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[The influence of downregulation of ezrin expression by RNA interference on the growth and metastasis of hepatocellular carcinoma: experiment in vitro].

OBJECTIVE
To investigate the effect of membrane-cytoskeleton linker ezrin on the growth and metastasis of human hepatocellular carcinoma cell (HCC) lines.


METHODS
Human HCC cells of the lines SF/SMMC7721, MHCC97-H, MHCC-1, and HepG2 were cultured. Four pairs of small interfering RNA (siRNA) targeting erzin were designed and transfected into the HCC cells. 48 h after transfection the cell total RNA was extracted and 72 h later the total cell protein was extracted. RT-PCR and Western blotting were used to detect the transfection rates so as to screen the most effective siRNA to be transfected into the HCC cells. HCC cells were collected every day for 7 days to extract the total RNA and protein. Real-time PCR and Western blotting were used to detect the downregulation rate of erzin at different times. MTT method was used to detect the proliferation of the cells. Flow cytometry was used to detect the cell cycle and apoptosis. Scanning electron microscopy was used to observe the cell pseudopods. Transwell test was used to detect the invasion ability of the transfected HCC cells.


RESULTS
Real-time PCR and western-blotting revealed that ezrin siRNA notably down-regulated ezrin expression at both mRNA and protein levels. Down-regulation of ezrin expression distinctly decreased the proliferation rates of these 4 kinds of HCC line. After RNAi treatment the cell proportion in G(2)-M phase decreased from 28.07% to 21.53% in the SF/SMMC7721 cells, from 24.94% to 13.92% in the MHCC97-H cells, from 19.30% to 13.2% in the MHCC-1cells, and from 7.73% to 4.24% in the HepG2 cells. After RNAi treatment, the number of pseudopods decreased from 20.8 +/- 3.0 to 13.2 +/- 2.4 in the SF/SMMC7721: cells (P < 0.05), from 18.4 +/- 2.7 to 14.0 +/- 2.9 in the MHCC97-H cells (P < 0.01), from 22.6 +/- 3.5 to 13.3 +/- 1.9 in the MHCC-1: cells (P < 0.01), and from 31.0 +/- 2.9 to 17.8 +/- 2.3 in the HepG2 cells (P < 0.01); and the motility and invasiveness decreased from 49.9 +/- 7.7 to 31.9 +/- 5.2 in the SF/SMMC7721 cells (P < 0.05), from 58.5 +/- 4.2 to 33.0 +/- 3.3 in the MHCC97-H cells (P < 0.01), from 57.6 +/- 6.1 to 28.3 +/- 3.4 in the MHCC-1 cells (P < 0.01), and from 37.3 +/- 3.0 to 25.3 +/- 2.3 in the HepG2 cells (P < 0.01). The pseudopods of the HCC cells remarkably shortened and decreased in number (for the SF/SMMC7721 cells: t = 4.95, P < 0.05, for the MHCC97-H cells: t = 5.88, P < 0.01, for the MHCC-1 cells: t = 5.56, P < 0.01, and for the HepG2 cells: t = 5.71, P < 0.01) after siRNA interference.


CONCLUSION
Ezrin is necessary for HCC proliferation and invasion. It is probably an important factor to inhibit tumor reoccurrence and metastasis.