Regulation Mechanism of Glypican-3 on Hippo Signaling Pathway and Its Effects on Biological Behavior of Hepatocellular Carcinoma Huh7 Cells_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

 YANGMin ¹ , LIYuan ² ,  LEIChang-jiang ² , LILei ² , FENGJia-rui ² , GONGXiao-jun ² , YANGRui ²

1. Integrated Department, The Second Affiliated Hospital of Jianghan University, Wuhan 430050, Hubei Province, China;
2. Department of General Surgery, The Second Affiliated Hospital of Jianghan University, Wuhan 430050, Hubei Province, China;

Corresponding?author：

LEIChang-jiang, Email: hhsskux123@126.com

Keywords：

Glypican-3; Hippo signaling pathway; Hepatocellular carcinoma

DOI：

10.7507/1007-9424.20160338

Video：

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Objective To investigate regulation mechanism of glypican-3 (GPC3) on Hippo signaling pathway and its effects on biological behavior of hepatocellular carcinoma Huh7 cells. Methods Short hairpin RNAs (shRNA) targeting GPC3 and Yes-associated protein 1 (YAP1) genes were constructed. All of the shRNAs were transfected into Huh7 cells by liposome transfection in order to screen out the stable expression cell lines. The expressions of GPC3 and YAP1 in Huh7 cells were detected by PCR and Western blot in order to screen out the effective shRNAs. The proliferation, invasion, and apoptosis of Huh7 cells were detected by Edu cell proliferation assay, transwell, and flow cytometry. GPC3 shRNA transfection experiments were divided into 6 groups:non-transfection group, empty vector group, GPC3-714-shRNA group, GPC3-647-shRNA group, GPC3-1718-shRNA group, and GPC3-2134-shRNA group. YAP1 shRNA transfection experiments were divided into 6 groups:non-transfection group, empty vector group, YAP1-906-shRNA group, YAP1-1363-shRNA group, YAP1-1666-shRNA group, and YAP1-2895-shRNA group. GPC3 regulation experiments were divided into 5 groups:non-transfection group, empty vector group, GPC3-1718-shRNA group, GPC3-1718-shRNA+ rhYAP1 group, and YAP1-1666-shRNA group. Results ① GPC3-1718-shRNA and YAP1-1666-shRNA plasmids were successfully constructed to silence the expressions of GPC3 and YAP1. ② The expressions of GPC3 mRNA and protein in each transfection group were significantly lower than those in the non-transfection group (P<0.05) and the empty vector group (P<0.05), while which in the GPC3-1718-shRNA group was significantly lower than those in all the other transfection groups (P<0.05). The expressions of YAP1 mRNA and protein in each transfection group were significantly lower than those in the non-transfection group and empty vector group (P<0.05), while which in the YAP1-1666-shRNA group was significantly lower than those in all the other transfection groups (P<0.05). ③ The expressions of YAP1 mRNA and protein in the GPC3-1718-shRNA group and the YAP1-1666-shRNA group were significantly lower than those in the non-transfection group (P<0.05) and the empty vector group (P<0.05), while which in the GPC3-1718-shRNA+rhYAP1 group were significantly higher than those in the GPC3-1718-shRNA group (P<0.05) and the YAP1-1666-shRNA group (P<0.05). ④ Compared with the non-transfection group and the empty vector group, the abilities of cell proliferation and invasion in the GPC3-1718-shRNA group and the YAP1-1666-shRNA group were significantly decreased, and the cell apoptosis was significantly increased (P<0.05); The cell proliferation, invasion, and apoptosis in the GPC3-1718-shRNA+rhYAP1 group were significantly improved (P<0.05). Conclusion GPC3 is likely to affect biological behavior of hepatocellular carcinoma Huh7 cells through regulation of YAP1 in Hippo signaling pathway.

Citation： YANGMin, LIYuan, LEIChang-jiang, LILei, FENGJia-rui, GONGXiao-jun, YANGRui. Regulation Mechanism of Glypican-3 on Hippo Signaling Pathway and Its Effects on Biological Behavior of Hepatocellular Carcinoma Huh7 Cells. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2016, 23(11): 1315-1320. doi: 10.7507/1007-9424.20160338 Copy

