Objective To explore the mechanism of TPT1-AS1 targeting miR-324/TWIST1 axis to regulate the proliferation, invasion, migration and angiogenesis of epithelial ovarian cancer (EOC) cells, thereby affecting ovarian cancer (OC) progression. Methods RT-qPCR was used to detect the expression of TPT1-AS1 and miR-324 in 29 OC lesions and adjacent tissue samples. The two OC cell models of TPT1-AS1 overexpression and miRNA324 knockdown were constructed, and the cell proliferation, invasion and migration abilities were detected by CCK-8, Transwell^TM and scratch test. Western blot analysis was used to detect the protein expression levels of TWIST1, epithelial cadherin (E-cadherin), Vimentin, and vascular endothelial growth factor A (VEGF-A) in OC cells. Fluorescence in situ hybridization (FISH) and RNA pull-down experiments were used to verify the interaction between TPT1-AS1 and miR-324. Immunohistochemistry and Targetscan bioinformatics analysis were used to verify the negative regulatory role of miR-324 in the epithelial-mesenchymal transition (EMT) process. Results The TPT1-AS1 expression was significantly higher in OC tissues than that in para-cancerous tissues, while the miR-324 expression was significantly lower. In SKOV3 cells with TPT1-AS1 overexpression, the miR-324 expression decreased significantly, and TPT1-AS1 was negatively correlated with miR-324. It was also found that TPT1-AS1 and miR-324 were co-expressed in OC cells, and there was a direct binding relationship between them. Down-regulation of miR-324 significantly promoted the proliferation, invasion and migration of SKOV3 cells. Further studies revealed that miR-324 had a binding site at the 3'-UTR end of the TWIST1, a key transcription factor for EMT. Inhibiting miR-324 expression increased the transcription level of TWIST1, leading to a decrease in E-cadherin protein expression and an increase in Vimentin protein expression. Additionally, the downregulation of miR-324 resulted in an increased expression level of VEGF-A protein, which in turn enhanced angiogenesis of OC. Conclusion TPT1-AS1 promotes EOC cell proliferation, invasion, migration and angiogenesis by negatively regulating the miR-324/TWIST1 axis, thus promoting the development of OC. These findings provide new potential targets for the diagnosis and treatment of OC.
Humans ; MicroRNAs/metabolism* ; Female ; Cell Movement/genetics* ; Ovarian Neoplasms/blood supply* ; Twist-Related Protein 1/metabolism* ; Cell Line, Tumor ; Neovascularization, Pathologic/genetics* ; Neoplasm Invasiveness ; Carcinoma, Ovarian Epithelial/metabolism* ; Nuclear Proteins/metabolism* ; Cell Proliferation/genetics* ; Epithelial-Mesenchymal Transition/genetics* ; Gene Expression Regulation, Neoplastic ; RNA, Long Noncoding/metabolism* ; Cadherins/genetics* ; Vascular Endothelial Growth Factor A/genetics* ; Vimentin/genetics* ; Angiogenesis

