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Mechanism of FGF7 Gene Silencing in Regulating Viability, Apoptosis, Invasion of Retinoblastoma Cell Line HXO-Rb44 and Angiogenesis

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Mechanism of FGF7 Gene Silencing in Regulating Viability, Apoptosis, Invasion of Retinoblastoma Cell Line HXO-Rb44 and Angiogenesis European Review for Medical and Pharmacological Sciences 2020; 24: 3538-3547 Mechanism of FGF7 gene silencing in regulating viability, apoptosis, invasion of retinoblastoma cell line HXO-Rb44 and angiogenesis W.-J. CHEN1, C.-X. SUN2, W. LI3 1Department of Orthopedics Surgery, Tongji Hospital Affiliated with Tongji Medical College of Huazhong University of Science & Technology, Wuhan, Hubei Province, China 2Department of Ophthalmology, Yueyang Huashahengkang Hospital, Yueyang, Hunan Province, China 3Department of Ophthalmology, Union Hospital Affiliated with Tongji Medical College of Huazhong University of Science & Technology, Wuhan, Hubei Province, China Abstract. – OBJECTIVE: To explore the mech- Key Words: anism of fibroblast growth factor 7 (FGF7) gene Gene silencing, Retinoblastoma, Apoptosis, Inva- silencing in regulating viability, apoptosis, inva- sion. sion of retinoblastoma (RB) cell line HXO-Rb44 and angiogenesis. MATERIALS AND METHODS: Human normal retinal vascular endothelial cells ACBRI-181 was set as the normal group. The cultured RB cell lines Introduction HXO-Rb44 were divided into three groups: the blank group (without plasmid transfection), nega- Retinoblastoma (RB) is a common primary tive control group (transfection of FGF7 plasmid), intraocular malignant cancer in children that aris- and the si-FGF7 group (transfection of FGF7 siR- NA plasmid). Quantitative Real Time-Polymerase es mainly in the embryonal nuclear layer of the Chain Reaction and Western blot were used to retina. Retinoblastoma is often accompanied by measure the mRNA and protein expression of strabismus and leukocoria in children1. Early di- B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X agnosis and treatment of RB is the key to improve protein (Bax), vascular endothelial growth fac- eye retention rate. However, many children with tor (VEGF), basic fibroblast growth factor (bF- RB in China are at the middle and advanced stage GF), angiopoietin-2 (Ang-2), and proliferating upon diagnosis and can only survive through enu- cell nuclear antigen (PCNA) in each group. The 2 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoli- cleation of the eye . Current treatments for RB um bromide (MTT) assay, transwell invasion as- mainly include local treatment modalities, such as say, and flow cytometry were used, respectively, enucleation, interventional therapy, cryotherapy to assess cell viability, invasive capability, and and laser photocoagulation, chemotherapy, and cell apoptosis in each group. 3 RESULTS: gene therapy . Gene therapy is a new approach to The mRNA and protein expression the treatment of tumors in recent years. It mainly of FGF7, Bcl-2, VEGF, bFGF, Ang-2, and PCNA were significantly decreased, and the mRNA uses genes as therapeutic targets to selectively kill 4 and protein expression of Bax were significantly tumor cells and promote their apoptosis . Most increased in the si-FGF7 group than in the blank tumors exhibit the inactivation of tumor suppres- group (all p<0.05). Compared with the blank sor genes and significant activation of proto-on- group, the si-FGF7 group had significantly de- cogenes5,6. Jia et al7 confirmed that the inhibition creased cells invasive capability, cell viability at of ABCG2 gene expression can significantly sup- 48 h and 72 h and proliferation, and significantly increased apoptosis rate (all p<0.05). press the growth of RB. Angiogenesis plays an CONCLUSIONS: FGF7 gene silencing can in- important role in the growth of RB. Therefore, hibit the viability and invasion of RB cells and the inhibition of neovascularization is a poten- the expression of angiogenesis-related factors tially effective approach for the treatment of RB8. and can promote RB apoptosis. Yang et al9 confirmed by in vitro research that 3538 Corresponding Author: Wan Li, MD; e-mail: [email protected] Effects of FGF7 gene silencing on retinoblastoma the increase of the expression of pigment epithe- used in all experiments. Human normal retinal lium-derived factor (PEDF) gene can effectively vascular endothelial cells ACBRI-181 were set reduce the angiogenesis of RB and promote the as the normal group. RB cell lines HXO-Rb44 apoptosis of RB cells. Fibroblast growth factor 7 were divided into three groups: the blank group (FGF7), also known as keratinocyte growth fac- (without plasmid transfection), negative control tor (KGF) for its effect of promoting the mitosis (NC) group (transfection of FGF7 plasmid), and of keratinocytes, was first isolated and purified the si-FGF7 group (transfection of FGF7 siRNA by Rubin et al10 from the supernatant of human plasmid). embryonic lung fibroblasts. Since KGF needs to FGF7 siRNA (5’-CCCTGAGCGACACA- bind to its receptor, which is mainly distributed CAAGAAGTTAT-3’) and negative control se- in epithelial cells, to perform its function, KGF is quence (5’-CCCGCGACACAAACAGAAGTGT- also considered to mainly act on epithelial cells11. TAT-3’) were designed by