Limits...
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.

Show MeSH

Related in: MedlinePlus

HPSE miRNAs inhibited expression of IL8 and CXCL1 and its probable mechanism.(A) Differentially expressed chemokine genes in the HPSE-miRNA1 and HPSE-miRNA2 groups compared to the Neg-miRNA group (log2 ratio≥1 or log2 ratio≤−1 and P<0.05). (B) Pathways including chemokines activity modulated by HPSE-miRNA1 or HPSE-miRNA2 were confirmed to be significant by gene-set enrichment analysis (P<0.05). (C) Both the mRNA and protein levels of IL8 and CXCL1 in HPSE miRNA transfected A375 cells were decreased compared to either control group. (†P<0.001, compared with the parental cells; *P<0.001, compared with the Neg-miRNA transfected cells). (D) Attenuation of the HPSE-induced phosphorylation of MAPKs by HPSE miRNA. Phosphorylation of MAPK p38 (second and third panel), JNK/SAPK (fourth and fifth panel), and ERK1/2 (sixth and seventh panel) was monitored by western blotting.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3376136&req=5

pone-0038659-g003: HPSE miRNAs inhibited expression of IL8 and CXCL1 and its probable mechanism.(A) Differentially expressed chemokine genes in the HPSE-miRNA1 and HPSE-miRNA2 groups compared to the Neg-miRNA group (log2 ratio≥1 or log2 ratio≤−1 and P<0.05). (B) Pathways including chemokines activity modulated by HPSE-miRNA1 or HPSE-miRNA2 were confirmed to be significant by gene-set enrichment analysis (P<0.05). (C) Both the mRNA and protein levels of IL8 and CXCL1 in HPSE miRNA transfected A375 cells were decreased compared to either control group. (†P<0.001, compared with the parental cells; *P<0.001, compared with the Neg-miRNA transfected cells). (D) Attenuation of the HPSE-induced phosphorylation of MAPKs by HPSE miRNA. Phosphorylation of MAPK p38 (second and third panel), JNK/SAPK (fourth and fifth panel), and ERK1/2 (sixth and seventh panel) was monitored by western blotting.

Mentions: Adhesion to Matrigel was evaluated by MTT assay at the indicated time point. The A570 value in both control groups (0.916±0.087 and 0.916±0.142) differed significantly compared to HPSE-miRNA1 (0.398±0.022) and HPSE-miRNA2 transfected A375 cells (0.416±0.068) (P<0.001, Figure 2E). Furthermore, the number of A375 cells transfected with HPSE-miRNA1 or HPSE-miRNA2 that migrated to the lower surface of the transwell chambers and invaded through Matrigel at 24 hours was significantly lower than that of the A375 cells in the control groups (P<0.001, Figure 3F, G and H). In addition, HPSE-miRNA1 could also attenuate the adhesion, migration, and invasion ability of HeLa cells (Figure S3C, D, E and F).


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)

HPSE miRNAs inhibited expression of IL8 and CXCL1 and its probable mechanism.(A) Differentially expressed chemokine genes in the HPSE-miRNA1 and HPSE-miRNA2 groups compared to the Neg-miRNA group (log2 ratio≥1 or log2 ratio≤−1 and P<0.05). (B) Pathways including chemokines activity modulated by HPSE-miRNA1 or HPSE-miRNA2 were confirmed to be significant by gene-set enrichment analysis (P<0.05). (C) Both the mRNA and protein levels of IL8 and CXCL1 in HPSE miRNA transfected A375 cells were decreased compared to either control group. (†P<0.001, compared with the parental cells; *P<0.001, compared with the Neg-miRNA transfected cells). (D) Attenuation of the HPSE-induced phosphorylation of MAPKs by HPSE miRNA. Phosphorylation of MAPK p38 (second and third panel), JNK/SAPK (fourth and fifth panel), and ERK1/2 (sixth and seventh panel) was monitored by western blotting.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3376136&req=5

pone-0038659-g003: HPSE miRNAs inhibited expression of IL8 and CXCL1 and its probable mechanism.(A) Differentially expressed chemokine genes in the HPSE-miRNA1 and HPSE-miRNA2 groups compared to the Neg-miRNA group (log2 ratio≥1 or log2 ratio≤−1 and P<0.05). (B) Pathways including chemokines activity modulated by HPSE-miRNA1 or HPSE-miRNA2 were confirmed to be significant by gene-set enrichment analysis (P<0.05). (C) Both the mRNA and protein levels of IL8 and CXCL1 in HPSE miRNA transfected A375 cells were decreased compared to either control group. (†P<0.001, compared with the parental cells; *P<0.001, compared with the Neg-miRNA transfected cells). (D) Attenuation of the HPSE-induced phosphorylation of MAPKs by HPSE miRNA. Phosphorylation of MAPK p38 (second and third panel), JNK/SAPK (fourth and fifth panel), and ERK1/2 (sixth and seventh panel) was monitored by western blotting.
Mentions: Adhesion to Matrigel was evaluated by MTT assay at the indicated time point. The A570 value in both control groups (0.916±0.087 and 0.916±0.142) differed significantly compared to HPSE-miRNA1 (0.398±0.022) and HPSE-miRNA2 transfected A375 cells (0.416±0.068) (P<0.001, Figure 2E). Furthermore, the number of A375 cells transfected with HPSE-miRNA1 or HPSE-miRNA2 that migrated to the lower surface of the transwell chambers and invaded through Matrigel at 24 hours was significantly lower than that of the A375 cells in the control groups (P<0.001, Figure 3F, G and H). In addition, HPSE-miRNA1 could also attenuate the adhesion, migration, and invasion ability of HeLa cells (Figure S3C, D, E and F).

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