Limits...
Oxidative Stress Alters miRNA and Gene Expression Profiles in Villous First Trimester Trophoblasts.

Cross CE, Tolba MF, Rondelli CM, Xu M, Abdel-Rahman SZ - Biomed Res Int (2015)

Bottom Line: Cytotoxicity, determined using the SRB assay, was used to calculate the IC50 of H2O2.H2O2 exerted a concentration- and time-dependent cytotoxicity on 3A trophoblast cells.Tool for annotations of microRNAs (TAM) analysis indicated that these altered miRNAs fall into 43 clusters and 34 families, with 41 functions identified.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, The University of Texas Medical Branch, Galveston, TX 77555-1066, USA.

ABSTRACT
The relationship between oxidative stress and miRNA changes in placenta as a potential mechanism involved in preeclampsia (PE) is not fully elucidated. We investigated the impact of oxidative stress on miRNAs and mRNA expression profiles of genes associated with PE in villous 3A first trimester trophoblast cells exposed to H2O2 at 12 different concentrations (0-1 mM) for 0.5, 4, 24, and 48 h. Cytotoxicity, determined using the SRB assay, was used to calculate the IC50 of H2O2. RNA was extracted after 4 h exposure to H2O2 for miRNA and gene expression profiling. H2O2 exerted a concentration- and time-dependent cytotoxicity on 3A trophoblast cells. Short-term exposure of 3A cells to low concentration of H2O2 (5% of IC50) significantly altered miRNA profile as evidenced by significant changes in 195 out of 595 evaluable miRNAs. Tool for annotations of microRNAs (TAM) analysis indicated that these altered miRNAs fall into 43 clusters and 34 families, with 41 functions identified. Exposure to H2O2 altered mRNA expression of 22 out of 84 key genes involved in dysregulation of placental development. In conclusion, short-term exposure of villous first trimester trophoblasts to low concentrations of H2O2 significantly alters miRNA profile and expression of genes implicated in placental development.

No MeSH data available.


Related in: MedlinePlus

IC50 calculation for H2O2 after 30 min, 4 h, 24 hr, or 48 hr in villous 3A trophoblasts: 3A cells were exposed to varying concentrations (0, 2, 3, 6, 10, 20, 30, 60, 100, 200, 300, and 1000 μM) of H2O2 and IC50 determined using the SRB assay. Data represent n = 3 for each time point and concentration. IC50 was calculated using SigmaPlot v12.3.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4538339&req=5

fig1: IC50 calculation for H2O2 after 30 min, 4 h, 24 hr, or 48 hr in villous 3A trophoblasts: 3A cells were exposed to varying concentrations (0, 2, 3, 6, 10, 20, 30, 60, 100, 200, 300, and 1000 μM) of H2O2 and IC50 determined using the SRB assay. Data represent n = 3 for each time point and concentration. IC50 was calculated using SigmaPlot v12.3.

Mentions: In order to determine the appropriate H2O2 concentration for further experiments, cells were exposed to varying micromolar concentrations of H2O2 in complete medium for up to 48 h and cytotoxicity was determined using the SRB assay. Exposure of 3A placental cells to H2O2 resulted in a time- and concentration-dependent cytotoxic effect. The IC50 values for H2O2 were 592, 487, 90, and 15 μM after 30 min, 4, 24, and 48 h exposures, respectively (Figure 1). A concentration equivalent to 5% of the IC50 concentration was used for the miRNAs and mRNA expression profiling experiments. The 4-hour exposure was selected for short-term exposure studies of the effect of H2O2 on miRNA and mRNA expression profile. The levels of GSH as well as SOD activity were assessed after exposure of the cells to 25 μM H2O2 for 4 h as markers for oxidative stress status [22, 23]. GSH level in H2O2 challenged cells was significantly reduced by 30% (from 361.44 ± 10.01 mg/mg protein in untreated cells to 251.35 ± 39.23 in the treated cells; P < 0.01). Similarly, SOD activity in cells exposed to H2O2 was significantly reduced by over 35% (208.9 ± 5.12 U/mg protein in treated cells compared to 321.71 ± 6.78 in the untreated cells; P < 0.01).


