miR-195-5p靶向FGF2抑制子宫内膜癌HEC-1B细胞恶性生物学行为
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摘要
目的:探讨miR-195-5p通过靶向FGF2抑制子宫内膜癌HEC-1B细胞增殖、凋亡、侵袭和迁移的分子机制。方法:HEC-1B细胞培养与转染完成后分为4组:HEC-1B组、miR-195-5p mimic组、p LV-FGF2组和miR-195-5p+FGF2组。q RT-PCR检测细胞miR-195-5p和FGF2 m RNA水平,荧光素酶实验验证miR-195-5p与FGF2的靶向关系,Western blotting检测FGF2表达水平,CCK-8法检测HEC-1B细胞增殖水平,流式细胞术检测HEC-1B细胞凋亡率,Transwell实验检测HEC-1B细胞侵袭能力,划痕实验检测HEC-1B细胞迁移能力。结果:与HEC-1B组相比,miR-195-5p mimic组miR-195-5p表达升高、FGF2 m RNA水平下降(均P<0.01);miR-195-5p可直接靶向FGF2。与HEC-1B组相比,miR-195-5p mimic组FGF2的蛋白表达水平下降,p LV-FGF2组FGF2的蛋白水平明显上升,且miR-195-5p+FGF2组FGF2的蛋白表达水平低于p LV-FGF2组(均P<0.01)。miR-195-5p mimic组细胞增殖水平低于HEC-1B组,p LV-FGF2组细胞增殖水平高于HEC-1B组(均P<0.01)。与HEC-1B组相比,miR-195-5p mimic组细胞凋亡率增加,p LV-FGF2组细胞凋亡率降低,且miR-195-5p+FGF2组细胞凋亡率高于p LV-FGF2组(均P<0.01)。与HEC-1B组相比,miR-195-5p mimic组每个视野下的侵袭细胞数和划痕愈合率下降,p LV-FGF2组每个视野下的侵袭细胞数和划痕愈合率上升,且miR-195-5p+FGF2组每个视野下的侵袭细胞数和划痕愈合率低于p LV-FGF2组(均P<0.01)。结论:miR-195-5p通过靶向FGF2抑制子宫内膜癌HEC-1B细胞的增殖、侵袭和迁移并促进细胞凋亡,其作为子宫内膜癌的治疗靶点。
Objective: To explore the molecular mechanism of miR-195-5 p targeting FGF2 to inhibit the proliferation, apoptosis, invasion and migration of endometrial cancer HEC-1 B cells. Methods: After culture and transfection, HEC-1 B cells were divided into 4 groups: HEC-1 B group, miR-195-5 p mimic group, p LV-FGF2 group and miR-195-5 p + FGF2 group. The expressions of miR-195-5 p and m RNA levels of FGF2 were detected by q RT-PCR. The targeted relationship of miR-195-5 p and FGF2 was verified by luciferase assay. The protein expression of FGF2 was examined by Western blotting; Proliferation of HEC-1 B cells was measured by CCK-8;Apoptosis was tested by flow cytometry; HEC-1 B cell invasion was detected by transwell, and migration was measured by scratch assay. Results: Compared with HEC-1 B group, the expression of miR-195-5 p in miR-195-5 p mimic group was elevated while FGF2 m RNA level was declined(all P<0.01). Luciferase assay indicated that FGF2 was a target of miR-195-5 p. Compared with HEC-1 B group, the protein level of FGF2 in miR-195-5 p mimic group was decreased, and the protein levels of FGF2 in p LV-FGF2 group were enhanced(P<0.01). The protein levels of FGF2 in miR-195-5 p+FGF2 group were lower than that in p LV-FGF2 group(all P<0.01). The proliferation in miR-195-5 p mimic group was lower than HEC-1 B group(P<0.01), while the proliferation in p LV-FGF2 group was higher than that in HEC-1 B group(all P<0.01). Compared with HEC-1 B group, apoptosis in miR-195-5 p mimic group was increased,and apoptosis in p LV-FGF2 group was decreased(P<0.01); moreover, apoptosis in miR-195-5 p + FGF2 group was higher than that in p LV-FGF2 group(P<0.01). Compared with HEC-1 B group, the number of invasive cells per field and the rate of wound healing in miR-195-5 p mimic group were decreased, while those in p LV-FGF2 group was enhanced(P<0.01); moreover, the number of invasive cells per field and the rate of wound healing in miR-195-5 p+FGF2 group was lower than those in p LV-FGF2 group(all P<0.01). Conclusion: miR-195-5 p inhibits proliferation, invasion and migration and promotes apoptosis of endometrial cancer HEC-1 B cells by target-ing FGF2, and could be used as a treatment target of endometrial cancer.
