RKIP影响人宫颈癌Hela细胞增殖、侵袭及其机制的研究
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摘要
目的观察上调Raf激酶抑制蛋白(RKIP)表达后人宫颈癌Hela细胞增殖侵袭能力及其机制探讨。方法培养人宫颈癌Hela细胞,利用脂质体介导方法,转染后将细胞分为空白对照组、转染空载体组及观察组,观察组转染pc DNA3. 1(+)-RKIP,空白对照组正常培养,转染空载体组转染真核表达载体pc DNA3. 1(+)。采用Western blot法检测RKIP蛋白;细胞培养24、48、72、96 h,采用MTT法测量各组在570 nm波长处的OD值。Transwell实验检测宫颈癌细胞株侵袭及迁移情况;并观察裸鼠成瘤情况。结果观察组RKIP蛋白表达含量明显增加,与空白对照组及转染空载体组比较,P <0. 05。细胞培养24、48、72、96 h时,OD值观察组较空白对照组及转染空载体组低(P <0. 05); Transwell实验结果显示观察组的宫颈癌细胞株侵袭能力弱于其余两组;裸鼠成瘤实验显示与对照组相比,观察组肿瘤生长速度较慢。结论 RKIP表达的上调在肿瘤发展迁移过程中起着抑制作用,其机制可能与其抑制肿瘤细胞增殖、迁移有关。
Objective To investigate the proliferation and invasiveness of human cervical cancer Hela cells induced by RKIP and its mechanism. Methods Human cervical cancer Hela cells were cultured and divided into blank control group,transfected with empty vector group and observation group by liposome-mediated method. The observation group was transfected with pcDNA3. 1( +)-RKIP,and the blank control group was cultured normally. The empty vector group was transfected with the eukaryotic expression vector pcDNA3. 1( +). After 48 h,three groups of cells were collected and RKIP protein was detected by Western blot. The threegroups of cells were cultured for 24,48,72 and 96 h. The OD value of each group at 570 nm was measured by MTT assay. The invasion and migration of cervical cancer cell lines were detected by Transwell assay. The growth of tumor in each group was observed. Results The expression of RKIP protein in the observation group was significantly increased,P < 0. 05. At24,48,72 and 96 hours,the OD value in observation group was lower than that in blank control group and empty vector group( P < 0. 05). The results of Transwell assay showed that the invasion and migration of cervical cancer cell line in observation group were weaker than those in the other two groups. The tumor formation in nude mice showed that the growth rate of tumor in observation group was slower than that in control group. Conclusion The up-regulation of RKIP expression may play an inhibitory role in the process of tumor development and migration,which may be related to the inhibition of tumor cell proliferation and migration.
Objective To investigate the proliferation and invasiveness of human cervical cancer Hela cells induced by RKIP and its mechanism. Methods Human cervical cancer Hela cells were cultured and divided into blank control group,transfected with empty vector group and observation group by liposome-mediated method. The observation group was transfected with pcDNA3. 1( +)-RKIP,and the blank control group was cultured normally. The empty vector group was transfected with the eukaryotic expression vector pcDNA3. 1( +). After 48 h,three groups of cells were collected and RKIP protein was detected by Western blot. The threegroups of cells were cultured for 24,48,72 and 96 h. The OD value of each group at 570 nm was measured by MTT assay. The invasion and migration of cervical cancer cell lines were detected by Transwell assay. The growth of tumor in each group was observed. Results The expression of RKIP protein in the observation group was significantly increased,P < 0. 05. At24,48,72 and 96 hours,the OD value in observation group was lower than that in blank control group and empty vector group( P < 0. 05). The results of Transwell assay showed that the invasion and migration of cervical cancer cell line in observation group were weaker than those in the other two groups. The tumor formation in nude mice showed that the growth rate of tumor in observation group was slower than that in control group. Conclusion The up-regulation of RKIP expression may play an inhibitory role in the process of tumor development and migration,which may be related to the inhibition of tumor cell proliferation and migration.
引文
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[15] Fu Z,KitagawaY,Shen R,et al. Metastasis suppressor gene Raf kinase inhibitor protein(RKIP)is a novel prognostic marker in Prostate cancer[J]. Prostate,2006,66(3):248-256.
[16] Schuierer MM,Bataille F,Hagan S,et al. Reduction in Raf kinase inhibitor protein expression is associated with increased rasextracellular signal-regulated kinase signaling in melanoma cell lines[J]. Cancer Res,2004,64(15):5186-5192.
[2] Howe SL,Vargas DE,Granada D,et al. Cervical cancer prevention in remote rural Nicaragua:a program evalulation[J]. Gyneol Oncol,2005,99(3 Suppl 1):S232-235.
[3] Sawaya GF,Huchko MJ. Cervical cancer screening[J]. Med Clin North Am,2017,101(4):743-753.
[4] Hwang TS,Jeong JK,Park M,et al. Detection and typing of HPV genotypes in various cervical lesions by HPV oligonucleotide microarray[J]. Gyneol Oncol,2003,90(1):51-56.
[5] Xu Q,Wang S,Xi L,et al. Effect s of human papillomavirus type16 E7 protein on the growth of cervical carcinoma cells and immunoescape through the TGF2β1 signaling pathway[J]. Gy neol Oncol,2006,101(1):132-140.
[6]胡新宙,王旭,张伟. Ra I激酶抑制蛋白(RKl P)的生物学功能研究[J].生命科学,2007,19(8):31-36.
[7] Bemier I,Jollea P. Purification and characterization of a basic 23k Da eytosolic protein from bovine brain[J]. Biochim Biophys Aeto,1984,790(2):174-181.
[8] Bemier I,Treaca JP,Jollès P. Ligand-binding studies with a 23k Da protein purified from bovine brain cytosol[J]. Biochim Biophys Acta,1986,871(1):19-23.
[9] Yeung K,Seitz T,Li S,et al. Suppression of Raf-1 kinase activity and MAP kinase signalling by RKIP[J]. Nature,1999,401(6749):173-177.
[10]顾晓梅,张玉泉.宫颈癌的流行病学高危因素研究进展[J].中国妇幼保健,2007,22(35):5073-5075.
[11] Maura G,Chaignot C,Weill A,et al,Cervical cancer screening and subsequent procedures in women under the age of 25 years between 2007 and 2013 in France:a nationwide French healthcare database study[J]. Eur J Cancer Prev,2018,27(5):479-485.
[12] Setiawan D,Luttjeboer J,Westra TA,et al. The cost-effectiveness of HPV vaccination in addition to screening:a Dutch perspective[J]. Expert Rev Vaccines,2015,14(4):589-604.
[13] Fang J,Zhang H,Jin S. Epigenetics and cervical cancer:from pathogenesis to therapy[J]. Tumour Biol,2014,35(6):5083-5093.
[14] Mruts KB,Gebremariam TB. Knowledge and perception towards cervical cancer among Female Debre Berhan University students[J]. Asian Pac J Cancer Prev,2018,19(7):1771-1777.
[15] Fu Z,KitagawaY,Shen R,et al. Metastasis suppressor gene Raf kinase inhibitor protein(RKIP)is a novel prognostic marker in Prostate cancer[J]. Prostate,2006,66(3):248-256.
[16] Schuierer MM,Bataille F,Hagan S,et al. Reduction in Raf kinase inhibitor protein expression is associated with increased rasextracellular signal-regulated kinase signaling in melanoma cell lines[J]. Cancer Res,2004,64(15):5186-5192.