4 FOXQ1 SMMC suggested that FOXQ1 was upregulated in HCC The abnormal expression of FOXQ1 related to the HCC progress and clinicopahology Inh

Transcription

1 ISSN CN / Q http / /cjbmb bjmu edu cn Chinese Journal of Biochemistry and Molecular Biology ~ 428 DOI /j cnki cjbmb FOXQ1 SMMC * Q1 forkhead box Q1 FOXQ1 qrt-pcr Western 24 L02 SMMC-7721 FOXQ1 mrna 68 FOXQ1 shrna-foxq1 shrna-nc SMMC-7721 shrna-foxq1 qrt-pcr Western FOXQ1 VEGF SMMC-7721 FOXQ1 mrna P < 0 05 FOXQ1 TNM P < 0 05 shrna-foxq1 shrna-nc P < 0 05 FOXQ1 VEGF shrna-nc P < 0 05 FOXQ1 FOXQ1 FOXQ1 R735 7 FOXQ1 Levels in SMMC-7721 Human Hepatocellular Carcinoma Cells and Clinical Samples DENG Da-Wei 1 KONG Xian-Bing 1 * WANG-Ping 1 YU Qing-San 1 WANG-Qiang 2 1 Department of Hepatobiliary Surgery 2 Department of Gastrointestinal Surgery First Hospital Affiliated to Chongqing Medical University Chongqing China Abstract To investigate the clinical significance of forkhead box Q1 FOXQ1 in human hepatocellular carcinoma HCC FOXQ1 mrna and protein expression were detected by qrt-pcr and Western blotting in 24 pairs of HCC tissues paracarcinoma tissues hepatic cells LO2 and SMMC-7721 cells Immunohistochemistry was used for the detection of FOXQ1 protein levels in 68 pairs of HCC and corresponding paraneoplastic tissues shrna- FOXQ1 and shrna-negative control NC were constructed and transfected into SMMC-7721 cells The effect was detected by angiogenesis-related in vitro assay The expression of FOXQ1 mrna and protein in HCC were significantly higher than in paraneoplastic liver tissues The expression of FOXQ1 in SMMC-7721 cells was also higher than that in LO2 liver cells P < 0 05 The FOXQ1 protein level was correlated with TNM stage tumor differentiation tumor numbers and tumor size P < 0 05 FOXQ1 and VEGF expression were decreased following shfoxq1 transfection as compared with the shrna-nc group P < 0 05 These findings No * Tel @ qq com Received July Accepted October Supported by Science and Technology Project of Chongqing No * Corresponding author Tel @ qq com

2 4 FOXQ1 SMMC suggested that FOXQ1 was upregulated in HCC The abnormal expression of FOXQ1 related to the HCC progress and clinicopahology Inhibition of FOXQ1 expression might suppress hepatoma cell angiopoiesis Key words hepatocellular carcinoma forkhead box Q1 FOXQ1 transcription factor clinicopahology angiopoiesis FOXQ1 forkhead box Q1 FOXQ GC PCR WB 1 10% 1 68 FOXQ1 SP FOXQ ~ FOXQ > EMT TNM 3-6 Ⅰ ~ Ⅱ 44 Ⅲ ~ Ⅳ FOXQ1 qrt-pcr Western FOXQ1 shrna-nc shrna-nc FOXQ1 FOXQ1 SMMC Trizol RNA TaKaRa cdna CFX96 VEGF 1 1 5'-TGCTGTGGACTTGAGTTGGGAG-3' SMMC-7721 L02 5'-CCTGGCCTTGCACATTCCTG-3' FOXQ1 5'-TGATTTCTTGCTATTGACCGATGC-3' RPMI 1640 DMEM 5'-GCCCAAGGAGACCACAGTTAGAG-3' GAPDH 10% 37 5% CO 2 5'-CTTTGGTATCGTGGAAGGACTC-3' Hyclone Trizol PCR 5'-GTAGAGGCAGGGATGATGTTCT-3' TaKaRa PCR TaKaRa s 95 5 s 56 ~ 60 Western 30 s 72 1 min FOXQ1 Santa Cruz 10 min 2 - ΔΔCt VEGF Western ABgent 1 2 SMMC shrna-foxq1 shrna- BCA 40 μg 2 h PVDF 5% h β-tublin ECL 2 cm 3

