This study was aimed to investigate the molecular effect of lidocaine and procaine on adipose stem cells by miRNA expression array. Lidocaine and procaine presented different alteration patterns of miRNA expression. Few studies have identified miRNA expression patterns in adipose stem cells associated with local anesthetics.
The lidocaine treatment on adipose stem cells showed only 4 miRNA changes in expression profile, miR-9*
, 29a, 296-5p and 373, of which were associated with stem cell function including lipoapoptosis [13
]. This means that the clinically available concentration with 30 minutes of treatment provide a little molecular effects associated with miRNA. This result is different from the previous report molecularly that is devastating results on chondrocytes after lidocaine treatment. Exposing the chondrocytes to a 50/50 mixture of culture medium and local anesthetics substantially decreased cytotoxicity but still showed high toxicity when compared with the saline group (90% dead cells for lidocaine 2%, P = 0.047) [15
]. In this study, the high-toxicity was not presented, considering a 50/50 mixture of culture medium and 2% lidocaine was used. There is a report that low concentrations of lidocaine as would be seen in plasma after spinal, epidural, or plexus anesthesia did not significantly affect multiplication of fibroblasts and higher concentrations, as would be seen after tissue infiltration, severely inhibit fibroblast multiplication and thus may impair wound healing [10
]. In this study, the expression of multiplication-associated miRNAs was not significantly changed. Local anesthetics used in this study contained sodium chloride as preservative, which would affect the results presented and there has been no published result to date about the relationship between miRNA and lidocaine, further future study is needed for verification of this result.
Otherwise, the procaine showed significant upregulation of 32 miRNAs expression. Among these, stem cell differentiation-related miRNAs were let-7s, miR-10b, 19a, 27b, 125a, 135b, 145, 155, and 200b. let-7s showed 1.52-1.75 fold, which are negative regulators of stem cell differentiation and essential for maintenance of an early developmental lineage [16
]. Besides, let-7d regulates cocaine-induced plasticity [17
]. The miRNA-200b expression negatively regulates epithelial to mesenchymal transition that facilitates tissue remodeling during embryonic development and is viewed as an essential early step in tumor metastasis by targeting ZEB1 and SIP1 [18
]. miR-373 significantly up-regulates tumor migration and invasion as metastasis-promoting miRNAs [19
A usual prediction with any one of the algorithms (PicTar, TargetScan, miRBase Targets) will yield hundreds of predicted targets for each miRNA. A list of selected target genes, which are of particular interest or significance in this study, for the differentially expressed miRNAs from adipose stem cell before and after treatment of anesthetics, are presented in Table 2. Most putative targets predicted in this study are homeo-box genes (HOX family), transcription factors (PPAR, RGS7BP, NR3C2, DFF45, EF2, ESR1, UBE2), growth, and apoptotic genes (RAS, BCL2, APC, p53, PTEN).
The various kinds of differentiation-associated miRNA were up-regulated. They are osteogenic differentiation (miR-135b) [20
], monocytic differentiation (miR-155, 222) [21
], miRNAs (miR-16, 133a, 145, 149, 186, 204) which cause apoptosis by targeting BCL2 [22
], the DNA fragmentation factor-45 (DFF45) [23
], and P2X7 [24
]. Immunity-related miRNAs were miR-132, -142-3p, -155, -222. miR-155 is a critical regulator of immune cell development [25
] and miR-132 potentiates cholinergic anti-inflammatory signaling [26
]. Hormonal-related miRNAs were miR-22, 125a, 135a. The miRNA, miR-135a, can participate in the regulation of renin-angiotensinaldosterone system involved in blood pressure regulation by repression of the mineralocorticoid receptor gene NR3C2
]. The up-regulation of miR-194, whose potential role in this study cannot be assumed, was reported as an hepatocyte marker [28
]. miR-143 (1.66 fold) and -200b (1.86 fold) was expressed highly, which have functioned in accelerating the rate of fat cell formation in preadipocytes [29
]. The miRNA, miR-143, is involved in adipocyte differentiation and may act through the target gene ERK5, normally promotes adipocyte differentiation. Interestingly, miR-27b (1.58) which has the function of anti-adipogenesis and muscle differentiation was also expressed simultaneously [30
Although the miRNA expression is cell or tissue specific and is multifunctional with multitargets [5
], therefore, the alteration of miRNA expression herein can speculate the cytotoxicity of local anesthetics on adipose stem cells.
In conclusion, these results suggests that local anesthetics affect miRNA expression on adipose stem cells and the effect of procaine is more marked than that of lidocaine, considering alteration of miRNAs' expression. Procaine can down-regulate stem cell differentiation, immunity, and apoptosis up-regulation by expressing miRNAs on stem cells. Especially miRNA-132 might have an important role in procaine activity. As lidocaine has been frequently used in local anesthesia and miRNA affect critical pathway of several biological processes, further study will be needed about the effect of lidocaine on stem cells including function of miR-9*, 29a, 296-5p and 373.