RNA结合蛋白PCBP1在肺腺癌转移中的作用与机制研究

背景和目的：

肺腺癌（LUAD）是肺癌中最常见的组织学亚型，患者的5年生存率低于30%。找到肺腺癌进展转移的调控机制有利于发现肺腺癌治疗的新靶点，控制肿瘤进展。PolyC-RNA-binding protein 1（PCBP1）是异质核糖核蛋白复合物的一种，可以通过与RNA和蛋白相互作用从不同层面调控基因表达发挥肿瘤抑制功能。然而，PCBP1在肺腺癌中的具体作用及机制仍不明确。本文旨在揭示PCBP1在肺腺癌转移中的功能机制。

方法与结果

方法：首先，通过癌症基因组图谱（TCGA）筛选，发现PCBP1是肺腺癌的重要生物标记物，并通过免疫组化和RT-PCR在临床样本中进行了验证。通过细胞划痕实验和Transwell实验，我们确认了PCBP1与肺腺癌细胞的迁移密切相关。接着，通过小鼠肺转移模型进一步验证了PCBP1与肺腺癌肿瘤转移的关系。机制上，通过对PCBP1敲除的肿瘤细胞及对照细胞进行转录组测序发现下游关键基因DKK1。使用蛋白质印迹、RT-PCR、RIP、RNA Pull-Down、mRNA稳定性等实验验证PCBP1与DKK1和β-catenin的调控关系。最后，对临床样本进行免疫组化的检测，分析PCBP1、DKK1和β-catenin三者之间的相关性。

结果：通过对数据库的分析和临床样本检测发现与正常组织相比，PCBP1在肺腺癌肿瘤组织中表达下降，且与肺腺癌患者预后呈正相关。然后通过体外迁移实验发现PCBP1的表达降低能够促进肺腺癌细胞迁移，而小鼠肺转移模型进一步验证了PCBP1能够显著抑制肺腺癌的转移。接着通过探究PCBP1抑制肺腺癌转移的机制，发现PCBP1能够与DKK1的mRNA直接结合，通过提高DKK1的mRNA稳定性而上调其表达，DKK1的表达上调抑制了WNT/β-catenin通路的激活。最后，对肺腺癌临床样本进行检测进一步确认PCBP1、DKK1与β-catenin之间的调控关系，发现PCBP1与DKK1的表达呈显著正相关，与β-catenin呈显著负相关，且三者均是肺腺癌预后的预测分子。

结论

PCBP1通过上调DKK1的表达失活WNT/β-catenin通路抑制肺腺癌的转移，PCBP1是潜在的肺腺癌肿瘤治疗的新靶点。

转录因子KLF12通过调控CD8⁺T细胞介导肿瘤anti-PD-1治疗机制研究背景和目的

以anti-PD-1治疗为代表的免疫检查点抑制剂（Immune Checkpoint Inhibitors，ICIs）疗法已经彻底改变了现有的肿瘤治疗格局，显著提高了患者的5年生存率。然而，仅有20–25%患者能够获得持久的免疫应答，大部分肿瘤患者会出现原发或者继发性耐药。anti-PD-1治疗耐药是一个极其复杂的、多分子间参与的过程，探寻anti-PD-1免疫治疗耐药的分子机制，降低其耐药性，提高免疫治疗效果，对提升患者生存率尤为重要。转录因子KLF12能够与靶基因启动子区结合发挥转录抑制功能。迄今为止，关于KLF12的研究主要集中在其对肿瘤转移和血管生成等调节的方面，关于KLF12对肿瘤浸润免疫细胞的调控作用尚未被报道。本论文旨在揭示KLF12对肿瘤微环境中免疫细胞浸润和功能的影响；探究KLF12影响肿瘤免疫的分子机制；明确KLF12与anti-PD-1疗效之间的关系，寻找改善anti-PD-1治疗效果的策略。

