Abstract：
BACKGROUND: Tropomyosin 2 (TPM2) has been linked to the advancement of various tumor types, exhibiting distinct impacts on tumor progression. In our investigation, the primary objective was to identify the potential involvement of TPM2 in the development of colitis-associated cancer (CAC) using a mice model.
METHODS: This study used lentiviral vector complex for TPM2 knockdown (sh-TPM2) and the corresponding negative control lentiviral vector complex (sh-NC) for genetic interference in mice. CAC was induced in mice using azoxymethane (AOM) and dextran sulfate sodium salt (DSS). This study included 6 groups of mice models: Control, Control+sh-NC, Control+sh-TPM2, CAC, CAC+sh-NC, and CAC+sh-TPM2. Subsequently, colon tissues were collected and assessed using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for TPM2 mRNA levels and flow cytometry for infiltrating immune cells. Tumor number, size, and weight within colon tissues from CAC mice were measured and recorded. The hematoxylin-eosin staining was used for observing tissue pathology changes. The intestinal epithelial cells (IECs) were isolated and analyzed for cell proliferation. This analysis included examining the levels of 5-bromo-2-deoxyuridine (BrdU) and Ki-67 using immunohistochemistry. Additionally, the mRNA levels of proliferating cell nuclear antigen (PCNA) and Ki-67 were detected by qRT-PCR. This study also investigated the activation of the c-Jun N-terminal kinase (JNK) pathway using western blot analysis. Immunogenicity analyses were conducted using immunohistochemistry for F4/80 and flow cytometry.
RESULTS: In 8-week-old mice, AOM injections and three cycles of DSS treatment induced TPM2 upregulation in tumor tissues compared to normal tissues (p < 0.05). Fluorescence-activated cell sorting (FACS)-isolated lamina CAC adenomas revealed macrophages and dendritic cells as primary TPM2 contributors (p < 0.001). Lentiviral TPM2 gene knockdown significantly reduced tumor numbers and sizes in CAC mice (p < 0.01, and p < 0.001), without invasive cancer cells. TPM2 suppression resulted in decreased IEC proliferation (p < 0.001) and reduced PCNA and Ki-67 expression (p < 0.05). Western blot analysis indicated reduced JNK pathway activation in TPM2-knockdown CAC mice (p < 0.05, p < 0.001). TPM2 knockdown decreased tumor-associated macrophage infiltration (p < 0.01) and increased CD3+ and CD8+ T cells (p < 0.01, and p < 0.001), with increased levels of regulator of inflammatory cytokines (CD44+, CD107a+) (p < 0.01, and p < 0.001), decreased levels of PD-1+ and anti-inflammatory factor (IL10+) (p < 0.01, and p < 0.001).
CONCLUSIONS: Our results demonstrated that TPM2 knockdown suppressed the proliferation of CAC IECs, enhanced immune suppression on CAC IECs, and inhibited the JNK signaling pathway within the framework of CAC. These findings suggest TPM2 can serve as a potential therapeutic target for CAC treatment.
METHODS: This study used lentiviral vector complex for TPM2 knockdown (sh-TPM2) and the corresponding negative control lentiviral vector complex (sh-NC) for genetic interference in mice. CAC was induced in mice using azoxymethane (AOM) and dextran sulfate sodium salt (DSS). This study included 6 groups of mice models: Control, Control+sh-NC, Control+sh-TPM2, CAC, CAC+sh-NC, and CAC+sh-TPM2. Subsequently, colon tissues were collected and assessed using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for TPM2 mRNA levels and flow cytometry for infiltrating immune cells. Tumor number, size, and weight within colon tissues from CAC mice were measured and recorded. The hematoxylin-eosin staining was used for observing tissue pathology changes. The intestinal epithelial cells (IECs) were isolated and analyzed for cell proliferation. This analysis included examining the levels of 5-bromo-2-deoxyuridine (BrdU) and Ki-67 using immunohistochemistry. Additionally, the mRNA levels of proliferating cell nuclear antigen (PCNA) and Ki-67 were detected by qRT-PCR. This study also investigated the activation of the c-Jun N-terminal kinase (JNK) pathway using western blot analysis. Immunogenicity analyses were conducted using immunohistochemistry for F4/80 and flow cytometry.
RESULTS: In 8-week-old mice, AOM injections and three cycles of DSS treatment induced TPM2 upregulation in tumor tissues compared to normal tissues (p < 0.05). Fluorescence-activated cell sorting (FACS)-isolated lamina CAC adenomas revealed macrophages and dendritic cells as primary TPM2 contributors (p < 0.001). Lentiviral TPM2 gene knockdown significantly reduced tumor numbers and sizes in CAC mice (p < 0.01, and p < 0.001), without invasive cancer cells. TPM2 suppression resulted in decreased IEC proliferation (p < 0.001) and reduced PCNA and Ki-67 expression (p < 0.05). Western blot analysis indicated reduced JNK pathway activation in TPM2-knockdown CAC mice (p < 0.05, p < 0.001). TPM2 knockdown decreased tumor-associated macrophage infiltration (p < 0.01) and increased CD3+ and CD8+ T cells (p < 0.01, and p < 0.001), with increased levels of regulator of inflammatory cytokines (CD44+, CD107a+) (p < 0.01, and p < 0.001), decreased levels of PD-1+ and anti-inflammatory factor (IL10+) (p < 0.01, and p < 0.001).
