The Role And Mechanism Of MiR-133b In Gastric Cancer

MicroRNAs (miRNAs) are a class of small non-coding RNAs in thesize range of19to25nucleotides. By entirely or partially binding tocomplementary sequences in the3’-untranslated region (3’UTR) of targetmRNAs, the miRNAs negatively regulate genes by triggering translationrepression or degradation of target mRNAs. Through these mechanisms,miRNAs regulate the expression of up to30%of human genes and playcrucial roles in various biological process, including development,differentiation, cell apoptosis, proliferation and migration. An increasingnumber of studies make it clear that miRNAs are associated with variousdiseases，including cancers. According to recent findings, miRNAs arefrequently dysregulated in many types of solid tumor and blood tumor. Itis clear that miRNAs can act as potential oncogenes or tumor suppressors,and dysregulation of miRNAs play an essential role in tumorigenesis andcancer progression. Therefore, identification of cancer specific miRNAsand their targets is critical for understanding their roles in tumorigenesisand may be important for defining novel therapeutic targets.Current findings reveal that a number of miRNAs are associated withgastric cancer. Some reports suggested that miR-133b might bedown-regulated in gastric cancer. miR-133b has recently been reported tobe deregulated in many kinds of cancer, such as colorectal, head and neck, squamous cell, lung and bladder cancer. miR-133b is generallyconsidered as a tumor suppressor mirna, as it negatively regulatespro-tumor genes such as fascin homolog1(FSCN-1), c-Met andepidermal growth factor receptor (EGFR) in several cancers. However, itsprecise role and relevance in gastric cancer are still largely unknown.This study investigated the expression of miR-133b in human gastriccancer tissues through quantitative RT-PCR. miR-133b was transfectedinto gastric cancer cell lines MKN-45and SGC-7901to examine the rolesof miR-133b in gastric cancer. The target gene of miR-133b, FGFR1, waspredicted by bioinformatics analysis, and identified by dual-luciferasereporter assay and western blot. FGFR1expression was examined ingastric cancer tissues and cell lines using western blot andimmunohistochemistry. Overexpression and knockdown of FGFR1wasperformed to analyze the roles of FGFR1in cell proliferation.The experiments as follows:(1) Expression of miR-133b in human gastric cancer tissues and celllinesmiR-133b levels in12pairs of gastric cancer tissue samples wasexamined using quantitative RT-PCR. The results showed that miR-133bexpression was significantly down-regulated in11/12of the tested gastriccancer tissues compared to adjacent non-tumor tissues. We also found that miR-133b was down-regulated overall but had different expressionlevels in all the tested gastric cancer cell lines including MGC-803,SGC-7901, MKN-45, BGC-823, MKN-74and AGS, compared to humangastric epithelial cell line GES-1. These results indicate that miR-133b isdown-regulated in human gastric cancer.(2) Roles of miR-133b in regulation of gastric cancer cell growthTo evaluate the biological significance of miR-133b in gastric cancer,we overexpressed miR-133b in gastric cancer cell lines MKN-45andSGC-7901, and evaluated cell proliferation using MTT assay. The resultsshowed that miR-133b significantly inhibited proliferation of gastriccancer cells in a dose-dependent manner. We further analyzed the colonyformation ability of miR-133b-transfected gastric cancer cells, and foundthat transfection of miR-133b caused a significant decrease in the numberof formed colonies. Our data clearly indicated that overexpression ofmiR-133b reduced cell proliferation of gastric cancer cells, suggestingthat miR-133b functions as a tumor suppressor in gastric cancer.(3) Target identification of miR-133b in gastric cancer cellsTo understand the possible mechanism of miR-133b, we predicted thetarget genes of miR-133b through bioinformatics analysis. We found thatfibroblast growth factor receptor1(FGFR1) was a potential target gene ofmiR-133b. We validated that miR-133b binded3’UTR of FGFR1mRNAand caused inhibition of FGFR1expression using dual-luciferase reporter assay. Transfection of miR-133b in MKN-45cells caused a significantdecrease of endogenous FGFR1protein levels in a dose-dependentmanner, but did not affect mRNA levels of FGFR1. It indicated thatmiR-133b negatively regulated FGFR1expression through translationrepression. Moreover, we found that FGFR1expression was up-regulatedin the12of14gastric cancer tissues compared to the adjacent non-tumortissues, and FGFR1expression inversely correlated with miR-133bexpression in gastric cancer. Detection of miR-133b and FGFR1in thesame paired tissues using in situ hybridization and immunohistochemistryassay respectively also showed a reverse correlation of miR-133b andFGFR1. These results clearly indicated that miR-133b targeted FGFR1and negatively regulated its expression in gastric cancer.(4) Roles of FGFR1in gastric cancer cellsTo evaluate the relevance of FGFR1in gastric cancer, we repressed itsexpression, and examined proliferation of gastric cancer cell using MTTassay. Our results showed that siRNA targeting FGFR1efficientlysuppressed FGFR1expression and inhibited gastric cancer cellproliferation in a dose-dependent manner. In contrast, overexpression ofFGFR1in gastric epithelial cell line GES-1could promote cell growthnot only in normal culture condition but also in FGF-2treated condition.These results indicated that FGFR1promoted cell proliferation in gastriccancer and took important part in gastric cancer with miR-133b down-regulation.Conclusions:(1) miR-133b is down-regulated in human gastric cancer tissues andcell lines.(2) miR-133b acts as a tumor suppressor miRNA, and overexpressionof miR-133b inhibited proliferation of gastric cancer cells.(3) FGFR1is a direct target of miR-133b and was negatively regulatedby miR-133b through translation repression.(4) FGFR1protein expression is inversely correlated with miR-133bexpression in human gastric cancer tissues.(5) Knockdown of FGFR1in gastric cancer cells inhibits cell proliferation,while overexpression of FGFR1promotes proliferation of GES-1gastric epithelial cells.