AIM: To observe the level of vascular endothelial growth factor (VEGF) secreted by monocytes cultured with electrical burn serum, and to explore the effect of VEGF on monocyte-endothelial cell adhesion.METH-ODS:The electrical burn serum of the rat was prepared.The normal serum from the rats without treating electric current was also collected for control.The contents of VEGF and its soluble receptor sFlt-1 in electrical burn group were determined by double-antibody sandwich ELISA.THP-1 cells were randomly divided into normal serum group and electrical burn serum group.The contents of VEGF and sFlt-1 in the culture supernatants were measured by double-antibody sandwich ELISA. THP-1 cells were also randomly divided into another 4 groups:normal serum group, electrical burn serum group, normal serum +inhibitor group and electrical burn serum +inhibitor group.THP-1 cells, which were incubated with the serum for 3 h and 6 h, were labeled with calcein-AM and then were added into the well with monolayer of endothelial cell line EA.hy926 to detect monocyte-endothelial cell adhesion.RESULTS:The levels of serum VEGF of the rats with electrical burns were significantly increased, the levels of serum sFlt-1 were significantly decreased as compared with the controls. The levels of VEGF secreted by THP-1 cells cultured with electrical burn serum were significantly increased, the levels of sFlt-1 were decreased correspondingly.Electrical burn serum enhanced monocyte-endothelial cell adhesion, sFlt-1 inhibi-ted the adhesion between monocytes and endothelial cells.CONCLUSION:The monocytes exposed to the electrical burn serum secrete VEGF, which enhance the adhesion between monocytes and endothelial cells.Blockage of VEGF activity may effectively inhibit monocyte-endothelial cell adhesion.

Objective To investigate the expression changes of microRNAs and VEGF-NOTCH in renal ischemic injury in mice, and to explore the potential mechanism associated with renal angiogenesis.Method Male Balb/c mice were subjected to a standard renal ischemia to induce acute kidney injury (AKI) after 45 min of bilateral renal artery clamping. Following 4 h, 24 h of reperfusion or sham operation, kindey tissues were collected and subjected to detect the expression changes of microRNAs which relatived with angiogenesis and VEGF, Flk-1, Notch1 mRNA by Quantitative Real-time RT-PCR. Flk-1 protein was detected by Western blotting analysis at 24 h and 72 h following Ischemia/Reperfusion(I/R) injury. The expression of CD31 was examined in tissue sections by immunohistochemistry staining, and the microvessels in ischemic region of each group were counted. Results miRNA-210 and miRNA-92a expression increased significantly, with prominent changes at 4 h and 24 h after reperfusion( P ＜ 0.05 ). VEGF and Flk-1 mRNA expression and Flk-1 protein were increased in renal I/R compared with control group respectively (P＜0.05 ).Immunohistochemistry staining results of CD31 showed a significant increase of microvessels in renal ischemic region. Conclusion This study first reported the changes in miRNAs expression in response to kidney I/R in mouse. our results implied that miRNAs may be involved in targeting VEGF-Notch pathway signaling to regulate angiogenesis after renal I/R injury. It provided novel insights into the angiogenesis mechanism of renal ischemic injury.

OBJECTIVETo explore the molecular mechanism of microRNA-21 in myocardial damage of rats in the early stage of severe scald injury by observing the expression of microRNA-21 and programmed cell death 4 (PDCD4) in myocardial tissue of rat and to validate the relationship between them in cell model.

METHODS(1) Forty SD rats were divided into sham injury group (n =8, sham injured) and scald injury group (n =32, inflicted with 30% TBSA full-thickness scald on the back) according to the random number table. The left ventricular tissue was collected from rats in sham injury group at post injury hour 1 without any fluid infusion. Rats in scald injury group were given an intraperitoneal injection of lactic acid Ringer's solution and 8 rats were respectively sacrificed at post injury hour 3, 6, 12, 24 to harvest left ventricular tissue. The expression of microRNA-21 in myocardial tissue was assessed by real-time fluorescent quantitative RT-PCR. The protein expression of PDCD4 in myocardial tissue was assessed by Western blotting. (2) Rat myocardial cell line H9C2 was divided into microRNA-21 inhibitor group (cells were transfected with microRNA-21 inhibitor) and negative transfection control group (cells were transfected with negative control of microRNA inhibitor) according to the random number table. At post transfection hour 48, real-time fluorescent quantitative RT-PCR and Western blotting were performed respectively to determine the mRNA and protein expression levels of PDCD4 in cells. Data were processed with one-way analysis of variance, LSD-t and two independent samples t test. The relationship between microRNA-21 expression and PDCD4 protein level in myocardial tissue of rats was assessed by linear correlation analysis.

