POU2F1 Inhibits miR-29b1/a Cluster-Mediated Suppression of PIK3R1 and PIK3R3 Expression to Regulate Gastric Cancer Cell Invasion and Migration
Authors:
Yizhi Xiao1,2, Ping Yang1, Wushuang Xiao1, Zhen Yu1, Jiaying Li1, Xiaofeng Li2, Jianjiao Lin3, Jieming Zhang1, Miaomiao Pei1, Linjie Hong1, Juanying Yang1, Zhizhao Lin1, Ping Jiang1, Li Xiang3, Guoxin Li4, Xinbo Ai5, Weiyu Dai1,6, Weimei Tang1, Jide Wang1,3
Affiliations:
1Department of Gastroenterology, Guangdong Provincial Key Laboratory of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, China;
2Department of Gastroenterology, Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, Guangdong 519000, China;
3Department of The Second Affiliated Hospital, School of Medicine, The Chinese University of Hong Kong, Shenzhen & Longgang District People’s Hospital of Shenzhen, Shenzhen, Guangdong 518172, China;
4Department of General Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, China;
5Department of Gastroenterology, Zhuhai People’s Hospital (Zhuhai Clinical Medical College of Jinan University), Zhuhai, Guangdong 519000, China;
6Department of Gastroenterology, Guangdong Provincial People’s Hospital (Guangdong Academy of Medical Sciences), Southern Medical University, Guangzhou, Guangdong 510080, China.
Abstract
Gastric cancer (GC) remains a leading cause of cancer-related mortality worldwide, necessitating a deeper understanding of its molecular mechanisms. This study investigates the role of the transcription factor POU2F1 in regulating the miR-29b1/a cluster and its downstream targets, PIK3R1 and PIK3R3, in GC progression. POU2F1 is overexpressed in GC and directly binds to the miR-29b1/a cluster promoter, repressing its expression. Downregulation of miR-29b-3p and miR-29a-3p promotes GC metastasis by upregulating PIK3R1 and PIK3R3, which activate the PI3K/AKT/mTOR signaling pathway. In vitro and in vivo experiments demonstrate that POU2F1 enhances GC cell invasion and migration through this regulatory axis. These findings highlight the POU2F1-miR-29b-3p/miR-29a-3p-PIK3R1/PIK3R3 signaling pathway as a potential therapeutic target for GC.
Introduction
Gastric cancer (GC) is a major global health burden, with high morbidity and mortality rates. Despite advances in treatment, the molecular mechanisms driving GC progression remain poorly understood. Transcription factors and microRNAs (miRNAs) play critical roles in regulating gene expression and cancer development. POU2F1, a member of the POU domain transcription factor family, has been implicated in various cancers, including hepatocellular carcinoma and head and neck cancer. However, its role in GC and its interaction with miRNA clusters remain unexplored.
The miR-29 family, comprising miR-29a, miR-29b, and miR-29c, is known to function as tumor suppressors in multiple cancers. These miRNAs are encoded by two clusters, miR-29b1~a and miR-29b2~c, and share identical seed sequences, allowing them to target overlapping sets of genes. In GC, miR-29 family members are frequently downregulated, suggesting their involvement in tumor suppression. However, the regulatory mechanisms controlling miR-29b1/a cluster expression in GC are not well understood.
Phosphoinositide-3-kinase regulatory subunits 1 and 3 (PIK3R1 and PIK3R3) are key components of the PI3K/AKT/mTOR signaling pathway, which is often dysregulated in cancer. Overexpression of PIK3R1 and PIK3R3 has been associated with poor prognosis in GC. This study aims to elucidate the regulatory axis involving POU2F1, the miR-29b1/a cluster, and PIK3R1/PIK3R3 in GC progression.
Methods
Cell Lines and Tissue Samples
Six GC cell lines (AGS, MKN45, SGC-7901, HGC-27, MGC-803, BGC-823) and a normal gastric epithelial cell line (GES-1) were used. Human GC tissues and adjacent non-tumor tissues were collected from 80 patients.
RNA Sequencing and miRNA Profiling
Small RNA sequencing was performed on AGS cells transfected with POU2F1 siRNA or scrambled siRNA. Differentially expressed miRNAs were identified using fold change analysis.
