Low-level Expression Of Let-7α In Gastric Cancer And Its Involvement In Tumorigenesis By Targeting RAB40C

BackgroundGastric cancer is the fourth most common cancer and the second leading cause of cancer mortality worldwide. It remains an important public health burden worldwide, especially in developing countries. In China, gastric cancer has the highest mortality among all cancers and the overall mortality rate has increased steadily in the past 20 years. However, the molecular mechanisms involved in gastric cancer are diverse, complex and not fully understood.New opportunities in the study of cancer molecular mechanisms have been provided by the discovery of microRNAs (miRNAs), a class of short, non-coding endogenous RNAs that function as negative regulators of gene expression. Recently acquired evidence demonstrates that miRNAs can be regulators in carcinogenesis. It has been showed that more than 50% of the known mature human miRNA genes are located in cancer-associated genomic regions or in fragile sites, suggesting that miRNAs might have an important role in the pathogenesis of human cancers. Moreover, different cancer types have distinct miRNA expression profiles, and an increasing number of miRNAs have been suggested to have important roles in tumor progression or in tumor suppression.Among all human cancer-related miRNAs, the let-7 family has attracted the most interest because its family members have been noted to express aberrantly in human cancers. The family was discovered initially in Caenorhabditis elegans and is currently one of the most important members of the miRNA family. The let-7 family consists of 11 very closely related genes and many human let-7 genes map to regions that are altered or deleted in human tumors, indicating that these genes might function as tumor suppressors. However, the data for the relationship between gastric carcinogenesis and the expression of let-7a miRNA are very limited.Evidence collected to date shows let-7a was linked to the modulation of different target genes, the most well-known being the RAS family. The RAS proteins function as the critical molecular switch for various signaling pathways controlling the diverse biological processes. RAB40C is a member of the RAS family, with the help of a bioinformatic analysis, RAB40C has been known to contain the let-7a binding site and be evolutionarily conserved. To our knowledge, there is no report of work investigating the role of let-7a or a possible correlation between RAB40C and let-7a miRNA in gastric cancers.The objective of this study was to explore the role of let-7a miRNA in gastric tumorigenesis and the possible correlation between RAB40C and let-7a miRNA in gastric cancer.Materials and MethodsTotal RNA from tissue samples and cell lines was obtained with the TRIzol(?) isolation reagent. The quantitative real-time (qRT)-PCR reaction for detecting the expression of let-7a in 27 pairs of gastric cancer tissues and matched normal gastric tissues and gastric cancer cell linesusing the TaqMan MicroRNA assay together with the TaqMan Universal PCR Master Mix were done with an Applied Biosystems 7500 real-time PCR system. The SYBR Premix Ex TaqTM Kit was used for detecting the expression of RAB40C mRNA. The relative quantification of let-7a and RAB40C mRNA was calculated using the 2-ΔΔCt method normalized with respect to RNU6B as the internal control, and relative to a calibrator sample as the external control.The cell proliferation assay was done with a cell counting kit (CCK-8) at 24 h after transfection. Cells were harvested at 24 h after transfection and fixed in 70% ice-cold ethanol, treated with RNAse A, stained with 50 mg/ml propidium iodide and 0.1 mg/ml RNase A for DNA content analysis by flow cytometry with a FACS Calibur system. The percentage of cell population in each phase was calculated with FlowJo FACS analysis software. We examined the malignant transformation by soft agar assay and by xenograft study. Ki-67 protein expression in the cancer tissues of nude mice was detected using the streptavidin-peroxidase complex method with a Histostain-plus kit.A bioinformatic analysis identified RAB40C as a hypothetical target gene of let-7a, as identified by TargetScan alghorithms (http://www.targetscan.org/). The protein and mRNA levels of RAB40C were determined by western blotting and by quantitative PCR. To confirm the direct interaction between let-7a and its binding site within RAB40C mRNA, a human RAB40C 3'-UTR fragment containing a wild type or mutant let-7a-binding sequence was cloned downstream of the luciferase