Objective: To explore the molecular mechanism of miR-195-5 p targeting FGF2 to inhibit the proliferation, apoptosis, invasion and migration of endometrial cancer HEC-1 B cells. Methods: After culture and transfection, HEC-1 B cells were divided into 4 groups: HEC-1 B group, miR-195-5 p mimic group, p LV-FGF2 group and miR-195-5 p + FGF2 group. The expressions of miR-195-5 p and m RNA levels of FGF2 were detected by q RT-PCR. The targeted relationship of miR-195-5 p and FGF2 was verified by luciferase assay. The protein expression of FGF2 was examined by Western blotting; Proliferation of HEC-1 B cells was measured by CCK-8;Apoptosis was tested by flow cytometry; HEC-1 B cell invasion was detected by transwell, and migration was measured by scratch assay. Results: Compared with HEC-1 B group, the expression of miR-195-5 p in miR-195-5 p mimic group was elevated while FGF2 m RNA level was declined(all P<0.01). Luciferase assay indicated that FGF2 was a target of miR-195-5 p. Compared with HEC-1 B group, the protein level of FGF2 in miR-195-5 p mimic group was decreased, and the protein levels of FGF2 in p LV-FGF2 group were enhanced(P<0.01). The protein levels of FGF2 in miR-195-5 p+FGF2 group were lower than that in p LV-FGF2 group(all P<0.01). The proliferation in miR-195-5 p mimic group was lower than HEC-1 B group(P<0.01), while the proliferation in p LV-FGF2 group was higher than that in HEC-1 B group(all P<0.01). Compared with HEC-1 B group, apoptosis in miR-195-5 p mimic group was increased,and apoptosis in p LV-FGF2 group was decreased(P<0.01); moreover, apoptosis in miR-195-5 p + FGF2 group was higher than that in p LV-FGF2 group(P<0.01). Compared with HEC-1 B group, the number of invasive cells per field and the rate of wound healing in miR-195-5 p mimic group were decreased, while those in p LV-FGF2 group was enhanced(P<0.01); moreover, the number of invasive cells per field and the rate of wound healing in miR-195-5 p+FGF2 group was lower than those in p LV-FGF2 group(all P<0.01). Conclusion: miR-195-5 p inhibits proliferation, invasion and migration and promotes apoptosis of endometrial cancer HEC-1 B cells by target-ing FGF2, and could be used as a treatment target of endometrial cancer.
引文
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[23]QU Q,CHU X,WANG P.Micro RNA-195-5p suppresses osteosarcoma cell proliferation and invasion by suppressing naked cuticle homolog 1[J].Cell Biol Int,2017,41(3):287-295.DOI:10.1002/cbin.10723.
[24]LUO Q,ZHANG Z,DAI Z,et al.Tumor-suppressive micro RNA-195-5p regulates cell growth and inhibits cell cycle by targeting cyclin dependent kinase 8 in colon cancer[J].Am J Transl Res,2016,8(5):2088-2096.
[25]WU J,JI A,WANG X,et al.Micro RNA-195-5p,a new regulator of Fra-1,suppresses the migration and invasion of prostate cancer cells[J].J Transl Med,2015,13(4):289-294.DOI:10.1186/s12967-015-0650-6.
[26]WANG M,ZHANG J,TONG L,et al.Mi R-195 is a key negative regulator of hepatocellular carcinoma metastasis by targeting FGF2and VEGFA[J].Int J Clin Exp Pathol,2015,8(11):14110-14120.
[27]GUO H,LI W,ZHENG T,et al.Mi R-195 targets HDGF to inhibit proliferation and invasion of NSCLC cells[J].Tumour Biol,2014,35(9):8861-8866.DOI:10.1007/s13277-014-2153-0.
