proliferation of normal human L-02 cells.

Effects of sodium arsenite on the expression of microRNA-191 and tissue inhibitor of metalloproteinase 3 in L-02 cells / 中华地方病学杂志

Objectives To investigate the effects of sodium arsenite (NaAsO2) on the expression ot microRNA-191 (miR-191) and tissue inhibitor of metalloproteinase 3 (TIMP-3) in human normal hepatic cells (L-02 cells).Methods L-02 cells were exposed to different doses of NaAsO2 [0 (control group),5,25,50 and 75 μmol/L]for 24 h,or treated with 5 and 25 μmol/L NaAs02 for 0 (control group),12,24 and 48 h.The miR-191 inhibitor was used to suppress the expression of miR-191.qRT-PCR was performed to detect the expression level of miR-191 and TIMP-3 mRNA,and the protein level of TIMP-3 was analyzed by Western blotting.Results Dose-effect study:There were significant differences in the expressions of miR-191,TIMP-3 mRNA and protein between the 5 groups (F =85.674,20.952,123.393,all P ＜ 0.05).The expressions of miR-191 in all groups (1.702 ± 0.124,2.077 ±0.234,2.145 ± 0.105,2.003 ± 0.077) were higher than that of control group (0.990 ± 0.035,all P ＜ 0.05);the mRNA expressions of TIMP-3 in 25,50,75 μmol/L groups (0.848 ± 0.067,0.804 ± 0.081,0.813 ± 0.076) were all lower than that of control group (0.996 ± 0.007,all P ＜ 0.05),but there was no significant difference in the mRNA expression of TIMP-3 between the 5 μmol/L group and control group (0.939 ± 0.133 vs 0.996 ± 0.007,P＞ 0.05),and the protein expressions of TIMP-3 in all groups (0.846 ± 0.093,0.611 ± 0.123,0.554 ± 0.098,0.529 ± 0.067) were lower than that of control group (1.006 ± 0.003,all P ＜ 0.05).Time-effect study:there were significant differences in the expressions of miR-191,TIMP-3 mRNA and protein between the exposure groups of 5 and 25 μmol/L (For 5 μmol/L:F =86.355,16.404,22.898,all P ＜ 0.05;For 25 μmol/L:F =104.321,20.123,52.321,all P ＜ 0.05).The expressions of miR-191 in all exposure groups of 5 and 25 μmol/L (1.392 ± 0.152,1.691 ± 0.167,2.018 ± 0.130 and 1.456 ± 0.167,1.946 ± 0.178,2.259 ± 0.256) were higher than those of control groups (1.001 ± 0.014,1.008 ±0.027,all P ＜ 0.05);the mRNA expressions of TIMP-3 in 48 h exposure group of 5 μmol/L and all exposure groups of 25 μmol/L (0.824 ± 0.093 and 0.897 ± 0.033,0.815 ± 0.089,0.709 ± 0.103) were lower than those of control groups (1.004 ± 0.018,0.997 ± 0.057,all P ＜ 0.05),but there were no significant differences in the mRNA expressions of TIMP-3 between the 12,24 h exposure groups of 5 μmol/L and control group (0.952 ± 0.072,0.929 ± 0.121 vs1.004 ± 0.018,all P ＞ 0.05);the protein expressions of TIMP-3 in all exposure groups of 5 and 25 μmol/L (0.857 ±0.068,0.832 ± 0.106,0.691 ± 0.112 and 0.785 ± 0.097,0.620 ± 0.066,0.453 ± 0.075) were lower than those of control groups (1.006 ± 0.045,1.004 ± 0.078,all P ＜ 0.05).The treatment of miR-191 inhibitor:there were significant differences in the expressions of miR-191 and TIMP-3 protein between different groups (F =104.306,67.015,all P ＜ 0.05).The elevated expression level of miR-191 induced by NaAsO2 was significantly suppressed after transfected with miR-191 inhibitor (0.314 ± 0.094 vs 2.051 ± 0.371,P ＜ 0.05),which in turn up-regulated the protein expression of TIMP-3 (1.965 ± 0.277 vs 0.541 ± 0.183,P ＜ 0.05).Conclusion The expression level of miR-191 is elevated in response to NaAsO2 exposure,and miR-191 has subsequently suppressed the expression of TIMP-3,a potential target of miR-191.

