Pien Tze Huang Increases 5-Fluorouracil Sensitivity And Inhibits Migration In Colorectal Cancer Cells Via Regulation Of MiR-494-3p

Aims:The purpose of this thesis is to study the influence of Pien Tze Huang (PZH) on the expression level of miR-494-3p and its downstream targets. The study will contribute to understanding the mechanism of the inhibitory effect of PZH on 5-Fluorouracil (5-FU) drug-resistance and metastasis of colorectal cancer cells, and lay a foundation for the clinical application of PZH.Methods:5-FU drug-resistant colorectal cancer cell line HCT-8/5-FU and its parental cell line HCT-8 have been used in this study. The 5-FU resistance of HCT-8/5-FU cell have been confirmed by MTT assay. The expression level of miR-494-3p in the PZH treated/untreated HCT-8/5-FU and HCT-8 cells have been analyzed by RT-PCR. To change the expression level of miR-494-3p in HCT-8/5-FU and HCT-8 cells, the miR-494-3p mimic and inhibitor has been transfected into two cells respectively. The efficiency of transfection has been confirmed by RT-PCR, and the 5-FU resistance of transfected cells has been analyzed by MTT. Typan Blue staining cell viability assay has been processed to study the effect of PZH treatment on the cell survival rate of miR-494-3p down-regulated HCT-8 cell. Colony-forming assay has been used to research the effect of PZH treatment on the colony formation of miR-494-3p down-regulated HCT-8 cell. The influence of PZH treatment on cell migration ability have been studied by Transwell assay. mRNA and protein level of miR-494-3p targeted genes have been analyzed by RT-PCR and Western Blot.Results:1. PZH increase the 5-FU sensitivity of colorectal cancer cells by manipulating expression level of miR-494-3p.5-FU resistance of HCT-8/5-FU cells was confirmed by MTT assay. RT-PCR experiment showed that the expression level of miR-494-3p was down-regulated in HCT-8/5-FU cells, and its expression level could be recovered by the PZH treatment. miR-494-3p mimic and inhibitor was transfected into HCT-8/5-FU or HCT-8 cells respectively. After 48 hours of 5-FU treatment, we found that upregulation of miR-494-3p expression level increased of the 5-FU sensitivity of HCT-8/5-FU cells. On contrary, inhibition of miR-494-3p expression level decreased the 5-FU sensitivity of HCT-8 cells. Typan Blue assay showed that 5-FU, PZH and the combination of the two all caused a lower survival rate of HCT-8 cells in which miR-494-3p was down-regulated. However, the combination of 5-FU and PZH showed the strongest impact. Colony forming assay showed a lower number of colony of HCT-8 cells after treatment with drugs mentioned above, and also the combination of 5-FU and PZH have the most dramatic effect. RT-PCR and Western blot assay showed that expression of DPYD and c-Myc was upregulated when miR-494-3p was down-regulated in HCT-8 cells. This increased expression of DPYD and c-Myc could be complemented by the treatment of 5-FU, PZH and the combination of the two, and the combination treatment have the strongest effect.2. PZH inhibits the metastasis of colorectal cancer cells by manipulating expression level of miR-494-3p.Transwell assay found that the metastasis of HCT-8/5-FU cells was inhibited after upregulation of miR-494-3p in the cell; on the other hand, down-regulation of miR-494-3p in the HCT-8 cell showed the opposite effect. Transwell assay also showed that PZH inhibits the metastasis of the miR-494-3p down-regulated HCT-8 cell in a dose dependent manner. Western blot assay showed that expression of TGF-P and ZEB2 was upregulated when miR-494-3p was down-regulated in HCT-8 cells. This increased expression of TGF-β and ZEB2 could be well complemented by the treatment of PZH with a concentration of 0.5mg/ml、 0.75mg/ml.Conclusions:MiR-494-3p and its downstream targets are critical factors involved in the inhibitory effect of PZH on 5-FU drug resistance and metastasis of the colorectal cancer cell. PZH affected the colorectal cancer cell likely through manipulating the expression level of these factors.