1.	Jemal A, Bray F, Center MM, et al. Global cancer statistics. CA Cancer J Clin, 2011, 61(2):69-90.
2.	龔忠發, 宋艷艷. 惡性腫瘤發病機理研究新進展. 中華腫瘤防治雜志, 2001, 8(5):449-451.
3.	Mínguez B, Tovar V, Chiang D, et al. Pathogenesis of hepatocellular carcinoma and molecular therapies. Curr Opin Gastroenterol, 2009, 25(3):186-194.
4.	謝春梅, 徐偉文. GPC3在肝癌診治中的價值及存在問題. 分子診斷與治療雜志, 2016, 8(1):1-6.
5.	Pan Z, Chen C, Long H, et al. Overexpression of GPC3 inhibits hepatocellular carcinoma cell proliferation and invasion through induction of apoptosis. Mol Med Rep, 2013, 7(3):969-974.
6.	Filmus J, Capurro M. Glypican-3:a marker and a therapeutic target in hepatocellular carcinoma. FEBS J, 2013, 280(10):2471-2476.
7.	Ahn EY, Kim JS, Kim GJ, et al. RASSF1A-mediated regulation of AREG via the Hippo pathway in hepatocellular carcinoma. Mol Cancer Res, 2013, 11(7):748-758.
8.	Sohn BH, Shim JJ, Kim SB, et al. Inactivation of Hippo pathway is significantly associated with poor prognosis in hepatocellular carcinoma. Clin Cancer Res, 2016, 22(5):1256-1264.
9.	Moroishi T, Park HW, Qin B, et al. A YAP/TAZ-induced feedback mechanism regulates Hippo pathway homeostasis. Genes Dev, 2015, 29(12):1271-1284.
10.	Xu Z, Shen L, Chen J. Clinical significance of genetic variations of Hippo-YAP pathway-related genes in hepatocellular carcinoma. Int J Mol Med, 2015, 36:S34.
11.	Miao HL, Pan ZJ, Lei CJ, et al. Knockdown of GPC3 inhibits the proliferation of Huh7 hepatocellular carcinoma cells through down-regulation of YAP. J Cell Biochem, 2013, 114(3):625-631.
12.	王琪. shRNA沉默YAP表達對肝癌細胞SMMC7721增殖和凋亡的影響. 山西醫科大學, 2013.
13.	Yu D, Dong Z, Yao M, et al. Targeted glypican-3 gene transcription inhibited the proliferation of human hepatoma cells by specific short hairpin RNA. Tumour Biol, 2013, 34(2):661-668.
14.	Wierzbicki PM, Rybarczyk A. The Hippo pathway in colorectal cancer. Folia Histochem Cytobiol, 2015, 53(2):105-119.
15.	戴朝六, 趙陽. 原發性肝癌的綜合治療. 中國普外基礎與臨床雜志, 2014, 21(2):133-137.
16.	王慧利, 趙金滿. 磷脂酰肌醇蛋白多糖-3在肝癌中的表達及在治療中的作用研究進展. 胃腸病學和肝病學雜志, 2015, 24(9):1035-1038.
17.	張愛英, 趙元順, 張永宏, 等. GPC3在肝細胞癌診斷及治療方面的研究進展. 臨床輸血與檢驗, 2015, 17(3):279-283.
18.	Jia X, Liu J, Gao Y, et al. Diagnosis accuracy of serum glypican-3 in patients with hepatocellular carcinoma:a systematic review with meta-analysis. Arch Med Res, 2014, 45(7):580-588.
19.	Feng M, Ho M. Glypican-3 antibodies:a new therapeutic target for liver cancer. FEBS Lett, 2014, 588(2):377-382.
20.	曹風華, 汪順才, 馬潔, 等. 血清中磷酯酰肌醇蛋白聚糖-3及高爾基體蛋白-73異常表達對肝癌的診斷價值. 南京醫科大學學報:自然科學版, 2015, 35(3):393-397.
21.	Capurro MI, Xu P, Shi W, et al. Glypican-3 inhibits Hedgehog signaling during development by competing with patched for Hedgehog binding. Dev Cell, 2008, 14(5):700-711.
22.	Midorikawa Y, Ishikawa S, Iwanari H, et al. Glypican-3, overexpre-ssed in hepatocellular carcinoma, modulates FGF2 and BMP-7 signaling. Int J Cancer, 2003, 103(4):455-465.
23.	Cheng W, Tseng CJ, Lin TT, et al. Glypican-3-mediated oncogenesis involves the insulin-like growth factorsignaling pathway. Carcino-genesis, 2008, 29(7):1319-1326.
24.	Huo XY, Zhang Q, Angela M, et al. Overexpression of Yes-associated protein confers doxorubicin resistance in hepatocellullar carcinoma. Oncol Rep, 2013, 29(2):840-846.
25.	雷長江, 龍浩成, 李磊, 等. MiR-132靶向YAP抑制肝癌Huh7細胞的生長. 中華臨床醫師雜志:電子版, 2014, 8(11):79-82.

