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An artificial miRNA against HPSE suppresses melanoma invasion properties, correlating with a down-regulation of chemokines and MAPK phosphorylation.

Liu X, Fang H, Chen H, Jiang X, Fang D, Wang Y, Zhu D - PLoS ONE (2012)

Bottom Line: Ribonucleic acid interference (RNAi) based on microRNA (miRNA) context may provide an efficient and safe therapeutic knockdown effect and can be driven by ribonucleic acid polymerase II (RNAP II).Further study on its probable mechanism declared that down-regulation of IL8 and CXCL1 by HPSE-miRNA may be correlated with reduced growth-factor simulated mitogen-activated kinase (MAPK) phosphorylation including p38 MAPK, c-Jun N-terminal kinase (JNK) and extracellular-signal-regulated kinase (ERK) 1 and 2, which could be rescued by miRNA incompatible mutated HPSE cDNA.Furthermore, miRNA-based RNAi was also a powerful tool for gene function study.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, People's Republic of China.

ABSTRACT
Ribonucleic acid interference (RNAi) based on microRNA (miRNA) context may provide an efficient and safe therapeutic knockdown effect and can be driven by ribonucleic acid polymerase II (RNAP II). In this study, we designed and synthesized miR155-based artificial miRNAs against heparanase (HPSE) constructed with BLOCK-iT™ Pol II miR RNAi Expression Vector Kit. The expression levels of HPSE declined significantly in both the mRNA and protein levels in HPSE-miRNA transfected melanoma cells that exhibited reduction of adhesion, migration, and invasion ability in vitro and in vivo. We also observed that HPSE miRNA could inhibit the expressions of chemokines of interleukin-8 (IL8) and chemokine (C-X-C motif) ligand 1 (CXCL1), at both the transcriptional and translational levels. Further study on its probable mechanism declared that down-regulation of IL8 and CXCL1 by HPSE-miRNA may be correlated with reduced growth-factor simulated mitogen-activated kinase (MAPK) phosphorylation including p38 MAPK, c-Jun N-terminal kinase (JNK) and extracellular-signal-regulated kinase (ERK) 1 and 2, which could be rescued by miRNA incompatible mutated HPSE cDNA. In conclusion, we demonstrated that artificial miRNAs against HPSE might serve as an alterative mean of therapy to low HPSE expression and to block the adhesion, invasion, and metastasis of melanoma cells. Furthermore, miRNA-based RNAi was also a powerful tool for gene function study.

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Construction of of miR-155-based HPSE miRNAs and their impact on HPSE expression levels in A375 cells.A375 cells were transfected with HPSE-miRNAs or Neg-miRNA for 48 hours. (A) The sequences and predicted secondary structures of three designed pre-miRNAs targeting HPSE (HPSE-miRNA1, HPSE-miRNA2, and HPSE-miRNA3) and a negative control miRNA (Neg-miRNA), and the precise regions of the HPSE mRNA that they targeted. (B) Pre-miRNA double-stranded oligo inserted into the miRNA expression vector-pcDNA6.2-GW/EmGFP-miR. (C) Schematic representation of the process of ligation and transformation. (D) Inhibitory effects of HPSE miRNAs on HPSE protein expression. Representative blots are shown from three independent experiments with identical results. The expressions of HPSE protein in A375 cells transfected with HPSE miRNAs were obviously down-regulated obviously compared to the parental cells and the Neg-miRNA group. (E) Inhibitory effects of HPSE miRNAs on HPSE mRNA expression. Calculation of the respective HPSE mRNA expression in each group was relative to the Neg-miRNA group (%). Quantitative real-time PCR results showed that the expression of HPSE mRNA in A375 cells transfected with HPSE miRNAs were down-regulated compared to the parental cells or the Neg-miRNA transfected cells. (†P<0.05, compared with the parental cells; *P<0.05, compared with the Neg-miRNA transfected cells).
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pone-0038659-g001: Construction of of miR-155-based HPSE miRNAs and their impact on HPSE expression levels in A375 cells.A375 cells were transfected with HPSE-miRNAs or Neg-miRNA for 48 hours. (A) The sequences and predicted secondary structures of three designed pre-miRNAs targeting HPSE (HPSE-miRNA1, HPSE-miRNA2, and HPSE-miRNA3) and a negative control miRNA (Neg-miRNA), and the precise regions of the HPSE mRNA that they targeted. (B) Pre-miRNA double-stranded oligo inserted into the miRNA expression vector-pcDNA6.2-GW/EmGFP-miR. (C) Schematic representation of the process of ligation and transformation. (D) Inhibitory effects of HPSE miRNAs on HPSE protein expression. Representative blots are shown from three independent experiments with identical results. The expressions of HPSE protein in A375 cells transfected with HPSE miRNAs were obviously down-regulated obviously compared to the parental cells and the Neg-miRNA group. (E) Inhibitory effects of HPSE miRNAs on HPSE mRNA expression. Calculation of the respective HPSE mRNA expression in each group was relative to the Neg-miRNA group (%). Quantitative real-time PCR results showed that the expression of HPSE mRNA in A375 cells transfected with HPSE miRNAs were down-regulated compared to the parental cells or the Neg-miRNA transfected cells. (†P<0.05, compared with the parental cells; *P<0.05, compared with the Neg-miRNA transfected cells).

