Kepple Daniel D, Thornburg Thomas E, Beckman Micaela F, Bahrani Mougeot Farah, Mougeot Jean-Luc C
Translational Research Laboratories, Department of Oral Medicine/Oral & Maxillofacial Surgery, Atrium Health Carolinas Medical Center, Charlotte, NC 28203, USA.
Department of Otolaryngology/Head & Neck Surgery, Wake Forest University School of Medicine, Winston-Salem, NC 27101, USA.
Int J Mol Sci. 2025 Jun 19;26(12):5881. doi: 10.3390/ijms26125881.
Sjögren's disease (SjD) is an autoimmune disease of exocrine tissues. Prior research has shown that ETS proto-oncogene 1 (ETS1), STAT1, and IL33 may contribute to the disease's pathology. However, the regulatory mechanisms of these genes remain poorly characterized. Our objective was to explore the mechanisms of SjD pathology and to identify dysfunctional regulators of these genes by immunostimulation of SjD and relevant cell lines. We used immortalized salivary gland epithelial cell lines (iSGECs) from Sjögren's disease (pSS1) and (nSS2) patients, previously developed in our lab, and control cell line A253 to dose with immunostimulants IFN-γ or poly(I:C) (0 to 1000 ng/mL and 0 to 1000 µg/mL, respectively) over a 72 h time course. Gene expression was determined using qRT-PCR delta-delta-CT method based on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for mRNA and U6 small nuclear RNA 1 (U6) for miRNA, using normalized relative fold changes 48 h post-immunostimulation. Protein expression was quantified 72 h post-stimulation by Western blotting. Reference-based RNA-seq of immunostimulated pSS1 and nSS2 cells was performed to characterize the reactome of genes conserved across all used doses. The expression of ETS1 and STAT1 protein was upregulated ( < 0.05) in IFN-γ-treated pSS1 and nSS2, as compared to A253 cells. IFN-γ-treated nSS2 cell showed significant IL33 upregulation. Also, IL33 had a correlated ( < 0.01) U-shaped response for low-mid-range doses for IFN-γ- and poly(I:C)-treated pSS1 cells. RNA-seq showed 175 conserved differentially expressed (DE) genes between nSS2 and pSS1 immunostimulated cells. Of these, 44 were shown to interact and 39 were more abundant ( < 0.05) in pSS1 cells. Western blotting demonstrated nSS2 cells expressing ETS1 uniformly across treatments compared to pSS1 cells, despite similar mRNA abundance. miR-145b and miR-193b were significantly under-expressed in IFN-γ-treated nSS2 cells compared to pSS1 cells ( < 0.01). ETS1 and IL33 showed disproportionate mRNA and protein abundances between immunostimulated Sjögren's disease-derived (pSS1), and sicca-derived (nSS2) cell lines. Such differences could be explained by higher levels of miR-145b and miR-193b present in pSS1 cells. Also, RNA-seq results suggested an increased sensitivity of pSS1 cells to immunostimulation. These results reflect current pathobiology aspects, confirming the relevance of immortalized salivary gland epithelial cell lines.
干燥综合征（SjD）是一种外分泌组织的自身免疫性疾病。先前的研究表明，ETS原癌基因1（ETS1）、信号转导和转录激活因子1（STAT1）以及白细胞介素33（IL33）可能与该疾病的病理过程有关。然而，这些基因的调控机制仍不清楚。我们的目的是通过对干燥综合征患者及相关细胞系进行免疫刺激，探索干燥综合征的病理机制，并确定这些基因的功能失调调节因子。我们使用了我们实验室先前建立的来自干燥综合征患者（原发性干燥综合征1，pSS1）和非干燥综合征患者（非干燥综合征2，nSS2）的永生化唾液腺上皮细胞系，以及对照细胞系A253，在72小时的时间内分别用免疫刺激剂干扰素-γ（IFN-γ）或聚肌苷酸-聚胞苷酸（poly(I:C)）（分别为0至1000 ng/mL和0至1000 μg/mL）进行处理。基于甘油醛-3-磷酸脱氢酶（GAPDH）用于mRNA定量，U6小核RNA 1（U6）用于miRNA定量，采用qRT-PCR ΔΔCT法，在免疫刺激后48小时测定基因表达，以标准化相对倍数变化表示。刺激后72小时通过蛋白质印迹法定量蛋白质表达。对免疫刺激后的pSS1和nSS2细胞进行基于参考的RNA测序，以表征所有使用剂量下保守基因的反应组。与A253细胞相比，IFN-γ处理的pSS1和nSS2细胞中ETS1和STAT1蛋白表达上调（P<0.05）。IFN-γ处理的nSS2细胞显示IL33显著上调。此外，对于IFN-γ和poly(I:C)处理的pSS1细胞，IL33在低-中剂量范围内呈现相关的（P<0.01）U形反应。RNA测序显示，nSS2和pSS1免疫刺激细胞之间有175个保守的差异表达（DE）基因。其中，44个显示有相互作用，39个在pSS1细胞中更为丰富（P<0.05）。蛋白质印迹法显示，尽管mRNA丰度相似，但与pSS1细胞相比，nSS2细胞在所有处理中均均匀表达ETS1。与pSS1细胞相比，miR-145b和miR-193b在IFN-γ处理的nSS2细胞中显著低表达（P<0.01）。在免疫刺激的干燥综合征来源（pSS1）和干燥症状来源（nSS2）细胞系之间，ETS1和IL33的mRNA和蛋白质丰度不成比例。这种差异可以用pSS1细胞中较高水平的miR-145b和miR-193b来解释。此外，RNA测序结果表明pSS1细胞对免疫刺激的敏感性增加。这些结果反映了当前的病理生物学情况，证实了永生化唾液腺上皮细胞系的相关性。