BLOCK-iTTM RNAi Husseman et al12 also confirmed that FGF7 can Designer and synthesized by The Beijing Ge- promote the proliferation of vascular endothelial nomics Institute (BGI, Beijing, China). These cells and accelerate neovascularization. Current sequences were respectively constructed into research on FGF7 demonstrates that it plays a the plasmid vector pcDNA3.1 with Hind III and catalytic role in the progression of a variety of XHo I restriction enzyme sites at 16°C overnight. tumors. Huang et al13 found that FGF7 and its The recombinant pcDNA3.1-FGF7 plasmids were receptor FGFR2 were significantly increased in added to DH5ɑ for transformation. Antibiotic-re- gastric cancer tissues, and the in vitro studies sistant colonies were screened and identified by found that the overexpression of FGF7 could pro- colony digestion PCR and amplified, and the plas- mote the migration and invasion of gastric cancer mids were extracted and stored at -20°C. cells. Fan et al14 found that FGF7 overexpression The cells in each group were inoculated into is an independent risk factor for bladder cancer a 24-well plate, with five replicate wells for each and urothelial carcinoma. In addition, Shang et group. When the confluence reached 50%-60%, al15 observed that miR-381-3p can specifically transfection was performed using Lipofect- inhibit the expression of FGF7 gene and, there- amine™ 2000 Transfection Reagent (Invitrogen, fore, suppress the proliferation of cervical cancer Carlsbad, CA, USA) according to its instruc- cells. However, the association between the FGF7 tions. Liposomes and target RNA mixture were gene and the pathogenesis of RB remains unclear. prepared in two separate sterile Eppendorf (EP; From the perspective of gene-targeted therapy, we Hamburg, Germany) tubes. The liposomes were studied the effect of FGF7 gene expression on the prepared by mixing 1 μl Lipofectamine 2000 with biological characteristics of RB cells and the tu- 100 μl serum-free medium and were kept at room mor angiogenesis by silencing FGF7 gene using temperature for 5 min. The target RNA mixture RNA interference technology, aiming at provid- was prepared by mixing 20 pmol target RNA with ing theoretical basis and experimental evidence 50 μl serum-free medium. The RNA mixture was for RB gene therapy targeting FGF7. then mixed with liposomes to allow the formation of RNA-liposome complex. The complex was then kept at room temperature for 20 min before being Materials and Methods added to the cell cultures and incubated at 37°C in a 5% CO2 incubator. After 6-8 h of incubation, Cell Grouping and Transfection these cells were grown in complete growth medi- RB cell line HXO-Rb44 and human normal um for 24-48 h before the performance of subse- retinal vascular endothelial cell line ACBRI-181 quent experiments. All plasmids were purchased were purchased from the cell center of Xiangya from RiboBio Co., (Guangzhou, China). School of Medicine, Central South University. The cell lines were cultured as adherent cells mRNA Expression of Genes in Roswell Memorial Park Institute (RMPI- by Quantitative Real Time-Polymerase 1640) medium (Thermo Fisher Scientific, Inc., Chain Reaction (qRT-PCR) Waltham, MA, USA) supplemented with 10% Cells were collected 48 hours after transfec- fetal bovine serum (FBS; Thermo Fisher Scien- tion, and centrifuged at 2,000 r/min. The superna- tific, Inc., Waltham, MA, USA) at 37°C in a 5% tants were discarded, followed by RNA extraction CO2 incubator. The cells were routinely passaged for cells in each group according to the instruc- every 3 days and the cells in the log phase were tions of TRIzol reagent (Thermo Fisher Scien- 3539 W.-J. Chen, C.-X. Sun, W. Li tific, Inc., Waltham, MA, USA). The primers for VEGF, basic fibroblast growth factor (bFGF), an- FGF7, B-cell lymphoma 2 (Bcl-2), Bcl-2-associat- giopoietin-2 (Ang-2), and PCNA were normalized ed X protein (Bax), vascular endothelial growth to the GAPDH internal control. The relative tran- factor (VEGF), proliferating cell nuclear antigen scription levels of the target genes were calculated (PCNA), and glyceraldehyde 3-phosphate dehy- using the relative mRNA quantification, the 2-ΔΔCt 16 drogenase (GAPDH) were designed and synthe- method: ΔΔCT = ΔCt (target gene) – ΔCt (internal control) . sized by TaKaRa Bio Inc. (Dalian, China), as Relative transcription levels of target genes = shown in Table I. Reverse transcription of RNA 2-ΔΔCt. was performed using PrimeScript RT reagent kit (RR036A; TaKaRa Bio Inc., Dalian, China). The Protein Expression of Genes reverse transcription was carried out according to by Western Blot the manufacturer’s instructions of the kit based Cells were collected 48 hours after transfec- on 10 μl reactions. Each reaction was incubated at tion and 1 ml of lysate was added with shaking 37°C for three times, each time 15 min, followed once every 10 min. The mixtures were then cen- by inactivation of reverse transcriptase at 85°C for trifuged at 4°C for 20 min at a speed of 4,000 r/ 5 min. The reaction solutions were then harvest- min. The levels of the proteins were determined ed to carry out quantitative fluorescence PCR ac- by BCA Protein Assay Kit (Shanghai Yeasen Bio- cording to the instructions of SYBR® Premix Ex technology Co.
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