RESULTS(1) The expression levels of microRNA-21 in myocardial tissue of rats in sham injury group at post injury hour 1 and in scald injury group at post injury hour 3, 6, 12, 24 were respectively 0. 96 ± 0. 13, 0. 44 ± 0. 08, 0. 42 ± 0. 10, 0.33 +0.07, and 0.61 0.10 (F = 27.331, P <0.001). Compared with that in myocardial tissue of rats in sham injury group at post injury hour 1, expression level of microRNA-21 was significantly decreased in scald injury group at post injury hour 3, 6, 12, 24 (with t values from 4. 558 to 9.410, P values below 0.01). The protein expression levels of PDCD4 in myocardial tissue of rats in sham injury group at post injury hour 1 and in scald injury group at post injury hour 3, 6, 12, 24 were respectively 0.44 ± 0.05, 0.60 ± 0.09, 0.92 ± 0. 15, 0. 86 ± 0.11, and 0.57 ± 0. 10 (F =8.622, P =0.003). Compared with that in sham injury group at post injury hour 1, protein expression level of PDCD4 was significantly increased in scald injury group at post injury hour 6 and 12 (with t values respectively 4. 968 and 4. 122, P values below 0.01). A significant negative correlation between the expression of microRNA-21 and PDCD4 protein in myocardial tissue of rats of scald injury group was observed at each time point (r = -0. 572, P = 0. 026). (2) The mRNA and protein expression levels of PDCD4 of myocardial cells in microRNA-21 inhibitor group were respectively 1.73 ± 0. 29 and 0. 38 ± 0. 08, which were significantly higher than those in negative transfection control group (0.95 ± 0.14 and 0.23 ± 0.03, with t values respectively 4. 857 and 3.356, P <0.05 or P <0.01).

CONCLUSIONSExpression of microRNA-21 was decreased, while expression of PDCD4 was increased, in myocardial tissue of rats in the early stage of severe scald injury. MicroRNA-21 might participate in myocardial damage in the early stage of scald injury by negatively regulating expression of PDCD4.

Animals ; Apoptosis Regulatory Proteins ; metabolism ; Blotting, Western ; Burns ; metabolism ; pathology ; MicroRNAs ; genetics ; metabolism ; Myocardium ; metabolism ; pathology ; Myocytes, Cardiac ; metabolism ; pathology ; Rats ; Reverse Transcriptase Polymerase Chain Reaction ; Soft Tissue Injuries

OBJECTIVE: To investigate the expression and significance of muscle segment homeobox2 (Msx2) and topo II-alpha in sinonasal inverted papilloma (SNIP), and the relationship in the process of malignant transformation of SNIP. METHOD: Immunohistochemical method was used to detect the expression of Msx2 and topo II-alpha in 32 cases of SNIP, 30 cases of inflammatory nasal polyp (INP) and 30 cases of SNIP with carcinoma. According to the pathology results, SNIP were divided into mild atypical hyperplasia, moderate atypical hyperplasia and severe atypical hyperplasia. RESULT: The mean optical density of Msx2 in SNIP and SNIP with carcinoma tissues were 0.2183 +/- 0.0598 and 0.2521 +/- 0.0761,which were significantly higher than 0.1878 +/- 0. 0372 in the INP tissue (P<0.05 or 0.01). The mean optical density of topo II-alpha in SNIP and SNIP with carcinoma tissues were 0.2303 +/- 0.0397 and 0.2666 +/- 0.0483, which were significantly higher than 0.1978 +/- 0.0388 in the NIP tissue (P<0.01). There were significant difference of Msx2 and topo II-alpha in SNIP between any two of the three groups divided according to pathological morphology (P<0.01 or 0.05). The expression of Msx2 and topo II-alpha in SNIP were positively correlated (P<0.05). CONCLUSION Msx2 and topo II-alpha may play an important role in the occurrence and development of SNIP. So it can be used as new therapeutic targets.
Antigens, Neoplasm ; genetics ; metabolism ; DNA Topoisomerases, Type II ; genetics ; metabolism ; DNA-Binding Proteins ; genetics ; metabolism ; Female ; Homeodomain Proteins ; genetics ; metabolism ; Humans ; Male ; Middle Aged ; Nose Neoplasms ; genetics ; metabolism ; pathology ; Papilloma, Inverted ; genetics ; metabolism ; pathology