Mentions: Pre-miRNA sequences for HPSE (NM_006665.3) were designed by Invitrogen’s RNAi Designer (sequences were shown in Figure 1A). The synthesized complementary DNA oligos (TaKaRa Biotechnology Co. Ltd., Dalian, China) were annealed to generate a double-stranded oligo and cloned into the linearized pcDNA™ 6.2-GW/EmGFP-miR vector (Invitrogen Corp., Carlsbad, CA, USA) using T4 DNA ligase (Figure 1B and C). The Neg-miRNA control plasmid was included in the Block-iT™-Pol II miR RNAi Expression Vector Kit (sequences were shown in Figure 1A). All of the vectors were transformed into One Shot® TOP10 Chemically Competent E. coli (Invitrogen Corp.), and the colonies containing spectinomycin-resistant transformants were analyzed for the desired expression clones. The recombinant vectors were purified with a purification kit (Qiagen Inc., Valencia, CA, USA) and confirmed by sequencing (TaKaRa).


An artificial miRNA against HPSE suppresses melanoma invasion properties, correlating with a down-regulation of chemokines and MAPK phosphorylation.

Liu X, Fang H, Chen H, Jiang X, Fang D, Wang Y, Zhu D - PLoS ONE (2012)

Construction of of miR-155-based HPSE miRNAs and their impact on HPSE expression levels in A375 cells.A375 cells were transfected with HPSE-miRNAs or Neg-miRNA for 48 hours. (A) The sequences and predicted secondary structures of three designed pre-miRNAs targeting HPSE (HPSE-miRNA1, HPSE-miRNA2, and HPSE-miRNA3) and a negative control miRNA (Neg-miRNA), and the precise regions of the HPSE mRNA that they targeted. (B) Pre-miRNA double-stranded oligo inserted into the miRNA expression vector-pcDNA6.2-GW/EmGFP-miR. (C) Schematic representation of the process of ligation and transformation. (D) Inhibitory effects of HPSE miRNAs on HPSE protein expression. Representative blots are shown from three independent experiments with identical results. The expressions of HPSE protein in A375 cells transfected with HPSE miRNAs were obviously down-regulated obviously compared to the parental cells and the Neg-miRNA group. (E) Inhibitory effects of HPSE miRNAs on HPSE mRNA expression. Calculation of the respective HPSE mRNA expression in each group was relative to the Neg-miRNA group (%). Quantitative real-time PCR results showed that the expression of HPSE mRNA in A375 cells transfected with HPSE miRNAs were down-regulated compared to the parental cells or the Neg-miRNA transfected cells. (†P<0.05, compared with the parental cells; *P<0.05, compared with the Neg-miRNA transfected cells).
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Related In: Results  -  Collection

