潜在转化生长因子β结合蛋白4影响肿瘤细胞伪足形成抑制结直肠癌转移的分子机制研究  

Molecular mechanism of latent transforming growth factor beta binding protein 4 inhibiting metastasis of colorectal cancer by aff ecting pseudopod formation in tumor cells

作　　者：及松涛 王好甲 李昂 孙亚盟 卢瑗瑗 赵晓迪 王新 Ji Songtao;Wang Haojia;Li Ang;Sun Yameng;Lu Yuanyuan;Zhao Xiaodi;Wang Xin(Department of Gastroenterology,Tangdu Hospital,Air Force Military Medical University,Xi’an 710038,China;State Key Laboratory of Holistic Integrative Management of Gastrointestinal Cancers and National Clinical Research Center for Digestive Diseases,Xijing Hospital,Air Force Military Medical University,Xi’an 710032,China;Department of Military Medicine and Special Medicine,971st Hospital of Navy,Qingdao 266000,China)

机构地区：[1]空军军医大学唐都医院消化内科,陕西西安710038 [2]空军军医大学西京医院,国家消化系统疾病临床医学研究中心和消化系肿瘤整合防治全国重点实验室,陕西西安710032 [3]海军第971医院军事医学与特种学科,山东青岛266000

出　　处：《实用肿瘤杂志》2024年第2期140-148,共9页Journal of Practical Oncology

摘　　要：目的 探究潜在转化生长因子β结合蛋白4(latent transforming growth factor beta binding protein 4,LTBP4)通过影响肿瘤细胞伪足形成从而遏制结直肠癌细胞转移的分子机制。方法 在结直肠癌细胞株DiFi中使用小干扰si-LTBP4敲减LTBP4,在结直肠癌细胞株HCT15中使用LTBP4过表达质粒过表达LTBP4。使用人源慢病毒在DiFi细胞株中稳定敲减/不敲减LTBP4构建稳转细胞株shLTBP4和shNC。Western blot法检测LTBP4、原肌球蛋白4(tropomyosin 4,TPM4)、金属基质蛋白酶14(matrix metalloproteinase-14,MMP14)及酪氨酸激酶底物5(tyrosine kinase substrate 5,TKS5)等蛋白。实时荧光定量PCR(quantitative real-time PCR,qPCR)检测LTBP4及TPM4的表达。Transwell迁移实验检测敲低/过表达LTBP4后结直肠癌细胞的体外转移能力。使用裸鼠尾静脉注射shLTBP4或shNC稳转细胞株构建裸鼠肺转移模型及阴性对照,检测结直肠癌细胞敲减LTBP4后的体内转移能力。转录组测序检测差异基因表达情况。鬼笔环肽染色观察敲低LTBP4后细胞骨架改变及侵袭性伪足形成情况。结果 qPCR实验检测发现,LTBP4在DiFi、HCT8和KM12C细胞株中均高表达(均P<0.05),在HCT15和KM12SM细胞株中均低表达(均P<0.01)。Westen blot检测发现,LTBP4在DiFi、HCT8和KM12C细胞株中均高表达(均P<0.01),在HCT15、KM12SM及RKO细胞株中均低表达(均P<0.05)。在高表达LTBP4的DiFi细胞株中转染小干扰si-LTBP4,敲低LTBP4,转染后72 h后进行transwell迁移实验发现,肿瘤细胞迁移能力增强(P<0.01);而在低表达LTBP4的HCT15细胞株中过表达LTBP4,72 h后肿瘤细胞迁移能力减弱(P<0.01)。裸鼠肺转移模型在通过尾静脉注射稳转细胞株shLTBP4以及shNC 5周后进行小动物活体荧光成像发现,敲减LTBP4后,小鼠肺部荧光信号增强(P<0.01)。高通量转录组测序发现,在DiFi细胞株中敲减LTBP4使细胞骨架蛋白TPM4的表达量增加(P<0.01),维持细胞运动功能(如肌动蛋白丝束收缩、应力�Objective To investigate the molecular mechanism of latent transforming growth factor beta binding protein 4(LTBP4)in-hibiting the metastasis of colorectal cancer cells by influencing pseudopod formation in tumor cells.Methods In colorectal cancer DiFi cells, si-LTBP4 was used to knock down LTBP4, while in colorectal cancer HCT15 cells, LTBP4 was overexpressed. Stable cell lines shLTBP4 and shNC were constructed using LTBP4-knockdown and empty lentiviral vectors, respectively. Western blot was used to detect the expression of LTBP4, tropomyosin 4 (TPM4), matrix metalloproteinase-14 (MMP14), tyrosine kinase substrate 5 (TKS5), etc. Quanti- tative real-time PCR (qPCR) was used to measure LTBP4 and TPM4 expressions. Transwell migration assay was performed to assess the in vitro migration ability of colorectal cancer cells with altered LTBP4 expression. A lung metastasis mouse model was constructed by in- jecting shLTBP4 cells into the tail vein of nude mice to detect the in vivo metastasis ability of colorectal cancer cells after knocking down LTBP4, and mice injected with shNC cells was used as control. Transcriptome sequencing was conducted to analyze the changes in gene expression. Immunofluorescence staining was used to observe cytoskeleton changes and invadopodia formation in LTBP4-knockdown cells. Results qPCR results showed that LTBP4 was highly expressed in the DiFi, HCT8, and KM12C cell lines (all P<0.05), and was expressed at low levels in the HCT15 and KM12SM cell lines (both P<0.01). Western blot assay revealed high expression of LTBP4 in the DiFi, HCT8, and KM12C cell lines (all P<0.01), and low expression in the HCT15, KM12SM, and RKO cell lines (all P<0.05). Transfec- tion of small interfering si-LTBP4 in the DiFi cell line which had high LTBP4 expression resulted in the knockdown of LTBP4. Transwell migration assay conducted 72 hours post-transfection showed enhanced migration ability of cells (P<0.01). Conversely, LTBP4 overex- pression in the HCT15 cell line which had low LTBP4 expression led to we

关 键 词：结直肠癌 潜在转化生长因子β结合蛋白4 肿瘤转移 转录组测序 免疫荧光 侵袭性伪足 

分 类 号：R735.34[医药卫生—肿瘤]