慢病毒构建的细胞系中过表达FRK对人脑胶质瘤细胞增殖、侵袭及迁移的影响-现代医学

慢病毒构建的细胞系中过表达FRK对人脑胶质瘤细胞增殖、侵袭及迁移的影响

单位：1. 徐州医科大学研究生学院, 江苏徐州 221002;
2. 徐州医科大学神经系统疾病研究所, 江苏徐州 221002

分类号：R739.41

出版年·卷·期（页码）：2017·36·第四期（539-546）

摘要：

目的：探讨在慢病毒构建的稳定表达酪氨酸相关激酶（fyn-related kinase，FRK）的细胞系中，过表达FRK对人脑胶质瘤细胞增殖、侵袭及迁移能力的影响。方法：1.将FRK慢病毒质粒与辅助质粒共转染293T细胞进行慢病毒包装，然后用包装好的慢病毒感染胶质瘤U251、U87细胞，建立稳定表达FRK的细胞系，同时运用Western blotting （WB）技术检测FRK在U251、U87细胞系中的过表达情况；2.细胞划痕实验与Transwell迁移实验检测过表达FRK对脑胶质瘤细胞迁移能力的影响；3Transwell侵袭实验检测过表达FRK对脑胶质瘤细胞侵袭能力的影响；4.EdU实验与平板集落形成实验检测过表达FRK对脑胶质瘤细胞增殖能力的影响。结果：1.荧光显微镜下可见慢病毒感染U251、U87细胞的效率达到90%以上，WB结果显示外源性FRK蛋白在U251、U87细胞系中充分表达；2.细胞划痕实验显示：与空载体组相比，过表达FRK组U251细胞迁移的细胞数在24 h和48 h分别减少了（50.00±1.04）%和（44.14±7.05）%，差异有统计学意义（P<0.01）；而过表达FRK组U87细胞迁移的细胞数在24 h和48 h分别减少了（58.50±4.12）%和（44.67±3.82）%（P<0.001）；3.Transwell迁移实验显示：与空载体组相比，过表达FRK组U251、U87细胞穿过滤膜的细胞数分别减少了（67.76±4.88）%和（50.76±4.54）%（P<0.001）；4.Transwell侵袭实验显示：与空载体组相比，过表达FRK组U251、U87细胞穿过Matrigel基质胶及滤膜的细胞数分别减少了（50.94±4.83）%和（57.92±6.19）%（P<0.001）；5.EdU实验显示：与空载体组相比，过表达FRK组U251、U87细胞的EdU阳性细胞率分别下降了（28.06±4.31）%和（33.51±7.26）%（P<0.01）；6.平板集落形成实验结果显示：与空载体组相比，过表达FRK组U251、U87细胞集落形成分别减少了（35.94±6.76）%与（52.33±7.42）%（P<0.01）。结论：过表达FRK可以抑制脑胶质瘤细胞的增殖，并且可以抑制胶质瘤细胞的侵袭和迁移能力。

Objective: To investigate the effect of FRK on proliferation, invasion and migration of human glioma cells. Methods: 1. FRK overexpression lentivirus plasmid and the helper plasmids were packaged in 293T cells in order to establish stable glioma cell lines by lentivirus system. 2. Western blot was applied to detect FRK expression level in U251 and U87 cell lines. 3. The migratory ability was examined by wound healing assay and transwell migratory assay after FRK overexpression.4. The invasive ability was examined by transwell invasion assay after FRK overexpression.5. EdU assay and Plate Colony Formation was used to investigate the effects of FRK overexpression on the proliferation of U251 and U87 cells.Results: 1. The efficiency of infection on FRK-overexpressed U251 and U87 cell lines reached up to 90%, the exogenous FRK protein overexpressed very well. 2.Compared with the empty vector group, the number of the FRK-overexpressed U251 cells migrating at 24 h and 48 h was reduced by (50.00±1.04)% and (44.14±7.05)%, respectively (P<0.01); And the number of the FRK-overexpressed U87 cells migrating at 24 h and 48 h was reduced by (58.50±4.12)% and (44.67±3.82)%(P<0.001);3.Compared with the empty vector group, in the transwell migratory assay, both numbers of the FRK-overexpressed U251 and U87 cells migrating through membrane filter were dramatically decreased by(67.76±4.88)% and(50.76±4.54)% (P<0.001);4. Compared with the empty vector group, in the matrigel invasion assay, both the number of the FRK-overexpressed U251 and U87 cells invading through matrigel were dramatically decreased by (50.94±4.83)% and (57.92±6.19)%(P<0.001);5.Compared with the empty vector group, the EdU positive cells rate of the FRK-overexpressed U251 and U87 cells was reduced by (28.06±4.31)% and (33.51±7.26)%(P<0.01);6.Compared with the empty vector group, plate colony formation assay showed that the number of the FRK-overexpressed U251 and U87 cells was reduced by (35.94±6.76)% and (52.33±7.42)%(P<0.01). Conclusion: FRK can inhibit the proliferation the invasion and migration ability of glioma cells.