方法与结果

方法：首先构建了KLF12过表达和敲除的肿瘤细胞系，通过体外和体内实验观察KLF12对肿瘤微环境的影响。继而，通过转录组测序、RT-PCR、Western Blot阐明了KLF12调控的关键下游分子，并通过挽救实验确定了两者之间的调控关系。然后通过ChIP-PCR和双荧光素酶报告等实验，明确KLF12的直接转录抑制作用。最后，通过荷瘤小鼠给予治疗，观察治疗疗效，确定KLF12等分子对anti-PD-1治疗的影响，并利用多色免疫荧光对免疫治疗队列进行染色验证，探讨KLF12等分子对anti-PD-1治疗响应率的预测作用。

结果：首先通过体内外实验确定KLF12能够通过促进CD8⁺T细胞的浸润和功能。其次通过转录组测序、RT-PCR和Western Blot等实验发现Galectin-1（Gal-1）是KLF12的关键下游分子，并且通过挽救实验发现肿瘤中KLF12表达降低所引起的CD8⁺T细胞改变能够通过抑制Gal-1的表达得到逆转。然后通过ChIP-PCR和双荧光素酶报告结果明确KLF12抑制Gal-1转录，揭示了二者直接结合的具体区域。最后通过对荷瘤小鼠进行治疗干预证明了靶向KLF12/Gal-1通路能够提高anti-PD-1治疗疗效，并且通过检测免疫治疗样本发现anti-PD-1治疗响应率高的患者中，KLF12、CD8和PD-1表达较高，Gal-1的表达较低。

结论

肿瘤细胞中KLF12能够促进CD8⁺T细胞浸润，提高CD8⁺T细胞功能。KLF12直接靶向Gal-1的启动子区抑制Gal-1的转录和表达。靶向KLF12/Gal-1通路能够改善anti-PD-1治疗疗效。

Effect and mechanism of RNA binding protein PCBP1 in lung adenocarcinoma metastasis

Background and Aims

Lung adenocarcinoma (LUAD) is the most common histological subtype of lung cancer, with the 5-year survival rate of less than 30%. Finding the regulatory mechanism for the progression and metastasis of lung adenocarcinoma will provide new therapeutic targets for lung adenocarcinoma and inhibit tumor progression. PolyC-RNA binding protein 1 (PCBP1) is a type of heterogeneous nuclear ribonuclear protein that interacts with RNA and protein to regulate gene expression at various levels and perform tumor inhibitory functions. However, the role of PCBP1 in lung adenocarcinoma is still unclear. This article aims to reveal the functional mechanism of PCBP1 in lung adenocarcinoma metastasis.

Method and Results

Methods: First, PCBP1 was identified as an important biomarker of lung adenocarcinoma through The Cancer Genome Atlas (TCGA) project screening, and was verified in clinical samples by immunohistochemistry and RT-PCR. Through wound healing and Transwell assays, we confirmed that PCBP1 is closely related to the migration of lung adenocarcinoma cells. Subsequently, the relationship of PCBP1 with tumor metastasis was further validated using a mouse lung metastasis model. Mechanistically, the downstream key gene DKK1 was discovered by RNA sequencing of tumor cells with PCBP1 knockout and control. Using Western Blot, RT-PCR, RIP, RNA Pull-Down, mRNA stability assay, and other experiments to verify the relationship of PCBP1 with DKK1 and β-catenin. Finally, immunohistochemical was performed on clinical samples to analyze the correlation of PCBP1, DKK1 and β-catenin.

Results: Through database analysis and clinical sample testing, we found that PCBP1 expression decreased in lung adenocarcinoma tumor tissue compared with normal tissue, and was positively correlated with the prognosis of lung adenocarcinoma patients. Then in vitro, migration experiments showed that the reduced expression of PCBP1 can promote the migration of lung adenocarcinoma cell, and the mouse lung metastasis model further verified that PCBP1 can significantly inhibit the metastasis of lung adenocarcinoma. Next, by exploring the mechanism we found that PCBP1 can directly bind to the mRNA of DKK1, and upregulate its expression by improving the mRNA stability of DKK1. Upregulation of DKK1 expression inhibits WNT/β-catenin pathway activation. Finally, clinical samples of lung adenocarcinoma were used to further confirm the correlation between PCBP1, DKK1, and β-catenin. The results showed that PCBP1 was positively correlated with DKK1 expression, but negative correlation with β-catenin expression. And all the three were predictive molecules for the prognosis of lung adenocarcinoma.