OBJECTIVE: To investigate the effect of zedoarondiol on neovascularization of atherosclerotic (AS) plaque by exosomes experiment. METHODS: ApoE^-/- mice were fed with high-fat diet to establish AS model and treated with high- and low-dose (10, 5 mg/kg daily) of zedoarondiol, respectively. After 14 weeks, the expressions of anti-angiogenic protein thrombospondin 1 (THBS-1) and its receptor CD36 in plaques, as well as platelet activation rate and exosome-derived miR-let-7a were detected. Then, zedoarondiol was used to intervene in platelets in vitro, and miR-let-7a was detected in platelet-derived exosomes (Pexo). Finally, human umbilical vein endothelial cells (HUVECs) were transfected with miR-let-7a mimics and treated with Pexo to observe the effect of miR-let-7a in Pexo on tube formation. RESULTS: Animal experiments showed that after treating with zedoarondiol, the neovascularization density in plaques of AS mice was significantly reduced, THBS-1 and CD36 increased, the platelet activation rate was markedly reduced, and the miR-let-7a level in Pexo was reduced (P<0.01). In vitro experiments, the platelet activation rate and miR-let-7a levels in Pexo were significantly reduced after zedoarondiol's intervention. Cell experiments showed that after Pexo's intervention, the tube length increased, and the transfection of miR-let-7a minics further increased the tube length of cells, while reducing the expressions of THBS-1 and CD36. CONCLUSION Zedoarondiol has the effect of inhibiting neovascularization within plaque in AS mice, and its mechanism may be potentially related to inhibiting platelet activation and reducing the Pexo-derived miRNA-let-7a level.
Animals ; MicroRNAs/genetics* ; Exosomes/drug effects* ; Plaque, Atherosclerotic/genetics* ; Neovascularization, Pathologic/genetics* ; Human Umbilical Vein Endothelial Cells/metabolism* ; Humans ; Blood Platelets/drug effects* ; Apolipoproteins E/deficiency* ; Thrombospondin 1/metabolism* ; CD36 Antigens/metabolism* ; Platelet Activation/drug effects* ; Male ; Mice ; Mice, Inbred C57BL

OBJECTIVE: To study the expression profiles changes of miRNA in apheresis platelets after 1, 3 and 5 days of storage. METHODS: The apheresis platelets were collected from 20 volunteer blood donors. After mixing fully, the platelets were stored in a shaker with (22±2) ℃ horizontal oscillation. The samples were taken on the 1st, 3rd and 5th day, and used to sequence for miRNAs by DNA nanoball (DNB) sequencing technology, which were named as C_1, C_3 and C_5, respectively. The expression level of platelets miRNA was standardized by transcripts per kilobase million (TPM) algorithm. MiRNAs with P-value < 0.001 and the expression difference of more than two times were considered as significant difference between two groups. The expression of miRNAs was verified by real-time fluorescence quantitative PCR (RT-qPCR). RESULTS: By DNB sequencing, there were 688, 730, and 679 platelet miRNAs expressed in C_1, C_3 and C_5 group, respectively. Cluster analysis showed that the expression profile of miRNAs changed significantly. The expression level of the first 20 high abundance miRNAs was about 4/5 of the total amounts of expressed miRNAs in each group, which the top five miRNAs were miR-21-5p, miR-26a-5p, miR-199a-3p, miR-126-3p, and let-7f-5p. The correlation of high abundance platelet miRNAs among the three groups was high (R2=0.876, R2=0.979, R2=0.937, respectively) and the differences were not statistically significant (P>0.05). Compared with the differential expression of platelet miRNAs with more than 1 000 TPM in the C_3 and C_1 group, there were 6 differentially expressed miRNAs, including 3 up-regulated (miR-146a-5p, miR-379-5p, and miR-486-5p) and 3 down-regulated (miR-652-3p, miR-142-5p, and miR-7-5p). While in the C_5 and C_1 group, there were 4 differentially expressed miRNAs, including 2 up-regulated (miR-146a-5p and let-7b-5p) and 2 down-regulated (miR-30d-5p and miR-142-5p). Compared with the differentially expression of platelet miRNAs between 1-1 000 TPM in the C_3 and C_1 group, there were 133 differentially expressed miRNAs, in which 99 were up-regulated and 34 were down-regulated. While in the C_5 and C_1 group, there were 77 differentially expressed miRNAs, in which 31 were up-regulated and 46 were down-regulated. The six selected differentially expressed miRNAs verified by RT-qPCR were consistent with those of sequencing. CONCLUSION The expression profiles of platelets miRNAs change significantly among 1, 3, and 5 d of storage in vitro.
Blood Component Removal ; Blood Platelets ; Cluster Analysis ; Gene Expression Profiling ; Humans ; MicroRNAs/genetics*

