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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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Restoration of HPSE functionality by HPSE RNAi rescue.(A) The sequencing results of the wild type and multisite mutant HPSE cDNA. Three nucleotide substitutions were introduced into the HPSE-miRNA2 hybridizing sequence (5′-CCTTTGCAGCTGGCTTTATGT-3′), which was verified by sequencing. (B-D) The levels of both HPSE mRNA and protein were restored in cells of the HPSE-miRNA2 group transfected with mutant HPSE cDNA, similar to those of Neg-miRNA cells transfected with the wild type or mutant HPSE cDNA. Furthermore, mutant cDNA transfection for miRNA rescue also increased IL8 and CXCL1 mRNA and protein levels. However, in HPSE-miRNA1 stably transfected cells, neither the wild type HPSE cDNA nor the mutant HPSE cDNA could restore the expressions of HPSE, IL8 and CXCL1. (*P<0.05 compared with the respective non-cotransfected cells). (E) Restoration of the HPSE-induced phosphorylation of MAPKs in the cells of the HPSE-miRNA2 group by mutant HPSE cDNA. Phosphorylation of MAPK p38 (first and second panel), JNK/SAPK (third and fourth panel), and ERK1/2 (fifth and sixth panel) was monitored by western blotting.
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pone-0038659-g005: Restoration of HPSE functionality by HPSE RNAi rescue.(A) The sequencing results of the wild type and multisite mutant HPSE cDNA. Three nucleotide substitutions were introduced into the HPSE-miRNA2 hybridizing sequence (5′-CCTTTGCAGCTGGCTTTATGT-3′), which was verified by sequencing. (B-D) The levels of both HPSE mRNA and protein were restored in cells of the HPSE-miRNA2 group transfected with mutant HPSE cDNA, similar to those of Neg-miRNA cells transfected with the wild type or mutant HPSE cDNA. Furthermore, mutant cDNA transfection for miRNA rescue also increased IL8 and CXCL1 mRNA and protein levels. However, in HPSE-miRNA1 stably transfected cells, neither the wild type HPSE cDNA nor the mutant HPSE cDNA could restore the expressions of HPSE, IL8 and CXCL1. (*P<0.05 compared with the respective non-cotransfected cells). (E) Restoration of the HPSE-induced phosphorylation of MAPKs in the cells of the HPSE-miRNA2 group by mutant HPSE cDNA. Phosphorylation of MAPK p38 (first and second panel), JNK/SAPK (third and fourth panel), and ERK1/2 (fifth and sixth panel) was monitored by western blotting.

Mentions: RNA interference rescue was used [22] to normalize artificial miRNA-induced depletion of HPSE. We used multisite-directed mutagenesis and introduced three nucleotide substitutions within the HPSE-miRNA2 hybridizing sequence, while retaining the amino acid identity of the wild-type protein (Figure 5A). As shown in Figure 5B and C, introduction of the mutated HPSE cDNA in HPSE-miRNA2 stably transfected cells not only rescued the miRNA-induced HPSE inhibition but also increased HPSE mRNA levels (P<0.0001), similar to those of Neg-miRNA cells transfected with wild type or mutant HPSE cDNA (P>0.05). Furthermore, the transfection of cDNA-mut for miRNA rescue also increased IL8 and CXCL1 mRNA levels (P<0.01), indicating HPSE could regulate IL8 and CXCL1 at the level of transcription, or prior to translation. With respect to a regulatory mechanism, HPSE-induced phosphorylation of MAPKs was also restored in cells of the HPSE-miRNA2 group transfected with mutant HPSE cDNA, but not with the wild type HPSE cDNA (Figure 5E). However, in the HPSE-miRNA1 stably transfected cells, neither the original HPSE cDNA nor the mutant HPSE cDNA could restore the expression of HPSE, and subsequently, to rescue the levels IL8 or CXCL1, and phosphorylation of MAPKs (Figure 5B–E).