3 sh-rna 1 0 1% ~ 33% 1 34% ~ 66% 2 > plkd CMV GFP U6shRNA FOXQ1-sh1 5'- 67% = 1 = 2 = CGC GGACUUUGCACUUUGA-3' Negative control 3 = NC shrna 5'-TTCTCCGAACGTGTCACGT-3' ~ ~ 4 MOI 30 ShRNA-Gli ~ shrna-nc 5 μg /ml 1 9 polybrene 72 h SPSS13 0 ± X - ± SD 1 7 t χ 2 SMMC-7721 Fisher s 72 h 1 10 P < μl min FOXQ1 5 " FOXQ ml 48 h qrt-pcr Fig 1A % FOXQ1 mrna 6 FOXQ1 mrna 1 8 Western Fig 1B % FOXQ1 DAB % PBS t 2 FOXQ1 mrna P < 0 05 Fig 1 Expression of FOXQ1 mrna and protein between paracarcinoma tissues and HCC tissues The total mrna and protein were extracted from paracarcinoma tissues and hepatocellular carcinoma tissues The expression level of FOXQ1 mrna and protein were detected by qrt-pcr A and Western blot B in paracarcinoma tissues and hepatocellular carcinoma tissues respectively N paracarcinoma tissues T hepatocellular carcinoma tissues GAPDH mrna was the internal control β-tublin staining was used to ensure loading of the proteins The data represent one of three independent experiments Data represent mean ± SD derived from three independent experiments * P < FOXQ1 qrt-pcr 2 2 FOXQ1 Western Fig 2 FOXQ1 FOXQ1 SMMC-7721 mrna Fig 3 68 FOXQ1 42 HepG2 LO2 61 7% % 68 FOXQ %

4 4 FOXQ1 SMMC χ 2 FOXQ1 P < 0 05 Fig 2 Expression of FOXQ1 mrna and protein between LO2 and SMMC-7721 cells The FOXQ1 protein expressions in normal cells LO2 and SMMC-7721 cells were analyzed by qrt-pcr A and Western blot B GAPDH mrna was the internal standard β-tublin staining was used to ensure loading of the protein The data represent one of three independent experiments Data represent mean ± SD derived from three independent experiments * P < shrna-foxq1 shrna-foxq h 9 FOXQ1 FOX FOXQ1 Fig 4A Fig 4B Fig 4C 120 shrna-foxq1 SMMC-7721 EMT 85% 2 4 FOXQ1 SMMC-7721 VEGF BCL Qin 16 FOXQ1 FOXQ h FOXQ1 ZEB2 Fig 5 shrna-foxq1 HUVECs VersicanV1 EMT 17 FOXQ1 shrna-nc P < FOXQ1 VEGF shrna-foxq1 SMMC h qrt-pcr Fig 6A Western Fig 6B SMMC-7721 FOXQ1 VEGF mrna shrna-nc 19 P < 0 05 FOXQ1 Wang 2 6 FOXQ1 HCC % FOXQ1 χ 2 HCC FOXQ1 FOXQ1 α-fetoprotein AFP portal FOXQ1 HCC vein tumor thrombus PVTT RNAi FOXQ1 TNM tumor node metastasis TNM FOXQ1 P < 0 05 Table 1 3 FOX Kaneda 15 FOXQ1 FOXQ1 2 SMMC-7721 SMMC-7721 VEGF FOXQ1 FOX forkhead box 110 DNA

5 Fig 3 Detection of FOXQ1 protein expression was used by immunohistochemistry in paracarcinoma tissues and HCC tissues Paraffin-embedded sections were deparaffinized re-hydrated and blocked endogenous peroxidase activity The tissues were incubation with anti-foxq1 antibodies The results revealed that protein expression of FOXQ1 in HCC B were significantly higher than that in paracarcinoma tissues A The representative images were shown 200 Fig 4 Transfection efficiency of the shrna-foxq1 in SMMC-7721 cells After infected for 72 hours SMMC-7721 cells with green fluorescent protein were observed under fluorescence microscope 120 A SMMC-7721 cells were observed under white light microscope120 B SMMC-7721 cells with green fluorescent protein and all SMMC-7721 cells were overlapped 120 C Fig 5 Angiogenesis in vitro assay The number of tube formation of HUVECs in various groups were counted by optical microscopies 120 The number of tube formation of shrna-foxq1 group C was less than that of shrna-nc group A and blank group B significantly Data represent mean ± SD * P < 0 05

