人肝癌细胞株中PTPRD表达变化及其对HuH-7细胞增殖、迁移的影响和机制
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摘要
目的观察人肝癌细胞株HuH-7中蛋白酪氨酸磷酸酶D(PTPRD)的表达变化及其对细胞增殖和迁移的影响,并探讨相关机制。方法采用实时荧光定量PCR法、Western blotting法分别检测人正常肝细胞株LO2及肝癌细胞株HepG2、HuH-7中的PTPRD mRNA和蛋白。将HuH-7细胞分为PTPRD上调组和阴性对照组,分别转染PTPRD过表达腺病毒和阴性对照腺病毒。采用MTT实验检测两组HuH-7细胞的增殖能力,采用细胞划痕实验检测细胞迁移能力,采用Western blotting法检测两组细胞中的蛋白激酶B(Akt)、磷酸化蛋白激酶B(p-Akt)、雷帕霉素靶蛋白(mTOR)、磷酸化雷帕霉素靶蛋白(p-mTOR)。结果 HepG2细胞和HuH-7细胞中PTPRD mRNA及蛋白相对表达量均低于LO2细胞(P均<0.05)。PTPRD上调组细胞贴壁24、48、72 h时细胞增殖能力低于阴性对照组。PTPRD上调组划痕48 h后细胞迁移率低于阴性对照组(P<0.01)。PTPRD上调组细胞p-Akt/Akt、p-mTOR/mTOR均低于阴性对照组(P均<0.01)。结论 HuH-7细胞中PTPRD呈低表达;PTPRD基因过表达后可抑制HuH-7细胞的增殖和迁移能力,其机制可能与抑制PI3K-Akt-mTOR信号通路有关。
Objective To observe the expression changes of protein tyrosine phosphate receptor type delta(PTPRD)in the hepatocellular carcinoma cell line HuH-7 and its effect on cell proliferation and migration and its mechanism.Methods The mRNA and protein expression of PTPRD in the normal liver cells LO2, hepatocellular carcinoma cells HepG2 and HuH-7 was detected by RT-PCR and Western blotting. PTPRD overexpression and negative control vector adenovirus were used to transfect the HuH-7 cells, which were taken as the PTPRD overexpression group and negative control group. Methyl thiazolyl tetrazolium(MTT) assay and cell scratch assay were used to detect the proliferation and migration abilities of HuH-7 cells in the two groups. The expression levels of protein kinase B(Akt), phosphorylated protein kinase B(p-Akt), rapamycin target protein(mTOR), and phosphorylated rapamycin target protein(p-mTOR) were detected by Western blotting. Results The mRNA and protein levels of PTPRD in the HepG2 and HuH-7 cells were significantly lower than those in the LO2 cells(all P<0.05). The ability of cell proliferation in the PTPRD overexpression group was lower than that in the negative control group at 24, 48 and 72 h after cell adhesion. The cell migration rate of the PTPRD overexpression group was lower than that of the negative control group(P<0.01). The p-Akt/Akt and p-mTOR/mTOR in the PTPRD overexpression group were lower than those in the negative control group(both P<0.01). Conclusion PTPRD is low expressed in HuH-7 cells; the overexpression of PTPRD gene can inhibit the proliferation and migration of HuH-7 cells by, and its mechanism may be related to the inhibition of PI3 K-Akt-mTOR signaling pathway.
Objective To observe the expression changes of protein tyrosine phosphate receptor type delta(PTPRD)in the hepatocellular carcinoma cell line HuH-7 and its effect on cell proliferation and migration and its mechanism.Methods The mRNA and protein expression of PTPRD in the normal liver cells LO2, hepatocellular carcinoma cells HepG2 and HuH-7 was detected by RT-PCR and Western blotting. PTPRD overexpression and negative control vector adenovirus were used to transfect the HuH-7 cells, which were taken as the PTPRD overexpression group and negative control group. Methyl thiazolyl tetrazolium(MTT) assay and cell scratch assay were used to detect the proliferation and migration abilities of HuH-7 cells in the two groups. The expression levels of protein kinase B(Akt), phosphorylated protein kinase B(p-Akt), rapamycin target protein(mTOR), and phosphorylated rapamycin target protein(p-mTOR) were detected by Western blotting. Results The mRNA and protein levels of PTPRD in the HepG2 and HuH-7 cells were significantly lower than those in the LO2 cells(all P<0.05). The ability of cell proliferation in the PTPRD overexpression group was lower than that in the negative control group at 24, 48 and 72 h after cell adhesion. The cell migration rate of the PTPRD overexpression group was lower than that of the negative control group(P<0.01). The p-Akt/Akt and p-mTOR/mTOR in the PTPRD overexpression group were lower than those in the negative control group(both P<0.01). Conclusion PTPRD is low expressed in HuH-7 cells; the overexpression of PTPRD gene can inhibit the proliferation and migration of HuH-7 cells by, and its mechanism may be related to the inhibition of PI3 K-Akt-mTOR signaling pathway.
引文
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[13] Zhou Q, Lui VW, Yeo W. Targeting the PI3K/Akt/mTOR pathway in hepatocellular carcinoma[J]. Future Oncol, 2011,7(10):1149-1167.
