Downregulation of Notch1 inhibits the invasion of human hepatocellular carcinoma HepG2 and MHCC97H cells through the regulation of PTEN and FAK

  • Authors:
    • Yan-Jian Hu
    • Hong-Ying Li
    • Kai-Jie Qiu
    • Da-Chuan Li
    • Jia-Hui Zhou
    • Yan-Hua Hu
    • Feng-Min Zhang
  • View Affiliations

  • Published online on: August 8, 2014     https://doi.org/10.3892/ijmm.2014.1889
  • Pages: 1081-1086
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Abstract

Tumor invasion and metastasis are the main causes of mortality in patients with hepatocellular carcinoma (HCC). Thus, the effective inhibition of these tumorigenic processes is critical in order for HCC therapy to be effective. Previous studies have demonstrated that Notch1 is associated with metastasis in several human malignancies. However, the exact molecular mechanisms underlying the Notch1-mediated induction of the invasion of HCC cells remain poorly understood. In the present study, we demonstrate that, compared to the normal liver cell line, L02, Notch1 is highly expressed in the human HCC cell lines, HepG2 and MHCC97H. Using small interfering RNA (siRNA), we knocked down the expression of Notch1 in the cell lines. Notch1 expression in the HCC cell lines was also measured following transfection with siRNA using RT-PCR and western blot analysis. In addition, a migration and invasion assay was performed to determine the effects of Notch1 knockdown on cell migration and invasion. Our results demonstrated that the downregulation of Notch1 by small interfering RNA (siRNA) significantly inhibited the migration and invasion of both HCC cell lines. Additionally, we demonstrated that the knockdown of Notch1 in both HCC cell lines increased both the total expression of phosphatase and tensin homolog (PTEN) and its phosphorylated form. By contrast, focal adhesion kinase (FAK) and phospho-FAK expression was decreased following Notch1 depletion. Taken together, our data suggest that targeting Notch1 may be a useful therapeutic approach to inhibiting the metastasis of HCC cells.

Introduction

In males, liver cancer is the fifth most frequently diagnosed cancer worldwide and the second most frequent cause of cancer-related mortality. In females, it is the seventh most commonly diagnosed cancer and the sixth leading cause of cancer-related mortality. Among primary liver cancers, hepatocellular carcinoma (HCC) is the major histological subtype, accounting for 70–85% of the total liver cancer burden worldwide (1). Although the prevalence of the disease remains the highest in Eastern Asia and Africa, the incidence of liver cancer has steadily increased in the Western world over the last 30–50 years (2). Over the past several years, the diagnosis and management of HCC have greatly improved. The primary curative treatment for HCC is surgical resection. However, many patients present with advanced stages of the disease, making surgery more difficult and less effective. This is due to the fact that the late stages of HCC are generally associated with greater invasion and metastasis, two characteristics associated with a significantly worse patient prognosis. Thus, the effective prevention of invasion and metastasis in HCC would likely be of great therapeutic value.

Increasing evidence suggests that the inhibition of cell signaling pathways can greatly influence the invasion and metastasis of HCC cells and may aid in the regulation of the disease (35). Previously, several independent research groups have demonstrated that Notch signaling regulates tumor cell invasion and metastasis (6,7). Other studies have also indicated that Notch signaling influences the invasion of HCC cells (8,9). Notch1 is a receptor that tends to be overexpressed in human HCC. Thus, Notch1 may be useful as an immunohistochemical biomarker for the detection of patients at high-risk for recurrence and with a shorter disease-specific survival (10). However, to date, the mechanisms governing the Notch1-mediated induction of the invasion of HCC cells remain poorly understood.

In the present study, we examined the mRNA expression levels of Notch1 both in the human liver non-tumorigenic cell line, L02, and in the HCC cell lines, HepG2 and MHCC97H. Notch1 was more highly expressed in the HCC lines compared to the normal liver cell line; thus, Notch1 may play an oncogenic role in HCC. We inhibited Notch1 expression using small interfering RNA (siRNA) and assessed the effects on HCC cell line biology. Notch1 knockdown inhibited the migration and invasion of both HCC cell lines. Notch1 knockdown was also associated with the increased expression of phosphatase and tensin homolog (PTEN), both the total and phosphorylated forms, and the decreased expression of both the total and phosphorylated forms of focal adhesion kinase (FAK). Our data suggest that the Notch1-PTEN-FAK pathway may provide a new means of inhibiting the metastasis of HCC cells.