Objective: To observe the clinical effects of recombinant human growth hormone (rhGH) on children with severe burn. Methods: Clinical data of 94 children with severe burn, hospitalized in our burn unit from April 2012 to December 2016, conforming to the study criteria, were retrospectively analyzed. According to the use of rhGH, children were divided into rhGH group (n=50) and control group (n=44). Children in control group received conventional treatment, while children in rhGH group received both conventional and rhGH treatment. The rhGH treatment was started 3 to 5 days post injury in dosage of 0.2-0.4 U·kg^-1·d^-1, by way of subcutaneous injection, and the course of treatment was (11±5) d. The plasma albumin and prealbumin levels, heart rate, alanine aminotransferase (ALT), and serum creatinine level in 2 weeks post injury, times of skin grafting operation, hospitalization time, total hospitalization treatment cost, and sepsis and death of children were compared between the 2 groups. Data were processed with independent sample t test, Mann-Whitney U test, and Fisher′s exact test. Results: (1) In 2 weeks post injury, the plasma albumin level [(36±4) g/L] and prealbumin level [(94±34) g/L] of children in rhGH group were significantly higher than those in control group [(33±4) and (73±20) g/L, t=3.666, 3.401, P<0.05]. (2) In 2 weeks post injury, the heart rate of children in rhGH group was (123±11) times per minute, which was slower than (130±14) times per minute of children in control group (t=2.839, P<0.05). There was no significant difference in ALT level of children between the 2 groups (Z=0.868, P>0.05). The blood creatinine levels of children in the 2 groups were within normal range. (3) The times of skin grafting operation of children in rhGH group was 0.3±0.5, which was significantly less than 0.5±0.6 in control group (Z=2.234, P<0.05). The hospitalization time of children in rhGH group was (22±8) days, which was shorter than (28±10) days in control group (t=2.837, P<0.05). The total hospitalization treatment cost of children in rhGH group was (41±15) thousand yuan, which was significantly less than (53±25) thousand yuan in control group (t=2.878, P<0.05). (4) There were 2 cases of sepsis in control group and 1 case of sepsis in rhGH group, with no significant difference between the 2 groups (P>0.05). No children died in the 2 groups. Conclusions rhGH treatment of children with severe burn can correct post-injury hypoproteinemia, improve cardiac function, reduce the times of skin grafting operation and hospitalization treatment cost, shorten hospitalization time, with no significant effect on kidney and liver function, sepsis, and death.