CONCLUSIONS: Our results demonstrated that TPM2 knockdown suppressed the proliferation of CAC IECs, enhanced immune suppression on CAC IECs, and inhibited the JNK signaling pathway within the framework of CAC. These findings suggest TPM2 can serve as a potential therapeutic target for CAC treatment.
摘要：
背景：原肌球蛋白2（TPM2）与各种肿瘤类型的进展有关，对肿瘤进展表现出明显的影响。在我们的调查中,主要目的是使用小鼠模型确定TPM2在结肠炎相关癌症(CAC)发展中的潜在参与.
方法：本研究使用用于TPM2敲低的慢病毒载体复合物（sh-TPM2）和相应的阴性对照慢病毒载体复合物（sh-NC）用于小鼠的遗传干扰。使用偶氮甲烷(AOM)和葡聚糖硫酸钠盐(DSS)在小鼠中诱导CAC。本研究包括6组小鼠模型：对照，Control+sh-NC,控制+sh-TPM2,CAC,CAC+sh-NC,和CAC+sh-TPM2。随后，收集结肠组织并使用定量逆转录-聚合酶链反应(qRT-PCR)评估TPM2mRNA水平和流式细胞术评估浸润免疫细胞。肿瘤数量,尺寸,测量并记录来自CAC小鼠的结肠组织内的重量。采用苏木精-伊红染色观察组织病理变化。分离肠上皮细胞(IECs)并分析细胞增殖。该分析包括使用免疫组织化学检查5-溴-2-脱氧尿苷(BrdU)和Ki-67的水平。此外,qRT-PCR检测增殖细胞核抗原（PCNA）和Ki-67的mRNA水平。该研究还使用蛋白质印迹分析研究了c-JunN末端激酶(JNK)途径的激活。使用F4/80的免疫组织化学和流式细胞术进行免疫原性分析。
结果：在8周龄小鼠中，与正常组织相比,AOM注射和三个周期的DSS治疗在肿瘤组织中诱导TPM2上调(p<0.05)。荧光激活细胞分选(FACS)-分离的层CAC腺瘤显示巨噬细胞和树突状细胞是主要的TPM2贡献者(p<0.001)。慢病毒TPM2基因敲低显著减少CAC小鼠的肿瘤数量和大小（p<0.01，p<0.001），没有侵袭性癌细胞.TPM2抑制导致IEC增殖降低(p<0.001)和PCNA和Ki-67表达降低(p<0.05)。Western印迹分析表明TPM2敲低CAC小鼠中JNK途径活化降低(p<0.05,p<0.001)。TPM2敲低降低肿瘤相关巨噬细胞浸润（p<0.01），增加CD3+和CD8+T细胞（p<0.01和p<0.001），随着炎症细胞因子（CD44+，CD107a）（p<0.01，p<0.001），PD-1+和抗炎因子(IL10+)水平降低(p<0.01,p<0.001)。
结论：我们的结果表明TPM2敲低抑制了CACIECs的增殖，增强对CACIEC的免疫抑制，并在CAC框架内抑制JNK信号通路。这些发现表明TPM2可以作为CAC治疗的潜在治疗靶标。
方法：本研究使用用于TPM2敲低的慢病毒载体复合物（sh-TPM2）和相应的阴性对照慢病毒载体复合物（sh-NC）用于小鼠的遗传干扰。使用偶氮甲烷(AOM)和葡聚糖硫酸钠盐(DSS)在小鼠中诱导CAC。本研究包括6组小鼠模型：对照，Control+sh-NC,控制+sh-TPM2,CAC,CAC+sh-NC,和CAC+sh-TPM2。随后，收集结肠组织并使用定量逆转录-聚合酶链反应(qRT-PCR)评估TPM2mRNA水平和流式细胞术评估浸润免疫细胞。肿瘤数量,尺寸,测量并记录来自CAC小鼠的结肠组织内的重量。采用苏木精-伊红染色观察组织病理变化。分离肠上皮细胞(IECs)并分析细胞增殖。该分析包括使用免疫组织化学检查5-溴-2-脱氧尿苷(BrdU)和Ki-67的水平。此外,qRT-PCR检测增殖细胞核抗原（PCNA）和Ki-67的mRNA水平。该研究还使用蛋白质印迹分析研究了c-JunN末端激酶(JNK)途径的激活。使用F4/80的免疫组织化学和流式细胞术进行免疫原性分析。
结果：在8周龄小鼠中，与正常组织相比,AOM注射和三个周期的DSS治疗在肿瘤组织中诱导TPM2上调(p<0.05)。荧光激活细胞分选(FACS)-分离的层CAC腺瘤显示巨噬细胞和树突状细胞是主要的TPM2贡献者(p<0.001)。慢病毒TPM2基因敲低显著减少CAC小鼠的肿瘤数量和大小（p<0.01，p<0.001），没有侵袭性癌细胞.TPM2抑制导致IEC增殖降低(p<0.001)和PCNA和Ki-67表达降低(p<0.05)。Western印迹分析表明TPM2敲低CAC小鼠中JNK途径活化降低(p<0.05,p<0.001)。TPM2敲低降低肿瘤相关巨噬细胞浸润（p<0.01），增加CD3+和CD8+T细胞（p<0.01和p<0.001），随着炎症细胞因子（CD44+，CD107a）（p<0.01，p<0.001），PD-1+和抗炎因子(IL10+)水平降低(p<0.01,p<0.001)。
结论：我们的结果表明TPM2敲低抑制了CACIECs的增殖，增强对CACIEC的免疫抑制，并在CAC框架内抑制JNK信号通路。这些发现表明TPM2可以作为CAC治疗的潜在治疗靶标。