OBJECTIVE: To investigate the changes of gene sequencing and proteomics of apheresis platelet (AP) exosomes in different storage periods and predict the function of AP exosomes in different storage periods. METHODS: Platelets at different storage periods of 0 day (D0), 3 day (D3) and 5 day (D5) were collected, exosomes were extracted with Gradient centrifugation; gene sequencing and proteomic analysis were used to analyze the exosomes, and biological functions of platelet exosomes were analyzed and predicted by bioinformatics. Liquid mass spectrometry (LMS) was used to detect the changes and function prediction of exosomes proteins. The small RNA sequencing library was prepared, and the constructed library was sequenced and bioinformatics technology was used for data analysis. RESULTS: AP exosome iTRAQ protein analysis showed that AP exosomes stored in D3 with 55 up-regulated proteins and 94 down-regulated proteins (P<0.05, FC<0.83 or FC>1.2), while AP exosomes stored in D5 with 292 up-regulated proteins and 53 down-regulated proteins (P<0.05, FC<0.83 or FC>1.2) as compared with D0. KEGG pathway analysis showed that the proteins were mainly involved in transport and metabolism, immune system, cancer, membrane transport and other processes. There were statistically significant differences between AP exosome miRNAs in different storage days (P<0.01). The number of miRNA up-regulated and down-regulated was 374 and 255 as compared with the number of platelet exosomes miRNA stored in D3 and D0, while that was 297 and 242 in D5 and D0, and 252 and 327 in D5 and D3, respectively. The target genes of differential platelet exosome miRNAs were analyzed by GO enrichment. Target genes of differential miRNA were mainly involved in membrane composition, mainly played molecular functions binding to proteins, and participated in biological processes of transcriptional regulation. CONCLUSION The exosome differential proteins and miRNAs in D5 are significantly different from those in the D0 of APs, and they are involved in various biological processes.
Blood Component Removal ; Blood Platelets/metabolism* ; Exosomes/metabolism* ; Humans ; MicroRNAs/genetics* ; Proteomics

OBJECTIVE: To study the relationship between serum microRNA-122 (miR-122) and insulin resistance in obese children. METHODS: Forty-seven children with severely obesity aged 7-14 years and 45 age- and gender matched healthy children with normal weight (control group) were enrolled. The levels of height, weight, waistline, hip circumference, fasting blood glucose (FBG), fasting insulin (FINS), triglyceride (TG), total cholesterol (TC), free fatty acid (FFA), interleukin-6 (IL-6) and miR-122 in the two groups were measured. Body mass index (BMI), waist-hip ratio (WHR) and insulin resistance index (HOMA-IR) were calculated. RESULTS: Compared with the control group, the height, weight, BMI, WHR, FINS, HOMA-IR, TG, FFA, IL-6, and miR-122 levels in the obese group were significantly increased (P<0.05). MiR-122 levels in the obese group were positively correlated with FINS, HOMA-IR and IL-6 levels (r=0.408, 0.442, and 0.464 respectively, P<0.05). The changes of miR-122 have a linear regression relationship with IL-6 (b'=0.318, P<0.05). CONCLUSIONS The elevated serum miR-122 levels may be correlated with insulin resistance in obese children. The mechanism needs to be further studied.
Adolescent ; Blood Glucose ; Body Mass Index ; Child ; Humans ; Insulin ; Insulin Resistance ; MicroRNAs ; genetics ; Obesity ; Waist-Hip Ratio

