LncRNA NONRATT021972 siRNA attenuates P2X7 receptor expression and inflammatory cytokine production induced by combined high glucose and free fatty acids in PC12 cells

Abstract

Diabetic neuropathy (DNP) is a frequent chronic complication of diabetes mellitus with potentially life-threatening outcomes. High glucose and elevated free fatty acids (FFAs) have been recently recognized as major causes of nervous system damage in diabetes. Our previous study has indicated extracellular stimuli, such as high glucose and/or FFA stress, may activate the p38 mitogen-activated protein kinase (MAPK) signaling pathway and induce a p38 MAPK-dependent sensitization of the P2X7 receptor and release of inflammatory factors in PC12 cells, while the mechanisms underlying remain to be elucidated. Long noncoding RNAs (lncRNAs) play important roles in diverse biological processes, including activation of a series of pathway signalings. Here, we showed combined high D-glucose and FFAs (HGHF) induced an increment of lncRNA-NONRATT021972 (NONCODE ID, nc021972) in PC12 cells. Nc021972 small interference RNA (siRNA) alleviated HGHF-induced activation of p38 MAPK, expression of the P2X7 receptor, and [Ca(2+)]i increment upon P2X7 receptor activation. Further experiments showed that there existed a crosstalk between nc021972 and the p38 MAPK signaling pathway. Inhibition of p38 MAPK signaling decreased nc021972-induced expression of the P2X7 receptor and [Ca(2+)]i increment upon P2X7 receptor activation. Also, nc021972 siRNA inhibited HGHF-induced PC12 release of TNF-α and IL-6 and rescued decreased cell viability mediated by the P2X7 receptor. Therefore, inhibition of nc021972 may serve as a novel therapeutic strategy for diabetes complicated with nervous inflammatory diseases.

Keywords: Diabetic neuropathy; High fatty acids; High glucose; Long noncoding RNA; P2X7 receptor; PC12 cell.

Fig. 1

Nc021972 was up-regulated in PC12 cells treated with high d-glucose and high FFAs. a The expression of nc021972 determined by quantitative real-time PCR in PC12 cells treated with control and different high concentrations of d-glucose (HG 50 mM and HG 75 mM) for 5 days. The experiment was done in triplicate and repeated three times. One asterisk p < 0.05 vs control at 1 day, two number signs p < 0.01 vs control at 3 days, two ampersands p < 0.01 vs 75 mM at 1 day. b The expression of nc021972 determined by quantitative real-time PCR in PC12 cells treated with control and different high concentrations of FFAs (HF 0.25 mM, HF 0.75 mM, and HF 1 mM) for 5 days. The experiment was done in triplicate and repeated three times. One asterisk p < 0.05 vs control at 1 day, two number signs p < 0.01 vs control at 3 days, two ampersands p < 0.01 vs HG 75 mM at 1 day. c The expression of nc021972 determined by quantitative real-time PCR in PC12 cells cultured with HG 75 mM and/or HF 0.75 mM for 3 days. The experiment was done in triplicate and repeated three times. Two asterisks p < 0.01 vs control, one number sign p < 0.05 vs HG 75 mM group, one ampersand p < 0.05 vs HF 0.75 mM group

Fig. 2

Silencing efficiency of nc021972 induced in PC12 cell lines cultured with HGHF. a Silencing efficiency in nc021972 at 24, 48, and 72 h after transfection with nc021972 siRNA by real-time PCR. b Transfer efficiency of nc021972 siRNA at 72 h as shown by the fluorescence of cy3-NCsi transferred in the cells. Two asterisks p < 0.01 vs control, one number sign p < 0.05, two number signs p < 0.01 vs HGHF

Fig. 3

Silencing nc021972 suppressed the activation of the P2X7 receptor evoked by HGHF in PC12 cells. a mRNA expression of the P2X7 receptor determined by real-time PCR. The protein level of the P2X7 receptor was tested by Western blotting analysis. Representative images of Western blotting (top) are indicated, and the corresponding relative protein levels of the P2X7 receptor were quantified by densitometry (bottom) (b). Representative images of Ca²⁺ signals promoted by BzATP in PC12 cells are shown (c). After basal fluorescence had been measured, BzATP was added to the suspension to observe the fluorescence. Then, the BzATP-induced Ca²⁺ signal was detected until it dropped to the basal fluorescence. The histogram (d) shows the difference value (D value) between the peak value of fluo-3 fluorescence and the initial value of fluo-3 fluorescence in each group. The experiment was done in triplicate and repeated three times. One asterisk p < 0.05 and two asterisks p < 0.01 vs control, one number sign p < 0.05 and two number signs p < 0.01 vs HGHF

Fig. 4

Silencing nc021972 prevented the activation of the P2X7 receptor induced by HGHF via p38 MAPK signaling in PC12 cells. The protein level of phosphorylation of p38 was tested by Western blotting analysis. Representative images of Western blotting (a). The relative protein level of p-p38 was quantified by densitometry (b). Two asterisks p < 0.01 vs control, two number signs p < 0.01 vs HGHF. Overexpression of nc021972 (pcDNA3-nc021972) was induced by transfection of nc021972 into PC12 cells pretreated with SB-203580 or P2X7 receptor siRNA (P2X7si), for 72 h. The levels of nc021972 were tested by real-time PCR analysis (c). Representative images of Western blotting (top) are indicated, and the corresponding relative protein levels of P2X7 receptor were quantified by densitometry (bottom) (d). Representative images of Ca²⁺ signals promoted by BzATP in PC12 cells are shown (e). After basal fluorescence had been measured, BzATP was added to the suspension to observe the fluorescence. Then, the BzATP-induced Ca²⁺ signal was detected until it dropped to the basal fluorescence. The histogram (f) shows the difference value (D value) between the peak value of fluo-3 fluorescence and the initial value of fluo-3 fluorescence in each group. The experiment was done in triplicate and repeated three times. Two asterisks p < 0.01 vs pcDNA3, two number signs p < 0.01 vs pcDNA3-nc021972

Fig. 5

Silencing nc021972 prevented P2X7 receptor-mediated PC12 neuroinflammation and cell viability induced by HGHF in PC12 cells. Basal and BzATP-induced IL-6 (a) and TNF-α (b) release and cell viability of PC12 cells (c) were recorded after tranfection of nc021972 siRNA. The experiment was done in triplicate and repeated three times. One asterisk p < 0.05 and two asterisks p < 0.01 vs control, one number sign p < 0.05 and two number signs p < 0.01 vs HGHF. Overexpression of nc021972 (pcDNA3-nc021972) was induced by transfection of nc021972 into PC12 cells pretreated with SB-203580 or P2X7 receptor siRNA (P2X7si), for 72 h. Basal and BzATP-evoked IL-6 (d) and TNF-α (e) release in PC12 cells and cell viability (f) were recorded. The experiment was done in triplicate and repeated three times. One asterisk p < 0.05 and two asterisks p < 0.01 vs pcDNA3, one number sign p < 0.05 and two number signs p < 0.01 vs pcDNA3-nc021972