Journal: Bioengineering & Translational Medicine

Article Title: A novel platform using homobifunctional hydrazide for enrichment and isolation of urinary circulating RNAs

doi: 10.1002/btm2.10348

Figure Lengend Snippet: Clinical utility of circulating miRNA via HAZIS‐CirR. (a–f) Relative quantification (RQ) analysis of (a) hsa‐miR‐21‐5p , (b) hsa‐miR‐141‐3p , (c) hsa‐miR‐375‐3p , (d) hsa‐miR‐148a‐3p , (e) hsa‐miR‐483‐5p , and (f) hsa‐miR‐574‐3p using a total of 62 samples (30 PCa, 22 BPH, and 10 controls). Significant differences between groups are indicated: * p < 0.05, ** p < 0.01, and *** p < 0.001. PCa, prostate cancer; BPH, benign prostatic hyperplasia

Article Snippet: Briefly, a solution (1 ml) containing hsa‐mir‐21‐5p mimics (Shanghai GenePharma Co., Ltd., Shanghai, China) was used for the optimization of HAZIS‐CirR.

Techniques:

Journal: Bioengineering & Translational Medicine

Article Title: A novel platform using homobifunctional hydrazide for enrichment and isolation of urinary circulating RNAs

doi: 10.1002/btm2.10348

Figure Lengend Snippet: Three miRNAs panels of circulating miRNAs via HAZIS‐CirR. Panel 1 consists of miR‐141‐3p , miR‐375‐3p , miR‐483‐5p , and miR‐574‐3p for PCa diagnosis and differentiation from patients with BPH and controls. Panel 2 consists of miR‐21‐5p and miR‐148a‐3p for PCa diagnosis and differentiation from controls. Panel 3 consists of miR‐21‐5p , miR‐141‐3p , miR‐148a‐3p , miR‐375‐3p , miR‐483‐5p , and miR‐574‐3p for BPH diagnosis and differentiation from controls. This image was created using BioRender ( https://biorender.com ). PCa, prostate cancer; BPH, benign prostatic hyperplasia

Article Snippet: Briefly, a solution (1 ml) containing hsa‐mir‐21‐5p mimics (Shanghai GenePharma Co., Ltd., Shanghai, China) was used for the optimization of HAZIS‐CirR.

Techniques:

Journal: PLoS ONE

Article Title: miR-10a Regulates Proliferation of Human Cardiomyocyte Progenitor Cells by Targeting GATA6

doi: 10.1371/journal.pone.0103097

Figure Lengend Snippet: A. miR-10a mimics decrease the BrdU incorporation rate of hCMPCs, whereas a miR-10a inhibitor does not significantly affect the process. *P<0.05, n = 6. B. miR-10a inhibits EdU incorporation of hCMPCs. Representative image of hCMPCs transfected with mock, miR-10a mimics or inhibitor stained with DAPI (blue) and EdU (red) (×200). *P<0.05, n = 5. C. Representative flow cytometry results of hCMPC manipulated with mock, miR-10a mimics or miR-10a inhibitor. hCMPCs overexpressing miR-10a show G1/S blocking, and the inhibition of miR-10a promotes G1/S transition compare with mock. D. Data collected from C. *P<0.05, n = 3. E. Relative expression of cell cycle regulatory genes in hCMPCs transfected with miR-10a mimics. F. The same set of genes was measured in hCMPCs with inhibited miR-10a. The expression level of GAPDH was used as the control. *P<0.05, n = 5.

Article Snippet: hsa-miR-10a mimics (5′-UACCCUGUAGAUCCGAAUUUGUG-3′ and 5′-CAAAUUCGGAUCUACAGGGUAUU-3′), inhibitor (5′-CACAAAUUCGGAUCUACAGGGUA-3′) and negative scramble (mock) were purchased from Genepharm.

Techniques: BrdU Incorporation Assay, Transfection, Staining, Flow Cytometry, Blocking Assay, Inhibition, Expressing, Control

Journal: PLoS ONE

Article Title: miR-10a Regulates Proliferation of Human Cardiomyocyte Progenitor Cells by Targeting GATA6

doi: 10.1371/journal.pone.0103097

Figure Lengend Snippet: A. Representative image of differentiating hCMPCs with manipulated expression of miR-10a or mock. Cells were stained with DAPI, α-actinin and Trop I (×200). B. Data collected from A. *P<0.05, n = 6. C. Relative expression of cardiomyocyte markers in differentiating hCMPCs transfected with miR-10a mimics. D. The same markers were tested in hCMPCs with inhibited miR-10a. The expression level of GAPDH was used as the control. *P<0.05, n = 6.