Materials and methods

Cell culture and reagents

The normal liver cell line, L02, was kindly provided by No. 3 People’s Hospital Affiliated with Shanghai Jiao Tong University, Shanghai, China. The MHCC97H metastatic HCC cell line was obtained from the Liver Cancer Institute of Zhong Shan Hospital Affiliated with Fudan University, Shanghai, China. The HepG2 HCC line was obtained from the Experiment Center of the Second Affiliated Hospital of Harbin Medical University, Harbin, China. All cell lines were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Biowest SAS, Nuaillé, France) and incubated in 5% CO2 at 37°C. Primary antibodies for Notch1, PTEN, phospho-PTEN, FAK and phospho-FAK were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). All secondary antibodies were obtained from Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd. (Beijing, China). Notch1 small interfering RNA (Notch1-siRNA), control siRNA and Lipofectamine RNAiMAX were purchased from Invitrogen (Carlsbad, CA, USA). All other chemicals and solutions were purchased from Sigma-Aldrich, unless otherwise indicated.

siRNA transfection

Three putative Notch1 candidate sequences and one control sequence were designed using Oligoengine software, as previously described (11). The sequences of the siRNAs were as follows: Notch1 sequence 1 forward primer, 5′-AAC AUC AAC GAG UGG UCC AGC dTdT-3′) and reverse primer, 5′-GCU GGA GCA CUC CUU GAU GUU-3′); Notch1 sequence 2 forward primer, 5′-GGG CUA ACA AAG AUA UGC ATT dTdT-3′ and reverse primer, 5′-UGC AUA UCU UUG UUA GCC CTT-3′; Notch1 sequence 3 forward primer, 5′-CAG GGA GCA UGU GUA ACA UTT dTdT-3′ and reverse primer, 5′-AUG UUA CAC AUG CUC CCU GTT-3′; and control sequence forward primer, 5′-CGU GCC AAC AAG UCG UAC AGA dTdT-3′ and reverse primer, 5′-UGU GUA GUA CCC AGU GUU GCC-3′. All siRNA molecules were synthesized by Invitrogen (Shanghai, China). Transfection with siRNA was carried out using Lipofectamine RNAiMAX according to the manufacturer’s instructions. Cells transfected with Notch1-siRNA were seeded into 6-well culture plates at a density of 1×105 cells/well. Cells were allowed to grow for 24–48 h and were then harvested for analysis. Irrelevant control siRNA was used as a negative control (mock group) under similar conditions.

Reverse transcription polymerase chain reaction (RT-PCR)

Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions and quantified by UV spectroscopy. To prepare RNA for PCR analysis, 2 μg total RNA was converted into cDNA using SuperScript II reverse transcriptase (Invitrogen) with oligo(dT) (Promega, Madison, WI, USA) and random hexamer primers (Promega). PCR was performed using Taq DNA polymerase (Invitrogen). All PCR experiments were performed using the PCR system TC-XP-G (Bioer Technology Co., Ltd., Hangzhou, China). β-actin was used as an internal control for normalization. All reactions were carried out for 30 cycles. The primers used in the present study were as follows: Notch1 forward, 5′-CGA CGT CAA CGC CGT AGA T-3′ and reverse, 5′-CTC CTC CCT GTT GTT CTG CATAT-3′; β-actin forward, 5′-GTC AGG TCA TCA CTA TCG GCA AT-3′ and reverse, 5′-AGA GGT CTT TAC GGA TGT CAA CGT-3′. Products were analyzed by polyacrylamide gel electrophoresis.