[28]SUN M,SONG H,WANG S,et al.Integrated analysis identifies micro RNA-195 as a suppressor of Hippo-YAP pathway in colorectal cancer[J].J Hematol Oncol,2017,10(1):79-86.DOI:10.1186/s13045-017-0445-8.
[29]ZHOU S,YU L,XIONG M,et al.Lnc RNASNHG12 promotes tumorigenesis and metastasis in osteosarcoma by upregulating Notch2 by sponging mi R-195-5p[J].Biochem Biophys Res Commun,2018,495(2):1822-1832.DOI:10.1016/j.bbrc.2017.12.047.
[30]LIU C,GUAN H,WANG Y,et al.Mir-195 inhibits emt by targeting FGF2 in prostate cancer cells[J/OL].PLo S One,2015,10(12):e0144073[2018-04-06].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4674136/.DOI:10.1371/journal.pone.0144073.
[2]BINDER P S,MUTCH D G.Update on prognostic markers for endometrial cancer[J].Womens Health(Lond),2014,10(3):277-288.DOI:10.2217/whe.14.13.
[3]BELL D W.Novel genetic targets in endometrial cancer[J].Expert Opin Ther Targets,2014,18(7):725-730.DOI:10.1517/14728222.2014.909414.
[4]ACUNZO M,ROMANO G,WERNICKE D,et al.Micro RNA and cancer--a brief overview[J].Adv Biol Regul,2015,57(1):1-9.DOI:10.1016/j.jbior.2014.09.013.
[5]VASILATOU D,SIOULAS V D,PAPPA V,et al.The role of mi R-NAs in endometrial cancer[J].Epigenomics,2015,7(6):951-959.DOI:10.2217/epi.15.41.
[6]YANOKURA M,BANNO K,IIDA M,et al.Micrornas in endometrial cancer:Recent advances and potential clinical applications[J].Excli J,2015,14(5):190-198.DOI:10.17179/excli2014-590.
[7]JAYARAMAN M,RADHAKRISHNAN R,MATHEWS C A,et al.Identification of novel diagnostic and prognostic mi RNA signatures in endometrial cancer[J].Genes Cancer,2017,8(5/6):566-576.DOI:10.18632/genesandcancer.144.
[8]CAI C,CHEN Q B,HAN Z D,et al.mi R-195inhibitstumorprogression by targetingrps6kb1 in humanprostatecancer[J].Clin Cancer Res,2015,21(21):4922-4934.DOI:10.1158/1078-0432.CCR-15-0217.
[9]WANG F,JIANG C,SUN Q,et al.Mi R-195 is a key regulator of Raf1 in thyroid cancer[J/OL].Onco Targets Ther,2015,8:3021-3028[2018-04-06].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4621222/.DOI:10.2147/OTT.S90710.
[10]ZHAO C,QI L,CHEN M,et al.Micro RNA-195 inhibits cell proliferation in bladder cancer via inhibition of cell division control protein 42 homolog/signal transducer and activator of transcription-3signaling[J].Exp Ther Med,2015,10(3):1103-1108.DOI:10.3892/etm.2015.2633.
[11]CONWAY J L,LUKOVIC J,LAFRAMBOISE S,et al.Brachy-ing unresectableendometrialcancers with magneticresonanceguidance[J/OL].Cureus,2018,10(3):e2274[2018-04-06].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5935429/.DOI:10.7759/cureus.2274.
[12]顾星,曹方,胡永伟,等.小脑锌指结构1基因在人子宫内膜癌中的表达及其预后预测价值[J].中国肿瘤生物治疗杂志,2017,24(8):875-879.DOI:10.3872/j.issn.1007-385X.2017.08.010.
[13]SILVA J L,PAULINO E,DIAS M F,et al.Endometrial cancer:redefining the molecular-targeted approach[J].Cancer Chem Pharmacol,2015,76(1):1-11.DOI:10.1007/s00280-015-2758-z.
[14]LHEUREUX S,OZA A M.Endometrial cancer-targeted therapies myth or reality?Review of current targeted treatments[J].Eur JCancer,2016,59(2):99-108.DOI:10.1016/j.ejca.2016.02.016.
[15]HAYES J,PERUZZI P P,LAWLER S.Micro RNAs in cancer:biomarkers,functions and therapy[J].Trends Mol Med,2014,20(8):460-469.DOI:10.1016/j.molmed.2014.06.005.