Article Snippet: hsa-miR-10a mimics (5′-UACCCUGUAGAUCCGAAUUUGUG-3′ and 5′-CAAAUUCGGAUCUACAGGGUAUU-3′), inhibitor (5′-CACAAAUUCGGAUCUACAGGGUA-3′) and negative scramble (mock) were purchased from Genepharm.

Techniques: Expressing, Staining, Transfection, Control

Journal: PLoS ONE

Article Title: miR-10a Regulates Proliferation of Human Cardiomyocyte Progenitor Cells by Targeting GATA6

doi: 10.1371/journal.pone.0103097

Figure Lengend Snippet: A. Pmir- glo dual luciferase plasmid containing a wild type or mutant GATA6-3′UTR was cotransfected with miR-10a mimics or mock into 293 cells. The relative luciferase activity was shown. *P<0.05, n = 5. B. Western blot shows the expression of GATA6 in hCMPCs transfected with mock and miR-10a mimics. C. The same experiment with mock and inhibitor. Quantification of protein expression was normalized to GAPDH. *P<0.05, n = 3. D. Overexpression of GATA6 rescues the miR-10a inhibitory effect on hCMPC proliferation. Protein expression of GATA6 with different treatment was showed. BrdU absorbance of hCMPCs transfected with mock, miR-10a mimics and siRNA that downregulated GATA6 are shown. Similar to miR-10a, the downregulation GATA6 in hCMPCs reduces proliferation. miR-10a mimics and a plasmid that overexpresses GATA6 were cotransfected into hCMPCs, and a reduced BrdU absorbance was not observed. *P<0.05, n = 6.

Article Snippet: hsa-miR-10a mimics (5′-UACCCUGUAGAUCCGAAUUUGUG-3′ and 5′-CAAAUUCGGAUCUACAGGGUAUU-3′), inhibitor (5′-CACAAAUUCGGAUCUACAGGGUA-3′) and negative scramble (mock) were purchased from Genepharm.

Techniques: Luciferase, Plasmid Preparation, Mutagenesis, Activity Assay, Western Blot, Expressing, Transfection, Over Expression

Journal: PLoS ONE

Article Title: miR-10a Regulates Proliferation of Human Cardiomyocyte Progenitor Cells by Targeting GATA6

doi: 10.1371/journal.pone.0103097

Figure Lengend Snippet: Hearts of different stages of cardiogenesis were collected for miR-10a expression analysis. Quantification of miR-10a level was related to U6. *P<0.05, n = 6.

Article Snippet: hsa-miR-10a mimics (5′-UACCCUGUAGAUCCGAAUUUGUG-3′ and 5′-CAAAUUCGGAUCUACAGGGUAUU-3′), inhibitor (5′-CACAAAUUCGGAUCUACAGGGUA-3′) and negative scramble (mock) were purchased from Genepharm.

Techniques: Expressing

Journal: Thyroid

Article Title: MicroRNA-146b-3p Promotes Cell Metastasis by Directly Targeting NF2 in Human Papillary Thyroid Cancer

doi: 10.1089/thy.2017.0626

Figure Lengend Snippet: MiR-146b-5p and miR146b-3p expression is significantly increased in PTC tissues and further enhanced in MLN. (A) The histological changes of different thyroid tissues and LN tissues stained with H&E (magnification × 100). (B) Relative expression levels of mature miR-146b-5p (left panels) and miR-146b-3p (right panels) in 72 samples, including PTC, matched normal thyroid tissue, MLN, and LN, were determined by qRT-PCR and normalized against an endogenous control RUN6B. Data were analyzed using a ΔΔCt approach and expressed as miR-146b/RUN6B ratio (−2ΔCt[miR-146b-RUN6B]). (C) Fold change of expression of mature miR-146b-5p (left panels) and miR-146b-3p (right panels) in 14 pairs of MLN and their corresponding primary PTC. Data were analyzed using log2 fold change (ΔΔCt [MLN/each corresponding PTC]). Significant upregulation of miR-146b in paired samples was defined as log2 fold change >1, which means twofold higher. (D) Relative expression levels of miR-146b-5p and miR-146b-3p in PTC patients with different aggressive abilities (PTC with LNM [PTC-M] vs. PTC without LNM [PTC-NM]) by qRT-PCR and normalized against an endogenous control RUN6B. NT, normal thyroid tissues, PTC, papillary thyroid cancer; MLN, metastatic lymph nodes; LN, lymph node; H&E, hematoxylin and eosin; qRT-PCR, quantitative reverse transcription polymerase chain reaction. #p < 0.05; ##p < 0.005. Color images available online at www.liebertpub.com/thy

