The Influence Of Pinobanksin-3-acetate On The MicroRNA Expression In Colorectal Cancer Cell SW480

To study the effects of propolis flavonoid pinobanksin- 3-acetate(PB3A) on the mRNA expression and protein expression levels of RGS2 and GEM in the human colorectal cancer SW480, discuss the anticancer molecular mechanism of PB3 A and the relevated signal pathway, to confirm the previous mRNA microarray result. To discuss the influence of PB3 A on the microRNA expression profile in human colorectal cancer SW480, screen the differentially expressed microRNAs and find these microRNAs` target genes from online websites, furthur discuss the possible anticancer molecular mechanism of PB3 A on colorectal cancer SW480. This article mainly includes the following two parts. 1. The influence of Pinobanksin-3-acetate on the gene expression of RGS2 and GEM in colorectal cancer SW480Used PB3A(100μg/mL) to intervene colon cancer SW480 which is at its exponential phase(for 24 hours), After the treatment of PB3 A on sw480, used inverted photomicroscopy to observe the morphological changes and growth situation of SW480 cells between control group and experimental group with; Analyzed the mRNA transcriptional levels by Real-Time qunatitative RT-PCR technology, amplified each molecular target in the samples, β-actin was the loading control to draw the standard curve; detected the changes of mRNA transcriptional level of RGS2 and GEM genes between control group andexperimental group(PB3A intervented group). Detected the protein expression level of RGS2 and GEM genes,by using protein immunoblot( Western Blot) technology, used β-actin as a loading control, compare the differences of protein expression in the experimental groups and control groups.The real-time fluorescence quantitative RT-PCR results showed that PB3 A can increase the mRNA transcription level of RGS2 and GEM in human colon cancer cell SW480, found that there was a significant difference when compared with control group(?Ct comparison,P<0.01)。The mRNA change pattern was in consistent with the result of the previous mRNA gene chip array, which validate the reliability and accuracy of the chip array.Western Blot results showed that PB3 A increased the RGS2 and GEM protein expression level.The relative ratio of the RGS2 and β-actin in control group was 0.917±0.01, when compared with the relative ratio(2.318±0.02)in SW480 cells with PB3 A treatment, and the difference was statistically significant(P<0.05); while for GEM the relative ratio(2.168±0.02) in experiment group was significantly higher when compared with the control group(1.442±0.03),and the difference was statistically significant(P<0.05). From all the results, it is obvious that the trend of protein expression levels of RGS2 and GEM is in consistent with the results of qRT-PCR and mRNA gene chip array. 2. The influence of Pinobanksin-3-acetate on the microRNA expression in colorectal cancer cell SW480Used PB3A(100μg/mL) to intervene colon cancer SW480 which is at its exponential phase(for 24 hours), After the treatment of PB3 A on sw480, made the microRNA expression gene chip(miRCURYTM LNA Array(v.18.0),Exiqon), selected the differentially expressed mciroRNAs(fold change≧2)in control group and experimental group, in addition, predicted the target genes of the differentially expressed microRNAs(fold change≧10) through online websites.Use miRCURYTM LNA Array(v.18.0)(Exiqon) microarray to detect the microRNA profile in human colon cancer cell SW480 after PB3 A treatment, use the image analyse software to digitilize and analyze the microarray scanning image, results showed that there are 267 differentially expressed microRNAs(fold change≧2) in PB3 A treated colon cancer cell SW480; 218 of these 267 microRNAs are upregulated microRNAs and 49 microRNAs are downregulated. Results showed that 121 microRNAs are upregulated microRNAs and 8 microRNAs are downregulated(fold change≧3); 28 microRNAs are upregulated microRNAs and 8 microRNAs are downregulated(fold change≧10).Predicted the target gene of 30 differentially expressed microRNAs(fold change≧10) on online websites;results showed that the target genes of 22 microRNA can be found, but the rest 8 of them have not any target genes online websites. Conclusion:According to the results of this study, we confirmed that the anticancer effect of PB3 A on sw480 maybe involved gene expression change level of RGS2 and GEM. These two gene RGS2 and GEM maybe closely realted to the carcinogenesis and development of colorectal cancer, they are maybe potential target gene marker in colorectal cancer.From the miRCURYTM LNA Array(v.18.0)(Exiqon) microarray results,initially confirmed the differentially expressed microRNAs after the treatment of PB3 A on sw480, predicted the target gene of 30 differentially expressed microRNAs(fold change≧10) on online websites for the analyzing anticancer effect of PB3 A and its development as anticancer drug.