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pone-0038659-g001: Construction of of miR-155-based HPSE miRNAs and their impact on HPSE expression levels in A375 cells.A375 cells were transfected with HPSE-miRNAs or Neg-miRNA for 48 hours. (A) The sequences and predicted secondary structures of three designed pre-miRNAs targeting HPSE (HPSE-miRNA1, HPSE-miRNA2, and HPSE-miRNA3) and a negative control miRNA (Neg-miRNA), and the precise regions of the HPSE mRNA that they targeted. (B) Pre-miRNA double-stranded oligo inserted into the miRNA expression vector-pcDNA6.2-GW/EmGFP-miR. (C) Schematic representation of the process of ligation and transformation. (D) Inhibitory effects of HPSE miRNAs on HPSE protein expression. Representative blots are shown from three independent experiments with identical results. The expressions of HPSE protein in A375 cells transfected with HPSE miRNAs were obviously down-regulated obviously compared to the parental cells and the Neg-miRNA group. (E) Inhibitory effects of HPSE miRNAs on HPSE mRNA expression. Calculation of the respective HPSE mRNA expression in each group was relative to the Neg-miRNA group (%). Quantitative real-time PCR results showed that the expression of HPSE mRNA in A375 cells transfected with HPSE miRNAs were down-regulated compared to the parental cells or the Neg-miRNA transfected cells. (†P<0.05, compared with the parental cells; *P<0.05, compared with the Neg-miRNA transfected cells).
Mentions: Pre-miRNA sequences for HPSE (NM_006665.3) were designed by Invitrogen’s RNAi Designer (sequences were shown in Figure 1A). The synthesized complementary DNA oligos (TaKaRa Biotechnology Co. Ltd., Dalian, China) were annealed to generate a double-stranded oligo and cloned into the linearized pcDNA™ 6.2-GW/EmGFP-miR vector (Invitrogen Corp., Carlsbad, CA, USA) using T4 DNA ligase (Figure 1B and C). The Neg-miRNA control plasmid was included in the Block-iT™-Pol II miR RNAi Expression Vector Kit (sequences were shown in Figure 1A). All of the vectors were transformed into One Shot® TOP10 Chemically Competent E. coli (Invitrogen Corp.), and the colonies containing spectinomycin-resistant transformants were analyzed for the desired expression clones. The recombinant vectors were purified with a purification kit (Qiagen Inc., Valencia, CA, USA) and confirmed by sequencing (TaKaRa).

Bottom Line: Ribonucleic acid interference (RNAi) based on microRNA (miRNA) context may provide an efficient and safe therapeutic knockdown effect and can be driven by ribonucleic acid polymerase II (RNAP II).Further study on its probable mechanism declared that down-regulation of IL8 and CXCL1 by HPSE-miRNA may be correlated with reduced growth-factor simulated mitogen-activated kinase (MAPK) phosphorylation including p38 MAPK, c-Jun N-terminal kinase (JNK) and extracellular-signal-regulated kinase (ERK) 1 and 2, which could be rescued by miRNA incompatible mutated HPSE cDNA.Furthermore, miRNA-based RNAi was also a powerful tool for gene function study.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, People's Republic of China.

ABSTRACT
Ribonucleic acid interference (RNAi) based on microRNA (miRNA) context may provide an efficient and safe therapeutic knockdown effect and can be driven by ribonucleic acid polymerase II (RNAP II). In this study, we designed and synthesized miR155-based artificial miRNAs against heparanase (HPSE) constructed with BLOCK-iT™ Pol II miR RNAi Expression Vector Kit. The expression levels of HPSE declined significantly in both the mRNA and protein levels in HPSE-miRNA transfected melanoma cells that exhibited reduction of adhesion, migration, and invasion ability in vitro and in vivo. We also observed that HPSE miRNA could inhibit the expressions of chemokines of interleukin-8 (IL8) and chemokine (C-X-C motif) ligand 1 (CXCL1), at both the transcriptional and translational levels. Further study on its probable mechanism declared that down-regulation of IL8 and CXCL1 by HPSE-miRNA may be correlated with reduced growth-factor simulated mitogen-activated kinase (MAPK) phosphorylation including p38 MAPK, c-Jun N-terminal kinase (JNK) and extracellular-signal-regulated kinase (ERK) 1 and 2, which could be rescued by miRNA incompatible mutated HPSE cDNA. In conclusion, we demonstrated that artificial miRNAs against HPSE might serve as an alterative mean of therapy to low HPSE expression and to block the adhesion, invasion, and metastasis of melanoma cells. Furthermore, miRNA-based RNAi was also a powerful tool for gene function study.

Show MeSH
Related in: MedlinePlus