Article Snippet: The successfully xenografted nude mice were randomly divided into two groups: hsa-miR-146b-3p mimics (miR-146b-3p agomir) or agomir Ncontrol (NC agomir; RiboBio Co.) were directly injected into the implanted tumor at the dose of 2.5 nmol in 100 μL PBS per mouse twice per week for a total of eight times.

Techniques: Expressing, Staining, Quantitative RT-PCR, Control, Reverse Transcription, Polymerase Chain Reaction

Journal: Thyroid

Article Title: MicroRNA-146b-3p Promotes Cell Metastasis by Directly Targeting NF2 in Human Papillary Thyroid Cancer

doi: 10.1089/thy.2017.0626

Figure Lengend Snippet: MiR-146b-3p promotes tumor metastasis in vivo. (A) Schematic of male BALB/c PTC intramuscular xenograft nude mice model. (B) Body weight of miR-146b-3p agomir or NC agomir (control) treated BHP10-3SCmluc inoculated nude mice (n = 5, 10). (C) For spontaneous tumor metastasis images were taken from three representative miR-146b-3p agomir or NC agomir (control) treated model separately. Bioluminescence images were obtained from the chest side of the mice in vivo (upper), and fluorescence images were obtained from the lungs and lymph nodes of sacrificed mice ex vivo (lower) at the end time point. (D) The size and morphology of the miR-146b-3p agomir or NC agomir treated BHP10-3SCmluc xenografts in nude mice. (E) Ratio of metastasis of miR-146b-3p agomir or NC agomir (control) treated BHP10-3SCmluc inoculated nude mice. (F) Histopathological characteristics of a representative intramuscular xenograft tumor, pulmonary and lymph node metastases of sacrificed mouse stained with H&E (magnification × 100). The expression of miR-146b-3p (G) and protein levels of merlin (H) were determined by qRT-PCR and Western blot assay separately. The tissues were derived from xenograft mouse tumors treated with miR-146b-3p agomir or NC agomir. **p < 0.001. Color images available online at www.liebertpub.com/thy

Article Snippet: The successfully xenografted nude mice were randomly divided into two groups: hsa-miR-146b-3p mimics (miR-146b-3p agomir) or agomir Ncontrol (NC agomir; RiboBio Co.) were directly injected into the implanted tumor at the dose of 2.5 nmol in 100 μL PBS per mouse twice per week for a total of eight times.

Techniques: In Vivo, Control, Fluorescence, Ex Vivo, Staining, Expressing, Quantitative RT-PCR, Western Blot, Derivative Assay

Journal: Thyroid

Article Title: MicroRNA-146b-3p Promotes Cell Metastasis by Directly Targeting NF2 in Human Papillary Thyroid Cancer

doi: 10.1089/thy.2017.0626

Figure Lengend Snippet: Correlation Analysis Between miR-146b-3p or miR-146b-5p and Clinicopathologic Features

Article Snippet: The successfully xenografted nude mice were randomly divided into two groups: hsa-miR-146b-3p mimics (miR-146b-3p agomir) or agomir Ncontrol (NC agomir; RiboBio Co.) were directly injected into the implanted tumor at the dose of 2.5 nmol in 100 μL PBS per mouse twice per week for a total of eight times.