Objective: To explore differential expression of microRNAs in serum of patients with severe burn and analysis of the signaling pathway at early stage. Methods: In this study, we included three healthy adult volunteers and three patients with severe burn, conforming to the inclusion criteria and hospitalized in Tongren Hospital of Wuhan University & Wuhan Third Hospital in July 2015. Venous whole blood of 6 mL of each burn patient and healthy volunteer was collected at 24 to 48 h post injury of burn patients. The whole blood was divided into burn group and healthy control group. Whole blood of 2 mL of each one was used to determine white blood cell count and neutrophile granulocyte content. Serum was separated from the other whole blood of 4 mL of each one. Half of serum was used to determine content of blood glucose, total protein, and albumin; another half of serum was used to extract total RNA with Trizol method. The differentially expressed microRNA, with differential expression ratio larger than or equal to 1.500 between 2 groups, were screened by microRNA chip technique. Then cluster analysis and functional enrichment analysis of Kyoto encyclopedia of genes and genomes (KEGG) signaling pathway were performed on the differentially expressed microRNAs. Data were processed with t test. Results: (1) Content of white blood cell count, neutrophile granulocyte of whole blood, and blood glucose of serum of patients in burn group was obviously higher than that in healthy control group (with t values from 4.27 to 7.83, P<0.05 or P<0.01). Content of total protein and albumin of serum of patients in burn group was significantly lower than that in healthy control group (with t values respectively -12.80 and -12.36, P values below 0.01). (2)Compared with those in serum of healthy control group, differential expression ratios of 48 microRNAs in serum of burn group were larger than 1.500, with 22 up-regulated microRNAs and 26 down-regulated microRNAs. MicroRNA expression profile in serum of burn group was different from that of healthy control group. (3)Functional enrichment analysis of KEGG signaling pathway showed that compared with those in serum of healthy control group, microRNAs of differential expression in serum of burn group took part in tumor transcription misregulation signaling pathway, tumor proteoglycan signaling pathway, long-term potentiation signaling pathway, tumor associated microRNAs signaling pathway, citrate cycle signaling pathway, tumor necrosis factor signaling pathway, focal adhesion signaling pathway, endocytosis signaling pathway, insulin secretion signaling pathway, and estrogen signaling pathway. Conclusions MicroRNA expression profile in serum of patients with severe burn is different from that in serum of healthy adults. MicroRNAs of differential expression may take part in important pathophysiological process of energy metabolism, inflammatory response, and regulation of blood glucose at early stage of severe burn.

Objective: To explore the differential expression of microRNAs in the serum among patients with electrical burn or thermal burn and healthy persons and to explore the significance. Methods: In this study we included three patients with electrical burn and three patients with thermal burn, conforming to the inclusion criteria and hospitalized in our burn ward from June to August 2015, and three healthy adult volunteers. Their serum samples were separated from whole blood and divided into electrical burn group, thermal burn group, and normal control group. Total RNA was extracted from their serum samples using Trizol method. The differentially expressed microRNAs (with differential ratio larger than or equal to 2.000, less than or equal to 0.500) among the three groups were screened by microRNA chip technique. Then cluster and Venn diagram analysis of the differentially expressed microRNAs were performed. Enrichment analysis of Kyoto encyclopedia of genes and genomes (KEGG) signaling pathway was performed on the distinctly changed microRNAs (with differential ratio larger than or equal to 5.000, less than or equal to 0.500). Results: There were 220 differentially expressed microRNAs among serum of the three groups. MicroRNA expression profiles in serum of electrical burn and thermal burn groups were different from that in serum of normal control group. Compared with those in serum of normal control group, the expressions of 59 microRNAs changed more than 2.000 times in serum of electrical burn group, with 50 up-regulated microRNAs and 9 down-regulated microRNAs; the expressions of 40 microRNAs changed more than 2.000 times in serum of thermal burn group, with 21 up-regulated microRNAs and 19 down-regulated microRNAs. Compared with those in serum of thermal burn group, the expressions of 167 microRNAs changed more than 2.000 times in serum of electrical burn group. There were 17 exclusively expressed microRNAs in serum of thermal burn group and 26 exclusively expressed microRNAs in serum of electrical burn group, compared with those in serum of normal control group. Enrichment analysis of KEGG signaling pathway showed that compared with those in serum of normal control group, microRNAs which changed distinctly in serum of electrical burn group took part in the insulin secretion signaling pathway, arrhythmogenic right ventricular cardiomyopathy signaling pathway, hypertrophic cardiomyopathy signaling pathway, glutamatergic synapse signaling pathway, calcium signaling pathway, cyclic adenosine monophosphate signaling pathway, glycerophospholipid metabolism, pyrimidine metabolism, serotonergic synapse signaling pathway, etc, while microRNAs which changed distinctly in serum of thermal burn group took part in the tumor transcription misregulation signaling pathway, proteoglycans in tumor signaling pathway, microRNAs in tumor signaling pathway, long-term potentiation signaling pathway, citrate cycle signaling pathway, tumor necrosis factor signaling pathway, focal adhesion signaling pathway, endocytosis signaling pathway, insulin secretion signaling pathway, p53 signaling pathway, and estrogen signaling pathway, etc. Conclusions MicroRNA expression profiles in serum of electrical and thermal burn are different from that in serum of healthy adult. The signaling pathways enriched with target genes which are regulated by the differentially expressed microRNAs are related to the pathological changes and clinical manifestations after electrical or thermal burn.