reporter gene. To confirm that the let-7a effect on cell proliferation and anchorage-independent growth is associated with its modulation of RAB40C, GES-1 cells were transfected with siRNA targeting RAB40C or control siRNA.All statistical analysis was done with SPSS13.0 software. Differences between groups were analyzed by Student's t-test or the non-parametric Mann-Whitney U test for comparison of two groups and one-way ANOVA or the non-parametric Kruskal-Wallis H test for multiple comparisons. Correlations between groups were evaluated with Pearson's correlation test. All experiments were done at least in triplicate and the level of statistical significance was set at P<0.05 for all tests.ResultsWe found that expression of let-7a is reduced in human gastric cancer tissues and cell lines and there was a significant correlation between the level of let-7a expression and the stage of differentiation. AGS and BGC-823 cells transfected with the let-7a mimic showed a significant reduction in the number of metabolically active cells compared to those transfected with mimic non-specific control miRNAs (mimic NC) or the untransfected control. An increase of 58.8% in cell growth was observed in GES-1 cells at 48 h after transfection with a let-7a inhibitor compared with inhibitor non-specific control miRNA (inhibitor NC). DNA content analysis by flow cytometry revealed let-7a restoration induced an accumulation of AGS cells in the percentage of cells at the G1 phase and a reduction of cells in S phase and this effect on cell cycle was observed also for BGC-823 cells. In contrast, the percentage of GES-1 cells transfected with let-7a inhibitor in the G1 phase was decreased, whereas the percentage of cells in the S phase was increased. Over-expression of let-7a significantly suppressed anchorage-dependent growth in vitro and the tumorigenicity of gastric cancer cells in a nude mouse xenograft model. Additionally, the expression of the proliferation marker Ki-67 was significantly lower in tumor xenografts of the let-7a mimic group compared to the mimic NC and untransfected groups.Nextly, we found over-expression of let-7a in AGS and BGC-823 cells effectively decreased the level of the RAB40C protein. A blocking strategy was further adapted by introducing the let-7a inhibitor into GES-1 cells, which increased the level of the RAB40C protein. Neither the let-7a mimic nor the inhibitor affected the mRNA of RAB40C, suggesting post-transcriptional regulation of RAB40C by let-7a in gastric cancer cell lines. A further hint about the potential role of let-7a in the regulation of RAB40C expression came from the analysis of five different gastric cancer cell lines that showed a significant inverse correlation between let-7a levels and the RAB40C protein. To assess the clinical relevance of these findings, we examined the correlation between the level of the RAB40C protein with let-7a expression in the 27 matched normal and cancer tissues. There was a significant inverse correlation between the level of the RAB40C protein and let-7a expression. The result of luciferase reporter assay showed a direct interaction between let-7a and RAB40C mRNA and indicated that let-7a might suppress gene expression through the let-7a binding sequence at the 3'-UTR of RAB40C.Transfection of AGS cells with siRNA for RAB40C effectively suppressed RAB40C mRNA expression and RAB40C protein. A similar effect was observed in BGC-823 cells. RAB40C depletion significantly alleviated the anti-proliferative effect of let-7a up-regulation in AGS cells and BGC-823 cells as determined by the CCK-8 assay. Furthermore, RAB40C siRNA attenuated the plating efficiency of cells in soft agar, which was decreased upon the transfection of let-7a mimic in AGS cells and BGC-823 cells. These data provided further evidence that RAB40C was targeted by let-7a and therefore suggested that RAB40C mediates the let-7a effect on cell proliferation and anchorage-independent growth in gastric cancer cells.ConclusionsThis study revealed that let-7a is significant in suppressing gastric cancer growth in vivo and in vitro. Furthermore, we demonstrated that RAB40C is regulated directly by let-7a and plays an essential role as a mediator of the biological effects of let-7a in gastric tumorigenesis. It provided the first evidence that RAB40C is negatively regulated by let-7a at the post-transcriptional level via binding to the 3'-untranslated region of RAB40C mRNA in gastric cancer. The results of this study suggest that let-7a and RAB40C are potentially useful targets for gastric cancer diagnosis and therapy.