Techniques: Biomarker Discovery

Journal: Thyroid

Article Title: MicroRNA-146b-3p Promotes Cell Metastasis by Directly Targeting NF2 in Human Papillary Thyroid Cancer

doi: 10.1089/thy.2017.0626

Figure Lengend Snippet: Expression of miRNA-146b is significantly increased in PTC cell lines and positively correlated with the ability of cell invasion. (A) Transwell™ assay was performed to determine the migration or invasive ability of BHP10-3, BHP10-3SC mouse and K1 cells. Representative images showed migrated or invasive cells in the lower chamber stained with hematoxylin. (B) Cell count of Transwell™ assay for above three cells was statistically analyzed. (C) Fold changes of miR-146b-5p and -3p expressions in PTC cells with different invasive abilities (BHP10-3 and BHP10-3SCmice) were calculated relative to miR-146b-5p expression in PHT by qRT-PCR and normalized against an endogenous control RUN6B. PHT, primary normal human thyrocytes. ##p < 0.005 vs. BHP10-3SCmice; **p < 0.005 vs. PHT. Color images available online at www.liebertpub.com/thy

Article Snippet: The successfully xenografted nude mice were randomly divided into two groups: hsa-miR-146b-3p mimics (miR-146b-3p agomir) or agomir Ncontrol (NC agomir; RiboBio Co.) were directly injected into the implanted tumor at the dose of 2.5 nmol in 100 μL PBS per mouse twice per week for a total of eight times.

Techniques: Expressing, Transwell Assay, Migration, Staining, Cell Counting, Quantitative RT-PCR, Control

Journal: Thyroid

Article Title: MicroRNA-146b-3p Promotes Cell Metastasis by Directly Targeting NF2 in Human Papillary Thyroid Cancer

doi: 10.1089/thy.2017.0626

Figure Lengend Snippet: MiR-146b-3p enhances the migratory and invasive abilities more obviously than miR-146b-5p in BHP10-3 cells. Inhibiting miR-146b-3p expression can reduce the ability of metastasis in BHP10-3SC mouse cells. (A) Expression of miR-146b-5p or -3p was confirmed by real-time PCR after BHP10-3 cells were transfected with miR146b-5p mimics or -3p mimics. (B and C) BHP10-3 cells were transiently transfected with miR-146b-3p mimics (50 nM), miR-146b-5p mimics (50 nM), or negative control (miR-NC; 50 nM). Real-time measurements were performed in 24 h for the migration assay or 96 h for the invasion assay using the real-time cell analyzer system. (D) Transwell™ assay was performed to determine the migration or invasive ability of BHP10-3 cells after being transfected with miR-146b-3p mimics, miR-146b-5p mimics, or miR-NC for 24 or 48 h. Representative images showed migrated or invasive BHP10-3 cells in the lower chamber stained with hematoxylin. (E) Cell count of Transwell™ assay for the above three treatments was statistically analyzed. (F) Expression of miR-146b-5p or -3p was confirmed by real-time PCR after BHP10-3SC mouse cells were transfected with miR146b-5p inhibitor or -3p inhibitor. (G) Transwell™ assay was performed to determine the migration or invasive ability of BHP10-3SC mouse cells after transfection with miR-146b-3p inhibitor, miR-146b-5p inhibitor, or anti-NC for 24 or 48 h. Representative images show migrated or invasive BHP10-3SC mouse cells in the lower chamber stained with hematoxylin. (H) Cell count of Transwell™ assay for the above two treatments was statistically analyzed. #p < 0.05, ##p < 0.005 vs. miR-NC or anti-NC. Color images available online at www.liebertpub.com/thy

Article Snippet: The successfully xenografted nude mice were randomly divided into two groups: hsa-miR-146b-3p mimics (miR-146b-3p agomir) or agomir Ncontrol (NC agomir; RiboBio Co.) were directly injected into the implanted tumor at the dose of 2.5 nmol in 100 μL PBS per mouse twice per week for a total of eight times.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, Negative Control, Migration, Invasion Assay, Transwell Assay, Staining, Cell Counting

Journal: Thyroid

Article Title: MicroRNA-146b-3p Promotes Cell Metastasis by Directly Targeting NF2 in Human Papillary Thyroid Cancer

