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MicroRNA-29b attenuates non-small cell lung cancer metastasis by targeting matrix metalloproteinase 2 and PTEN.

Wang H, Guan X, Tu Y, Zheng S, Long J, Li S, Qi C, Xie X, Zhang H, Zhang Y - J. Exp. Clin. Cancer Res. (2015)

Bottom Line: Our pilot study using miRNA PCR array found that miRNA-29b (miR-29b) is differentially expressed in primary cultured CD133-positive A549 cells compared with CD133-negative A549 cells.Clinicopathological analysis demonstrated that miR-29b had significant negative correlation with lymphatic metastasis.Western blotting and quantitative RT-PCR indicated that miR-29b down-regulated the expression of MMP2 at the protein and mRNA levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, School of Basic Medical Science, Guangzhou Medical University, 195# Dongfeng West Road, Guangzhou, Guangdong, 510182, People's Republic of China. wanghongyan75@126.com.

ABSTRACT

Background: Our pilot study using miRNA PCR array found that miRNA-29b (miR-29b) is differentially expressed in primary cultured CD133-positive A549 cells compared with CD133-negative A549 cells.

Methods: Ten human non-small cell lung cancer (NSCLC) cell lines and samples from thirty patients with NSCLC were analyzed for the expression of miR-29b by quantitative RT-PCR. Bioinformatics analysis combined with tumor metastasis PCR array showed the potential target genes for miR-29b. miR-29b lentivirus and inhibitors were transfected into NSCLC cells to investigate its role on regulating cell proliferation which was measured by CCK-8 assay in vitro and nude mice xenograft tumor assay in vivo. Cell motility ability was evaluated by transwell assay. The target genes of miR-29b were determined by luciferase assay, quantitative RT-PCR and western blot.

Results: Bioinformatics analysis combined with tumor metastasis PCR array showed that matrix metalloproteinase 2 (MMP2) and PTEN could be important target genes of miR-29b. The expression of miR-29b was down regulated in NSCLC tissues compared to the normal tissues. Clinicopathological analysis demonstrated that miR-29b had significant negative correlation with lymphatic metastasis. The gain-of-function studies revealed that ectopic expression of miR-29b decreased cell proliferation, migration and invasion abilities of NSCLC cells. In contrasts, loss-of-function studies showed that inhibition of miR-29b promoted cell proliferation, migration and invasion of NSCLC cells in vitro. Nude mice xenograft tumor assay confirmed that miR-29b inhibited lung cancer growth in vivo. High-invasion (A549-H) and low-invasion (A549-L) NSCLC cell sublines from A549 cells were created by using the repeated transwell assay aimed to confirm the effect of miR-29b on migration and invasion of NSCLC. Furthermore, the dual-luciferase reporter assay demonstrated that miR-29b inhibited the expression of the luciferase gene containing the 3'-UTRs of MMP2 and PTEN mRNA. Western blotting and quantitative RT-PCR indicated that miR-29b down-regulated the expression of MMP2 at the protein and mRNA levels.

Conclusion: Taken together, our results demonstrate that miR-29b serves as a tumor metastasis suppressor, which suppresses NSCLC cell metastasis by directly inhibiting MMP2 expression. The results show that miR-29b may be a novel therapeutic candidate target to slow NSCLC metastasis.

No MeSH data available.


Related in: MedlinePlus

Expression of miR-29b in NSCLC cell lines and paired NSCLC tissues. a, Quantitative RT-PCR analysis of miR-29b expression levels in NSCLC cell lines and immortalized human bronchial epithelial cell line were shown relative to U6 snRNA as an internal control. b, Quantitative RT-PCR analysis of miR-29b levels in 20 pairs of paraffin-embedded NSCLC tissues. c, Quantitative RT-PCR analysis of miR-29b levels in 10 pairs of fresh NSCLC tissues
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Fig2: Expression of miR-29b in NSCLC cell lines and paired NSCLC tissues. a, Quantitative RT-PCR analysis of miR-29b expression levels in NSCLC cell lines and immortalized human bronchial epithelial cell line were shown relative to U6 snRNA as an internal control. b, Quantitative RT-PCR analysis of miR-29b levels in 20 pairs of paraffin-embedded NSCLC tissues. c, Quantitative RT-PCR analysis of miR-29b levels in 10 pairs of fresh NSCLC tissues

Mentions: Quantitative RT-PCR results revealed that the expression levels of miR-29b were significantly higher in the H460 and 95C cell lines compared to 16HBE cell line, while the expression levels were lower in the PGCL3, PAa, H520, A549, H1299 and 95D cell lines (Fig. 2a). Twenty pairs of paraffin-embedded NSCLC tissues and normal tissues (Fig. 2b) and ten pairs of fresh NSCLC tissues and normal adjacent tissues (Fig. 2c) were also chosen to detect the expression levels of miR-29b, the results showed that the expression level of miR-29b in twenty cases of paraffin NSCLC tissues was (−1.893 ± 1.367), significantly lower than that in the adjacent lung tissue (−0.605 ± 0.639; P = 0.001, t = −3.817). The expression level of miR-29b in ten cases of fresh non-small cell lung cancer tissues was (−1.996 ± 0.460), significantly lower than that in the adjacent lung tissue (−0.463 ± 0.257; P < 0.001, t = −9.016).Fig. 2


MicroRNA-29b attenuates non-small cell lung cancer metastasis by targeting matrix metalloproteinase 2 and PTEN.

