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ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 4  |  Page : 844-850

Promotion of Insulin-like growth factor II in cell proliferation and epithelial–mesenchymal transition in hepatocellular carcinoma


Department of Gastroenterology, The First Affiliated Hospital of Jinan University, Guangzhou, China

Date of Web Publication27-Jun-2018

Correspondence Address:
Wei Huang
Department of Gastroenterology, The First Affiliated Hospital of Jinan University, Guangzhou, 510630
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_605_17

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 > Abstract 


Aim: This study aims to investigate the involvement of insulin-like growth factor II (IGF-II) in human hepatocellular carcinoma (HCC) proliferation and metastasis.
Materials and Methods: The effects of IGF-II on cell proliferation, cell cycle, apoptosis, cell migration, and invasion in HCC Huh7 cells were investigated in the study.
Results: IGF-II promoted cell proliferation and colony formation, suppressed cell apoptosis in Huh7 cells by promoting cell cycle progression, induced epithelial–mesenchymal transition (EMT) phenotypes, and enhanced the metastatic potential of HCC in vitro.
Conclusion: Our results revealed that IGF-II promotes cell proliferation and EMT in HCC cells.

Keywords: Cell proliferation, epithelial–mesenchymal transition, hepatocellular carcinoma, insulin-like growth factor II, metastasis


How to cite this article:
Ma Y, Chen Y, Chen L, Liu Z, Ieong ML, Gao F, Huang W. Promotion of Insulin-like growth factor II in cell proliferation and epithelial–mesenchymal transition in hepatocellular carcinoma. J Can Res Ther 2018;14:844-50

How to cite this URL:
Ma Y, Chen Y, Chen L, Liu Z, Ieong ML, Gao F, Huang W. Promotion of Insulin-like growth factor II in cell proliferation and epithelial–mesenchymal transition in hepatocellular carcinoma. J Can Res Ther [serial online] 2018 [cited 2019 Nov 17];14:844-50. Available from: http://www.cancerjournal.net/text.asp?2018/14/4/844/235092




 > Introduction Top


Hepatocellular carcinoma (HCC) is one of the most common aggressive tumors worldwide, and its treatment remains highly challenging due to poor prognosis and its potential for recurrence and metastasis even after surgical resection.[1],[2],[3] Epithelial–mesenchymal transition (EMT), a traditional phenomenon revealed in embryonic development, has been gradually accepted as a potential mechanism underlying cancer progression and metastasis. Evidences suggest that EMT plays critical roles in different aspects of cancer progression such as metastasis, stem cell features, and chemoresistance.[4],[5],[6] Hence, it is important to understand the molecular changes associated with HCC progression and metastasis. Among these changes, the activation of oncogenes and inactivation of tumor suppressor genes may play important roles in tumor formation and development.[7],[8],[9],[10],[11]

Insulin-like growth factor II (IGF-II) plays a key role in mammalian growth and influences fetal cell division and differentiation, and possibly metabolic regulation.[12] Furthermore, it has been reported that IGF-II is related to a series of pathological processes in various cancers such as invasion, metastasis, and prognosis.[13],[14],[15] Since the roles of IGF-II in the pathogenesis of HCC are not well elucidated, these aforementioned findings prompted the investigators to investigate whether IGF-II is associated with the cell growth, apoptosis, and migration of human HCC.


 > Materials and Methods Top


Cell lines and cell culture

Human HCC Huh7 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum in a humidified incubator with 5% CO2 at 37°C.

MTT assay

Cells (1 × 103) were plated onto 96-well plates in 80% growth medium and allowed to adhere overnight. At different time points (day 0, 1, 2, and 3), the culture medium was removed and replaced with a culture medium containing MTT (Methylthiazolyldiphenyl-tetrazolium bromide) dye (5 mg/ml). After incubation at 37°C for 4 h, the MTT solution was removed, and dimethyl sulfoxide was added to dissolve the formazan crystals. Spectrometric absorbance at 570 nm was measured using a microplate photometer (Thermo, Waltham, MA, USA).

Colony formation assay

Cells were plated in 6-well plates at 200 per well, and grown for 2 weeks. After 2 weeks, cells were washed twice with phosphate-buffered saline (PBS), fixed with methanol/acetic acid (3:1, v/v), and stained with hematoxylin. Then, the number of colonies was counted.

Annexin V-PE apoptosis detection kit

Cells were spun down to remove the supernatant and resuspended in 100 μl of Annexin V Binding Buffer. Then, 5 μl of Annexin V-PE and 5 μl of 7-AAD from the Annexin V-PE Apoptosis Detection Kit (Keygen Biotech, Nanjing, Jiangsu, China) were added into the solution, according to the instructions of the kit. After 15 min of incubation, 400 μl of Annexin V Binding Buffer was added. The Annexin V-PE and 7-AAD stained cells were analyzed through the FL2 (Ex= 488 nm; Em= 578 nm) and FL4 channels (Ex= 546 nm; Em= 647 nm).