[14] Sharma Y, Ahmad A, Bashir S, et al. Implication of protein tyrosine phosphatase SHP-1 in cancer-related signaling pathways[J]. Future Oncol, 2016,12(10):1287-1298.
[15] Zhang Z, Chen Q, Zhang J, et al. Associations of genetic polymorphisms in pTEN/AKT/mTOR signaling pathway genes with cancer risk: A meta-analysis in Asian population[J]. Scient Rep, 2017,7(1):17844.
[16] Riquelme I, Tapia O, Espinoza JA, et al. The gene expression status of the PI3K/AKT/mTOR pathway in gastric cancer tissues and cell lines[J]. Pathol Oncol Res, 2016,22(4):797-805.
[17] Costa R, Han HS, Gradishar WJ. Targeting the PI3K/AKT/mTOR pathway in triple-negative breast cancer: a review[J]. Breast Cancer Res Treat, 2018,169(6):1-10.
[18] King D, Yeomanson D, Bryant HE. PI3King the Lock: Targeting the PI3K/Akt/mTOR pathway as a novel therapeutic strategy in neuroblastoma[J]. J Pediat Hematol Oncol, 2015,37(4):245.
[19] Ocana A, Verabadillo F, Almubarak M, et al. Activation of the PI3K/mTOR/AKT pathway and survival in solid tumors: systematic review and meta-analysis[J]. PLoS One, 2014,9(4):e95219.
[2] Moudgil V, Redhu D, Dhanda S, et al. A review of molecular mechanisms in the development of hepatocellular carcinoma by aflatoxin and hepatitis B and C viruses[J]. J Environ Pathol Toxicol Oncol, 2013,32(2):165-175.
[3] Norris K, Norris F, Kono DH, et al. Expression of protein-tyrosine phosphatases in the major insulin target tissues[J]. Febs Letters, 1997,415(3):243-248.
[4] Ostman A, Hellberg C, Bohmer FD. Protein-tyrosine phosphatases and cancer[J]. Nat Rev Cancer, 2006,6(4):307.
[5] Ortiz B, Fabius AW, Wu WH, et al. Loss of the tyrosine phosphatase PTPRD leads to aberrant STAT3 activation and promotes gliomagenesis[J]. Proc Nat Acad Sci U S A, 2014,111(22):8149-8154.
[6] Kohno T, Otsuka A, Girard L, et al. A catalog of genes homozygously deleted in human lung cancer and the candidacy of PTPRD, as a tumor suppressor gene[J]. Genes Chrom Cancer, 2010,49(4):342-352.
[7] Walia V, Prickett TD, Kim JS, et al. Mutational and functional analysis of the tumor-suppressor PTPRD in human melanoma[J]. Hum Mutat, 2015,35(11):1301-1310.
[8] Acun T, Demir K, Oztas E, et al. PTPRD is homozygously deleted and epigenetically downregulated in human hepatocellular carcinomas[J]. OMICS, 2015,19(4):220-229.
[9] Matter MS, Decaens T, Andersen JB, et al. Targeting the mTOR pathway in hepatocellular carcinoma: current state and future trends[J]. J Hepatol, 2014,60(4):855-865.
[10] Sacco R, Gadaletacaldarola G, Galati G, et al. EASL HCC summit: liver cancer management[J]. Future Oncol, 2014,10(7):1129-1132.
[11] Sangiovanni A, Colombo M. Treatment of hepatocellular carcinoma: beyond international guidelines[J]. J Clin Hepatol, 2016,36(S1):124-129.
[12] Bruix J, Reig M, Sherman M. Evidence-based diagnosis, staging, and treatment of patients with hepatocellular carcinoma[J]. Gastroenterology, 2016,150(4):835-853.
[13] Zhou Q, Lui VW, Yeo W. Targeting the PI3K/Akt/mTOR pathway in hepatocellular carcinoma[J]. Future Oncol, 2011,7(10):1149-1167.
[14] Sharma Y, Ahmad A, Bashir S, et al. Implication of protein tyrosine phosphatase SHP-1 in cancer-related signaling pathways[J]. Future Oncol, 2016,12(10):1287-1298.
[15] Zhang Z, Chen Q, Zhang J, et al. Associations of genetic polymorphisms in pTEN/AKT/mTOR signaling pathway genes with cancer risk: A meta-analysis in Asian population[J]. Scient Rep, 2017,7(1):17844.
[16] Riquelme I, Tapia O, Espinoza JA, et al. The gene expression status of the PI3K/AKT/mTOR pathway in gastric cancer tissues and cell lines[J]. Pathol Oncol Res, 2016,22(4):797-805.
[17] Costa R, Han HS, Gradishar WJ. Targeting the PI3K/AKT/mTOR pathway in triple-negative breast cancer: a review[J]. Breast Cancer Res Treat, 2018,169(6):1-10.
[18] King D, Yeomanson D, Bryant HE. PI3King the Lock: Targeting the PI3K/Akt/mTOR pathway as a novel therapeutic strategy in neuroblastoma[J]. J Pediat Hematol Oncol, 2015,37(4):245.
[19] Ocana A, Verabadillo F, Almubarak M, et al. Activation of the PI3K/mTOR/AKT pathway and survival in solid tumors: systematic review and meta-analysis[J]. PLoS One, 2014,9(4):e95219.