[Intervention of glypican-3 genetic transcription on anti-proliferative effect of hepatoma cells with high metastatic potentiality].

Abstract

OBJECTIVE:

To explore the silencing glypican-3 (GPC-3) gene transcription by specific small hairpin RNA (shRNA) on the inhibition of hepatoma cells with high metastatic potentiality and hepatoma growth.

METHODS:

After MHCC-97H cells were transfected with higher effective GPC-3-shRNA, GPC-3 mRNA was analyzed by multiple FG-RT-PCR or protein by Western blot. Cell proliferation was detected by 5-ethynyl-2'-deoxyuridine and sulforhodamine B assay, its migratory metastasis and invasiveness by wound healing or transwell chamber system and cell apoptosis was detected by Caspase-Glo(®) 3/7 Luminescence assay. Nude mice were subcutaneously injected with stable MHCC-97H cells for observing the forming time or volume of xenograft tumors. And the expressions of GPC-3, β-catenin, p-GSK3β and CyclinD1 were analyzed by immunohistochemistry.

RESULTS:

After shRNA1 transfection with high efficiency (>80%), the expression of GPC-3 was down-regulated to 75.6% (t = 15.473, P < 0.001) at mRNA level in accordance with its protein, inhibiting cell proliferation (71.1%, t = 10.468, P < 0.001) notably, decreasing its migration (80.1%, t = 32.697, P < 0.001) and invasiveness (69.1%, t = 39.647, P < 0.001). β-catenin was down-regulated (67.7%, t = 18.4, P < 0.001) and Gli1 increased (53.5%, t = -4.824, P = 0.008) with its protein. The average forming time of subcutaneous tumors was 11.2 days (d) in the shRNA group and it was significantly longer (P < 0.01) than that in the control (5.3 d) or shRNA-neg (5.5 d) group. And the average volume (65.5 mm(3)) of tumors with decreased GPC-3, β-catenin, p-GSK3β, and cyclinD1 expressions in the shRNA group was significantly smaller (P < 0.01) than that in the shRNA-neg (365.7 mm(3)) or control (404.8 mm(3)) group, respectively.

CONCLUSION:

Specific shRNA might intervene effectively the GPC-3 gene transcription and inhibit invasion and tumor growth. Thus GPC-3 may become a potential molecular target for hepatoma gene therapy.

• Authors: Min Yao, Li Wang, Yun Shi, Qi Qian, Dandan Yu, Yang Shi, Shaolin Lu, Dengfu Yao
• Journal: Zhonghua yi xue za zhi (Zhonghua Yi Xue Za Zhi) Vol. 94 Issue 32 Pg. 2544-8 (Aug 26 2014) ISSN: 0376-2491 [Print] China
• PMID: 25410930 (Publication Type: Journal Article)

Chemical References

• Glypicans
• RNA, Messenger
• RNA, Small Interfering
• beta Catenin
• 5-ethynyl-2'-deoxyuridine
• Deoxyuridine

Topics

• Animals
• Apoptosis
• Carcinoma, Hepatocellular
• Cell Line
• Cell Line, Tumor
• Cell Proliferation
• Deoxyuridine (analogs & derivatives)
• Down-Regulation
• Gene Silencing
• Glypicans
• Humans
• Liver Neoplasms
• Mice
• Mice, Nude
• Neoplasm Invasiveness
• Neoplasm Metastasis
• RNA, Messenger
• RNA, Small Interfering
• Transcription, Genetic
• Transfection
• beta Catenin