title: [Abnormal expression of insulin-like growth factor-I receptor and inhibitory effect of its transcription intervention on nude mice xenograft tumor].
abstract: Objective:To investigate the expression of insulin-like growth factor-I receptor (IGF-IR) in liver cancer and the inhibitory effect of its transcription intervention on nude mice xenograft tumor.Methods:A total of 40 patients with primary liver cancer were enrolled, and 40 samples of cancer lesions, peri-cancerous tissues (with a distance of 2 cm to the margin of cancer lesion), or distal liver tissues (with a distance of 5 cm to the margin of cancer lesion), with a weight of 200 mg, were collected after surgery. Some of these samples were used for pathological examination, and the rest were stored at -85°C. A total of 18 BALB/c nude mice aged 4-6 weeks with a body weight of 18-20 g (9 male and 9 female mice) were randomly divided into control group, negative control group, and co-intervention group, with 6 mice in each group, and fed under specific pathogen-free conditions. The cell line was cultured in the dimethyl sulfoxide complete medium containing 10% fetal bovine serum in a CO2incubator at 37°C. When the cell confluence reached 90% after cell inoculation, shRNA was divided into co-intervention group, negative control group, and untreated control group and were transfected to hepatoma cells using PolyJetTM transfection reagent. Stable cell clones obtained by G418 screening and used for the in vivo study. Immunohistochemistry, Western blotting, and quantitative real-time PCR were used to analyze the expression of IGF-IR in the human hepatoma tissue and cell line. The IGF-IR shRNA eukaryotic expression plasmids were established and screened for the most effective sequence; they were transfected to PLC/PRF/5 hepatoma cells, and the CCK-8 assay was used to analyze the changes in cell proliferation. The stable cell line screened out by G418 was inoculated to establish the subcutaneous xenograft tumor in nude mice. The tumor growth curve was plotted and histological examination was performed. Graphpad Prism 5.0 and SPSS 18.0 were used for plotting and data analysis; the variance test and Q test were used for comparison of means between multiple samples, the t-test was used for comparison of means between any two samples, the chi-square test or Fisher's exact test was used for comparison of rates between samples, and a rank correlation analysis was performed for expression intensity.Results:The liver cancer group had a significantly higher positive rate of IGF-IR than the peri-cancerous group and distal tissue group (82.5% vs 42.5%/10%,χ2= 13.653 and 42.29, bothP< 0.01), as well as significantly higher expression intensity than these two groups (Z= 4.771 and 6.579, bothP< 0.01). IGF-IR was not significantly expressed in the L02 cell line and was strongly expressed in the PLC/PRF/5 hepatoma cells, and the expression intensity of IGF-IR in the PLC/PRF/5 hepatoma cells was 4 and 5 times that in Bel-7404 cells and HepG2 cells, respectively. After the PLC/PRF/5 hepatoma cells were transfected with shRNA4 with the best co-intervention effect, the mean inhibition rate of tumor cell growth reached 63.9% at 72 hours, and the mean inhibition rate of IGF-IR transcription reached 59.6%. Tumor cells were arrested in G1 phase, and there was a significant increase in apoptosis rate. As for the subcutaneous hepatoma xenograft in nude mice, the intervention group had significantly slower tumor growth than the blank control group and negative control group (143±24 mm3 vs 372±46 mm3/350±50 mm3,t= 10.776 and 9.142, bothP< 0.01); the intervention group had significantly downregulated IGF-IR expression, which was significantly lower than that in the blank control group and negative control group (t= 11.184 and 9.450, bothP< 0.01).Conclusion:Intervention of IGF-IR transcription can effectively inhibit the growth of xenograft tumor in nude mice, suggesting that IGF-IR gene might become a new potential target for the treatment of liver cancer.
authors.affiliation: Research Center of Clinical Medicine, Affiliated Hospital of Nantong University, Nantong Jiangsu 226001, China.
authors.forename: M
authors.initials: M
authors.lastname: Yao
authors.forename: X D
authors.initials: XD
authors.lastname: Yan
authors.forename: Y
authors.initials: Y
authors.lastname: Cai
authors.forename: J J
authors.initials: JJ
authors.lastname: Gu
authors.forename: X L
authors.initials: XL
authors.lastname: Yang
authors.forename: L H
authors.initials: LH
authors.lastname: Pan
authors.forename: L
authors.initials: L
authors.lastname: Wang
authors.forename: D F
authors.initials: DF
authors.lastname: Yao
mesh_terms: D000818:Animals; D017209:Apoptosis; D006528:Carcinoma, Hepatocellular; D002453:Cell Cycle; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D015536:Down-Regulation / Q000235:genetics*; D005260:Female; D015316:Genetic Therapy / Q000379:methods; D056945:Hep G2 Cells / Q000378:metabolism*; D064593:Heterografts; D006801:Humans; D008113:Liver Neoplasms; D008297:Male; D051379:Mice; D008819:Mice, Nude; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D017526:Receptor, IGF Type 1 / Q000235:genetics* / Q000378:metabolism*; D013002:Somatomedins; D014162:Transfection; D023041:Xenograft Model Antitumor Assays
chemical_list: D012333:RNA, Messenger; D034741:RNA, Small Interfering; D013002:Somatomedins; D017526:Receptor, IGF Type 1