Migration and invasion assay

A wound-healing assay was performed to assess the effects on migration. HCC cells (1×105) were seeded in a fibronectin (Fn)-coated 6-well plate. These cells were incubated for 24 h. The cell monolayer was then disrupted with a pipette tip followed by 6 washes with DMEM medium to wash away any floating cells. The cells were then cultured in DMEM medium containing 2% FBS, and images were captured at time 0 and 24 h after the scratch was made using an inverted microscope. Six fields for each point were recorded. For the invasion assay, Transwell assays were performed. The membranes had an 8 μm diameter pore (Corning Inc., New York, NY, USA) and was coated with 200 μl Matrigel at 200 μg/ml. The membranes were incubated overnight at 4°C. Cells (2×104) in 0.20 ml serum-free DMEM were seeded in the upper chamber. The lower chamber was filled with 0.75 ml DMEM containing 10% FBS. After 48 h of incubation, the cells were removed from the upper surface of the filter by scraping with a cotton swab. Cells that had invaded and adhered to the bottom of the membrane were fixed with methanol and stained with crystal violet solution. The number of invaded cells was determined by counting the mean cell number of 5 randomly selected fields. Experiments were carried out in triplicate.

Western blot analysis

Cells were lysed in buffer containing 50 mmol/l Tris-Cl (pH 8.0), 0.02% sodium azide, 1 mg/l aprotinin, 1% nonidet P-40, and 100 mg/l phenylmethylsulfonyl fluoride. Final protein concentrations were determined using the BCA protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer’s specifications. Equal amounts of protein were separated by 10% SDS-polyacrylamide gel electrophoresis. Proteins were transferred to a nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ, USA) and blocked for 2 h in 5% fat-free dry milk, 0.1% Tween-20, 150 mmol/l sodium chloride, and 50 mmol/l Tris. The membranes were incubated overnight at 4°C with primary antibodies. Immunocomplexes were incubated with horseradish peroxidase-conjugated polyclonal anti-mouse or anti-rabbit IgG for 1 h at room temperature (diluted at 1:500) and visualized using an ECL kit (Amersham Biosciences) based on the manufacturer’s instructions.

Statistical analysis

Each experiment was repeated at least 3 times. The data are presented as the means ± standard deviation (SD). The results were analyzed by one-way analysis of variance. All statistical analyses were performed using SPSS 13.0 software (SPSS Inc., Chicago, IL, USA). A value of P<0.05 was considered to indicate a statistically significant difference.

Results

Notch1 expression is elevated in HCC cells

We first examined the baseline mRNA expression level of Notch1 in the L02, HepG2 and MHCC97H cell lines by RT-PCR. The Notch1 transcript was highly expressed in the HCC cells compared to the normal liver cell line (Fig. 1). Based on the gene expression data, we hypothesized that Notch1 expression may be associated with the invasion of HCC cells.

Notch1 silencing using siRNA

The HepG2 and MHCC97H cells, which have relatively high expression levels of Notch1, were transiently transfected with Notch1-siRNA or mock siRNA. We designed 3 candidate Notch1-specific sequences and one control sequence (mock). RT-PCR was performed to assess the knockdown efficiency of the candidate siRNAs. As illustrated in Fig. 2, the candidate sequence 1 most effectively inhibited Notch1 mRNA expression compared to the control. Thus, this siRNA was selected for use in the subsequent experiments. Notch1 mRNA and protein expression was quantified and analyzed by RT-PCR and western blot analysis, respectively, 72 h following transfection with siRNA. Compared to the control (no siRNA) and mock-transfected cells (negative control siRNA), Notch1 mRNA and protein expression was markedly decreased in the cells transfected with Notch1-siRNA (Fig. 3).