1. Jemal A, Bray F, Center MM, et al. Global cancer statistics. CA Cancer J Clin, 2011, 61(2):69-90.
2. 龔忠發, 宋艷艷. 惡性腫瘤發病機理研究新進展. 中華腫瘤防治雜志, 2001, 8(5):449-451.
3. Mínguez B, Tovar V, Chiang D, et al. Pathogenesis of hepatocellular carcinoma and molecular therapies. Curr Opin Gastroenterol, 2009, 25(3):186-194.
4. 謝春梅, 徐偉文. GPC3在肝癌診治中的價值及存在問題. 分子診斷與治療雜志, 2016, 8(1):1-6.
5. Pan Z, Chen C, Long H, et al. Overexpression of GPC3 inhibits hepatocellular carcinoma cell proliferation and invasion through induction of apoptosis. Mol Med Rep, 2013, 7(3):969-974.
6. Filmus J, Capurro M. Glypican-3:a marker and a therapeutic target in hepatocellular carcinoma. FEBS J, 2013, 280(10):2471-2476.
7. Ahn EY, Kim JS, Kim GJ, et al. RASSF1A-mediated regulation of AREG via the Hippo pathway in hepatocellular carcinoma. Mol Cancer Res, 2013, 11(7):748-758.
8. Sohn BH, Shim JJ, Kim SB, et al. Inactivation of Hippo pathway is significantly associated with poor prognosis in hepatocellular carcinoma. Clin Cancer Res, 2016, 22(5):1256-1264.
9. Moroishi T, Park HW, Qin B, et al. A YAP/TAZ-induced feedback mechanism regulates Hippo pathway homeostasis. Genes Dev, 2015, 29(12):1271-1284.
10. Xu Z, Shen L, Chen J. Clinical significance of genetic variations of Hippo-YAP pathway-related genes in hepatocellular carcinoma. Int J Mol Med, 2015, 36:S34.
11. Miao HL, Pan ZJ, Lei CJ, et al. Knockdown of GPC3 inhibits the proliferation of Huh7 hepatocellular carcinoma cells through down-regulation of YAP. J Cell Biochem, 2013, 114(3):625-631.
12. 王琪. shRNA沉默YAP表達對肝癌細胞SMMC7721增殖和凋亡的影響. 山西醫科大學, 2013.
13. Yu D, Dong Z, Yao M, et al. Targeted glypican-3 gene transcription inhibited the proliferation of human hepatoma cells by specific short hairpin RNA. Tumour Biol, 2013, 34(2):661-668.
14. Wierzbicki PM, Rybarczyk A. The Hippo pathway in colorectal cancer. Folia Histochem Cytobiol, 2015, 53(2):105-119.
15. 戴朝六, 趙陽. 原發性肝癌的綜合治療. 中國普外基礎與臨床雜志, 2014, 21(2):133-137.
16. 王慧利, 趙金滿. 磷脂酰肌醇蛋白多糖-3在肝癌中的表達及在治療中的作用研究進展. 胃腸病學和肝病學雜志, 2015, 24(9):1035-1038.
17. 張愛英, 趙元順, 張永宏, 等. GPC3在肝細胞癌診斷及治療方面的研究進展. 臨床輸血與檢驗, 2015, 17(3):279-283.
18. Jia X, Liu J, Gao Y, et al. Diagnosis accuracy of serum glypican-3 in patients with hepatocellular carcinoma:a systematic review with meta-analysis. Arch Med Res, 2014, 45(7):580-588.
19. Feng M, Ho M. Glypican-3 antibodies:a new therapeutic target for liver cancer. FEBS Lett, 2014, 588(2):377-382.
20. 曹風華, 汪順才, 馬潔, 等. 血清中磷酯酰肌醇蛋白聚糖-3及高爾基體蛋白-73異常表達對肝癌的診斷價值. 南京醫科大學學報:自然科學版, 2015, 35(3):393-397.
21. Capurro MI, Xu P, Shi W, et al. Glypican-3 inhibits Hedgehog signaling during development by competing with patched for Hedgehog binding. Dev Cell, 2008, 14(5):700-711.
22. Midorikawa Y, Ishikawa S, Iwanari H, et al. Glypican-3, overexpre-ssed in hepatocellular carcinoma, modulates FGF2 and BMP-7 signaling. Int J Cancer, 2003, 103(4):455-465.
23. Cheng W, Tseng CJ, Lin TT, et al. Glypican-3-mediated oncogenesis involves the insulin-like growth factorsignaling pathway. Carcino-genesis, 2008, 29(7):1319-1326.
24. Huo XY, Zhang Q, Angela M, et al. Overexpression of Yes-associated protein confers doxorubicin resistance in hepatocellullar carcinoma. Oncol Rep, 2013, 29(2):840-846.
25. 雷長江, 龍浩成, 李磊, 等. MiR-132靶向YAP抑制肝癌Huh7細胞的生長. 中華臨床醫師雜志:電子版, 2014, 8(11):79-82.

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CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Regulation Mechanism of Glypican-3 on Hippo Signaling Pathway and Its Effects on Biological Behavior of Hepatocellular Carcinoma Huh7 Cells

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