[16]ZHANG X,TAO T,LIU C,et al.Downregulation of mi R-195 promotes prostate cancer progression by targeting HMGA1[J].OncolRep,2016,36(1):376-382.DOI:10.3892/or.2016.4797.
[17]WANG Y,ZHANG X,ZOU C,et al.mi R-195 inhibits tumor growth and angiogenesis through modulating IRS1 in breast cancer[J].Biomed Pharmacother,2016,80(2):95-101.DOI:10.1016/j.biopha.2016.03.007.
[18]LI Z,WANG H,WANG Z,et al.Mi R-195 inhibits the proliferation of human cervical cancer cells by directly targeting cyclin D1[J].Tumour Biol,2016,37(5):6457-6463.DOI:10.1007/s13277-015-4540-6.
[19]ZHANG X,XU J,JIANG T,et al.Micro RNA-195 suppresses colorectal cancer cells proliferation via targeting FGF2 and regulating Wnt/β-catenin pathway[J].Am J Cancer Res,2016,6(11):2631-2640.
[20]FEI X,QI M,WU B,et al.Micro RNA-195-5p suppresses glucose uptake and proliferation of human bladder cancer T24 cells by regulating GLUT3 expression[J].FEBS Lett,2012,586(4):392-397.DOI:10.1016/j.febslet.2012.01.006.
[21]CHEN S,WANG L,YAO X,et al.mi R-195-5p is critical in REGγ-mediated regulation of wnt/β-catenin pathway in renal cell carcinoma[J].Oncotarget,2017,8(38):63986-64000.DOI:10.18632/oncotarget.19256.
[22]WANG T,REN Y,LIU R,et al.mi R-195-5psuppresses the proliferation,migration,and invasion of oralsquamouscellcarcinoma by targeting TRIM14[J/OL].Biomed Res Int,2017,2017:7378148[2018-04-06].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5674489/.DOI:10.1155/2017/7378148.
[23]QU Q,CHU X,WANG P.Micro RNA-195-5p suppresses osteosarcoma cell proliferation and invasion by suppressing naked cuticle homolog 1[J].Cell Biol Int,2017,41(3):287-295.DOI:10.1002/cbin.10723.
[24]LUO Q,ZHANG Z,DAI Z,et al.Tumor-suppressive micro RNA-195-5p regulates cell growth and inhibits cell cycle by targeting cyclin dependent kinase 8 in colon cancer[J].Am J Transl Res,2016,8(5):2088-2096.
[25]WU J,JI A,WANG X,et al.Micro RNA-195-5p,a new regulator of Fra-1,suppresses the migration and invasion of prostate cancer cells[J].J Transl Med,2015,13(4):289-294.DOI:10.1186/s12967-015-0650-6.
[26]WANG M,ZHANG J,TONG L,et al.Mi R-195 is a key negative regulator of hepatocellular carcinoma metastasis by targeting FGF2and VEGFA[J].Int J Clin Exp Pathol,2015,8(11):14110-14120.
[27]GUO H,LI W,ZHENG T,et al.Mi R-195 targets HDGF to inhibit proliferation and invasion of NSCLC cells[J].Tumour Biol,2014,35(9):8861-8866.DOI:10.1007/s13277-014-2153-0.
[28]SUN M,SONG H,WANG S,et al.Integrated analysis identifies micro RNA-195 as a suppressor of Hippo-YAP pathway in colorectal cancer[J].J Hematol Oncol,2017,10(1):79-86.DOI:10.1186/s13045-017-0445-8.
[29]ZHOU S,YU L,XIONG M,et al.Lnc RNASNHG12 promotes tumorigenesis and metastasis in osteosarcoma by upregulating Notch2 by sponging mi R-195-5p[J].Biochem Biophys Res Commun,2018,495(2):1822-1832.DOI:10.1016/j.bbrc.2017.12.047.
[30]LIU C,GUAN H,WANG Y,et al.Mir-195 inhibits emt by targeting FGF2 in prostate cancer cells[J/OL].PLo S One,2015,10(12):e0144073[2018-04-06].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4674136/.DOI:10.1371/journal.pone.0144073.