Increased expression of microRNA-191 as a potential serum biomarker for diagnosis and prognosis in human osteosarcoma.

BACKGROUND: MicroRNA (miR)-191 has been observed to be overexpressed in osteosarcoma cell lines in comparison with osteoblasts. OBJECTIVE: To investigate the clinical significance of miR-191 in human osteosarcomas. METHODS: Quantitative PCR was performed to detect miR-191 expression in osteosarcoma tissues and patients' sera. RESULTS: miR-191 expression levels, both in osteosarcoma tissues and patients' sera, were significantly higher than those in matched adjacent normal bone tissues and healthy controls (both P< 0.001). Importantly, miR-191 could efficiently screen osteosarcoma patients from healthy controls (Area under receiver operating characteristic curve, AUC = 0.808). Then, high serum miR-191 expression was significantly associated with advanced clinical stage (P = 0.001), large tumor size (P = 0.01) and positive distant metastasis (P = 0.001). Moreover, overall and disease-free survival durations in patients with high miR-191 expression were both shorter than those with low miR-191 expression. Multivariate analysis further identified serum miR-191 level as an independent and significant prognostic factor for both overall survival (P = 0.01) and disease-free survival (P = 0.02). CONCLUSIONS: Our data provide new insights for the involvement of miR-191 in osteosarcoma and suggest that the increased expression of miR-191 may be associated with aggressive tumor progression and adverse outcome. Of note, serum miR-191 quantification may be a promising biomarker for the diagnosis and prognosis in osteosarcoma.

Genomewide Study of Salivary MicroRNAs for Detection of Oral Cancer.

MicroRNAs (miRNAs) in human saliva have recently demonstrated to be potential biomarkers for diagnosis purposes. However, lack of well-characterized/matched clinical groups and lack of suitable endogenous control (EC) for salivary extracellular miRNA detection and normalization are among the restrictions of applying salivary-based miRNA biomarker discovery. In the present study, we examined the differential expression pattern of miRNAs among 4 groups of subjects-including patients with oral squamous cell carcinoma (OSCC), patients with OSCC in remission (OSCC-R), patients with oral lichen planus, and healthy controls (HCs)-using a genomewide high-throughput miRNA microarray. First, we systematically screened 10 pooling samples and 34 individual samples of different groups to find a proper EC miRNA. We then investigated the genomewide expression patterns of differentially expressed miRNAs in saliva of different groups using NanoString nCounter miRNA expression assay and real-time quantitative polymerase chain reaction, followed by construction of receiver operating characteristic curves to determine the sensitivity and specificity of the assay. We identified miRNA-191 as a suitable EC miRNA with minimal intergroup and intragroup variability, and we used it for normalization. Of more than 700 miRNAs tested, 13 were identified as being significantly deregulated in saliva of OSCC patients compared to HCs: 11 miRNAs were underexpressed (miRNA-136, miRNA-147, miRNA-1250, miRNA-148a, miRNA-632, miRNA-646, miRNA668, miRNA-877, miRNA-503, miRNA-220a, miRNA-323-5p), and 2 miRNAs were overexpressed (miRNA-24, miRNA-27b). MiRNA-136 was underexpressed in both OSCC vs. HCs and OSCC vs. OSCC-R. MiRNA-27b levels were significantly higher in OSCC patients compared to those found in HCs, patients with OSCC-R, and patients with oral lichen planus and served as a characteristic biomarker of OSCC. Receiver operating characteristic curve analyses showed that miRNA-27b could be a valuable biomarker for distinguishing OSCC patients from the other groups. Our novel findings established a reliable EC miRNA for salivary-based diagnostic and indicate that the salivary miRNA profiles are discriminatory in OSCC patients.