RNA isolation, reverse transcription and quantitative reverse transcription-polymerase chain reaction

For mRNA analyses, total RNA was extracted using the Trizol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's protocol. Total RNA was reverse transcribed using the PrimeScript RT reagent Kit (TaKaRa, Kyoto, Japan). For the analysis of mRNA expression, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed on a Stratagene Mx3005P qRT-PCR system using the SYBR Green qRT-PCR master mix (TaKaRa, Kyoto, Japan), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for normalization. All samples were normalized to internal controls, and fold changes were calculated through relative quantification (2△△Ct).

Western blot analysis

Protein lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresisSDS-PAGE, and electrophoretically transferred onto a polyvinylidene difluoride membrane (millipore). The membrane was incubated with antibodies, IGF-II (Bioss, Beijing, China), Vimentin (Abcam, Cambridge, MA, USA), E-cadherin (Abcam, Cambridge, MA, USA), and N-cadherin (Abcam, Cambridge, MA, USA). Then, the membrane was incubated with HRP-labeled goat-anti-mouse or rabbit IgG, and the proteins were detected by a high-sensitivity chemiluminescence imaging system (BIORAD, Hercules, CA, USA). GAPDH was used as the protein-loading control.

Cell migration assays

For the cell migration assay, 1 × 105 cells in 100 μl of RPMI 1640 medium without newborn calf serum were seeded onto a fibronectin-coated polycarbonate membrane inserted in a Transwell apparatus (Corning, Corning, NY, USA). In the lower chamber, 500 μl of RPMI 1640 with 10% NBCS was added as a chemoattractant. After cells were incubated for 20–24 h at 37°C in a 5% CO2 atmosphere, the inserted membrane was washed with PBS, and cells on the top surface of the insert were removed with a cotton swab. Cells that adhered to the lower surface were fixed with methanol, stained with hematoxylin, and counted under a microscope in five predetermined fields (200×). All assays were independently repeated at least thrice.

Scratch migration assay

Cells were scratched using the tip of a sterile 10-μl pipette (width: Approximately 1 mm) in each well. The plates were washed twice with PBS to remove the detached cells, and incubated at 37°C in 5% CO2. Wound closure was monitored at various time points by observation under a microscope, and the degree of cell migration was quantified by the ratio of the gap distance at 24 h to that at 0 h. The experiment was performed in triplicate.

Ethics approval

This study was conducted in accordance with the Declaration of Helsinki. This study was conducted with approval from the Ethics Committee of the First Affiliated Hospital of Jinan University.

Written informed consent was obtained from all participants.


 > Results Top


Insulin-like growth factor II promotes cell growth in human hepatocellular carcinoma Huh7 cells

To explore the effect of IGF-II on cell growth, HCC Huh7 cells were transfected with pcDNA3.1(+)-IGF-II (IGF-II) or pLVX-shRNA2-IGF-II (siIGF-II), respectively, RNA and protein were extracted 48 h after the transfection. As shown in [Figure 1]a and [Figure 1]b, real-time PCR and western blot results revealed that IGF-II was successfully overexpressed or silenced. Furthermore, MTT assay results revealed that IGF-II promoted cell proliferation in Huh7 cells, compared with IGF-II control (IGF-II con), while IGF-II siRNA inhibited cell proliferation in HCC cells [Figure 1]c, (P< 0.05), compared with IGF-II siRNA control (siIGF-II con).
Figure 1: Insulin-like growth factor II promotes cell growth in human hepatocellular carcinoma Huh7 cells. (a) Insulin-like growth factor II expression detected by qPCR; (b) Insulin-like growth factor II expression detected by Western blot; (c) Effects of insulin-like growth factor II overexpression or inhibition on cell growth showed by MTT assay; (d and e) Effects of insulin-like growth factor II overexpression or inhibition on cell growth showed by colony formation assay P < 0.05

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[Figure 1]d and [Figure 1]e showed the results of the colony formation assay, IGF-II-overexpressed Huh7 cells displayed more and bigger colonies compared with control cells, while siIGF-II-knockdown cells displayed much fewer and smaller colonies compared with control cells (P< 0.05).