OBJECTIVETo observe the changes in the expressions of microRNA-126 in myocardial tissue and cardiac troponin I (cTnI) in serum of rats in the early stage of severe burn injury with analysis of their relationship, and to validate the relationship between microRNA-126 and myocardial damage in cellular level.

METHODS(1) Forty-eight SD rats were divided into sham injury group (n=8, without fluid therapy after sham injury) and burn injury group (n=40, inflicted with 30% TBSA full-thickness scald on the back, hereinafter referred to as burn, and received intraperitoneally injection of lactic acid Ringer's solution) according to the random number table. Blood was collected from abdominal aorta of rats in sham injury group at post injury hour (PIH) 1, and then these 8 rats were sacrificed for obtaining left ventricular tissue. Blood was respectively collected from abdominal aorta of 8 rats in burn injury group at PIH 3, 6, 12, 24, and 48, and then they were sacrificed and the left ventricular tissue was obtained at each time point. The expression of microRNA-126 in myocardial tissue was assessed by real-time fluorescent quantitative RT-PCR. Serum level of cTnI was assessed by ELISA. (2) Rat myocardial cell line H9C2 was divided into normal control group (NC, routinely cultured), stimulation group (S), negative transfection+stimulation group (NT+S), and transfection+stimulation group (T+S) according to the random number table. Cells in S group were treated with hypoxia for 24 h after being cultured with DMEM containing 10% burn serum obtained from rats in burn injury group at PIH 6 in experiment (1). Cells in NT+S group and T+S group were respectively transfected with the negative control of microRNA mimics and microRNA-126 mimics for 24 h, and then were given the same treatment as that of S group. The expression of microRNA-126 in myocardial cells was determined by real-time fluorescent quantitative RT-PCR (with the sample number of 3). Cell counting kit 8 was used to examine the vitality of myocardial cell (with the sample number of 4, denoted as absorbance value). Apoptotic rate of myocardial cells was determined by flow cytometer (with the sample number of 3). Data were processed with one-way analysis of variance and LSD-t test. The relationship between microRNA-126 expression in myocardial tissue and serum level of cTnI of rats was assessed by linear correlation analysis.

RESULTS(1) Compared with that of sham injury group at PIH 1, the expression levels of microRNA-126 in myocardial tissue of rats in burn injury group at PIH 3, 6, 12, 24, and 48 were significantly decreased (with t values from 5.68 to 9.79, P values below 0.01), reaching its nadir at PIH 24 (0.40 ± 0.08). Compared with that of sham injury group at PIH 1, the serum levels of cTnI of rats in burn injury group at PIH 3, 6, 12, 24, and 48 were significantly increased (with t values from 6.68 to 12.79, P values below 0.01), peaking at PIH 12 [(1 035 ± 177) pg/mL]. A significant negative correlation between the expression level of microRNA-126 in myocardial tissue and serum level of cTnI was observed in rats of burn injury group at each time point (r=-0.797, P<0.001). (2) Compared with those of NC group, the microRNA-126 expression levels in myocardial cells of S group and T+S group were respectively decreased and increased (with t values respectively 4.57 and 5.73, P<0.05 or P<0.01), the cell vitality levels were obviously decreased (with t values respectively 14.88 and 6.48, P values below 0.01), and the apoptotic rates were significantly increased (with t values respectively 13.82 and 6.96, P values below 0.01). Compared with that in NT+S group, the microRNA-126 expression level in myocardial cells of T+S group was significantly increased (t=6.77, P<0.01), the cell vitality level was obviously increased (t=8.23, P<0.001), and the apoptotic rate was significantly decreased (t=6.14, P<0.001).

CONCLUSIONSExpression level of microRNA-126 in myocardial tissue of rat was decreased in the early stage of severe burn injury. It may participate in regulating myocardial damage and play a protective role.

Animals ; Burns ; metabolism ; pathology ; Cell Line ; Enzyme-Linked Immunosorbent Assay ; Hypoxia ; MicroRNAs ; genetics ; metabolism ; Myocardium ; metabolism ; pathology ; Myocytes, Cardiac ; metabolism ; pathology ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Reverse Transcriptase Polymerase Chain Reaction ; Serum ; Soft Tissue Injuries ; Transfection ; Troponin I ; metabolism

OBJECTIVETo observe the change in phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signal pathway in monocytes as induced by serum of rats with electrical burn, and to explore the effects of PI3K/Akt pathway on monocyte-endothelial cell adhesion.

