miR‑875‑5p regulates IR and inflammation via targeting TXNRD1 in gestational diabetes rats

Abstract

Gestational diabetes mellitus (GDM) is a serious life‑threatening disease that affects the mother and fetus. However, the pathogenesis of GDM is still unclear. microRNAs (miRs) play vital roles in the regulation of various cell functions. The present study aimed to investigate the effects of miR‑875‑5p and thioredoxin reductase 1 cytoplasmic (TXNRD1) in GDM rats and analyze the associated underlying mechanism. A GDM rat model was induced using an intraperitoneal injection of streptozotocin. miR‑875‑5p knockdown plasmids or TXNRD1 knockdown plasmids were injected into the rats via the caudal vein. miR‑875‑5p and TXNRD1 expression in the serum were detected using reverse transcription‑quantitative PCR (RT‑qPCR) or western blot (WB) analyses. The fasting blood‑glucose (FBG), fasting serum insulin, triglyceride and high density lipoprotein levels were detected by specific commercial kits. The inflammatory response and the induction of oxidative stress were analyzed by assessing the expression of associated markers via WB, RT‑qPCR or commercial kits. The pancreatic and placental injuries were detected by hematoxylin and eosin staining. The results indicated that miR‑875‑5p expression levels were downregulated, whereas TXNRD1 levels were upregulated in GDM rats compared with normal pregnancy rats. miR‑875‑5p significantly regulated TXNRD1 expression in GDM rats. miR‑875‑5p silencing notably reduced FBG and insulin resistance, which was accompanied by reduced expression levels of blood lipid and pro‑inflammatory markers as well as reduced oxidative stress. However, the effects of miR‑875‑5p could be reversed by TXNRD1 silencing. Therefore, the present study indicated that miR‑875‑5p regulated IR and inflammation by targeting TXNRD1 in GDM rats. miR‑875‑5p and TXNRD1 may be considered as potential targets for treating GDM.

Keywords: gestational diabetes mellitus; microRNA‑875‑5p; thioredoxin reductase 1 cytoplasmic; inflammation.

Figure 1.

miR-875-5p knockdown reduces FBG, IR and blood lipid levels in GDM rats. (A) miR-875-5p levels in serum were significantly reduced in GDM rats, as determined by RT-qPCR. (B) Transfection with miR-875-5p inhibitor significantly reduced miR-875-5p expression in the serum of rats, as analyzed by RT-qPCR. (C) miR-875-5p inhibitor significantly reduced FBG, FINS and HOMA-IR blood levels. (D) Blood lipids levels were significantly affected by transfection with the miR-875-5p inhibitor. n=10, data are presented as the mean ± SD. *P<0.05, **P<0.01, ***P<0.001. miR, microRNA; GDM, gestational diabetes mellitus; RT-qPCR, reverse transcription-quantitative PCR; FBG, fasting blood glucose; FINS, serum fasting insulin; HOMA-IR, homeostatic model assessment of insulin resistance; TG, triglyceride; TC, total cholesterol; HDL, high-density lipoprotein.

Figure 2.

miR-875-5p inhibitor significantly reduces the levels of inflammatory markers and alters the levels of oxidative stress mediator. (A) Transfection with miR-875-5p inhibitor significantly reduced the protein expression of pro-inflammatory markers in the pancreatic islet tissues, as determined by western blotting. (B) Transfection with miR-875-5p inhibitor significantly reduced the mRNA expression of pro-inflammatory markers in the pancreatic islet tissues, as determined by reverse transcription-quantitative PCR. (C) Levels of oxidative stress markers were significantly altered following transfection with miR-875-5p inhibitor. n=10, data are presented as the mean ± SD. **P<0.01, ***P<0.001. miR, microRNA; GDM, gestational diabetes mellitus; CRP, C reactive protein; SOD, superoxide dismutase; CAT, catalase; MDA, malondialdehyde.

Figure 3.

miR-875-5p knockdown ameliorates pancreas and placenta injury. (A) Transfection with miR-875-5p inhibitor notably improved pancreatic tissue injury in GDM rats. Arrows indicate islet cells. (B) Transfection with miR-875-5p inhibitor notably improved placental tissue injury in GDM rats. Arrows indicate blood vessels and red blood cells. n=10. miR, microRNA; GDM, gestational diabetes mellitus.

Figure 4.

miR-875-5p knockdown upregulates the transcription and translation levels of TXNRD1. (A) Following transfection of INS-1 cells with miR-875-5p mimic, miR-875-5p expression significantly increased. (B) Transfection with miR-875-5p inhibitor significantly increased the TXNRD1 expression in the serum of GDM rats. (C) StarBase was used to predict that miR-875-5p can bind to the 3′ untranslated region of TXNRD1, and the luciferase activity was significantly reduced by transfection with the miR-875-5p mimic in the WT group. n=10, data are presented as the mean ± SD. **P<0.01, ***P<0.001 vs. miR-875-5p-NC. miR, microRNA; GDM, gestational diabetes mellitus; TXNRD1, thioredoxin reductase 1 cytoplasmic; WT, wild-type; MUT, mutant; NC, negative control.

Figure 5.

TXNRD1 knockdown reverses the effects of miR-875-5p inhibitor. (A and B) Transfection with sh-TXNRD1 suppressed the expression of TXNRD1 in the serum of GDM rats. (C) TXNRD1 knockdown increased FBG and IR in the blood of GDM rats. (D and E) TXNRD1 knockdown increased expression levels of pro-inflammatory markers in the pancreatic islet tissues. (F) Oxidative stress levels in the pancreatic islet tissues were altered by TXNRD1 knockdown. n=10, data are presented as the mean ± SD. *P<0.05, **P<0.01, ***P<0.001. miR, microRNA; GDM, gestational diabetes mellitus; sh-, short hairpin RNA; TXNRD1, thioredoxin reductase 1 cytoplasmic; FBG, fasting blood glucose; FINS, serum fasting insulin; HOMA-IR, homeostatic model assessment of insulin resistance; CRP, C reactive protein; SOD, superoxide dismutase; CAT, catalase; MDA, malondialdehyde; NC, negative control.

Figure 6.

TXNRD1 knockdown reverses the effects of miR-875-5p inhibitor on pancreas and placenta injury. (A) Transfection with miR-875-5p inhibitor notably increased pancreatic injury. Arrows indicate islet cells. (B) Transfection with miR-875-5p inhibitor induced placental injury. Arrows indicate blood vessels and red blood cells. miR, microRNA; GDM, gestational diabetes mellitus; sh-, short hairpin RNA; TXNRD1, thioredoxin reductase 1 cytoplasmic; NC, negative control.