FHIT对宫颈癌细胞增殖与诱导凋亡的影响
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摘要
目的研究脆性组氨酸三联体(FHIT)对宫颈癌细胞增殖和凋亡的影响。方法在宫颈癌细胞CaSki中转染FHIT过表达载体,筛选稳定转染的细胞株,用Real time PCR和Western blot检测转染效果。MTT法测定细胞的增殖活性,PI单染法测定细胞周期变化,Annexin V-FITC/PI双染法测定细胞凋亡变化,Western blot检测活化型Caspase-3(Cleaved Caspase-3)、活化型Caspase-9(Cleaved Caspase-9)、细胞周期蛋白D1(cyclin D1)、p21蛋白水平。结果转染FHIT过表达载体能上调宫颈癌细胞中FHIT mRNA和蛋白水平。高表达FHIT的宫颈癌细胞增殖活性降低,细胞G0/G1期比例升高,细胞凋亡率升高,细胞中Cleaved Caspase-3、Cleaved Caspase-9、p21蛋白水平升高,Cyclin D1蛋白水平降低,与未转染细胞比较,差异有统计学意义(P<0.05)。结论高表达FHIT能阻滞细胞周期和诱导细胞凋亡,降低宫颈癌细胞的增殖能力。
Objective To investigate the effects of fragile histidine triad(FHIT) on proliferation and apoptosis of cervical cancer cells.Methods The FHIT overexpression vector was transfected into human cervical cancer cell line CaSki. The transfection efficiency was detected by Real time PCR and Western blot. The proliferation activity of cells was measured by MTT method. The cell cycle changes were determined by PI single staining method. Apoptosis was measured by Annexin V-FITC/PI double staining. Western blot was also used to detect the levels of Cleaved Caspase-3, Cleaved Caspase-9, Cyclin D1 and p21 protein. Results Transfection of FHIT overexpression vector enhanced the mRNA and protein levels of FHIT in cervical cancer cells. Compared with cells without transfection, the proliferation activity of the cervical cancer cells with high expression of FHIT decreased. The proportion of G0/G1 phase increased, and the rate of apoptosis increased. The levels of Cleaved Caspase-3, Cleaved Caspase-9 and p21 protein increased, while the level of Cyclin D1 protein reduced, all with statistical differences(P<0.05). Conclusion High expression of FHIT decreases the proliferation of cervical cancer cells by blocking cell cycle and inducing apoptosis.
Objective To investigate the effects of fragile histidine triad(FHIT) on proliferation and apoptosis of cervical cancer cells.Methods The FHIT overexpression vector was transfected into human cervical cancer cell line CaSki. The transfection efficiency was detected by Real time PCR and Western blot. The proliferation activity of cells was measured by MTT method. The cell cycle changes were determined by PI single staining method. Apoptosis was measured by Annexin V-FITC/PI double staining. Western blot was also used to detect the levels of Cleaved Caspase-3, Cleaved Caspase-9, Cyclin D1 and p21 protein. Results Transfection of FHIT overexpression vector enhanced the mRNA and protein levels of FHIT in cervical cancer cells. Compared with cells without transfection, the proliferation activity of the cervical cancer cells with high expression of FHIT decreased. The proportion of G0/G1 phase increased, and the rate of apoptosis increased. The levels of Cleaved Caspase-3, Cleaved Caspase-9 and p21 protein increased, while the level of Cyclin D1 protein reduced, all with statistical differences(P<0.05). Conclusion High expression of FHIT decreases the proliferation of cervical cancer cells by blocking cell cycle and inducing apoptosis.
引文
[1]Ouh YT, Lee JK. Proposal for cervical cancer screening in the era of HPV vaccination[J]. Obstet Gynecol Sci, 2018,61(3):298-308. doi:10.5468/ogs.2018.61.3.298.
[2]Qian QP, Zhang X, Ding B, et al. Performance of P16/Ki67dual staining in triaging hr-HPV-positive population during cervical Cancer screening in the younger women[J]. Clin Chim Acta, 2018, 483:281-285. doi:10.1016/j. cca.2018.05.023.
[3]Rocco A, Schandl L, Chen J, et al. Loss of FHIT protein expression correlates with disease progression and poor differentiation in gastric cancer[J]. J Cancer Res Clin Oncol,2003, 129(2):84-88. doi:10.1007/s00432-002-0409-3.
