PO.MCB03.03 · 分子与细胞生物学
TPM4过表达改变分化型结肠上皮细胞的屏障完整性
TPM4 overexpression modifies differentiated colon epithelial cell barrier integrity
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
结肠上皮细胞从结肠隐窝干细胞区迁出对正常分化和生长停滞至关重要。收缩性和迁移特异性基因表达的改变可能破坏这些过程，并促成结肠肿瘤发生的早期阶段。其中一个基因——原肌球蛋白-4（TPM4）在结直肠癌的早期和晚期均过表达。确定TPM4过表达如何促成结肠细胞肿瘤发生可能鉴定出新的治疗靶点。我们假设TPM4过表达可能通过破坏细胞间连接并降低分化中结肠上皮细胞的上皮屏障完整性来促进结肠肿瘤发生。为验证这一点，我们利用Caco2细胞——一种源自人结肠腺癌的结肠上皮细胞分化体外模型。我们的方法是确定在TPM4正常受抑制的分化中Caco2细胞里，通过稳定转染人为增加TPM4表达是否会降低Caco-2细胞单层的完整性。为评估这一点，我们测量了跨上皮电阻（TEER，细胞间连接完整性的指标），比较TPM4过表达的分化Caco-2单层与对照转染子。第0天将100,000个Caco2细胞接种于6孔板中的6孔滤膜插入件。使用Millicell ERS 3.0数字电压欧姆计，从第6天至第24天每隔一天以三次重复记录TEER测量值。我们观察到两个TPM4过表达Caco2细胞克隆与对照转染子之间的TEER测量值存在显著差异。对照空载体稳定转染子的TEER值在1237–1445 ohm-sq. cm之间达到平台，而两个TPM4过表达稳定克隆的TEER值分别在464–898 ohm-sq. cm（克隆1）和1743–1868 ohm-sq. cm（克隆7）之间达到平台。克隆1的TEER值在与空载体对照相似的时间点（第12天）达到平台，而克隆7的TEER值在较晚（第16天）达到平台。此外，我们观察到TPM4过表达细胞相对于对照，编码连接复合体蛋白的基因表达发生改变。这些结果表明TPM4过表达在分化中结肠上皮细胞的体外模型中调节细胞间连接的完整性。未来实验将确定两个不同克隆之间观察到的差异是否可能由各克隆TPM4过表达的程度所致。我们的研究提示持续的TPM4过表达可能通过改变正常上皮屏障形成和连接完整性来破坏结肠上皮细胞分化并促进肿瘤发生。因此，我们鉴定了一种可能将TPM4过表达与早期结直肠肿瘤发生联系起来的潜在机制，并阐明了预防和治疗结肠癌（尤其是早期阶段）的可能新途径。
查看英文原文 English abstract
Colon epithelial cell migration out of the stem cell compartment in colon crypts is essential for normal differentiation and growth arrest. Altered expression of contractility- and migration-specific genes may disrupt these processes and contribute to early stages of colon tumorigenesis. One such gene, tropomyosin-4 ( TPM4 ), is overexpressed in early and late stages of colorectal cancer. Determining how TPM4 overexpression contributes to colon cell tumorigenesis may identify novel therapeutic targets. We hypothesized that TPM4 overexpression may promote colon tumorigenesis by disrupting cell-cell junctions and reducing epithelial barrier integrity in differentiating colon epithelial cells. To test this, we utilized Caco2 cells, an in vitro model of colon epithelial cell differentiation derived from a human colon adenocarcinoma. Our approach was to determine whether artificially increasing TPM4 expression by stable transfection in differentiating Caco2 cells, when TPM4 is normally suppressed, will decrease integrity of the Caco-2 cell monolayer. To assess this, we measured transepithelial electrical resistance (TEER), an indicator of cell-cell junction integrity, in differentiating TPM4 -overexpressing Caco-2 monolayers relative to control transfectants. 100,000 Caco2 cells were seeded in 6 well filter inserts in wells of a 6-well plate at day 0. TEER measurements were recorded in triplicate every other day from day 6 to 24 using the Millicell ERS 3.0 Digital Voltohmmeter. We observed significant differences between TEER measurements in two clones of TPM4 -overexpressing Caco2 cells and control transfectants. TEER values in control empty vector stable transfectants plateaued between 1237-1445 ohm-sq. cm, while those in two TPM4 -overexpressing stable clones plateaued between 464-898 ohm-sq. cm (clone 1) and 1743-1868 ohm-sq. cm (clone 7). Clone 1 TEER values plateaued at a similar time point (day 12) as empty vector control, whereas clone 7 TEER values plateaued later (day 16). In addition, we observed alterations in expression of genes encoding junctional complex proteins in TPM4-overexpressing cells relative to controls. These results indicate that TPM4 overexpression modulates the integrity of cell-cell junctions in an in vitro model of differentiating colon epithelial cells. Future experiments will determine whether differences observed between the two distinct clones may be due to the extent of TPM4 overexpression in each clone. Our study suggests that sustained TPM4 overexpression may disrupt colon epithelial cell differentiation and promote tumorigenesis by altering normal epithelial barrier formation and junctional integrity. Therefore, we identify a potential mechanism that may link TPM4 overexpression to early colorectal tumorigenesis and elucidate possible novel approaches to prevent and treat colon cancer, particularly at early stages.
利益披露 Disclosure
B. Backus, None..
M. Papetti, None.