doi: 10.1089/thy.2017.0626

Figure Lengend Snippet: NF2 is a direct downstream target for miR-146b-3p. (A) Diagram of miR-146b-3p putative seed sequences in the 3′ UTR of candidate mRNA. Based on bioinformation software, NF2 and MTSS1 were found to contain two possible candidate miR-146b-3p binding sites on 3′ UTR separately. (B and C) Relative luciferase activity analysis in BHP10-3 cells transiently co-transfected with 3′ UTR of wild-type or mutant recombinant NF2 or MTSS1 luciferase plasmids with miR-NC or miR-146b-3p mimics. The two wild-type recombinant plasmids of NF2 (NF2-P1 and NF2-P2) and two wild-type recombinant plasmids of MTSS1 (MTSS1-P1 and MTSS1-P2) were co-transfected into cells (B). The wild type of two NF2 3′ UTR and mutant of these NF2 3′ UTR were transfected into cells (C). (D) Merlin protein expression was detected by Western blot analysis after transfection of miR-146b-3p mimics in BHP10-3 cells. (E) Relative luciferase activity analysis in BHP10-3SC mouse cells transiently co-transfected with 3′ UTR of wild-type or mutant recombinant NF2 luciferase plasmids with anti-NC or miR-146b-3p inhibitor. (F) Merlin protein expression was detected by Western blot analysis after miR-146b-3p inhibition in BHP10-3SC mouse cells. Renilla luciferase vector was used as an internal control. The relative luciferase activities of group transfected with miR-NC or anti-NC were set as 1. UTR, untranslated region. #p < 0.05; ##p < 0.005 vs. miR-NC or anti-NC. Color images available online at www.liebertpub.com/thy

Article Snippet: The successfully xenografted nude mice were randomly divided into two groups: hsa-miR-146b-3p mimics (miR-146b-3p agomir) or agomir Ncontrol (NC agomir; RiboBio Co.) were directly injected into the implanted tumor at the dose of 2.5 nmol in 100 μL PBS per mouse twice per week for a total of eight times.

Techniques: Software, Binding Assay, Luciferase, Activity Assay, Transfection, Mutagenesis, Recombinant, Expressing, Western Blot, Inhibition, Plasmid Preparation, Control

Journal: Thyroid

Article Title: MicroRNA-146b-3p Promotes Cell Metastasis by Directly Targeting NF2 in Human Papillary Thyroid Cancer

doi: 10.1089/thy.2017.0626

Figure Lengend Snippet: Reintroduction of NF2 abrogates miR-146b-3p induced metastasis enhancement of BHP10-3 cells. BHP10-3 cells were co-transfected with miR-146b-3p mimics and pcDNA3 merlin or pcDNA3 (empty vector control), miR-NC (mimics control), or pcDNA3 merlin was respectively transfected into cells as control. (A) The protein level of endogenous merlin was detected by Western blot analysis after transfection. (B) The densitometric histogram of protein bands and results were expressed as ratio of corresponding protein to GAPDH. (C) Transwell™ assay was performed to determine the migration or invasive ability of BHP10-3 cells after being transfected for 24 or 48 h. Representative images show migrated or invasive BHP10-3 cells in the lower chamber stained with hematoxylin. (D) Cell count of Transwell™ assay for different treatment was statistically analyzed. (E and F) The migratory and invasive capacities of BHP10-3 cells were performed by RTCA assay after being transfected. #p < 0.05; ##p < 0.005 vs. miR-NC. Color images available online at www.liebertpub.com/thy

Article Snippet: The successfully xenografted nude mice were randomly divided into two groups: hsa-miR-146b-3p mimics (miR-146b-3p agomir) or agomir Ncontrol (NC agomir; RiboBio Co.) were directly injected into the implanted tumor at the dose of 2.5 nmol in 100 μL PBS per mouse twice per week for a total of eight times.

Techniques: Transfection, Plasmid Preparation, Control, Western Blot, Transwell Assay, Migration, Staining, Cell Counting

Journal: Molecular Medicine Reports

Article Title: miR-27b-3p and miR-607 cooperatively regulate BLM gene expression by directly targeting the 3′-UTR in PC3 cells

doi: 10.3892/mmr.2019.10135

Figure Lengend Snippet: miR screening method and designed mutation or deletion sites. (A) Several prediction and function analysis software programs were used to screen candidate miRs, including miR-27b-3p, miR-607, miR-361-3p, miR-628-5p and miR-338-3p as candidates. (B) miR-27b-3p and miR-607 interaction sites with BLM 3′-UTR and mutational sites where four interaction bases of miR-27b-3p are changed and edited sequence where the interaction region of miR-607 is deleted. Predicted miR binding sites of BLM 3′-UTR are displayed. miR, microRNA; WT, wild type; BLM, BLM, BLM RecQ like helicase; UTR, untranslated region; MUT, mutated.