METHODSSixty-four SD rats of clean grade were inflicted with electrical burn for the collection of serum of rats with electrical burn; another group of twenty-four SD rats were used to obtain normal serum without treatment. (1) Human monocyte line THP-1 was routinely cultured. The THP-1 cells in logarithmic phase were divided into normal serum group (resuspended in RPMI 1640 medium with 20% normal rat serum) and burn serum group (resuspended with RPMI 1640 medium with 20% serum of rats with electrical burn) according to the random number table, with 6 wells in each group. Morphology of THP-1 cells in normal serum group was observed at post culture hour (PCH) 24, and that in burn serum group at PCH 3, 6, 24. The contents of TNF-α in culture supernatant were determined by double-antibody sandwich ELISA at the corresponding time point in each group. The state of Akt activation was determined by Western blotting at PCH 3, 6, 24. (2) Another portion of THP-1 cells were divided into 4 groups according to the random number table, with 6 wells in each group. Cells in normal serum group and burn serum group were given with the same culture condition as above; cells in normal serum+inhibitor group and burn serum+inhibitor group were cultured with the same culture conditions as in the former two groups correspondingly with addition of 100 nmol/L wortmannin in the nutrient solution. At PCH 3 and 6, THP-1 cells were added into the well with a monolayer of endothelial cell line EA.hy926 to observe the monocyte-endothelial cell adhesion. Data were processed with one-way analysis of variance and LSD- t test.

RESULTS(1) In normal serum group, THP-1 cells showed growth in suspension, with uniform shape at PCH 24. In burn serum group, the cell shape became irregular though the membrane was complete at PCH 3; cellular size became irregular and cell membrane and cytoplasm were swollen at PCH 6; cell membrane was disrupted with death of cells at PCH 24. The contents of TNF-α in culture supernatant in normal serum group at PCH 24 and in burn serum group at PCH 3, 6, 24 were respectively (38.5 ± 1.4), (75.1 ± 1.5), (91.5 ± 1.8), (117.0 ± 1.4) pg/mL (F = 1 415.306, P < 0.01). The contents of TNF-α in culture supernatant in burn serum group at PCH 3, 6, 24 were all significantly higher than the content of TNF-α in normal serum group at PCH 24 (with t values respectively 29.614, 42.852, 63.485, P values below 0.01). The ratio values of phosphorylated Akt to Akt in burn serum group at PCH 3, 6, 24 were respectively 2.66, 3.69, 1.17 times of those in normal serum group at the corresponding time point. (2) In normal serum group, normal serum+inhibitor group, burn serum group, and burn serum+inhibitor group at PCH 3 and 6, the numbers of THP-1 cells adherent to endothelial cells were respectively (231 ± 45), (280 ± 47), (703 ± 169), (335 ± 85) per 100-time field; (219 ± 49), (235 ± 21), (562 ± 123), (226 ± 29) per 100-time field (with F values respectively 25.630 and 18.975, P values below 0.01). The number of THP-1 cells adhered to EA.hy926 cells was significantly more in burn serum group than in normal serum group at PCH 3 and 6 (with t values respectively 6.189 and 6.601, P values below 0.01). The number of THP-1 cells adherent to EA.hy926 cells was significantly fewer in burn serum+inhibitor group than in burn serum group at PCH 3 and 6 (with t values respectively 6.821 and 6.465, P values below 0.01).

CONCLUSIONSThe serum of rats suffering from electrical burn can induce the monocytes to secrete TNF-α, thus enhancing monocyte-endothelial cell adhesion, but it can be inhibited by blocking PI3K/Akt signal pathway.

Animals ; Burns, Electric ; blood ; Cell Line ; Humans ; Monocytes ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats ; Serum ; Signal Transduction ; Tissue Adhesions ; metabolism ; pathology ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo explore the effects of microRNA-21 on apoptosis of myocardial cell of rats as induced by ischemia and hypoxia, and to analyze the underlying mechanisms.

