1.Effect And Mechanism Of RNA Binding Protein PCBP1 In Lung Adenocarcinoma Metastasis 2.Transcription Factor KLF12 Mediates Anti-PD-1 Immunotherapy By Regulating CD8~+T Cells In Tumors

Background and AimsLung 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 ResultsMethods: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.ConclusionsPCBP1 inhibits LUAD development by upregulating DKK1 to inactivate the WNT/βcatenin pathway.Our findings highlight the potential of PCBP1 as a promising therapeutic target.Background and AimsIn 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 ResultsMethods: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 KLF 12 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 Gal1 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.ConclusionsKLF12 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.