参考文献：

[1] ROSKOSKI R,JR.Src protein-tyrosine kinase structure and regulation[J].Biochem Biophys Res Commun,2004,324(4):1155-1164.
[2] SHENG ZM,MARCHETTI A,BUTTITTA F,et al.Multiple regions of chromosome 6q affected by loss of heterozygosity in primary human breast carcinomas[J].Br J Cancer,1996,73(2):144-147.
[3] MEYER T,XU L,CHANG J,et al.Breast cancer cell line proliferation blocked by the Src-related Rak tyrosine kinase[J].Int J Cancer,2003,104(2):139-146.
[4] SERFAS MS,TYNER AL.Brk,Srm,Frk,and Src42A form a distinct family of intracellular Src-like tyrosine kinases[J].Oncol Res,2003,13(6-10):409-419.
[5] AKERBLOM B,ANNEREN C,WELSH M.A role of FRK in regulation of embryonal pancreatic beta cell formation[J].Mol Cell Endocrinol,2007,270(1-2):73-78.
[6] ZHOU X,HUA L,ZHANG W,et al.FRK controls migration and invasion of human glioma cells by regulating JNK/c-Jun signaling[J].J Neurooncol,2012,110(1):9-19.
[7] PAJER P,KARAFIAT V,PECENKA V,et al.Industasis,a promotion of tumor formation by nontumorigenic stray cells[J].Cancer Res,2009,69(11):4605-4612.
[8] CHEN JS,HUNG WS,CHAN HH,et al.In silico identification of oncogenic potential of fyn-related kinase in hepatocellular carcinoma[J].Bioinformatics,2013,29(4):420-427.
[9] JE DW,O YM,JI YG,et al.The inhibition of SRC family kinase suppresses pancreatic cancer cell proliferation,migration,and invasion[J].Pancreas,2014,43(5):768-776.
[10] YIM EK,SIWKO S,LIN SY.Exploring Rak tyrosine kinase function in breast cancer[J].Cell Cycle,2009,8(15):2360-2364.
[11] FRAME MC,FINCHAM VJ,CARRAGHER NO,et al.v-Src's hold over actin and cell adhesions[J].Nat Rev Mol Cell Biol,2002,3(4):233-245.
[12] CHANDRASEKHARAN S,QIU TH,ALKHAROUF N,et al.Characterization of mice deficient in the Src family nonreceptor tyrosine kinase Frk/rak[J].Mol Cell Biol,2002,22(14):5235-5247.
[13] YIM EK,PENG G,DAI H,et al.Rak functions as a tumor suppressor by regulating PTEN protein stability and function[J].Cancer Cell,2009,15(4):304-314.
[14] SHI Q,SONG X,WANG J,et al.FRK inhibits migration andinvasion of human glioma cells by promoting N-cadherin/beta-catenin complex formation[J].J Mol Neurosci,2015,55(1):32-41.
[15] KIM JL,HA GH,CAMPO L,et al.The role of Rak in the regulation of stability and function of BRCA1[J].Oncotarget,2015.
[16] CHAO Y,WANG Y,LIU X,et al.Mst1 regulates glioma cell proliferation via the AKT/mTOR signaling pathway[J].J Neurooncol,2015,121(2):279-288.

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