Downregulation of Notch1 expression suppresses HCC cell migration and invasion

To determine whether the down-regulation of Notch1 expression affects the migratory ability of the HepG2 and MHCC97H cells, we performed a wound-healing assay. The migration of HepG2 cells was significantly inhibited by Notch1 knockdown. The size of the wound in the Notch1-siRNA group was 0.78±0.09 mm, which was significantly larger than the size of the wound in either the mock-transfected group (0.32±0.11 mm) or the control group (0.29±0.07 mm) (P<0.01, n=6). Similar results were obtained for the MHCC97H cells; the size of the wound in the Notch1-siRNA group was 0.83±0.07 mm, compared to 0.44±0.13 mm in the mock group and 0.46±0.10 mm in the control group (P<0.01, n=6) (Fig. 4). These results demonstrate that the siRNA-mediated knockdown of Notch1 inhibits the migration of HepG2 and MHCC97H cells. The results from Transwell Matrigel invasion assays were consistent with our wound-healing assay results. As shown in Fig. 5, the number of HepG2 cells that successfully invaded through the chamber was lower in the Notch1-siRNA group (22±8.19) compared to both the mock group (47.67±3.51) and the control group (52.33±6.81) (P<0.01). The same was true for the MHCC97H cells (Notch1-siRNA, 36.33±5.51; mock, 79.67±8.51; control, 87.00±10.15; P<0.01). Taken together, our data support a role for Notch1 in the migratory and invasive capabilities of HepG2 and MHCC97H cells.

Downregulation of Notch1 alters the expression of PTEN and FAK

PTEN is a critical tumor suppressor gene located on human chromosome 10q23 (12). FAK (13) has been shown to be an important mediator of cell adhesion, growth, proliferation, survival, angiogenesis and migration, all of which are often disrupted in cancer cells. PTEN interacts with FAK and reduces its tyrosine phosphorylation (14). As shown in Fig. 6, the downregulation of Notch1 in HepG2 and MHCC97H cells increased the expression of both PTEN and phospho-PTEN and decreased the expression of FAK and phospho-FAK compared to the control and mock-transfected cells.

Discussion

Invasion and metastasis are the primary cause of mortality from HCC. Thus, novel therapies that specifically inhibit these processes are critical. The inhibition of cell signaling pathways for antitumor efficacy has shown great promise (15). It has recently been demonstrated that the persistent activation of Notch signaling is associated with liver malignancies (10). In humans, the Notch family of transmembrane proteins consists of four receptors (Notch1 through Notch4). Importantly, the high expression of Notch1 in HCC has been shown to correlate with an advanced TNM stage and blood vessel infiltration (8). In this study, we found that Notch1 expression was elevated in two HCC cell lines compared to normal liver cells. Thus, Notch1 may be a potential therapeutic target in HCC.

To elucidate the functional relevance of Notch1 in HCC, we modulated Notch1 expression levels in HCC cell lines using siRNA. Clinically, Notch signaling can be inhibited by one of three ways. First, the activation of the Notch receptor can be inhibited by the use of gamma-secretase inhibitors (GSIs). Second, ligand binding to the Notch receptor can be blocked by monoclonal antibodies. Finally, the transcriptional activity of the Notch intracellular domain can be inhibited using blocking peptides. Inhibition by siRNA, as used in the present study, is likely most similar to inhibition via the prevention of ligand binding. The use of siRNA tends to show greater specificity than GSIs, which are not cell-type specific. Moreover, GSIs have a considerable toxicity profile. Our results demonstrated that the downregulation of Notch1 expression in HepG2 and MHCC97H cells by siRNA suppressed HCC cell migration and invasion. Recent data provided by others supports our findings. For example, Zhou et al (9) demonstrated that GSIs suppress the invasion of HCC cells; however, Notch1 was not analyzed in their study. Our data support a new role for Notch1 in HCC cell invasion.