Article Snippet: The constructed plasmids (Promega Corporation, Madison, WI, USA), and miRNA mimics (negative control; hsa-miR-27b-3p mimics; hsa-miR-607 mimics; hsa-miR-628-5p mimic; miR-338-3p mimic; hsa-miR-361-3p mimics; GenePharma Co., Ltd., Shanghai, China) were transfected into PC3 cells using Lipofectamine ® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) when cells were ~60% confluent, with 2,500 ng plasmid and 5 μmol/l miRNA used per well of a 6-well-plate, according to the manufacturer's protocol.

Techniques: Mutagenesis, Software, Sequencing, Binding Assay

Journal: Molecular Medicine Reports

Article Title: miR-27b-3p and miR-607 cooperatively regulate BLM gene expression by directly targeting the 3′-UTR in PC3 cells

doi: 10.3892/mmr.2019.10135

Figure Lengend Snippet: miR reverse primers.

Article Snippet: The constructed plasmids (Promega Corporation, Madison, WI, USA), and miRNA mimics (negative control; hsa-miR-27b-3p mimics; hsa-miR-607 mimics; hsa-miR-628-5p mimic; miR-338-3p mimic; hsa-miR-361-3p mimics; GenePharma Co., Ltd., Shanghai, China) were transfected into PC3 cells using Lipofectamine ® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) when cells were ~60% confluent, with 2,500 ng plasmid and 5 μmol/l miRNA used per well of a 6-well-plate, according to the manufacturer's protocol.

Techniques: Sequencing

Journal: Molecular Medicine Reports

Article Title: miR-27b-3p and miR-607 cooperatively regulate BLM gene expression by directly targeting the 3′-UTR in PC3 cells

doi: 10.3892/mmr.2019.10135

Figure Lengend Snippet: Transfectional efficiency detection of miR mimic.

Article Snippet: The constructed plasmids (Promega Corporation, Madison, WI, USA), and miRNA mimics (negative control; hsa-miR-27b-3p mimics; hsa-miR-607 mimics; hsa-miR-628-5p mimic; miR-338-3p mimic; hsa-miR-361-3p mimics; GenePharma Co., Ltd., Shanghai, China) were transfected into PC3 cells using Lipofectamine ® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) when cells were ~60% confluent, with 2,500 ng plasmid and 5 μmol/l miRNA used per well of a 6-well-plate, according to the manufacturer's protocol.

Techniques:

Journal: Molecular Medicine Reports

Article Title: miR-27b-3p and miR-607 cooperatively regulate BLM gene expression by directly targeting the 3′-UTR in PC3 cells

doi: 10.3892/mmr.2019.10135

Figure Lengend Snippet: Amplification efficiencies and expression levels of BLM mRNA and five miRNAs in RWPE-2 and PC3 cell lines. BLM, miR-628-5p, miR-361-3p, miR-338-3p, miR-27b-3p and miR-607 expression was analyzed by reverse transcription-quantitative polymerase chain reaction and (A) standard curve equations of amplification are shown. (B) The relative mRNA and miRNA expression levels were normalized to GAPDH and U6 respectively and following Pfaffl's method was applied to calculate expression levels. n=3. *P<0.05, **P<0.01. BLM, BLM, BLM RecQ like helicase; miR, microRNA.

Article Snippet: The constructed plasmids (Promega Corporation, Madison, WI, USA), and miRNA mimics (negative control; hsa-miR-27b-3p mimics; hsa-miR-607 mimics; hsa-miR-628-5p mimic; miR-338-3p mimic; hsa-miR-361-3p mimics; GenePharma Co., Ltd., Shanghai, China) were transfected into PC3 cells using Lipofectamine ® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) when cells were ~60% confluent, with 2,500 ng plasmid and 5 μmol/l miRNA used per well of a 6-well-plate, according to the manufacturer's protocol.

