Primary Sjogren's Syndrome Associated with Lung Adenocarcinoma: Probing the Potential Common Pathogenic Mechanisms and Experimental Verification

Summary

This manuscript describes the operational procedures and precautions to probe the potential common pathogenic mechanisms linking primary Sjogren's syndrome and lung adenocarcinoma through bioinformatics analysis and experimental verification.

Abstract

This study aimed to probe the potential common pathogenic mechanisms linking primary Sjogren's syndrome (pSS) and lung adenocarcinoma (LUAD) through bioinformatics analysis and experimental verification. The relevant genes associated with pSS and LUAD were retrieved from the Gene Expression Omnibus (GEO) database and Genecard database. Subsequently, differentially expressed genes (DEGs) associated with pSS and LUAD were screened as pSS-LUAD-DEGs. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses were performed to elucidate the significant biological functions of pSS-LUAD-DEGs. Core targets were identified by constructing the protein-protein interaction (PPI) network, further assessing hub gene diagnostic accuracy through Receiver Operating Characteristic (ROC) curve analyses. In this study, NOD/Ltj mice served as pSS animal models and were stimulated with particulate matter 2.5 (PM2.5) to generate an inflammatory reaction. Quantitative real-time polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA), and western blotting were employed for relevant molecular biology experiment verification. The results revealed through KEGG and GO enrichment analyses indicate that inflammation plays a critical role in linking pSS and LUAD. IL6, CCNA2, JAK2, IL1B, ASPM, CCNB2, NUSAP1, and CEP55 were determined as key targets of pSS-LUAD. BALB/c mice and NOD/Ltj mice exhibited enhanced expression of inflammatory cytokines IL-6 and IL-1β in lung tissues following 21 days of stimulation with PM2.5, activating the JAK2/STAT3 signaling pathway and up-regulating the expression of tumor-associated genes CCNA2, CCNB2, and CEP55, with NOD/Ltj mice exhibiting more pronounced changes than BALB/c mice. This protocol demonstrates that carcinogenesis induced by the pulmonary inflammatory microenvironment may be a key reason for the high incidence of LUAD in pSS patients. Additionally, blocking-related mechanisms may help prevent the occurrence of LUAD in pSS patients.

Introduction

Primary Sjögren's syndrome (pSS) is an autoimmune disease characterized by lymphocytic infiltration of the exocrine glands and leads to the clinical symptoms of dry eyes (xerophthalmia) and dry mouth (xerostomia)^1,2. pSS is also usually accompanied by extra-glandular manifestations of involvement, including hyperglobulinemia³, interstitial lung disease⁴, renal tubular acidosis⁵, neurological damage⁶, and thrombocytopenia⁷, which constitute the main adverse prognostic factors. In rece....

Protocol

The experimental animals were housed in the animal facility of the China-Japan Friendship Hospital, where the housing conditions met the animal feeding environment in line with China's national standard, Laboratory Animal-Requirements of Environment and Housing Facilities (GB14925-2010). All animal care procedures and experiments complied with the ARRIVE guidelines and were based on the 3R principles (reduction, replacement, refinement), adhering to the guidelines of the National Animal Welfare Law of China. BALB/c m.......

Discussion

Although pSS is considered a disease primarily characterized by the invasion of exocrine glands, the damage of extra-glands cannot be ignored²⁴. The lungs represent a target organ for pSS, and lung involvement is a common extra-glandular manifestation of pSS, typically involving lymphocytic infiltration of the bronchial mucosa and pulmonary interstitium²⁵. Research indicates that at least 20% of pSS patients experience interstitial lung disease (ILD)²⁶

Materials

Name	Company	Catalog Number	Comments
3-Color Prestained Protein Marker	Epizyme	WJ103	Western Blot
Antibody Dilution Buffer	Epizyme	PS119	Western Blot
BCA Protein Quantification Kit	Epizyme	ZJ101	Western Blot
Cytoscape 3.7.1 software	National Institute of General Medical Sciences (NIGMS), National Institutes of Health (NIH)	Version 3.7.1.	Open-source software for biological network analysis and visualization
ECL Luminous Fluid	Epizyme	SQ203	Western Blot
Electrophoresis Buffer	Epizyme	PS105S	Western Blot
GraphPad Prism 10.0	GraphPad	Version 10.0	Data analysis
HRP-conjugated Goat anti-Rabbit IgG (H+L) (AS014)	abclonal	AS014	Western Blot
JAK2 Antibody	Cell Signaling Technology	3230T	Western Blot
Mouse IL-1β ELISA Kit	Beijing 4A Biotech Co., Ltd	CME0015	ELISA
Mouse IL-6 ELISA Kit	Beijing 4A Biotech Co., Ltd	CME0006	ELISA
Phosphatase Inhibitor Cocktail (100×)	Epizyme	GRF102	Western Blot
Phospho-JAK2 (Tyr1007/1008) Antibody	Cell Signaling Technology	3776S	Western Blot
Phospho-STAT3 (Tyr705) Antibody	Cell Signaling Technology	9145S	Western Blot
Protease Inhibitor Cocktail (100×)	Epizyme	GRF101	Western Blot
Protein Free Rapid Blocking Buffer (5×)	Epizyme	PS108	Western Blot
PVDF membrane	Millipore	IPVH00010	Western Blot
R software	R Foundation for Statistical Computing	Not Applicable	Statistical analysis software and programming language used for data analysis, visualization, and machine learning applications
Radio Immunoprecipitation Assay	Epizyme	PC101	Western Blot
Reverse Transcription System	Promega	A3500	PCR
SDS-PAGE	Epizyme	LK303	Western Blot
SDS-PAGE Protein Loading Buffer (5×)	Epizyme	LT103	Western Blot
STAT3 Antibody	Cell Signaling Technology	9139S	Western Blot
SYBR Green Realtime PCR Master Mix	TOYOBO	QPK-201	PCR
TBST (10×)	Epizyme	PS103	Western Blot
Western Blot Transfer Buffer (10×)	Epizyme	PS109	Western Blot
β-Actin Antibody	abclonal	AC026	Western Blot