Luciferase Reporter Assays
The miR-29b1/a cluster promoter was cloned into a luciferase reporter vector. Site-directed mutagenesis was used to identify POU2F1 binding sites. Luciferase activity was measured to assess promoter activity.
Chromatin Immunoprecipitation (ChIP)
ChIP assays were performed to confirm POU2F1 binding to the miR-29b1/a cluster promoter. Anti-POU2F1 antibody was used for immunoprecipitation, followed by PCR analysis.
Transwell Migration and Invasion Assays
GC cells were transfected with miR-29b-3p, miR-29a-3p mimics, or inhibitors. Migration and invasion were assessed using Transwell assays.
Western Blotting and qPCR
Protein and mRNA expression levels of POU2F1, miR-29b-3p, miR-29a-3p, PIK3R1, and PIK3R3 were analyzed using western blotting and quantitative PCR.
In Vivo Tumor Metastasis Model
AGS cells transfected with POU2F1, miR-29b1/a cluster, or control vectors were injected into nude mice. Lung metastasis was assessed by histopathology and immunohistochemistry.
Statistical Analysis
Data were analyzed using SPSS and GraphPad Prism. Survival analysis was performed using Kaplan-Meier curves. Correlations between gene and miRNA expression were assessed using Spearman’s correlation.
Results
POU2F1 Represses the miR-29b1/a Cluster
POU2F1 was overexpressed in GC cell lines compared to normal gastric cells. RNA sequencing revealed that POU2F1 knockdown upregulated miR-29b-3p and miR-29a-3p. Luciferase reporter assays and ChIP confirmed that POU2F1 binds to the miR-29b1/a cluster promoter, repressing its expression.
miR-29b-3p and miR-29a-3p Suppress GC Metastasis
miR-29b-3p and miR-29a-3p were downregulated in GC tissues and cell lines. Low expression of these miRNAs correlated with poor overall survival in GC patients. Transwell assays demonstrated that miR-29b-3p and miR-29a-3p overexpression inhibited GC cell migration and invasion.
PIK3R1 and PIK3R3 Are Direct Targets of miR-29b-3p and miR-29a-3p
Bioinformatics analysis identified PIK3R1 and PIK3R3 as potential targets of miR-29b-3p and miR-29a-3p. Luciferase reporter assays and Ago2-RIP confirmed that miR-29b-3p and miR-29a-3p directly target PIK3R1 and PIK3R3. Overexpression of PIK3R1 and PIK3R3 reversed the inhibitory effects of miR-29b-3p and miR-29a-3p on GC cell metastasis.
PIK3R1 and PIK3R3 Promote GC Metastasis
PIK3R1 and PIK3R3 were overexpressed in GC tissues and cell lines. Co-immunoprecipitation assays confirmed that PIK3R1 and PIK3R3 physically interact. Overexpression of PIK3R1 and PIK3R3 enhanced GC cell migration and invasion, while knockdown inhibited these effects.
The PI3K/AKT/mTOR Pathway Mediates GC Metastasis
miR-29b-3p and miR-29a-3p overexpression suppressed the PI3K/AKT/mTOR pathway, while inhibition of these miRNAs activated the pathway. The PI3K/mTOR inhibitor VS-5584 reversed the effects of PIK3R1 and PIK3R3 overexpression on GC cell metastasis.
POU2F1 Regulates PIK3R1 and PIK3R3 via miR-29b-3p and miR-29a-3p
POU2F1 expression positively correlated with PIK3R1 and PIK3R3 levels in GC tissues. POU2F1 knockdown reduced PIK3R1 and PIK3R3 expression, while miR-29b-3p and miR-29a-3p inhibition reversed this effect.
Discussion
This study identifies a novel regulatory axis in GC involving POU2F1, the miR-29b1/a cluster, and PIK3R1/PIK3R3. POU2F1 represses miR-29b-3p and miR-29a-3p expression, leading to upregulation of PIK3R1 and PIK3R3, which activate the PI3K/AKT/mTOR pathway and promote GC metastasis. These findings provide new insights into the molecular mechanisms of GC progression and highlight potential therapeutic targets.
Conclusion
The POU2F1-miR-29b-3p/miR-29a-3p-PIK3R1/PIK3R3 signaling axis plays a critical role in regulating GC cell invasion and migration. Targeting this pathway may offer new therapeutic strategies for GC treatment.
DOI:
doi.org/10.1097/CM9.0000000000003181
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