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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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The effect of HPSE miRNA on the in vivo lung metastasis of A375 cells.Cells (2×106) from the parental cells, Neg-miRNA or HPSE-miRNA2 transfected cells were injected into the tail veins of nude mice. (A-B) Xenograft mice were weight once a week for 6 weeks, and photos were taken at the end of the experiment on day 42, as shown in A. A weight increase was noted in the HPSE-miRNA2 group, compared to the control groups. (C) Representative lung tissue sections from each group (H&E staining, magnification of ×40, ×100 and ×400, respectively). (D) The number of lung metastases in HPSE-miRNA2 mice was decreased compared to that of mock control and Neg-miRNA control groups. (†P<0.05, compared with the parental cells; *P<0.05, compared with the Neg-miRNA transfected cells).
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pone-0038659-g004: The effect of HPSE miRNA on the in vivo lung metastasis of A375 cells.Cells (2×106) from the parental cells, Neg-miRNA or HPSE-miRNA2 transfected cells were injected into the tail veins of nude mice. (A-B) Xenograft mice were weight once a week for 6 weeks, and photos were taken at the end of the experiment on day 42, as shown in A. A weight increase was noted in the HPSE-miRNA2 group, compared to the control groups. (C) Representative lung tissue sections from each group (H&E staining, magnification of ×40, ×100 and ×400, respectively). (D) The number of lung metastases in HPSE-miRNA2 mice was decreased compared to that of mock control and Neg-miRNA control groups. (†P<0.05, compared with the parental cells; *P<0.05, compared with the Neg-miRNA transfected cells).

Mentions: At the start of the in vivo experiments, the weights of mice were not different amongst the groups (P>0.05). One days 21, 28, 35 and 42 after the inoculation of tumor cells, the weights of the mice in the HPSE-miRNA2 group were higher than both control groups (P<0.05, Figure 4A and B). Unexpectedly, in our experiments, there was no metastasis in the liver, another vulnerable metastatic site of melanoma (data not shown). At the end of the six weeks, the number of lung metastatic lesions in the HPSE-miRNA2 group (2.333±1.155) was much less than that in the control groups (10.667±2.216 and 11.000±4.000) (P<0.05, Figure 4C and D). Furthermore, the lung metastases in HPSE-miRNA2 group were grade I (≤20 cells) or grade II (20–50 cells), while those in the negative control group or mock group were grade III (50–100 cells) or grade IV (>100 cells) (Figure 4C).


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)

The effect of HPSE miRNA on the in vivo lung metastasis of A375 cells.Cells (2×106) from the parental cells, Neg-miRNA or HPSE-miRNA2 transfected cells were injected into the tail veins of nude mice. (A-B) Xenograft mice were weight once a week for 6 weeks, and photos were taken at the end of the experiment on day 42, as shown in A. A weight increase was noted in the HPSE-miRNA2 group, compared to the control groups. (C) Representative lung tissue sections from each group (H&E staining, magnification of ×40, ×100 and ×400, respectively). (D) The number of lung metastases in HPSE-miRNA2 mice was decreased compared to that of mock control and Neg-miRNA control groups. (†P<0.05, compared with the parental cells; *P<0.05, compared with the Neg-miRNA transfected cells).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038659-g004: The effect of HPSE miRNA on the in vivo lung metastasis of A375 cells.Cells (2×106) from the parental cells, Neg-miRNA or HPSE-miRNA2 transfected cells were injected into the tail veins of nude mice. (A-B) Xenograft mice were weight once a week for 6 weeks, and photos were taken at the end of the experiment on day 42, as shown in A. A weight increase was noted in the HPSE-miRNA2 group, compared to the control groups. (C) Representative lung tissue sections from each group (H&E staining, magnification of ×40, ×100 and ×400, respectively). (D) The number of lung metastases in HPSE-miRNA2 mice was decreased compared to that of mock control and Neg-miRNA control groups. (†P<0.05, compared with the parental cells; *P<0.05, compared with the Neg-miRNA transfected cells).
Mentions: At the start of the in vivo experiments, the weights of mice were not different amongst the groups (P>0.05). One days 21, 28, 35 and 42 after the inoculation of tumor cells, the weights of the mice in the HPSE-miRNA2 group were higher than both control groups (P<0.05, Figure 4A and B). Unexpectedly, in our experiments, there was no metastasis in the liver, another vulnerable metastatic site of melanoma (data not shown). At the end of the six weeks, the number of lung metastatic lesions in the HPSE-miRNA2 group (2.333±1.155) was much less than that in the control groups (10.667±2.216 and 11.000±4.000) (P<0.05, Figure 4C and D). Furthermore, the lung metastases in HPSE-miRNA2 group were grade I (≤20 cells) or grade II (20–50 cells), while those in the negative control group or mock group were grade III (50–100 cells) or grade IV (>100 cells) (Figure 4C).

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