Notably, we found that the total and phosphorylated levels of PTEN were increased in the HCC cells following Notch1 depletion. Consistent with this finding, GSI treatment has been shown to upregulate PTEN protein expression in the primary-like leukemia cell line, TAIL7 (16). Palomero et al (17) also reported that Notch1 negatively regulates PTEN at the transcriptional level. PTEN protein was originally identified as a potent tumor suppressor (1821). PTEN reduces the rates of migration through several mechanisms. One mechanism involves effects on cell adhesion. FAK is a key molecule implicated in integrin and growth factor-mediated signaling, and plays an important role in cell adhesion. FAK has also been shown to interact with PTEN to influence tumor cell invasion (14). FAK is an important tyrosine kinase that regulates tumor invasion and survival (2224), and it is significantly overexpressed in HCC (2527). Growing evidence indicates that the inhibition of FAK may be a useful therapy against cancer cell metastasis (2830). PTEN is a phosphatase that can negatively regulate FAK tyrosine phosphorylation (31,32). The decreased phosphorylation of FAK mediated by PTEN inhibits cellular migration, spreading and adhesion. In the present study, we demonstrated that the downregulation of Notch1 by siRNA in HepG2 and MHCC97H cells increased PTEN expression and decreased the expression of FAK and phospho-FAK. We hypothesized that the downregulation of Notch1 may inhibit HCC through the upregulation of PTEN and the subsequent inactivation of FAK. In conclusion, Notch1-siRNA affects the balance of phospho-FAK and FAK by increasing the levels of PTEN and phospho-PTEN; in effect, these molecular changes help suppress HCC invasion. Whether or not FAK phosphorylation is inversely correlated with PTEN levels in HCC cell lines transfected with Notch1-siRNA requires additional research. We suggest that the Notch1-PTEN-FAK signaling axis may be a critical determinant of liver cancer metastasis.

In conclusion, our results demonstrate that the downregulation of Notch1 by siRNA in HepG2 and MHCC97H cells decreases cell invasion. Furthermore, decreasing Notch1 expression upregulates PTEN and phospho-PTEN and downregulates FAK and phospho-FAK expression. The Notch1-PTEN-FAK signaling axis may be critical for HCC invasion and may represent a novel therapeutic target in the disease to inhibit metastasis.

Acknowledgements

We thank the No. 3 People’s Hospital Affiliated with Shanghai Jiao Tong University and the Liver Cancer Institute of Zhong Shan Hospital Affiliated with Fudan University for kindly providing the human liver non-tumor cell line (L02) and the HCC cell line (MHCC97H). The present study was supported by grants from the Postdoctoral Science Foundation of China (no. 2011M500686) and the Science and Technology Research Foundation of Heilongjiang Province Department of Education of China (no. 12521178).

References

1 

Siegel R, Ward E, Brawley O and Jemal A: Cancer statistics, 2011: the impact of eliminating socioeconomic and racial disparities on premature cancer deaths. CA Cancer J Clin. 61:212–236. 2011. View Article : Google Scholar : PubMed/NCBI

2 

Marquardt JU, Galle PR and Teufel A: Molecular diagnosis and therapy of hepatocellular carcinoma (HCC): an emerging field for advanced technologies. J Hepatol. 56:267–275. 2012. View Article : Google Scholar : PubMed/NCBI

3 

Wang YH, Dong YY, Wang WM, et al: Vascular endothelial cells facilitated HCC invasion and metastasis through the Akt and NF-κB pathways induced by paracrine cytokines. J Exp Clin Cancer Res. 32:512013.PubMed/NCBI

4 

Chen JS, Huang XH, Wang Q, et al: Sonic hedgehog signaling pathway induces cell migration and invasion through focal adhesion kinase/AKT signaling-mediated activation of matrix metalloproteinase (MMP)-2 and MMP-9 in liver cancer. Carcinogenesis. 34:10–19. 2013. View Article : Google Scholar

5 

Liu L, Dai Y, Chen J, et al: Maelstrom promotes hepatocellular carcinoma metastasis by inducing epithelial-mesenchymal transition by way of Akt/GSK-3β/Snail signaling. Hepatology. 59:531–543. 2014.PubMed/NCBI

6 

Bolós V, Grego-Bessa J and de la Pompa JL: Notch signaling in development and cancer. Endocr Rev. 28:339–363. 2007.