Insulin-like growth factor II suppresses cell apoptosis and promotes cell cycle progression

To determine the effect of IGF-II on the cell cycle, Flow cytometry was used to measure cell cycle distribution in IGF-II-overexpressed and IGF-II-silencing cells. These results revealed that the S-phase population significantly increased in IGF-II-overexpressed cells, when compared with IGF-II-silencing cells [Figure 2]a and [Figure 2]b, (P< 0.05).
Figure 2: Insulin-like growth factor II suppresses cell apoptosis and promotes cell cycle progression. (a and b) Effects of insulin-like growth factor II on cell cycle

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Then, we detected cell apoptosis using the Annexin V-PE Apoptosis Detection Kit. Results indicated that IGF-II suppressed cell apoptosis, while IGF-II siRNA promoted cell apoptosis [Figure 3]a and [Figure 3]b, (P< 0.05).
Figure 3: Insulin-like growth factor II suppresses cell apoptosis and promotes cell cycle progression. (a and b) Effects of insulin-like growth factor II overexpression or inhibition on cell apoptosis using Annexin V-PE Apoptosis Detection Kit P < 0.05

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Insulin-like growth factor II induces epithelial–mesenchymal transition phenotypes and enhances the metastatic potential of human hepatocellular carcinoma in vitro

To determine whether IGF-II induces EMT, EMT-related genes were detected with epithelial marker E-cadherin and mesenchymal marker N-cadherin and vimentin in HCC cells. As shown in [Figure 4]a and [Figure 4]b, IGF-II downregulated E-cadherin expression and the upregulated N-cadherin and vimentin, which exhibited a typical EMT phenotype.
Figure 4: Insulin-like growth factor II induces epithelial–mesenchymal transition phenotype and enhances the metastatic potential of human hepatocellular carcinoma in vitro. (a) Expression of epithelial–mesenchymal transition-related genes demonstrated by qPCR; (b) Expression of epithelial–mesenchymal transition-related genes demonstrated by Western blot; (c and d) Effects of insulin-like growth factor II on cell mobility detected by transwell invasion assays P < 0.01

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As shown in [Figure 4]c and [Figure 4]d, IGF-II-overexpressed HCC cells exhibited significantly increased mobility compared with control cells, while IGF-II-silencing cells decreased mobility, as detected by Transwell invasion assays (P< 0.01). This result was confirmed by scratch migration assay [Figure 5]a and [Figure 5]b.
Figure 5: Insulin-like growth factor II induces epithelial–mesenchymal transition phenotype and enhances the metastatic potential of human hepatocellular carcinoma in vitro. (a and b) Effects of insulin-like growth factor II on cell mobility detected by scratch migration assay P < 0.01

Click here to view



 > Discussion Top


HCC, also called malignant hepatoma, is the most common type of liver cancer. In HCC, multiple genetic alterations happen frequently include activation of oncogenes and inactivation of tumor suppressor genes. These are correlated with increased stages of carcinogenesis and further tumor progression with numerous characteristics such as fast infiltrating growth, early-stage metastasis, high-grade malignancy, and poor therapeutic efficacy.[16],[17]

IGF-II, a mitogenic polypeptide closely related to insulin, is a kind of fetal growth factor; and its gene has a complex transcription regulation that results in multiple mRNA transcripts through different promoters. IGF-II is highly expressed during hepatocellular carcinogenesis and increases vascular endothelial growth factor expression in a time-dependent manner in hepatoma cells.[18] In the present study, we found that IGF-II promoted HCC cell proliferation.

Apoptosis was initially described by its morphological characteristics including cell shrinkage, membrane bursting, chromatin condensation, and nuclear fragmentation. Studies have demonstrated that apoptosis contributes to the high rate of cell loss in malignant tumors and promote tumor progression, while the loss of apoptosis can impact tumor initiation, progression, and metastasis.[19],[20] Cells go through a cell cycle to grow and divide to produce new cells, and an uncontrolled cell proliferation is the hallmark of cancer. In this study, we revealed that IGF-II suppressed cell apoptosis in Huh7 cells by promoting cell cycle progression, which indicated the promotion of cell proliferation.

Metastasis is the basic biological characteristic of malignant tumors and a primary factor that affects the patient survival, which has become a challenge and focus in the field of cancer research. The most prominent feature of tumor metastasis is tissue specificity, in which different tumors can form metastases in the same or different parts of the human body.[21] EMT is a process characterized by the loss of cell adhesion, repression of E-cadherin expression, and increased cell motility.[22] EMT has been gradually accepted as a potential mechanism underlying cancer progression and metastasis. In this study, we found that IGF-II induced EMT phenotypes and enhanced the metastatic potential of HCC in vitro. E-cadherin is associated with poor overall survival in patients. Activated E-cadherin inhibits cells proliferation and EMT. Our results showed that IGF-II inhibited E-cadherin, which mediated proliferation and EMT in HCC.

IGF-II plays an important role in the initiation of HCC and is involved in the EMT progression of HCC. However, the complex regulatory mechanism of IGF-II during cancer cell proliferation and metastasis promotion has not been fully elucidated. Hence, the elucidation of the underlying signaling network that regulates these IGF-II-associated changes in biological behavior would provide important insights into the exact role of IGF-II in HCC.


 > Conclusion Top


IGF-II promotes cell proliferation and EMT, plays an important role in the development of HCC.

Acknowledgments

This research was supported by the Science and Technology Project of Guangdong Province (No. 2010B080701063).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]



 

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