Wang H, Guan X, Tu Y, Zheng S, Long J, Li S, Qi C, Xie X, Zhang H, Zhang Y - J. Exp. Clin. Cancer Res. (2015)

Expression of miR-29b in NSCLC cell lines and paired NSCLC tissues. a, Quantitative RT-PCR analysis of miR-29b expression levels in NSCLC cell lines and immortalized human bronchial epithelial cell line were shown relative to U6 snRNA as an internal control. b, Quantitative RT-PCR analysis of miR-29b levels in 20 pairs of paraffin-embedded NSCLC tissues. c, Quantitative RT-PCR analysis of miR-29b levels in 10 pairs of fresh NSCLC tissues
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4469413&req=5

Fig2: Expression of miR-29b in NSCLC cell lines and paired NSCLC tissues. a, Quantitative RT-PCR analysis of miR-29b expression levels in NSCLC cell lines and immortalized human bronchial epithelial cell line were shown relative to U6 snRNA as an internal control. b, Quantitative RT-PCR analysis of miR-29b levels in 20 pairs of paraffin-embedded NSCLC tissues. c, Quantitative RT-PCR analysis of miR-29b levels in 10 pairs of fresh NSCLC tissues
Mentions: Quantitative RT-PCR results revealed that the expression levels of miR-29b were significantly higher in the H460 and 95C cell lines compared to 16HBE cell line, while the expression levels were lower in the PGCL3, PAa, H520, A549, H1299 and 95D cell lines (Fig. 2a). Twenty pairs of paraffin-embedded NSCLC tissues and normal tissues (Fig. 2b) and ten pairs of fresh NSCLC tissues and normal adjacent tissues (Fig. 2c) were also chosen to detect the expression levels of miR-29b, the results showed that the expression level of miR-29b in twenty cases of paraffin NSCLC tissues was (−1.893 ± 1.367), significantly lower than that in the adjacent lung tissue (−0.605 ± 0.639; P = 0.001, t = −3.817). The expression level of miR-29b in ten cases of fresh non-small cell lung cancer tissues was (−1.996 ± 0.460), significantly lower than that in the adjacent lung tissue (−0.463 ± 0.257; P < 0.001, t = −9.016).Fig. 2

Bottom Line: Our pilot study using miRNA PCR array found that miRNA-29b (miR-29b) is differentially expressed in primary cultured CD133-positive A549 cells compared with CD133-negative A549 cells.Clinicopathological analysis demonstrated that miR-29b had significant negative correlation with lymphatic metastasis.Western blotting and quantitative RT-PCR indicated that miR-29b down-regulated the expression of MMP2 at the protein and mRNA levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, School of Basic Medical Science, Guangzhou Medical University, 195# Dongfeng West Road, Guangzhou, Guangdong, 510182, People's Republic of China. wanghongyan75@126.com.

ABSTRACT

Background: Our pilot study using miRNA PCR array found that miRNA-29b (miR-29b) is differentially expressed in primary cultured CD133-positive A549 cells compared with CD133-negative A549 cells.

Methods: Ten human non-small cell lung cancer (NSCLC) cell lines and samples from thirty patients with NSCLC were analyzed for the expression of miR-29b by quantitative RT-PCR. Bioinformatics analysis combined with tumor metastasis PCR array showed the potential target genes for miR-29b. miR-29b lentivirus and inhibitors were transfected into NSCLC cells to investigate its role on regulating cell proliferation which was measured by CCK-8 assay in vitro and nude mice xenograft tumor assay in vivo. Cell motility ability was evaluated by transwell assay. The target genes of miR-29b were determined by luciferase assay, quantitative RT-PCR and western blot.

Results: Bioinformatics analysis combined with tumor metastasis PCR array showed that matrix metalloproteinase 2 (MMP2) and PTEN could be important target genes of miR-29b. The expression of miR-29b was down regulated in NSCLC tissues compared to the normal tissues. Clinicopathological analysis demonstrated that miR-29b had significant negative correlation with lymphatic metastasis. The gain-of-function studies revealed that ectopic expression of miR-29b decreased cell proliferation, migration and invasion abilities of NSCLC cells. In contrasts, loss-of-function studies showed that inhibition of miR-29b promoted cell proliferation, migration and invasion of NSCLC cells in vitro. Nude mice xenograft tumor assay confirmed that miR-29b inhibited lung cancer growth in vivo. High-invasion (A549-H) and low-invasion (A549-L) NSCLC cell sublines from A549 cells were created by using the repeated transwell assay aimed to confirm the effect of miR-29b on migration and invasion of NSCLC. Furthermore, the dual-luciferase reporter assay demonstrated that miR-29b inhibited the expression of the luciferase gene containing the 3'-UTRs of MMP2 and PTEN mRNA. Western blotting and quantitative RT-PCR indicated that miR-29b down-regulated the expression of MMP2 at the protein and mRNA levels.

Conclusion: Taken together, our results demonstrate that miR-29b serves as a tumor metastasis suppressor, which suppresses NSCLC cell metastasis by directly inhibiting MMP2 expression. The results show that miR-29b may be a novel therapeutic candidate target to slow NSCLC metastasis.

No MeSH data available.


Related in: MedlinePlus