6 4 FOXQ1 SMMC Fig 6 Analysis of mrna and protein expression of FOXQ1 and VEGF in various groups The FOXQ1 and VEGF expression in various groups were analyzed by qrt-pcr A and Western blot B The GAPDH mrna was the internal standard β-tublin staining was used to ensure loading of the proteins The data represent one of three independent experiments Data represent mean ± SD derived from three independent experiments * P < 0 05 Table 1 Correlation of high FOXQ1 expression and clinicopathological characteristics in HCC patients Clinicopathological FOXQ1 protein expression characteristics + - χ 2 value P value α-fetoprotein μ g /L > Cirrrhosis Present 15 9 Absent Tumor diameter cm * > Portal vein invasion PVTT Stage grouping with TNM * I-II III-IV 21 3 Differentiation * Well 1 10 Moderate-poorly Tumor number * Single Multiple 10 1 * P < 0 05 References 1 Hannenhalli S Kaestner K H The evolution of Fox genes and their role in development and disease J Nat Rev Genet Feuerborn A Srivastava P K Kuffer S et althe Forkhead factor FoxQ1 influences epithelial differentiation J J Cell Physiol Christensen J Bentz S Sengstag T et al FOXQ1 a novel target of the Wnt pathway and a new marker for activation of Wnt signaling in solid tumors J PLoS One e Zhang H Meng F Liu G et al Forkhead transcription factor foxq1 promotes epithelial-mesenchymal transition and breast cancer metastasis J Cancer Res Law P T Wong N Emerging roles of microrna in the intracellular signaling networks of hepatocellular carcinoma J J Gastroenterol Hepatol Wu Y Qiao X Qiao S et al Targeting integrins in hepatocellular carcinoma J Expert Opin Ther Targets Ilhan A Gartner W Neziri D et al Angiogenic factors in plasma of brain tumour patients J Anticancer Res

7 Yegin E G Siykhymbayev A Eren F et al Prognostic implication of serum vascular endothelial growth factor in advanced hepatocellular carcinoma staging J Ann Hepatol Xia L Huang W Tian D et al Overexpression of forkhead box C1 promotes tumor metastasis and indicates poor prognosis in hepatocellular carcinoma J Hepatology Gao M Wu R C Herlinger A L et al Identification of the NAC1-regulated genes in ovarian cancer J Am J Pathol Qiao Y Jiang X Lee S T et al FOXQ1 regulates epithelialmesenchymal transition in human cancers J Cancer Res Zhu Z Zhu Z Pang Z et al Short hairpin RNA targeting FOXQ1 inhibits invasion and metastasis via the reversal of epithelial-mesenchymal transition in bladder cancer J Int J Oncol Sun H T Cheng S X Tu Y et al FoxQ1 promotes glioma cells proliferation and migration by regulating NRXN3 expression J PLoS One e Feng J Zhang X Zhu H et al FoxQ1 overexpression influences poor prognosis in non-small cell lung cancer associates with the phenomenon of EMT J PLoS One e Kaneda H Arao T Tanaka K et al FOXQ1 is overexpressed in colorectal cancer and enhances tumorigenicity and tumor growth J Cancer Res Qin J Xu Y Li X et al Effects of lentiviral-mediated Foxp1 and Foxq1 RNAi on the hepatocarcinoma cell J Exp Mol Pathol Xia L Huang W Tian D et al Forkhead box Q1 promotes hepatocellular carcinoma metastasis by transactivating ZEB2 and VersicanV1 expression J Hepatology Liang S H Yan X Z Wang B L et al Increased expression of FOXQ1 is a prognostic marker for patients with gastric cancer J Tumour Biol Fan L Wu Q Xing X et al MicroRNA-145 targets vascular endothelial growth factor and inhibits invasion and metastasis of osteosarcoma cells J Acta Biochim Biophys Sin Shanghai Wang W He S Ji J et al The prognostic significance of FOXQ1 oncogene overexpression in human hepatocellular carcinoma J Pathol Res Pract