Pien Tze Huang inhibits the proliferation of colorectal cancer cells by increasing the expression of miR-34c-5p

Abstract

MicroRNAs (miRNAs) are small, short endogenous non-coding RNA that act as oncogenes or tumor suppressors, and serve an important role in various human malignant cancers, including colorectal cancer (CRC). Evidence has indicated that miRNAs regulate the expression of various genes associated with human cancer, in particular the miR-34 family. A well-known traditional Chinese formula, Pien Tze Huang (PZH), has a significant clinical effect on CRC. Previous studies have demonstrated that PZH inhibits CRC growth in vitro and in vivo via multiple mechanisms, including the induction of apoptosis, inhibition of cell proliferation and tumor angiogenesis. To further elucidate the molecular mechanisms underlying the antitumor activity of PZH, in the present study its effects on cell proliferation and miRNA expression in human colon carcinoma (HCT)-8 cell lines was examined. It was observed that treatment with PZH inhibited cell viability and upregulated the expression of miR-34c-5p in HCT-8 cells. In addition, transfection with an miR-34c-5p mimic and treatment with PZH inhibited cell survival and arrested the cell cycle between the G0/G1 and S phase in HCT-8 cells. Furthermore, PZH treatment and transfection with miR-34c-5p downregulated the expression of cyclin-dependent kinase 4 and cMyc (a promoter of cell proliferation), and increased the expression of p53, which is a promoter of apoptosis. These results suggest that PZH may suppress proliferation in CRC cells by upregulating the expression of miR-34c-5p, which provides a novel perspective for understanding the mode of action of PZH.

Keywords: Pien Tze Huang; colorectal cancer; miR-34c; proliferation; traditional Chinese medicine.

Figure 1.

Effect of PZH on the suppression of cell viability. (A) HCT-8 cells were treated with the indicated concentrations of PZH for 24 h. Cell viability was determined using an MTT assay. (B) miRNA was extracted from HCT-8 cells, and the expression of miR-34c-5p was assayed by reverse transcription-quantitative polymerase chain reaction and normalized to a control. Data are expressed as the mean ± standard deviation of three independent experiments. PZH, Pien Tze Huang; HCT-8, human colorectal cancer-8. *P<0.05 vs. control (0 mg/ml).

Figure 2.

Effect of miR-34c-5p transfection and PZH treatment on the growth of human colorectal cancer-8 cells. (A) The morphology of cells treated with PZH or transfected with miR-34c-5p were observed using a phase-contrast microscope (magnification, ×200). (B) Live cells treated with PZH or transfected with miR-34c-5p were analyzed using Trypan Blue staining by Countstar. Data are expressed as the mean ± standard deviation of three independent experiments. PZH, Pien Tze Huang; NC, normal cells. *P<0.05 vs. NC; ^#P<0.05 vs. control.

Figure 3.

Effect of miR-34c-5p transfection and PZH treatment on the survival of human colorectal cancer-8 cells. (A) Cells were treated with 0.5 mg/ml PZH or transfected with an miR-34c-5p mimic (20 nm) for 24 h and cultured for 10 days. Colony formation analysis was employed to detect cell survival. Images are of three independent experiments. (B) Quantification of colony formation analysis. Data are expressed as the mean ± standard deviation of three independent experiments. PZH, Pien Tze Huang; NC, normal cells. *P<0.05 vs. NC; ^#P<0.05 vs. control.

Figure 4.

Effect of miR-34-5p transfection and PZH treatment on cell cycle progression. Human colorectal cancer-8 cells were (A) transfected with an miR-34c-5p mimic and (B) treated with indicated concentrations of PZH for 24 h, stained with propidium iodide (PI) and analyzed by flow cytometry. Data are expressed as the mean ± standard deviation from three independent experiments. PZH, Pien Tze Huang; NC, normal cells. *P<0.05 vs. NC; ^#P<0.05 vs. control.

Figure 5.

Effects of PZH treatment and miR-34c-5p transfection on the expression of CDK4, cMyc and p53 in human colorectal cancer-8 cells. (A) cMyc (B) CDK4 and (C) p53 mRNA expression levels were determined by reverse transcription-quantitative polymerase chain reaction. *P<0.05 vs. NC; ^#P<0.05 vs. control. (D) Protein expression levels were determined by western blot analysis. The results shown are representative of three independent experiments. PZH, Pien Tze Huang; NC, normal cells; cMyc, cyclin-dependent kinase 4.