MicroRNA-146a and microRNA-155 as novel crevicular fluid biomarkers for periodontitis in nondiabetic and type 2 diabetic patients

Abstract

Recent studies point at the crucial role of genetic and epigenetic mechanisms in the development of multifactorial diseases such as periodontitis and diabetes mellitus (DM) type 2. In addition, circulatory microRNAs (miRs) have emerged as novel biomarkers for various diseases. Aim of this study was to investigate the levels of miR-146a and miR-155 and superoxide dismutase (SOD) activity in gingival crevicular fluid (GCF), as well as levels of MMP-9, in periodontitis patients with (CPDM) and without DM type 2 (CP), as well as in periodontally healthy, control groups with or without DM type 2 (PHDM and PH, respectively). Ninety-six subjects (44 males and 52 females) were included in the study. Using a periodontal probe (PCPUNC 15, Hu Friedy, Chicago, USA), full-mouth periodontal evaluation was carried out. The following clinical parameters were recorded: probing pocket depth (PPD), clinical attachment level (CAL), bleeding on probing (BOP), gingival index (GI), and plaque index (PI). Baseline, GCF samples were collected from two representative sites per participant, 24h after the clinical measurments were recorded using paper strip, PerioPaper®, (Pro-Flow Corp., Amityville, NY, USA). Representative sites in periodontally healthy participants were mesiovestibular aspect of the right upper central incisor and right upper first molar. In patients with periodontitis, the two sites demonstrating the highest values of PPD and positive BOP were considered as representative. The clinical measurements were recorded, and GCF samples were taken from the sampling site at baseline and 6 weeks after the periodontal treatment. MiRNA modulation was analyzed using real-time quantitative reverse transcriptase- polymerase chain reaction qRT-PCR. SOD activity was measured spectrophotometrically, while the levels of MMP-9 was measured using enzyme-linked immunosorbent assay (ELISA). Mean values of full-mouth and sample sites’ PPD, CAL, BOP, GI, PI were statistically higher in CP and CPDM groups compared to periodontally healthy groups (PH and PHDM), baseline as well as 6 weeks after non-surgical therapy. (Anova and Bonferroni post hoc test. p lt 0.01). The upregulation of miR-146a and miR-155 was observed baseline in CP and CPDM patients, while the levels decreased six weeks after the non-surgical therapy to the levels comparable to PH and PHDM, respectively. (Mann-Whitney test, p lt 0.01)...

Nedavne studije pokazuju ključnu ulogu genetičkih i epigenetičkih mehanizama u razvoju multifaktorijalnih oboljenja kao što su parodontopatija i dijabetes melitus tip 2 (DM tip 2). Tako su se cirkulatorne miRNK pojavile kao potencijalni novi biomarkeri mnogih oboljenja i igraju važnu ulogu u regulaciji značajnih bioloških procesa među kojima je i inflamacija. Cilj ove studije je bio da se istraže vrednosti i značaj nivoa ekspresije molekula miRNK-146a i miRNK-155, nivo aktivnosti enzima SOD-e (superoksid dizmutaze), kao i vrednosti koncentracije enzima matriksne metaloproteinaze 9 (MMP-9) u gingivalnoj tečnosti ispitanika obolelih od hronične parodontopatije sistemski zdravih (HP grupa) i obolelih od DM tipa 2 (HPDM grupa) i ispitanika sa klinički zdravim parodoncijumom sistemski zdravih (Z grupa) i obolelih od DM tipa 2 (ZDM grupa). U istraživanje je bilo uključeno 96 ispitanika oba pola (44 muškaraca i 52 žene). Nivo oralne higijene i stanje parodontalnih tkiva verifikovano je pomoću graduisane parodontalne sonde (PCPUNC 15, Hu Friedy, Chicago, USA) merenjem sledećih kliničkih parametara: plak indeks, gingivalni indeks, krvarenje na provokaciju, dubina sondiranja, nivo pripojnog epitela i nivo ivice gingive. Uzorci gingivalne tečnosti su uzimani sa dva reprezentativna mesta filter papirima PerioPaper®, (Pro-Flow Corp., Amityville, NY, USA), 24h nakon merenja i beleženja kliničkih parametara. Ispitanicima sa hroničnom parodontopatijom uzorci su uzimani iz dva parodontalna džepa, na mestima sa najvećim vrednostima dubine sondiranja i pozitivnim vrednostima krvarenja na provokaciju, dok je ispitanicima sa zdravim parodoncijumom uzorak gingivalne tečnosti uziman u regiji gornjeg desnog centralnog sekutića i gornjeg desnog prvog molara (meziovestibularne površine). Klinička merenja i uzorkovanje gingivalne tečnosti su rađena dva puta: na prvom pregledu i šest nedelja nakon sprovedene kauzalne terapije obolelog parodoncijuma. Vrednosti nivoa ekspresije molekula miRNK su određivane lančanom reakcijom polimeraze kojoj prethodi reverzna transkripcija korišćenjem prajmera sa petljom, (eng. stem-loop) qRT-PCR (eng. real-time quantitative reverse transcriptase- polymerase chain reaction). Aktivnost enzima SOD-e je određivana spektrofotometrijskom metodom, dok su vrednosti koncentracije molekula MMP-9 mereni ELISA metodom (eng. enzyme-linked immunosorbent assay)...