7 

Hu YY, Zheng MH, Zhang R, Liang YM and Han H: Notch signaling pathway and cancer metastasis. Adv Exp Med Biol. 727:186–198. 2012. View Article : Google Scholar : PubMed/NCBI

8 

Wang XQ, Zhang W, Lui EL, et al: Notch1-Snail1-E-cadherin pathway in metastatic hepatocellular carcinoma. Int J Cancer. 131:E163–E172. 2012. View Article : Google Scholar : PubMed/NCBI

9 

Zhou L, Wang DS, Li QJ, Sun W, Zhang Y and Dou KF: Downregulation of the Notch signaling pathway inhibits hepatocellular carcinoma cell invasion by inactivation of matrix metalloproteinase-2 and -9 and vascular endothelial growth factor. Oncol Rep. 28:874–882. 2012.

10 

Ahn S, Hyeon J and Park CK: Notchl and Notch4 are markers for poor prognosis of hepatocellular carcinoma. Hepatobiliary Pancreat Dis Int. 12:286–294. 2013. View Article : Google Scholar : PubMed/NCBI

11 

Fox V, Gokhale PJ, Walsh JR, Matin M, Jones M and Andrews PW: Cell-cell signaling through NOTCH regulates human embryonic stem cell proliferation. Stem Cells. 26:715–723. 2008. View Article : Google Scholar : PubMed/NCBI

12 

Singh G and Chan AM: Post-translational modifications of PTEN and their potential therapeutic implications. Curr Cancer Drug Targets. 11:536–547. 2011. View Article : Google Scholar : PubMed/NCBI

13 

Golubovskaya VM: Focal adhesion kinase as a cancer therapy target. Anticancer Agents Med Chem. 10:735–741. 2010. View Article : Google Scholar : PubMed/NCBI

14 

Zhang L, Yu Q, He J and Zha X: Study of the PTEN gene expression and FAK phosphorylation in human hepatocarcinoma tissues and cell lines. Mol Cell Biochem. 262:25–33. 2004. View Article : Google Scholar : PubMed/NCBI

15 

Wong R, Frenette C and Gish R: Hepatocellular carcinoma: locoregional and targeted therapies. Gastroenterol Clin North Am. 40:599–610. 2011. View Article : Google Scholar : PubMed/NCBI

16 

Silva A, Jotta PY, Silveira AB, et al: Regulation of PTEN by CK2 and Notch1 in primary T-cell acute lymphoblastic leukemia: rationale for combined use of CK2-and γ-secretase inhibitors. Haematologica. 95:674–678. 2010.PubMed/NCBI

17 

Palomero T, Sulis ML, Cortina M, et al: Mutational loss of PTEN induces resistance to NOTCH1 inhibition in T-cell leukemia. Nat Med. 13:1203–1210. 2007. View Article : Google Scholar : PubMed/NCBI

18 

Wang L, Wang WL, Zhang Y, Guo SP, Zhang J and Li QL: Epigenetic and genetic alterations of PTEN in hepatocellular carcinoma. Hepatol Res. 37:389–396. 2007. View Article : Google Scholar : PubMed/NCBI

19 

Chow LM and Baker SJ: PTEN function in normal and neoplastic growth. Cancer Lett. 241:184–196. 2006. View Article : Google Scholar : PubMed/NCBI

20 

Hu TH, Huang CC, Lin PR, et al: Expression and prognostic role of tumor suppressor gene PTEN/MMAC1/TEP1 in hepatocellular carcinoma. Cancer. 97:1929–1940. 2003. View Article : Google Scholar : PubMed/NCBI

21 

Dong-Dong L, Xi-Ran Z and Xiang-Rong C: Expression and significance of new tumor suppressor gene PTEN in primary liver cancer. J Cell Mol Med. 7:67–71. 2003. View Article : Google Scholar : PubMed/NCBI

22 

Thanapprapasr D, Hu W, Sood AK and Coleman RL: Moving beyond VEGF for anti-angiogenesis strategies in g ynecologic cancer. Curr Pharm Des. 18:2713–2719. 2012. View Article : Google Scholar : PubMed/NCBI

23 

Bai X, Wang J, Zhang L, et al: Prostaglandin E2 receptor EP1-mediated phosphorylation of focal adhesion kinase enhances cell adhesion and migration in hepatocellular carcinoma cells. Int J Oncol. 42:1833–1841. 2013.