Oxidative Stress Alters miRNA and Gene Expression Profiles in Villous First Trimester Trophoblasts.

Cross CE, Tolba MF, Rondelli CM, Xu M, Abdel-Rahman SZ - Biomed Res Int (2015)

IC50 calculation for H2O2 after 30 min, 4 h, 24 hr, or 48 hr in villous 3A trophoblasts: 3A cells were exposed to varying concentrations (0, 2, 3, 6, 10, 20, 30, 60, 100, 200, 300, and 1000 μM) of H2O2 and IC50 determined using the SRB assay. Data represent n = 3 for each time point and concentration. IC50 was calculated using SigmaPlot v12.3.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4538339&req=5

fig1: IC50 calculation for H2O2 after 30 min, 4 h, 24 hr, or 48 hr in villous 3A trophoblasts: 3A cells were exposed to varying concentrations (0, 2, 3, 6, 10, 20, 30, 60, 100, 200, 300, and 1000 μM) of H2O2 and IC50 determined using the SRB assay. Data represent n = 3 for each time point and concentration. IC50 was calculated using SigmaPlot v12.3.
Mentions: In order to determine the appropriate H2O2 concentration for further experiments, cells were exposed to varying micromolar concentrations of H2O2 in complete medium for up to 48 h and cytotoxicity was determined using the SRB assay. Exposure of 3A placental cells to H2O2 resulted in a time- and concentration-dependent cytotoxic effect. The IC50 values for H2O2 were 592, 487, 90, and 15 μM after 30 min, 4, 24, and 48 h exposures, respectively (Figure 1). A concentration equivalent to 5% of the IC50 concentration was used for the miRNAs and mRNA expression profiling experiments. The 4-hour exposure was selected for short-term exposure studies of the effect of H2O2 on miRNA and mRNA expression profile. The levels of GSH as well as SOD activity were assessed after exposure of the cells to 25 μM H2O2 for 4 h as markers for oxidative stress status [22, 23]. GSH level in H2O2 challenged cells was significantly reduced by 30% (from 361.44 ± 10.01 mg/mg protein in untreated cells to 251.35 ± 39.23 in the treated cells; P < 0.01). Similarly, SOD activity in cells exposed to H2O2 was significantly reduced by over 35% (208.9 ± 5.12 U/mg protein in treated cells compared to 321.71 ± 6.78 in the untreated cells; P < 0.01).

Bottom Line: Cytotoxicity, determined using the SRB assay, was used to calculate the IC50 of H2O2.H2O2 exerted a concentration- and time-dependent cytotoxicity on 3A trophoblast cells.Tool for annotations of microRNAs (TAM) analysis indicated that these altered miRNAs fall into 43 clusters and 34 families, with 41 functions identified.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, The University of Texas Medical Branch, Galveston, TX 77555-1066, USA.

ABSTRACT
The relationship between oxidative stress and miRNA changes in placenta as a potential mechanism involved in preeclampsia (PE) is not fully elucidated. We investigated the impact of oxidative stress on miRNAs and mRNA expression profiles of genes associated with PE in villous 3A first trimester trophoblast cells exposed to H2O2 at 12 different concentrations (0-1 mM) for 0.5, 4, 24, and 48 h. Cytotoxicity, determined using the SRB assay, was used to calculate the IC50 of H2O2. RNA was extracted after 4 h exposure to H2O2 for miRNA and gene expression profiling. H2O2 exerted a concentration- and time-dependent cytotoxicity on 3A trophoblast cells. Short-term exposure of 3A cells to low concentration of H2O2 (5% of IC50) significantly altered miRNA profile as evidenced by significant changes in 195 out of 595 evaluable miRNAs. Tool for annotations of microRNAs (TAM) analysis indicated that these altered miRNAs fall into 43 clusters and 34 families, with 41 functions identified. Exposure to H2O2 altered mRNA expression of 22 out of 84 key genes involved in dysregulation of placental development. In conclusion, short-term exposure of villous first trimester trophoblasts to low concentrations of H2O2 significantly alters miRNA profile and expression of genes implicated in placental development.

No MeSH data available.


Related in: MedlinePlus