To explore the differential expression of serum miRNAs in patients of advanced non- small cell lung cancer (NSCLC) treated by gifitinib before and after acquiring drug resistance.
 Methods: A total of 4 patients with advanced NSCLC from Affiliated Hospital of Yueyang Vocational Technical College, who acquired drug resistance during gefitinib therapy from June 2013 to June 2015, were enrolled. Serum samples were collected before treatment and after acquiring drug resistance. MicroRNA (miRNA) microarray was used to assess the levels and compositions of miRNAs in serum. Real-time RT-PCR was used to validate the results of miRNAs with significant differences in expression. The candidate miRNAs inhibitors and mimics were transfected into lung cancer cells by liposome, and the sensitivity of lung cancer cells to gifitinib was detected.
 Results: The miRNA microarray showed that there were significantly differential expression of miRNAs in serum of NSCLC patients after acquiring drug resistance, and 24 miRNAs were changed in more than 2-fold. Among them, 19 miRNAs were up-regulated and 5 miRNAs were down- regulated (both P<0.05). Especially, the expression of miR-21 in serum of NSCLC patients after obtaining resistance was up-regulated more than 10-fold compared with that before treatment. The results of RT-PCR was consistent with the results of miRNA microarray. The up-regulation of miR-21 in lung cancer cells could elevate the half maximal inhibition concentration (IC50) of gefitinib, and the down-regulation of miR-21 in lung cancer cells could reduce the IC50 of gefitinib (both P<0.05).
 Conclusion: There is differential expression of miRNAs in serum of NSCLC patients before treatment and after acquiring drug resistance during gefitinib therapy. The up-regulation of miR-21 may be involved in regulating the acquiring drug resistance of gefitinib.
Antineoplastic Agents ; pharmacology ; therapeutic use ; Carcinoma, Non-Small-Cell Lung ; drug therapy ; Cell Line, Tumor ; Drug Resistance, Neoplasm ; drug effects ; genetics ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Lung Neoplasms ; drug therapy ; MicroRNAs ; blood ; genetics

Sex differences are widely observed under various circumstances ranging from physiological processes to therapeutic responses, and a myriad of sex-biased genes have been identified. In recent years, transcriptomic datasets of microRNAs (miRNAs), an important class of non-coding RNAs, become increasingly accessible. However, comprehensive analysis of sex difference in miRNA expression has not been performed. Here, we identified the differentially-expressed miRNAs between males and females by examining the transcriptomic datasets available in public databases and conducted a systemic analysis of their biological characteristics. Consequently, we identified 73 female-biased miRNAs (FmiRs) and 163 male-biased miRNAs (MmiRs) across four tissues including brain, colorectal mucosa, peripheral blood, and cord blood. Our results suggest that compared to FmiRs, MmiRs tend to be clustered in the human genome and exhibit higher evolutionary rate, higher expression tissue specificity, and lower disease spectrum width. In addition, functional enrichment analysis of miRNAs show that FmiR genes are significantly associated with metabolism process and cell cycle process, whereas MmiR genes tend to be enriched for functions like histone modification and circadian rhythm. In all, the identification and analysis of sex-biased miRNAs together could provide new insights into the biological differences between females and males and facilitate the exploration of sex-biased disease susceptibility and therapy.
Biological Evolution ; Female ; Genome, Human ; Humans ; Male ; MicroRNAs ; blood ; genetics ; Organ Specificity ; Sex Characteristics ; Transcriptome

OBJECTIVETo investigate whether miR-492 is involved in the post-transcriptional regulation of OK blood group antigen expression on red blood cells.

METHODSTwo 3'-UTR fragments of the BSG gene were synthesized with a chemical method, which respectively encompassed the BSG rs8259 TT or BSG rs8259 AA sites. The fragments were added with Xho I and Not I restriction enzyme cutting sites at both ends and cloned into a pUC57 vector, which in turn was constructed into a psiCHECK-2 vector and verified by sequencing. K562 cells were transfected with various combinations of miR-492 mimic and constructed psiCHECK2-BSG-T or psiCHECK2-BSG-A recombinant plasmid. A blank control group was set up. Each transfection experiment was repeated three times. The activity of Renilla reniformis luciferase was determined and normalized with that of firefly luciferase, and detected with a dual-luciferase reporter assay system. The data were subjected to statistical analysis.