[4]Foja S, Goldberg M, Schagdarsurengin U, et al. Promoter methylation and loss of coding exons of the fragile histidine triad(FHIT)gene in intrahepatic cholangiocarcinomas[J]. Liver Int, 2005, 25(6):1202-1208. doi:10.1111/j.1478-3231.2005.01174. x.
[5]Jahid S, Sun J, Gelincik O, et al. Inhibition of colorectal cancer genomic copy number alterations and chromosomal fragile site tumor suppressor FHIT and WWOX deletions by DNA mismatch repair[J]. Oncotarget, 2017, 8(42):71574-71586. doi:10.18632/oncotarget.17776.
[6]Sevinc ED, Cecener G, Ak S, et al. Expression and clinical significance of miRNAs that may be associated with the FHIT gene in breast cancer[J]. Gene, 2016, 590(2):278-284. doi:10.1016/j. gene.2016.05.033.
[7]Vural S, Simon R, Krushkal J. Correlation of gene expression and associated mutation profiles of APOBEC3A,APOBEC3B, REV1, UNG, and FHIT with chemosensitivity of cancer cell lines to drug treatment[J]. Hum Genomics,2018, 12(1):20. doi:10.1186/s40246-018-0150-x.
[8]Ki KD, Lee SK, Tong SY, et al. Role of 5'-CpG island hypermethylation of the FHIT gene in cervical carcinoma[J]. J Gynecol Oncol, 2008, 19(2):117-122. doi:10.3802/jgo.2008.19.2.117.
[9]Toledo G, Sola JJ, Lozano MD, et al. Loss of FHIT protein expression is related to high proliferation, low apoptosis and worse prognosis in non-small-cell lung cancer[J]. Mod Pathol, 2004,17(4):440-448. doi:10.1038/modpathol.3800081.
[10]Nakagawa Y, Akao Y. Fhit protein inhibits cell growth by attenuating the signaling mediated by nuclear factorkappa B in colon cancer cell lines[J]. Exp Cell Res, 2006,312(13):2433-2442. doi:10.1016/j. yexcr.2006.04.004.
[11]Gorlova OY, Demidenko EI, Amos CI, et al. Downstream targets of GWAS-detected genes for breast, lung, and prostate and colon cancer converge to G1/S transition pathway[J]. Hum Mol Genet, 2017, 26(8):1465-1471. doi:10.1093/hmg/ddx050.
[12]Zhang J, Zhu L, Fang J, et al. LRG1 modulates epithelialmesenchymal transition and angiogenesis in colorectal cancer via HIF-1αactivation[J]. J Exp Clin Cancer Res, 2016,35:29. doi:10.1186/s13046-016-0306-2.
[13]Luo J, Zhang C, Wang C, et al. Miz-1 promotes the proliferation of esophageal cancer cells via suppression of p21and release of p21-arrested cyclin D1[J]. Oncol Rep, 2016,35(6):3532-3540. doi:10.3892/or.2016.4731.
[14]Bragantini E, Barbi S, Beghelli S, et al. Loss of Fhit expression is associated with poorer survival in gastric cancer but is not an independent prognostic marker[J]. J Cancer Res Clin Oncol, 2006, 132(1):45-50. doi:10.1007/s00432-005-0045-9.
[15]Woenckhaus M, Merk J, Stoehr R, et al. Prognostic value of FHIT, CTNNB1, and MUC1 expression in non-small cell lung cancer[J]. Hum Pathol, 2008, 39(1):126-136. doi:10.1016/j. humpath.2007.05.027.
[16]Wu DW, Lee MC, Hsu NY, et al. FHIT loss confers cisplatin resistance in lung cancer via the AKT/NF-κB/Slugmediated PUMA reduction[J]. Oncogene, 2017, 36(38):5439. doi:10.1038/onc.2017.249.
[17]Semba S, Trapasso F, Fabbri M, et al. Fhit modulation of the Akt-survivin pathway in lung cancer cells:Fhittyrosine 114(Y114)is essential[J]. Oncogene, 2006, 25(20):2860-2872. doi:10.1038/sj. onc.1209323.
[2]Qian QP, Zhang X, Ding B, et al. Performance of P16/Ki67dual staining in triaging hr-HPV-positive population during cervical Cancer screening in the younger women[J]. Clin Chim Acta, 2018, 483:281-285. doi:10.1016/j. cca.2018.05.023.
