| Objective:To study the effects of Propolis flavonoid Pinobanksin-3-acetate (PB3A) on thechanges of differentially expressed genes of CXCL1, C3orf63, HSPA6and FOSmRNA transcription and protein expression levels in the human colon cancer cellsHCT116and SW480, discussion these differentially expressed genes and role andpossible molecular mechanisms of PB3A to inhibit the occurrence and developmentof human colon cancer.Methods:1. Using PB3A to intervene the colon cancer SW480and HCT116cells in exponentialphase for24hours; morphological changes and growth status of SW480and HCT116cells of intervention groups and control groups were observed by invertedphotomicroscopy;2. The gene expression profiles were analyzed by Real-time quantitative RT-PCR (realtime fluorescence quantitative PCR, FQ RT-PCR) technology, each molecular targetin the samples were amplified, β-actin as a loading control, to draw the standardcurve; and detecting the changes of CXCL1, C3orf63, HSPA6and FOS genes mRNAtranscription levels in the intervention group;3. By using protein immunoblot (Western Blot) technology to detect the proteinexpression level of CXCL1, HSPA6and FOS genes, β-actin as a loading control, tocompare the differences of protein expression in the intervention groups and controlgroups;4. Statistic analysis was performed with SPSS17.0Statistic Software, the results of△Ct value and relative intensity of protein expression of target genes in both of drugtreatment group and control group were analyzed by two independent samples t-test(two-sided) analysis. Experimental data were expressed as mean±SD, and0.05determine of standard statistical significance tests. Values of P＜0.05were consideredstatistically significant. Results:1. The real-time fluorescence quantitative RT-PCR results showed that, PB3A canreduce colon cancer SW480and HCT116cells CXCL1and C3orf63gene mRNAtranscription levels, and also have very significant difference compared with thecontrol group (△CT comparison, P <0.01), in both cell lines, levels of HSPA6andFOS genes mRNA are increased, the difference was statistically significant (△Ctcomparison, P <0.01), The experimental data is compared with the results of genechip array data, each gene changing trends of mRNA expression are in consistentwith the results of the gene chip array. The results further validate the accuracy of thegene chip array.2.Western Blot results showed that,colon cancer SW480cells treated with the drugafter (PB3A100μg/mL)24hours,when compared to the control group(0.24±0.03),the relative intensity ratio of the CXCL1protein and β-actin (protein expressionrelative intensity) was0.52±0.04, and the difference was statistically significant (P<0.05); while HSPA6protein expression levels (2.62±0.25),when compared to thecontrol group (1.67±0.05), was significantly higher; FOS protein expression relativeintensity ratio was1.43±0.16, when compared with the control group (0.58±0.07),there was a significant difference (p <0.05) for the corresponding control group; in thedrug treated HCT116cells CXCL1protein expression relative intensity ratio was(4.76±0.35), when compared to the control group(5.84±0.24) the expression levelswas decreased, have a significant difference (P <0.05); the expression of HSPA6protein (0.71±0.20) was significantly higher than control group (0.47±0.05)(P<0.05); FOS protein and β-actin ratio relative gray was2.92±0.34, and the controlgroup (8.64±0.36) compared to a significant difference in expression (P <0.05),andwhen compared to the control group (8.64±0.36), have a significant difference inexpression (P <0.05). The trend of protein expression levels is in consistent with theresults of RT-PCR and gene chip array.3. Correlation analysis of mRNA expression of differentially expressed genes showedthat, in the SW480cell lines,there were negative correlation between downregulated gene CXCL1and C3orf63（r=0.833，p<0.05）, but no correlation have been found withthe other gene (p>0.05); and there were have negative correlation between C3orf63and FOS gene（r=-0.714，p<0.05）; and there were also have high positive correlationbetween upregulated genes HSPA6and FOS（r=0.940，p<0.01）. In the HCT116celllines, have positive correlation between CXCL1and HSPA6（r=0.881，p<0.01）;downregulated gene C3orf63,CXCL1and upregulated gene HSPA6are showed anegative correlation（-0.810<r<-0.762，p<0.05）。4. CXCL1, C3orf63, HSPA6and FOS genes differentially expressed in drug treatedSW480and HCT116cell lines. Compared with the previous findings, this mayindicate that the effect of PB3A on the proliferation of SW480cells is stronger thanthe HCT116cells. According to the results of immunoblot band and△Ct value, theeffect of PB3A on gene expression in the two kinds of colon cancer cell lines hassignificant differences.Conclusion:After intervention of propolis flavonoids PB3A in the human colon cancer SW480and HCT116cells, which induced the changes of CXCL1, C3orf63, HSPA6and FOSgene expression levels, suggesting the role of Propolis flavonoid PB3A anti-humancolorectal cancer cells may be associated with the downregulation of theCXCL1,C3orf63genes and upregulation of the HSPA6,FOS genes. |
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