MicroRNA Profiling In Cutaneous Wounds Of Diabetic Rats And The Intervention Of GM-CSF

Objective To explore the differential expression profiling of microRNAs(miRNAs) in cutaneous wounds between diabetic rats and normal rats and the effect of recombinant human granulocyte macrophage colony stimulating factor on wound healing and miRNAs expression in diabetic rats. Further to provide experimental foundation for further understanding the mechanisms underlying the poor diabetic wound healing and investigation of mi RNA-based therapeutics associated with wound healing.Methods(1) The diabetic rat models were induced by intraperitoneal injection of 65 mg/kg of streptozotocin(STZ); after four weeks, create full-thickness excisional dorsal wounds in 6 established diabetic model rats and normal rats respectively; then wound tissues were harvested on day 3. Use Trizol to extract total RNA from the sample; purify it after quality inspection qualified; then to the procedures of sample labeling and hybridization. Now hybrid images can be obtained through scanning the washed microarray slides by Agilent Scanner. Analyze these images and extract the data by Agilent Feature Extraction Software; then normalize and analyze the data by Agilent GeneSpring software. The miRNA profiling results were validated by real-time reverse transcriptase polymerase chain reaction(Real-time PCR).(2) The diabetic rat models were induced and 4 full-thickness excisional dorsal wounds were created in 16 established diabetic model rats, respectively. Using paired design: 2 symmetrical wounds on the both sides of the spine as a pair, and then the wounds were divided into the rhGM-CSF gel treatment group(group A) and matrix control group(group B), according to the table of random number using blind method. All the rats were changed dressing 1 times daily until 14 days after operation. Postoperative day 3,7,14, the wound healing were observed, the wound healing rate was calculated, and the wound tissues were harvested for histological examination. The indicators of immunohistochemical detection were PCNA, CD31. On day 7 after the operation, the wound tissues were harvested for miRNAs microarray. The steps of miRNAs microarray and PCR experiment were the same as part one. Data were processed with paired t-test or two sample t-test.Results(1) A total of 83 miRNAs were differentially expressed in cutaneous wounds of diabetic rats compared with normal rats, among which 18 were up-regulated while 65 are down-regulated. The significantly up-regulated mi RNAs were rno-miR-496-5p, rno-miR-105, rno-miR-122-5p, etc; and the significantly down-regulated miRNAs were rno-miR-196a-5p, rno-miR-134-5p, rno-miR-31-3p, etc. The results of RT-PCR have good consistency with the microarray results. KEGG pathways enrichment analysis shows that miR-31、miR-222、 miR-449 a and other significant differential expressed miRNAs were respectively involved in mitogen-activated protein kinase(MAPK)、Wnt、Notch and other signaling pathways which link to wound healing.(2) ○1 Compared with the matrix control group, the wound granulation was healthier and more abundant, the number of epidermal cells, endothelial cells, fibroblasts and inflammatory cells were more abundant, the wound healing was faster in the rhGM-CSF gel treatment group. ○2 On day 3 and 7,the wound healing rates of experimental group were(41±5)% and(75±4)%,significantly higher than those of control group(31±9)% and(71±4)%(P<0.05). ○3 On day 3,7 and 14,the average optical values of PCNA positive cell in experimental group were significantly higher than those of control group(P<0.05). The average optical values of CD31 positive cell in experimental group were significantly higher than those of control group(P<0.05). ○4 MiRNAs microarray results showed that a total of 31 miRNAs were differentially expressed in cutaneous wounds between two groups, among which 18 were up-regulated while 13 are down-regulated. ○5 The results of RT-PCR have good consistency with the microarray results.Conclusion(1) There was significant differential miRNAs expression in cutaneous wounds between diabetic rats and normal rats, which may be closely related to the mechanisms of diabetic wound healing.(2) There was significant differential miRNAs expression in cutaneous wounds of diabetic rats after the rhGM-CSF gel intervention. It may be the gene transcription level target of rhGM-CSF gel which promote the wound healing, and may provide a theoretical basis and experimental foundation for further targeted treatment.