Conclusions

PCBP1 inhibits LUAD development by upregulating DKK1 to inactivate the WNT/β-catenin pathway. Our findings highlight the potential of PCBP1 as a promising therapeutic target.

Transcription factor KLF12 mediates anti-PD-1 immunotherapy by regulating CD8⁺T cells in tumors

Background and Aims

In recent years, immune checkpoint inhibitors (ICIs) therapy, represented by anti PD-1 therapy, has completely changed the current pattern of tumor treatment, and significantly improved the 5-year survival rate of cancer patients. However, only 20-25% of cancer patients can achieve a durable immune response, and most patients exhibit primary or secondary drug resistance. The drug resistance of anti-PD-1 therapy is an extremely complex process involving multi-molecules. Exploring the molecular mechanisms of anti PD-1 immunotherapy resistance, reducing resistance rate, and improving the effectiveness of immunotherapy are particularly important for improving patient survival. Transcription factor KLF12 can bind to the promoter region of target gene to exert transcriptional inhibition function. So far, research on KLF12 has mainly focused on its regulation of tumor metastasis and angiogenesis, but the regulatory effect of KLF12 on immune cells infiltration in tumor has not been reported. This study aims to uncover the effect of KLF12 on the infiltration and function of immune cells in tumor microenvironment, explore the molecular mechanism of KLF12 affecting tumor immunity, clarify the relationship between KLF12 and the therapeutic effect of anti-PD-1 and find strategies to improve the therapeutic effect of anti-PD-1 immunotherapy.

Method and Results

Methods: First constructed cell lines with KLF12 overexpression or knockout, and observed the effect of KLF12 on tumor microenvironment in vitro and in vivo. Subsequently, we elucidated the key downstream molecules regulated by KLF12 through RNA sequencing, RT-PCR and Western Blot, and determined the regulatory relationship between them by rescue experiments. Next, ChIP-PCR and Dual-Luciferase reporter assay determine the direct transcriptional inhibition of KLF12. Finally, we used mouse models to observe the therapeutic effect and clarify the effects of KLF12 on anti-PD-1 therapy. And multiplex immunohistochemistry was performed using immunotherapy cohort to explore the role of KLF12 and other molecules in anti PD-1 treatment.

Results: We first found KLF12 can inhibit tumor progression by promoting the infiltration and function of CD8⁺T cells in vitro and in vivo. Secondly, Galectin-1 (Gal-1) was found to be a key downstream molecule of KLF12 through experiments such as RNA sequencing, RT-PCR and Western Blot. And rescue experiments showed that CD8⁺T cell decrease caused by decreased KLF12 expression in tumors can be reversed by Gal-1 repression. Then, ChIP-PCR and Dual-Luciferase reporter assay confirmed that KLF12 bound to Gal-1 promoter region leading to its transcription inhibition. Finally, it was demonstrated that targeting KLF12/Gal-1 can improve the therapeutic efficacy of anti PD-1 through intervention therapy on tumor bearing mice. And the detection results of immunotherapy samples showed that KLF12, CD8 and PD-1 expressions were higher in patients with a high response rate to anti-PD-1 treatment, while the expression of Gal-1 was lower.

Conclusions

KLF12 can promote the infiltration of CD8⁺T cells in the tumor microenvironment and enhance the function of CD8⁺T cell. KLF12 binds to the promoter region of Gal-1 to inhibit its transcription and expression. Targeting KLF12/Gal-1 can significantly improve the therapeutic efficacy of anti-PD-1 therapy.