| BackgroundDiabetic kidney disease(DKD) is one of the main microvascular complications of type 1 and type 2 diabetes. At present, the renal failure caused by diabetes has growing in China,which will become the major cause of end-stage renal disease in the future.The pathogenesis of DKD is not completely clear, now which is generally accepted that genetic factors, renal hemodynamic changes caused by long-term sustained high blood sugar and inflammatory reaction, glucose and lipid metabolism disorder and other factors lead to diabetic nephropathy.Many studies have shown that the activation of the inflammatory response is closely associated with diabetic nephropathy, including TNF-?, IL-18, IL-6 and so on, and regulating inflammation has a certain role in preventing or treatment DKD.Micro RNAs(mi RNAs) are a family of short non-coding RNA molecules,which can target several genes by partial homology to sequence elements in the 3'UTR of genes,Their action as largely as post-transcriptional regulators of gene expression occurs at the level of RNA and protein translation.Mi RNAs largely conserved acrossmany species and implicated in many normal physiological and pathological processes.Reports have found that Tristetraprolin(TTP) was the prototype member of the zinc finger protein(ZFP36) RNA, which can combined with m RNA3'UTR region AU-rich region, and regulate the expression of related proteins.TTP could degraded many inflammatory cytokines such as TNF-?, IL-6 and other inflammatory cytokines and they could be combined with the TTP.The early stage of the experimental study on the blood and urine specimens of patients with DKD showed that the expression of TTP increase and the expression of IL-6 and TNF alpha decrease,both of which negatively correlated, which means TTP involved in diabetic kidney disease.Combining with the characteristics of mi RNAs, we suppose that whether mi RNAs can regulate the expression of TTP and further participate in the inflammation in diabetic kidney disease.Objective1.Exiqon mi RCURY LNATM mi RNA Array Chip technology to detect the plasma,urine sediment and kidney tissues mi RNAs expression by Shanghai biological engineering conpany.Screening mi RNAs which can combinate TTP m RNA:mi R-29 c and mi R-1207-5p.2.Detecting the expression level of Inflammatory cytokines IL- 6 and TNF-?in plasma and urine supernatant.3.Discuss the correlation between mi RNA-29c/1207-5p and inflammatory factor IL-6 in plasma of DKD patients.Methods1.100 cases of experimental blood and urine samples was select from the First Affiliated Hospital of Zhengzhou University,October 2014 to July 2015. Including normal group:35 cases was collected in Medical Department;32 patients with renal biopsy diagnosed as DKD,the rest of subjects were collected in Endocrinology.We also studied normal renal tissue from the nephrectomy specimen of 10 patients with renal cell carcinoma and biopsy-proven DKD renal tissue.2.By slected age and sex-matched patient with DKD,health control group by mi RCURY LNATM expression array screening(Kang Cheng company) two groups differneces mi RNAs in plasm, urine and renal tissue samples,further combined datebase analyse mi Rbase, Target Scan Human screened out that mi RNA-29 c and mi RNA-1207-5p can combinated with TTP m RNA.3.q RT-PCR detect the expression of mi R-29 c and mi R-1207-5p in plasma,urines ediment and kidney tissues,use 2- ? ? ctmuliple relationship with between analyse groups.ELISA detect the expression level of IL-6 and TNF-? in plasma and urine supernatant.Analysis the correlation of mi R-29 c and mi R-1207-5p expression levels with IL-6 in plasma.Result1.Exiqon mi RCURY LNATM micro RNA Array result:According to mi RNA Array singal intensity,there are more than two times between the two groups:(1) in the kidney tissues, DKD group than in the NC group hike mi RNAs 15,downregulated in micro RNAs 167;(2) in the plasma, DKD group than NC group raised 103 mi RNAs, down 200;(3) in the urine sediment, DKD group than NC group hike mi RNAs 770 downregulated in mi RNA 239 months.Applicating mi RBase,Target Scan database analysis in order to know the mi RNA-29 c and mi RNA-1207-5p can regulate TTP m RNA.2.The expression of mi RNA-29 c and mi RNA-1207-5p: Compared with NC group, the levels of plasma mi RNA-29 c was higher and mi RNA-1207-5p was significantly lower; the levels of urinary mi RNA-29 c was significantly lower and mi RNA-1207-5p was significantly higher in DKD and DM group(P<0.05).Compared with DM group,the plasma mi RNA-29 c expression increased and mi RNA-1207-5p expression reduced;urinary mi RNA-29 c downregualted and mi RNA-1207-5p upregulated in DKD group, both the difference is statistically significant(P <0.05).Compared with NC group, the expression of renal tissue mi RNA-29 c was reduced and mi RNA-1207-5p was increased in DKD group(P<0.05).3.The concentration of TNF-a and IL-6:Test patients plasma samples by ELISA showed IL-6 in NC group and DM group were 3.685±0.47pg/ml and4.159 ± 0.44pg/ml, significantly lower than the DKD group 70.397 ±2.196pg/ml,TNF- ? in NC group and DM group were 2.010 ± 0.28pg/ml and2.373 ± 0.13pg/ml, significantly lower than the DKD group 37.087 ±3.12pg/ml.urine samples NC group,DM group and DKD group, IL-18 was(2.373±0.58pg/ml),(4.242±0.69pg/ml) and(6.176±0.66pg/ml) respectively,TNF-a were(7.202 ± 1.42pg/ml)(11.241 ± 1.53pg/ml) and(22.639 ±1.47pg/ml), consistent with the results of the plasma specimen,the difference was statistically significant(P> 0.05).4.To further explore the relationship between mi RNA-29c/mi RNA-1207-5p and IL-6, we both correlation analysis showed that patients with DKD mi RNA-1207-5p were negatively correlated with IL-6, the correlation coefficient was-0.818(P <0.05),mi RNA-129 c were positively correlated with IL-6, the correlation coefficient was 0.879(P <0.05).Conclusion1.mi R-29 c and mi RNA-1207-5p expression exist differences in renal tissue,blood and urine specimens with DKD patients.2.mi R-29 c and mi R-1207-5p had correlation with IL-6,and involved in the inflammatory response in diabetic nephropathy. |
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