METHODS(1) Rat myocardial cell line H9C2 was cultured in a serum-free and low glucose DMEM medium using a hypoxic incubator which was filled with 1% oxygen, 5% carbon dioxide, and 94% nitrogen to simulate ischemic environment. The expression of microRNA-21 in normal myocardial cells and cells treated with low oxygen exposure for 6 and 24 h were assessed by real-time fluorescent quantitative RT-PCR. (2) Another portion of myocardial cells were divided into 4 groups according to the random number table: normal control group (NC, ordinary culture without any treatment), ischemia/hypoxia group (IH, treated with ischemia and hypoxia for 24 h), negative transfection control+ischemia/hypoxia group (NC+IH, treated with ischemia and hypoxia for 24 h after the transfection of microRNA mimics control for 24 h), microRNA-21+ischemia/hypoxia group (M+IH, treated with ischemia and hypoxia for 24 h after the transfection of microRNA-21 mimics for 24 h). The cells in the latter three groups were examined immediately after treatment, and cells in group NC were collected and examined at the same time point. Apoptosis rate of myocardial cells was determined by flow cytometer. The mRNA and protein expression levels of programmed cell death 4 (PDCD4) in myocardial cells were determined by real-time fluorescent quantitative RT-PCR and Western blotting respectively. The sample numbers in this experiment were 6 or 3. Data were processed with one-way analysis of variance and LSD- t test.

RESULTS(1) The expression level of microRNA-21 in normal myocardial cells and cells treated with ischemia and hypoxia for 6 and 24 h were respectively 1.09 ± 0.17, 0.75 ± 0.08, and 0.67 ± 0.08 (F = 11.280, P = 0.009). Compared with expression level of microRNA-21 in normal myocardial cells, those of cells treated for 24 h (t = 4.461, P = 0.004) and 6 h (t = 3.642, P = 0.011) were both lower, and the former was more obvious. Therefore all the ischemia and hypoxia treatment time of cells in the following experiment was 24 h. (2) The apoptosis rate of myocardial cells in group NC was (3.5 ± 0.7)%. After being treated with ischemia and hypoxia for 24 h, the apoptosis rates of myocardial cells in groups IH, NC+IH, and M+IH were respectively (17.3 ± 3.2)%, (16.4 ± 3.0)%, and (7.6 ± 2.0)% (F = 15.176, P = 0.001). Compared with that of group NC, the apoptosis rate of myocardial cells of group IH was significantly increased (t = 5.641, P < 0.001), while it was significantly decreased in group M+IH as compared with group NC+IH (t = 3.588, P = 0.007). The mRNA expression level of PDCD4 in group NC was 1.06 ± 0.21. After being treated with ischemia and hypoxia for 24 h, the mRNA expression levels of PDCD4 in groups IH, NC+IH, and M+IH were respectively 3.01 ± 0.34, 3.05 ± 0.25, and 1.48 ± 0.24 (F = 44.952, P < 0.001). Compared with that of group NC, the mRNA expression level of PDCD4 in group IH was higher (t = 8.945, P < 0.001), while it was significantly lower in group M+IH as compared with group NC+IH (t = 7.253, P < 0.001). The protein expression level of PDCD4 in group NC was 0.44 ± 0.08. After being treated with ischemia and hypoxia for 24 h, the protein expression levels of PDCD4 in groups IH, NC+IH, and M+IH were respectively 0.96 ± 0.13, 1.05 ± 0.12, and 0.58 ± 0.12 (F = 18.804, P = 0.008). Compared with that of group NC, the protein expression level of PDCD4 in group IH was higher (t = 5.429, P = 0.006), while it was significantly reduced in group M+IH as compared with group NC+IH (t = 4.903, P = 0.008).

CONCLUSIONSIschemia and hypoxia reduce the expression of microRNA-21 in myocardial cells, while increasing the expression of microRNA-21 can alleviate the ischemia/hypoxia-induced apoptosis by lowering the expression of PDCD4.

Animals ; Apoptosis ; genetics ; physiology ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Blotting, Western ; Cell Line ; Flow Cytometry ; Hypoxia ; Ischemia ; MicroRNAs ; genetics ; Myocardium ; Myocytes, Cardiac ; RNA, Messenger ; genetics ; Rats ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection