Abstract
As rates of type II diabetes mellitus (T2DM) and related cardiovascular disease (CVD) are becoming increasingly prevalent globally and in Aotearoa, New Zealand, the demand for accurate and affordable diagnostic tools is also on the rise. miRNAs, a type of small non-coding RNA, that are readily available in all bodily-fluids have been shown to be a valuable potential diagnostic biomarker in fluids such as blood-plasma. However, thus far no investigations have investigated whether salivary miRNAs are capable of being a biomarker of cardiovascular disease. This study aimed to establish the expression of four CVD-enhanced microRNAs – miR-21, miR-126, miR-133a and miR-208a in the saliva of young, healthy individuals. It then aimed to examine how these miRNAs differed in expression for participants with T2DM, that do not experience symptoms of CVD. The miRNAs expressed in saliva were compared to their expression in plasma, as these miRNAs have an expression in plasma that has been established.
Participants of different ethnicity groups - European, Māori, Pasifika and South Asian were recruited to the young, healthy cohort (n = 38). Participants with T2DM, without any known CVD disease, were recruited to the T2DM cohort. Alongside healthy participants as controls (NDb) (n = 40 total). For the T2DM cohort both Europeans and South Asians were recruited (n = 20 each). Plasma and saliva samples were collected from each participant, including numerous basic clinical markers of T2DM and CVD.
The study was able to compare the expression of the chosen CVD-enhanced miRNAs in both body fluids using reverse transcription (RT) and quantitative polymerase chain reaction techniques (qPCR). Using statistical analysis, we were able to compare the expression of salivary miRNAs to plasma miRNAs. What was found in the young healthy group was that the miRNAs were not expressed in a consistent manner between plasma and saliva. miRNAs in saliva were consistently shown to be expressed at a greater range than plasma. Additionally, when we included demographic determinants the expression of these miRNAs in both plasma and saliva was influenced by ethnicity and by age. These findings indicated that salivary miRNA is variable between individuals and that their use may be better suited to act as a biomarker if personalised.
The results of our study in participants with T2DM showed plasma miRNA expression that is indicative of early compensatory changes in the progression of CVD such as a decrease in plasma miR-21. The expression of salivary miRNAs was not able to differentiate between NDb participants and those with T2DM. Additionally, the correlation of plasma and salivary miRNAs were very poor in T2DM, suggestive of mechanisms that are inefficient in transporting systemic miRNA to saliva.
Interestingly secondary analysis showed South Asian participants to express elevated levels of miR-21 in saliva regardless of their T2DM status. What was also observed was that the South Asian cohort expressed no significant differences in hbA1c or CVD-related clinical markers between T2DM and NDb participants compared to the European participants. These findings may indicate that miRNA-21 may acts an early marker of metabolic disease in this South Asian cohort.
This was a preliminary study that aimed to assess the ability to use salivary miRNAs as non-invasive biomarker tools for the diagnosis of CVD, in individuals with T2DM. While salivary miRNAs did not show a strong ability to differentiate between individuals with T2DM and those without, this study highlights the unknowns of salivary miRNA transport and the unique expression of miRNA in this biofluid.