24 

Zhang C, He H, Zhang H, et al: The blockage of Ras/ERK pathway augments the sensitivity of SphK1 inhibitor SKI II in human hepatoma HepG2 cells. Biochem Biophys Res Commun. 434:35–41. 2013. View Article : Google Scholar : PubMed/NCBI

25 

Chen JS, Huang XH, Wang Q, et al: FAK is involved in invasion and metastasis of hepatocellular carcinoma. Clin Exp Metastasis. 27:71–82. 2010. View Article : Google Scholar : PubMed/NCBI

26 

Lau GM, Lau GM, Yu GL, et al: Expression of Src and FAK in hepatocellular carcinoma and the effect of Src inhibitors on hepatocellular carcinoma in vitro. Dig Dis Sci. 54:1465–1474. 2009. View Article : Google Scholar : PubMed/NCBI

27 

Han S, Han L, Yao Y, Sun H, Zan X and Liu Q: Activated hepatic stellate cells promote hepatocellular carcinoma cell migration and invasion via the activation of FAK-MMP9 signaling. Oncol Rep. 31:641–648. 2014.PubMed/NCBI

28 

Ko BS, Jan YJ, Chang TC, et al: Upregulation of focal adhesion kinase by 14-3-3ɛ via NFκB activation in hepatocellular carcinoma. Anticancer Agents Med Chem. 13:555–562. 2013.PubMed/NCBI

29 

Sheng SL, Liu JJ, Dai YH, Sun XG, Xiong XP and Huang G: Knockdown of lactate dehydrogenase A suppresses tumor growth and metastasis of human hepatocellular carcinoma. FEBS J. 279:3898–3910. 2012. View Article : Google Scholar : PubMed/NCBI

30 

Xu HY, Qian AR, Shang P, et al: siRNA targeted against HAb18G/CD147 inhibits MMP-2 secretion, actin and FAK expression in hepatocellular carcinoma cell line via ERK1/2 pathway. Cancer Lett. 247:336–344. 2007. View Article : Google Scholar : PubMed/NCBI

31 

Chetram MA and Hinton CV: PTEN regulation of ERK1/2 signaling in cancer. J Recept Signal Transduct Res. 32:190–195. 2012. View Article : Google Scholar : PubMed/NCBI

32 

Tamura M, Gu J, Danen EHJ, et al: PTEN interactions with focal adhesion kinase and suppression of the extracellular matrix-dependent phosphatidylinositol 3-kinase/Akt cell survival pathway. J Biol Chem. 274:20693–20703. 1999. View Article : Google Scholar : PubMed/NCBI

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October 2014
Volume 34 Issue 4

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Spandidos Publications style
Hu Y, Li H, Qiu K, Li D, Zhou J, Hu Y and Zhang F: Downregulation of Notch1 inhibits the invasion of human hepatocellular carcinoma HepG2 and MHCC97H cells through the regulation of PTEN and FAK. Int J Mol Med 34: 1081-1086, 2014
APA
Hu, Y., Li, H., Qiu, K., Li, D., Zhou, J., Hu, Y., & Zhang, F. (2014). Downregulation of Notch1 inhibits the invasion of human hepatocellular carcinoma HepG2 and MHCC97H cells through the regulation of PTEN and FAK. International Journal of Molecular Medicine, 34, 1081-1086. https://doi.org/10.3892/ijmm.2014.1889
MLA
Hu, Y., Li, H., Qiu, K., Li, D., Zhou, J., Hu, Y., Zhang, F."Downregulation of Notch1 inhibits the invasion of human hepatocellular carcinoma HepG2 and MHCC97H cells through the regulation of PTEN and FAK". International Journal of Molecular Medicine 34.4 (2014): 1081-1086.
Chicago
Hu, Y., Li, H., Qiu, K., Li, D., Zhou, J., Hu, Y., Zhang, F."Downregulation of Notch1 inhibits the invasion of human hepatocellular carcinoma HepG2 and MHCC97H cells through the regulation of PTEN and FAK". International Journal of Molecular Medicine 34, no. 4 (2014): 1081-1086. https://doi.org/10.3892/ijmm.2014.1889