RESULTSThe sequencing results confirmed that the recombinant psiCHECK2 plasmids containing the BSG rs8259 TT or rs8259 AA sites were constructed successfully. The results of dual-luciferase report gene detection showed that the miR-492 mimic could significantly inhibit psiCHECK2-BSG-T at a concentration over 100 nmol/L. However, it could not inhibit psiCHECK-BSG-A.

CONCLUSIONmiR-492 may be involved in the regulation of OK antigen expression on red blood cells with the BSG rs8259 TT genotype.

Basigin ; genetics ; Blood Group Antigens ; genetics ; Erythrocytes ; immunology ; Gene Expression Regulation ; Genotype ; Humans ; MicroRNAs ; physiology

OBJECTIVETo investigate the value of serum miR-17-92 cluster in the diagnosis of retinoblastoma (RB).

METHODSSerum samples were collected from 20 children with RB and 20 healthy controls. Quantitative real-time PCR was used to measure the expression of miR-17-92 cluster. The expression of miR-17-92 cluster was compared between children with different stages of RB and the changes in the expression of miR-17-92 cluster after multimodality therapy were analyzed. The receiver operating characteristic (ROC) curve was used to investigate the value of serum miR-17-92 cluster in the diagnosis of RB.

RESULTSCompared with the healthy controls, the children with RB had significantly higher relative expression of miR-17-3P, miR-17-5P, miR-18a, and miR-20a in serum (P<0.05), and miR-18a showed the greatest increase. There were no significant differences in the relative expression of miR-19a, miR-19b-1, and miR-92a-1 between children with RB and healthy controls (P>0.05). There were no significant differences in the expression of miR-17-5P, miR-17-3P, miR-18a, and miR-20a between the children with early-to-moderate stage of RB and those with advanced stage of RB (P>0.05), but there were significant reductions after multimodality therapy (P<0.05). In the diagnosis of RB, the areas under the ROC curve (AUCs) for serum miR-17-3P, miR-17-5P, miR-18a, and miR-20a were 0.770, 0.755, 0.828, and 0.665 respectively, and miR-18a had the largest AUC, with a sensitivity of 90% and a specificity of 65%.

CONCLUSIONSmiR-17-3P, miR-17-5P, miR-18a, and miR-20a are highly expressed in the serum of children with RB, and miR-18a may be used as a new marker for the diagnosis of RB.

Biomarkers, Tumor ; blood ; Child, Preschool ; Female ; Humans ; Infant ; Male ; MicroRNAs ; blood ; ROC Curve ; Retinoblastoma ; blood ; diagnosis ; genetics

Gastroenteropancreatic neuroendocrine neoplam (GEP-NEN) is a rare group of tumors with its incidence rising significantly in recent decades. Because of the late presentation of the disease and limitations in conventional biomarkers, about 50% of GEP-NEN patients manifests advanced disease when diagnosed. Therefore, it is vital to identify circulating biomarkers which can not only be used for early diagnosis but also accurately evaluating the biological behavior of GEP-NEN. This review summarizes the advances of circulating biomarkers in diagnosing and evaluating efficacy of treatment in GEP-NEN. Well-known circulating biomarkers include chromogranin A (CgA), pancreastatin (PST), chromogranin B (CgB), neuron-specific enolase (NSE) and pancreatic peptide(PP). Novel biomarkers including circulating tumor cell(CTC), microRNA and NETest are promising biomarkers with potential clinical benefit, but further researches are needed before their clinical applications.
Biomarkers, Tumor ; blood ; Chromogranin A ; blood ; Chromogranin B ; blood ; chemistry ; Gastrointestinal Neoplasms ; blood ; chemistry ; diagnosis ; genetics ; Humans ; MicroRNAs ; blood ; Neoplastic Cells, Circulating ; Neuroendocrine Tumors ; blood ; chemistry ; diagnosis ; genetics ; Pancreatic Neoplasms ; blood ; chemistry ; diagnosis ; genetics ; Pancreatic Polypeptide ; blood ; Phosphopyruvate Hydratase ; blood