[3]Rocco A, Schandl L, Chen J, et al. Loss of FHIT protein expression correlates with disease progression and poor differentiation in gastric cancer[J]. J Cancer Res Clin Oncol,2003, 129(2):84-88. doi:10.1007/s00432-002-0409-3.
[4]Foja S, Goldberg M, Schagdarsurengin U, et al. Promoter methylation and loss of coding exons of the fragile histidine triad(FHIT)gene in intrahepatic cholangiocarcinomas[J]. Liver Int, 2005, 25(6):1202-1208. doi:10.1111/j.1478-3231.2005.01174. x.
[5]Jahid S, Sun J, Gelincik O, et al. Inhibition of colorectal cancer genomic copy number alterations and chromosomal fragile site tumor suppressor FHIT and WWOX deletions by DNA mismatch repair[J]. Oncotarget, 2017, 8(42):71574-71586. doi:10.18632/oncotarget.17776.
[6]Sevinc ED, Cecener G, Ak S, et al. Expression and clinical significance of miRNAs that may be associated with the FHIT gene in breast cancer[J]. Gene, 2016, 590(2):278-284. doi:10.1016/j. gene.2016.05.033.
[7]Vural S, Simon R, Krushkal J. Correlation of gene expression and associated mutation profiles of APOBEC3A,APOBEC3B, REV1, UNG, and FHIT with chemosensitivity of cancer cell lines to drug treatment[J]. Hum Genomics,2018, 12(1):20. doi:10.1186/s40246-018-0150-x.
[8]Ki KD, Lee SK, Tong SY, et al. Role of 5'-CpG island hypermethylation of the FHIT gene in cervical carcinoma[J]. J Gynecol Oncol, 2008, 19(2):117-122. doi:10.3802/jgo.2008.19.2.117.
[9]Toledo G, Sola JJ, Lozano MD, et al. Loss of FHIT protein expression is related to high proliferation, low apoptosis and worse prognosis in non-small-cell lung cancer[J]. Mod Pathol, 2004,17(4):440-448. doi:10.1038/modpathol.3800081.
[10]Nakagawa Y, Akao Y. Fhit protein inhibits cell growth by attenuating the signaling mediated by nuclear factorkappa B in colon cancer cell lines[J]. Exp Cell Res, 2006,312(13):2433-2442. doi:10.1016/j. yexcr.2006.04.004.
[11]Gorlova OY, Demidenko EI, Amos CI, et al. Downstream targets of GWAS-detected genes for breast, lung, and prostate and colon cancer converge to G1/S transition pathway[J]. Hum Mol Genet, 2017, 26(8):1465-1471. doi:10.1093/hmg/ddx050.
[12]Zhang J, Zhu L, Fang J, et al. LRG1 modulates epithelialmesenchymal transition and angiogenesis in colorectal cancer via HIF-1αactivation[J]. J Exp Clin Cancer Res, 2016,35:29. doi:10.1186/s13046-016-0306-2.
[13]Luo J, Zhang C, Wang C, et al. Miz-1 promotes the proliferation of esophageal cancer cells via suppression of p21and release of p21-arrested cyclin D1[J]. Oncol Rep, 2016,35(6):3532-3540. doi:10.3892/or.2016.4731.
[14]Bragantini E, Barbi S, Beghelli S, et al. Loss of Fhit expression is associated with poorer survival in gastric cancer but is not an independent prognostic marker[J]. J Cancer Res Clin Oncol, 2006, 132(1):45-50. doi:10.1007/s00432-005-0045-9.
[15]Woenckhaus M, Merk J, Stoehr R, et al. Prognostic value of FHIT, CTNNB1, and MUC1 expression in non-small cell lung cancer[J]. Hum Pathol, 2008, 39(1):126-136. doi:10.1016/j. humpath.2007.05.027.
[16]Wu DW, Lee MC, Hsu NY, et al. FHIT loss confers cisplatin resistance in lung cancer via the AKT/NF-κB/Slugmediated PUMA reduction[J]. Oncogene, 2017, 36(38):5439. doi:10.1038/onc.2017.249.
[17]Semba S, Trapasso F, Fabbri M, et al. Fhit modulation of the Akt-survivin pathway in lung cancer cells:Fhittyrosine 114(Y114)is essential[J]. Oncogene, 2006, 25(20):2860-2872. doi:10.1038/sj. onc.1209323.