Techniques: Amplification, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: miR-27b-3p and miR-607 cooperatively regulate BLM gene expression by directly targeting the 3′-UTR in PC3 cells

doi: 10.3892/mmr.2019.10135

Figure Lengend Snippet: Effects of miRs on relative luciferase activity. PC3 cells were co-transfected with miR mimic and luciferase reporter vectors. Relative luciferase activity was detected at 24 h and the firefly luciferase activity of each sample was normalized to the Renilla luciferase activity. PGL-UTR-WT is a reporter vector containing the wild 3′-UTR sequence BLM mRNA at the 3′-terminal of the firefly luciferase sequence in the pmir-GLO vector. Similarly, PGL-UTR-MUT is a reporter vector containing a mutated BLM 3′-UTR containing an miR-27b-3p interaction region (changed at four bases). PGL-UTR-DEL is a reporter vector with the BLM 3′-UTR sequence containing a miR-607 interaction region partially deleted. (A) Relative luciferase activity results of PGL-UTR-WT + miR mimics. (B) Relative luciferase activity of PGL-UTR-MUT or PGL-UTR-DEL + miR mimics. n=6. *P<0.05, **P<0.01. BLM, BLM RecQ like helicase; PGL, pmir-GLO; UTR, untranslated region; WT, wild-type; NC, negative control; miR, microRNA; DEL, deletion; MUT, mutation.

Article Snippet: The constructed plasmids (Promega Corporation, Madison, WI, USA), and miRNA mimics (negative control; hsa-miR-27b-3p mimics; hsa-miR-607 mimics; hsa-miR-628-5p mimic; miR-338-3p mimic; hsa-miR-361-3p mimics; GenePharma Co., Ltd., Shanghai, China) were transfected into PC3 cells using Lipofectamine ® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) when cells were ~60% confluent, with 2,500 ng plasmid and 5 μmol/l miRNA used per well of a 6-well-plate, according to the manufacturer's protocol.

Techniques: Luciferase, Activity Assay, Transfection, Plasmid Preparation, Sequencing, Negative Control, Mutagenesis

Journal: Molecular Medicine Reports

Article Title: miR-27b-3p and miR-607 cooperatively regulate BLM gene expression by directly targeting the 3′-UTR in PC3 cells

doi: 10.3892/mmr.2019.10135

Figure Lengend Snippet: Response surface contour map. PGL-UTR-WT and different doses of miR-27b-3p, miR-607 and miR-338-3p were co-transfected into PC3 cells. The relative luciferase activity was detected to determine interactions among miRs targeting BLM. The contour map shows the 0 dose levels (5 µmol/l) of each miR to ease understanding of the interaction effect between miRs. (A) Interaction contour map between miR-27b-3p and miR-607 where the 0 dose level of miR-338-3p has been applied. (B) Interaction contour map between miR-338-3p and miR-607 where the 0 dose level of miR-27b-3p has been applied. (C) Interaction contour map between miR-27b-3p and miR-338-3p where the 0 dose level of miR-607 has been applied. n=6. PGL, pmir-GLO; UTR, untranslated region; WT, wild-type; Luc/Rluc, luciferase/ Renilla luciferase; miR, microRNA.

Article Snippet: The constructed plasmids (Promega Corporation, Madison, WI, USA), and miRNA mimics (negative control; hsa-miR-27b-3p mimics; hsa-miR-607 mimics; hsa-miR-628-5p mimic; miR-338-3p mimic; hsa-miR-361-3p mimics; GenePharma Co., Ltd., Shanghai, China) were transfected into PC3 cells using Lipofectamine ® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) when cells were ~60% confluent, with 2,500 ng plasmid and 5 μmol/l miRNA used per well of a 6-well-plate, according to the manufacturer's protocol.

Techniques: Transfection, Luciferase, Activity Assay

Journal: Molecular Medicine Reports

Article Title: miR-27b-3p and miR-607 cooperatively regulate BLM gene expression by directly targeting the 3′-UTR in PC3 cells

doi: 10.3892/mmr.2019.10135

Figure Lengend Snippet: Effects of miR on PC3 cell proliferation. PC3 cells were collected following transfection with miR-27b-3p, miR-607 or NC mimics and used to analyze cell proliferation in an MTT assay. n=4. **P<0.01. OD, optical density; miR, microRNA; NC, negative control.

Article Snippet: The constructed plasmids (Promega Corporation, Madison, WI, USA), and miRNA mimics (negative control; hsa-miR-27b-3p mimics; hsa-miR-607 mimics; hsa-miR-628-5p mimic; miR-338-3p mimic; hsa-miR-361-3p mimics; GenePharma Co., Ltd., Shanghai, China) were transfected into PC3 cells using Lipofectamine ® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) when cells were ~60% confluent, with 2,500 ng plasmid and 5 μmol/l miRNA used per well of a 6-well-plate, according to the manufacturer's protocol.

Techniques: Transfection, MTT Assay, Negative Control