Notch2通过上皮-间质转化促进生长激素腺瘤的侵袭

Notch2 regulated the invasion of somatotroph adenomas via epithelial-mesenchymal transition

目的探讨Notch2在生长激素腺瘤侵袭中的作用以及对上皮-间质转化(EMT)相关基因的影响。方法回顾性分析2013年1月至2015年12月首都医科大学附属北京天坛医院收治的62例生长激素腺瘤,按照Knosp分级分为侵袭组(28例)和非侵袭组(34例)。应用免疫组织化学染色检测Notch2和Vimentin在生长激素腺瘤中的表达情况,并分析两者的相关性。将小鼠垂体瘤细胞(GT1-1)分为对照组、空载体组和干扰组(Sh-A、Sh-B、Sh-C、ShD)。干扰组使用Sh-A、Sh-B、Sh-C、Sh-D 4种片段在GT1-1细胞中干扰Notch2的表达并选择干扰效果最佳的两种进行后续实验。采用MTS法检测GT1-1细胞增殖能力,采用小室实验检测GT1-1细胞的侵袭能力,酶联免疫吸附实验(ELISA)检测GT1-1细胞培养上清中生长激素(GH)水平,采用实时定量-PCR(RT-PCR)实验检测EMT相关基因的mRNA水平。结果免疫组织化学染色显示在生长激素腺瘤中,Notch2的表达水平与Vimentin呈正相关(r=0. 456,P=0. 027)。Notch2在侵袭组中的表达水平较非侵袭组明显升高(P <0. 05)。同样,侵袭组Vimentin表达水平较非侵袭组水平明显升高(P <0. 05)。RT-PCR和Western blotting实验筛选出干扰Notch2表达的有效片段为ShB和Sh-C。MTS实验显示,与对照组比较,Sh-B组细胞活力24 h、48 h和72 h时分别下降(6. 8±2. 1)%、(29. 2±4. 1)%和(43. 1±5. 2)%,差异均具有统计学意义(P <0. 05);Sh-C组分别下降(5. 2±2. 3)%、(17±3. 5)%和(26. 9±3. 8)%,差异均具有统计学意义(P <0. 05)。小室实验显示,对照组穿膜阳性细胞为(407±53)个/视野,Sh-B组为(126±35)个/视野,Sh-C组为(163±41)个/视野,差异均具有统计学意义(P <0. 05)。ELISA结果显示,处理72 h后,Sh-B和Sh-C的GH水平分别为对照组的(27. 7±7. 3)%和(36. 4±8. 5)%,差异具有统计学意义(P <0. 05)。Sh-B组及Sh-C组细胞E-cadherin较对照组升高,N-cadherin和Vimentin mRNA水平均较对照组明显降低,差异均具有统计学意义(P <0. 05)。结论 Notch2通过调控E-cadherin、N-Cadherin和vimentin的表达影响EMT,促进生长激素腺瘤侵袭性生长。

Objective To study the role and mechanism of Notch2 in the invasion of somatotroph adenomas,further to testify the effect of Notch2 on genes related to epithelial-mesenchymal transition( EMT). Methods Clinical data of 62 patients with somatotroph adenomas admitted in Beijing Tiantan Hospital,Capital Medical University from January 2013 to December 2015 were selected and retrospectively analyzed. These patients were divided into invasive group( 28 cases) and non-invasive group( 34 cases) according to Knosp staging. Immunohistochemical staining was used to detect the expression of Notch2 and Vimentin in somatotroph adenomas,and the correlation of them was analyzed. GT1-1 cells were divided into control group,vector group and knockdown group( Sh-A,Sh-B,Sh-C,Sh-D). Notch2 was knockdown in mouse pituitary adenomas cell line( GT1-1) by fragments of Sh-A,Sh-B,Sh-C,Sh-D,two efficient fragments were used for functional experiment according to RT-PCR and Western blotting. MTS experiment was used to detect cell proliferation,and chamber assay was used to detect the migration of tumor cells. Enzyme linked immunosorbent assay( ELISA) was used to measure the levels of growth hormone( GH) in cell culture. The mRNA levels of genes related to EMT were assayed by real time-PCR( RT-PCR). Results The results of immunohistochemical staining showed that there was positive correlation between Notch2 and Vimentin( r = 0. 456,P = 0. 027). The Notch2 level was significantly higher in invasive group compared to noninvasive group( P < 0. 05). Additionally,the Vimentin level was significantly higher in invasive group compared to noninvasive group( P < 0. 05). The Sh-B and Sh-C fragments of RNAi-Notch2 were filtered by RT-PCR and western blotting assay. MTS experiment showed that cell viability of Sh-B group decreased( 6. 8 ± 2. 1) %,( 29. 2 ± 4. 1) % and( 43. 1 ± 5. 2) % compared to those in control group after 24 h,48 h and 72 h( P < 0. 05),and( 5. 2 ± 2. 3) %,( 17 ± 3. 5) % and( 26. 9 ± 3. 8) % in Sh-C group( P < 0. 05). Chamber assay showed that the trans-membrane positive cells were( 407 ± 53)/field in control group,and( 126 ± 35)/field in Sh-B group and( 163 ± 41)/field in Sh-C group( P < 0. 05). ELISA results proved that the level of GH in Sh-B group and Sh-C group was( 27. 7 ± 7. 3) % and( 36. 4 ± 8. 5) % of that in control group( P < 0. 05). RT-PCR assay showed that mRNA levels of E-cadherin in the Sh-B and Sh-C group increased compared to that in control group,mRNA levels of N-cadherin and Vimentin reduced compared to those in the control group( P< 0. 05). Conclusion Notch2 can promote the invasive behavior of somatotroph adenoma through regulating the expression of E-cadherin,N-cadherin and vimentin.