The Role Of MicroRNAs And Target Gene In Uveal Melanoma And Gastric Cancer Development And Progression

MicroRNAs (miRNAs), a class of small endogenous noncoding RNAs, negatively regulate post-transcriptional gene expression by directly cleaving target mRNA or by inhibiting their translation. Although the underlying biological functions are not completely clear, it has been shown to play important roles in a variety of cellular processes including apoptosis, differentiation and cell proliferation. Previous study has shown that some special miRNAs can serve as both diagnostic markers and therapeutic targets for many different tumors including colon tumors, breast cancer, lung cancer and gastric cancer. Recently, it has been suggested that aberrant miRNAs expression was considered as accurate biomarker for metastatic risk in uveal melanoma.According to national health statistics, cancer ranks as the first major cause of death among urban population and third among rural population in China. Therefore, it is critical to improve understanding of the molecular pathogenesis of cancers and eventually develop new therapeutic strategy. In the present study, we focus on the role of miRNA in uveal melanoma, which is the most common primary malignant tumor of the eye in adults, and one of the commonest malignant tumors in the world—gastric cancer.PartⅠ: The effect of genistein in human uveal melanoma cells and mechanism of microRNAsGenistein, an isoflavone isolated from soybean, has been reported to inhibit the growth of various human cancers by affecting a wide variety of cellular processes and/or enzymes, such as cell cycle, apoptosis and angiogenesis, however, there is no related study between miRNAs-mediated transcriptional modulation and the inhibitory effects of genistein on carcinogenesis. MicroRNA-27a (MiR-27a) is identified as an oncogenic miRNA and has been reported to be widely expressed in various cancer cells. Moreover, Transfection of antisense miR-27a (as-miR-27a) led to increased Zinc finger and BTB domain containing 10 (ZBTB10) levels in breast cancer cells. MiR-9 has been found associated with tumor invasion and metastasis.In the present study, we explored the possible activity of genistein to inhibit human uveal melanoma cells growth, and investigated the possible role of genistein on miR-27a as well as its target gene–ZBTB10 expression levels. The role of miR-9 in uveal melanoma invasion was also conducted. MTT assay suggested a significant inhibition of uveal melanoma cell growth in a time and dose-related manner. When the treatment extended to 72 h, the 50% inhibitory concentration (IC50) value was 48.23μM, and at higher concentration (200μM), the cell viability was only 16.7%. In vivo study also indicated treatment groups with genistein could significantly inhibit the growth of xenograft (F = 8.849; P = 0.001). The"stem-loop"real time PCR results indicated that genistein markedly inhibited miR-27a expression in a concentration-dependent manner. After the cells were treated with 25, 50, 100, and 200μM of genistein for 48 h, compared with the control, the levels of miR-27a were decreased to 88.9%±6.9%, 72.9%±5.4%, 51.7%±3.2% and 42%±2.5%, respectively. To verify whether the decreased level of miR-27a could ultimately influence the target gene transcription activity, we examined the expression level of known target gene ZBTB10. After 48 h treatment with genistein, ZBTB10 expression in 200μM genistein group was significantly higher than that in control group (P = 0.037). Moreover, compared with the highly aggressive uveal melanoma C918 cells, miR-9 markedly upregulated in poor aggressive uveal melanoma OCM-1A cells (P < 0.001), and the expression of miR-9 in C918 cell lines was increased after treatment with genistein (P = 0.022). This suggested that reactivated miR-9 could affect the aggression of uveal melanoma.In conclusion, we for the first time reported a correlation between antitumor activity of genistein and miRs mediated regulatory mechanism.PartⅡ: The association between genetic polymorphism in pre-miR-27a and susceptibility to gastric cancerAberrant miRNAs expression is presently proposed to correlate with various human cancers. The high degree of phylogenetic conservation in miRNA sequences determines that the functional variation in miRNAs may influence various biological processes. Therefore, a mutation or single-nucleotide polymorphisms (SNP) in miRNA genes might influence the transcription of primary miRNAs (pri-miRNAs), maturation of miRNAs, or miRNA-mediated transcriptional regulation. However, whether miRNAs SNP alter gastric cancer susceptibility is still unclear.Here we first compared miR-27a and ZBTB10 expression between tumor and adjacent nontumorous tissues and investigated the possible role of a common A/G polymorphism (rs895819) within pre-miR-27a in the development or progression of gastric cancer. The effect of rs895819 on the expression of miR-27a and ZBTB10 was also assessed. Quantitative real-time PCR revealed that miR-27a was expressed at a higher level in tumor compared with the nontumorous tissues came from 12 gastric cancer cases. Relative Ct ratios were 73.42±4.80% versus 78.79%±5.90% (P = 0.023) (Lower Ct ratio represents a higher expression). While the mean expression levels of ZBTB10 mRNA was lower in tumor tissues than that in nontumor tissues. Relative Ct ratios were 110.25%±5.98% versus 100.30%±5.78% (P < 0.001).In hospital-based case-control study (304 gastric cancer patients and 304 age- and sex-matched controls), we found that the frequency of G alleles in cases was 39.97%, which was higher than in the controls (32.73%) (χ2 = 6.88; P = 0.009). The difference in rs895819 genotype distributions between the cases and controls was statistically significant (χ2 = 6.55; P = 0.038). The genotype distributions of the SNP among the controls (χ2 = 3.75; P = 0.053) and the cases (χ2 = 1.69; P = 0.194) were in Hardy–Weinberg equilibrium. Subjects with the variant genotypes (AG+GG) showed a significantly increased risk of gastric cancer relative to AA carriers (adjusted odds ratio, 1.48; 95% confidence interval, 1.06-2.05; P = 0.019). The elevated risk was especially evident in older subjects (age >58 years), males, nonsmokers and rural subjects. A significant association of pre-miR-27a variant genotypes with lymph node metastasis was also observed.To determine whether the identified mutation of pre-miR-27a SNP rs895819 could affect miR-27a expression, we screened 65 tumor tissues obtained from gastric cancer patients and found the expression of miR-27a in AG+GG cases compared with AA cases was significantly different, relative Ct ratios were 73.74%±4.34% versus 78.98%±7.31% (P < 0.001). However, the expression of ZBTB10 in AG+GG genotypes was significantly lower levels than AA genotype. Subjects with AG+GG genotypes displayed higher Ct ratios compared with AA carriers (107.56%±7.05% versus 101.68%±7.16%; P = 0.003). Our results showed that a common polymorphism (rs895819) in pre-miR-27a, by modulating miR-27a and ZBTB10 levels, acted as an important factor for the gastric cancer susceptibility.