title: MicroRNA-29b attenuates non-small cell lung cancer metastasis by targeting matrix metalloproteinase 2 and PTEN.
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.
  
  
  CONCLUSIONS
  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.
authors.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.
authors.forename: Hongyan
authors.initials: H
authors.lastname: Wang
authors.affiliation: Department of Pathology, School of Basic Medical Science, Guangzhou Medical University, 195# Dongfeng West Road, Guangzhou, Guangdong, 510182, People's Republic of China. gxy2012@163.com.
authors.forename: Xiaoying
authors.initials: X
authors.lastname: Guan
authors.affiliation: Department of Physiology, School of Basic Medical Sciences, Guangzhou Medical University, 195# Dongfeng West Road, Guangzhou, Guangdong, 510182, People's Republic of China. tuysh1976@163.com.
authors.forename: Yongsheng
authors.initials: Y
authors.lastname: Tu
authors.affiliation: Department of Pathology, School of Basic Medical Science, Guangzhou Medical University, 195# Dongfeng West Road, Guangzhou, Guangdong, 510182, People's Republic of China. 1258807@qq.com.
authors.forename: Shaoqiu
authors.initials: S
authors.lastname: Zheng
authors.affiliation: Department of Pathology, School of Basic Medical Science, Guangzhou Medical University, 195# Dongfeng West Road, Guangzhou, Guangdong, 510182, People's Republic of China. 7737499@qq.com.
authors.forename: Jie
authors.initials: J
authors.lastname: Long
authors.affiliation: Department of Pathology, School of Basic Medical Science, Guangzhou Medical University, 195# Dongfeng West Road, Guangzhou, Guangdong, 510182, People's Republic of China. 1760855032@qq.com.
authors.forename: Shuhua
authors.initials: S
authors.lastname: Li
authors.affiliation: Department of Pathology, School of Basic Medical Science, Guangzhou Medical University, 195# Dongfeng West Road, Guangzhou, Guangdong, 510182, People's Republic of China. GXYAZ@yeah.net.
authors.forename: Cuiling
authors.initials: C
authors.lastname: Qi
authors.affiliation: Department of Pathology, School of Basic Medical Science, Guangzhou Medical University, 195# Dongfeng West Road, Guangzhou, Guangdong, 510182, People's Republic of China. Xiaobin_xie97@126.com.
authors.forename: Xiaobin
authors.initials: X
authors.lastname: Xie
authors.affiliation: Department of Pathology, School of Basic Medical Science, Guangzhou Medical University, 195# Dongfeng West Road, Guangzhou, Guangdong, 510182, People's Republic of China. 7102981@qq.com.
authors.forename: Huiqiu
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Department of Pathology, School of Basic Medical Science, Guangzhou Medical University, 195# Dongfeng West Road, Guangzhou, Guangdong, 510182, People's Republic of China. yajie.zhang@163.com.
authors.forename: Yajie
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000818:Animals; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D002465:Cell Movement / Q000502:physiology; D064593:Heterografts; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D008297:Male; D020778:Matrix Metalloproteinase 2 / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D035683:MicroRNAs / Q000096:biosynthesis* / Q000235:genetics; D008875:Middle Aged; D009362:Neoplasm Metastasis; D051059:PTEN Phosphohydrolase / Q000235:genetics* / Q000378:metabolism; D010865:Pilot Projects; D014162:Transfection
chemical_list: C532045:MIRN29a microRNA, human; D035683:MicroRNAs; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human; C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2