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)

Restoration of HPSE functionality by HPSE RNAi rescue.(A) The sequencing results of the wild type and multisite mutant HPSE cDNA. Three nucleotide substitutions were introduced into the HPSE-miRNA2 hybridizing sequence (5′-CCTTTGCAGCTGGCTTTATGT-3′), which was verified by sequencing. (B-D) The levels of both HPSE mRNA and protein were restored in cells of the HPSE-miRNA2 group transfected with mutant HPSE cDNA, similar to those of Neg-miRNA cells transfected with the wild type or mutant HPSE cDNA. Furthermore, mutant cDNA transfection for miRNA rescue also increased IL8 and CXCL1 mRNA and protein levels. However, in HPSE-miRNA1 stably transfected cells, neither the wild type HPSE cDNA nor the mutant HPSE cDNA could restore the expressions of HPSE, IL8 and CXCL1. (*P<0.05 compared with the respective non-cotransfected cells). (E) Restoration of the HPSE-induced phosphorylation of MAPKs in the cells of the HPSE-miRNA2 group by mutant HPSE cDNA. Phosphorylation of MAPK p38 (first and second panel), JNK/SAPK (third and fourth panel), and ERK1/2 (fifth and sixth panel) was monitored by western blotting.
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pone-0038659-g005: Restoration of HPSE functionality by HPSE RNAi rescue.(A) The sequencing results of the wild type and multisite mutant HPSE cDNA. Three nucleotide substitutions were introduced into the HPSE-miRNA2 hybridizing sequence (5′-CCTTTGCAGCTGGCTTTATGT-3′), which was verified by sequencing. (B-D) The levels of both HPSE mRNA and protein were restored in cells of the HPSE-miRNA2 group transfected with mutant HPSE cDNA, similar to those of Neg-miRNA cells transfected with the wild type or mutant HPSE cDNA. Furthermore, mutant cDNA transfection for miRNA rescue also increased IL8 and CXCL1 mRNA and protein levels. However, in HPSE-miRNA1 stably transfected cells, neither the wild type HPSE cDNA nor the mutant HPSE cDNA could restore the expressions of HPSE, IL8 and CXCL1. (*P<0.05 compared with the respective non-cotransfected cells). (E) Restoration of the HPSE-induced phosphorylation of MAPKs in the cells of the HPSE-miRNA2 group by mutant HPSE cDNA. Phosphorylation of MAPK p38 (first and second panel), JNK/SAPK (third and fourth panel), and ERK1/2 (fifth and sixth panel) was monitored by western blotting.
Mentions: RNA interference rescue was used [22] to normalize artificial miRNA-induced depletion of HPSE. We used multisite-directed mutagenesis and introduced three nucleotide substitutions within the HPSE-miRNA2 hybridizing sequence, while retaining the amino acid identity of the wild-type protein (Figure 5A). As shown in Figure 5B and C, introduction of the mutated HPSE cDNA in HPSE-miRNA2 stably transfected cells not only rescued the miRNA-induced HPSE inhibition but also increased HPSE mRNA levels (P<0.0001), similar to those of Neg-miRNA cells transfected with wild type or mutant HPSE cDNA (P>0.05). Furthermore, the transfection of cDNA-mut for miRNA rescue also increased IL8 and CXCL1 mRNA levels (P<0.01), indicating HPSE could regulate IL8 and CXCL1 at the level of transcription, or prior to translation. With respect to a regulatory mechanism, HPSE-induced phosphorylation of MAPKs was also restored in cells of the HPSE-miRNA2 group transfected with mutant HPSE cDNA, but not with the wild type HPSE cDNA (Figure 5E). However, in the HPSE-miRNA1 stably transfected cells, neither the original HPSE cDNA nor the mutant HPSE cDNA could restore the expression of HPSE, and subsequently, to rescue the levels IL8 or CXCL1, and phosphorylation of MAPKs (Figure 5B–E).

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