Periodontitis is a common and frequent occurring disease in human oral cavity, which can cause gingival inflammation, alveolar bone resorption and teeth loosening and falling out. Plaque biofilm is the initiating factor that causes periodontitis, and excessive immune inflammatory response of the host also plays an important role in the occurrence and development of periodontitis . At present, some studies have confirmed that lncRNA may change the function of periodontal related cells in the microenvironment of periodontitis. Li et al. Found lncRNA SNHG1 associated with osteoblastic dysfunction in periodontitis can regulate the osteogenic differentiation of inflammatory periodontal ligament stem cells through EZH2-mediated H3K27me3 methylation of KLF2 promotor . In this study, 4 DElncRNAs (KIAA0125, Runx1-IT1, LOC100130476, LOC101929272) were screened out from the dataset GSE10334. The results showed that the expressions of KIAA0125, Runx1-IT1, and LOC10191929272 were significantly increased in periodontitis, while the expressions of LOC100130476 were decreased.
Next, we investigated the infiltration of immune cells in the periodontitis group and the normal group, and found that plasma cell infiltration was dominant. Besides, we found that four DElncRNAs have high correlation to B cell surface Markers CD19 and CD79A. qRT-PCR results also showed that CD19 and CD79A were up-regulated in periodontitis. Some studies have shown that T lymphocytes and B lymphocytes are dominant in the chronic inflammatory period of periodontitis, and up to 50% of B lymphocytes were detected in gingival tissue . B cells/plasma cells play a protective role in periodontitis. The antibody response against bacteria is beneficial to control the imbalance of microbial flora in the periodontal pocket and prevent bacteria from entering the connective tissue of the gingiva, thus limiting the inflammation and disease. While B cells secrete TNF-α, IL-1β, IL-17, MMPS or plasma cells secrete IgA, IgG, IgM and other antibodies to kill pathogens, they also promote the expression of RANKL to activate osteoclasts and accelerate alveolar bone absorption [23, 24]. Studies have shown that B lymphocytes are one of the main sources of RANKL in periodontal lesion tissues. Up to 90% of B lymphocytes in inflammatory tissues of periodontitis express RANKL . Currently, Suzuki et al. identified CD19 and CD79A as the molecular biomarker candidates and pathogenesis of chronic periodontitis . The above evidence suggested that B lymphocytes play an important role in the development of periodontitis.
In order to explore the regulatory network of DElncRNAs, we constructed the ceRNA network and a total of 1 lncRNA (KIAA0125), 3 miRNAs (miR-449c-5p, miR-125a-5p and miR-125b-5p) and 2 mRNAs (CYP24A1, BTG2) were involved in establishing the ceRNA network for periodontitis. KIAA0125 is a long non-coding RNA gene, which is located on chromosome 14q32.33. KIAA0125 was first reported in 1995, the mechanism of which under human physiological and pathological conditions has been rarely explored . Studies have found that lncRNA KIAA0125 was down-regulated in colorectal cancer and inhibited epithelial-mesenchymal transition (EMT) through Wnt/β-catenin signaling . However, Diniz et al. detected that the expression level of KIAA0125 transcript in the ameloblastoma group was higher than that of dental follicles, which may be involved in the pathobiological process of ameloblastoma . Few studies on KIAA0125 have been conducted in periodontitis. However, Guzeldemir-akcakanat et al. found that KIAA0125 was highly expressed in the chronic periodontitis group by whole-genome Transcriptomic . The specific mechanism of KIAA0125 involved in the pathogenic process of periodontitis needs to be further explored.
Through WGCNA analysis and transcriptomic sequencing results, we further found that CYP24A1 was the key mRNA. 1,25D is the main bioactive form of vitamin D, and its role in the prevention and treatment of osteoporosis, diabetes and periodontitis has been attracting people's attention. The main role of 1,25D is to regulate calcium and phosphorus metabolism. However, in recent years, as an immunomodulator, its role in anti-inflammatory and immunomodulatory has become a focus of research, especially its role in innate immunity. 1,25D can up-regulate the expression of antimicrobial peptides, urge phagocytes to kill pathogenic microorganisms, down-regulate the expression of inflammatory factors, and reduce the inflammatory response . Vitamin D 24-hydroxylase can add hydroxyl to the 24-bit carbon atom of 1,25D, thus greatly reducing the activity. It is an inhibitor of 1,25D activity in vivo and negatively regulates the biological effects of 1,25D, the gene encoded by which is CYP24A1 . Liu et al. found that the mRNA expression of CYP24A1 and RANKL in human gingival fibroblasts (hGF) and PDLCs significantly increased after treatment 1,25D . In addition, most studies suggested that the abnormal expression of CYP24A1 leads to excessive or insufficient 1,25D, which is associated with the occurrence and development of most cancers. Studies have found that the risk of colorectal cancer was negatively correlated with the status of vitamin D in patients, and vitamin D supplementation can reduce the incidence of colorectal cancer. The expression level of CYP24A1 increased during the occurrence of colorectal cancer, and the higher the level of CYP24A1, the higher the malignancy of colorectal cancer. At the same time, the expression level of Ki-67, a marker for cancer cell proliferation, also increased, suggesting that the overexpression of CYP24A1 reduced the local practicability and anti-tumor effect of 1,25D . Wang et al. demonstrated that knockdown of CYP24A1 can aggravate 1,25D to suppress EMT, proliferation and invasion, increase the expression of E-cadherin, and reduce the expression of N-cadherin, Vimentin, β-catenin and Snail in mouse ovarian epithelial cells . These above findings suggested that inhibition of CYP24A1 may activate the vitamin D pathway in prevention and treatment of diseases.
qRT-PCR results by us showed that both KIAA0125 and CYP24A1 were highly expressed in periodontitis and correlated with the clinical characteristics of PLI, PD and CAL. In addition, we found that both KIAA0125 and CYP24A1 were positively correlated with CD19 and CD79A expression levels. When treated with 1 μg/mL LPS for 24 h, KIAA0125 and CYP24A1 expression in hPDLCs were increased, and the results of KIAA0125 were statistically significant. Next, we explored the effects on the expression levels of key genes in an inflammatory environment. When treated with 1 μg/mL LPS and 10 nM 1,25D, the expressions of KIAA0125 and CYP24A1 were significantly increased, while the inflammatory factors IL1B and IL6 were relatively decreased, suggesting that 1,25D could alleviate inflammation. When we added low-dose ketoconazole, we found that the expression levels of KIAA0125 and CYP24A1 were further increased. With the increase of the expression levels, CD19, CD79A, IL1B and IL6 were relatively increased, which also indicated that CYP24A1 and KIAA0125 could reduce the anti-inflammatory effect of 1,25D. When we used high doses of ketoconazole, KIAA0125 and CYP24A1 were significantly inhibited, and as their expression decreased, so did the expression of inflammatory factors. Through vitro experiments, we found that 1,25D could alleviate the inflammation of LPS-induced hPDLCs, while the increased expression of KIAA0125 and CYP24A1 would antagonize the anti-inflammatory effect.
This study has some limitations. First of all, we screened out the key gene CYP24A1 through WGCNA analysis and transcriptional sequencing and made subsequent verification, but we did not conduct subsequent experiments on BTG2 and miRNAs in the ceRNA network. In addition, the specific mechanism of the co-expression of KIAA0125 and CYP24A1 remains to be further explored.