Ectopic expression of RMRP promoted lung adenocarcinoma cell proliferation, colony formation and invasion.

<p>(A) The expression of RMRP was measured in the H1299 cell after treated with RMRP vector. (B) Ectopic expression of RMRP promoted H1299 cell proliferation. (C) Overexpression of RMRP enhanced the cyclin D1 expression in the H1299 cell. (D) Ectopic expression of RMRP promoted ki-67 expression in the H1299 cell. (E) Overexpression of RMRP promoted the H1299 cell colony formation. (F) Overexpression of RMRP enhanced the H1299 cellinvasion. *p<0.05, **p<0.01 and ***p<0.001.</p

miR-206 expression was downregulated in the lung adenocarcinoma tissues.

<p>(A) The miR-206 expression was measured in lung adenocarcinoma cell lines (A549, SPC-A1, H1299 and H23) and the bronchial epithelial cell line using qRT-PCR. (B) The miR-206 expression was detected in lung adenocarcinoma tissues and the matched adjacent normal tissues by using qRT-PCR. (C) miR-206 expression was downregulated in 21 cases (21/35; 60%) compared to the adjacent normal tissues. (D) The expression of RMRP was negative correlated with the expression of miR-206 in lung adenocarcinoma tissues.</p

RMRP suppressed expression of miR-206 and increased the expression of KRAS, FMNL2 and SOX9.

<p>(A) Overexpression of RMRP inhibited theexpression of miR-206 in the H1299 cell. (B) Ectopic expression ofRMRP promoted the KRAS mRNA expression in the H1299 cell. (C) The protein expression of KRAS was determined using western blot. (D) Ectopic expression ofRMRP promoted the FMNL2 mRNA expression in the H1299 cell. (E) The protein expression ofFMNL2 was determined using western blot. (F) Ectopic expression ofRMRP promoted the SOX9 mRNA expression in the H1299 cell. (G) The protein expression ofSOX9 was determined using western blot.</p

RMRP exhibited an oncogenic activity through targeting miR-206.

<p>(A) miR-206 expression was upregulated in H1299 cell after treated with the miR-206 mimic. (B) miR-206 expression was decreased in the RMRP-induced H1299 cell after treated with RMRP vector. (C) Restoration of miR-206 suppressed cell proliferation in the RMRP-induced H1299 cell after treated with miR-206 mimic. (D) Restoration of miR-206 inhibited the cell invasion in the RMRP-induced H1299 cell after treated with miR-206 mimic.*p<0.05, **p<0.01 and ***p<0.001.</p

The expression of RMRP was upregulated in the lung adenocarcinoma tissues.

<p>(A) RMRP expression was measured in the lung adenocarcinoma tissues and the matched adjacent normal tissues using qRT-PCR. (B) The RMRP was upregulated in 25 cases (25/35; 71.4%) compared to the adjacent normal tissues. (C) The RMRP expression was upregulated in lung adenocarcinoma cell lines (A549, SPC-A1, H1299 and H23) compared to the bronchial epithelial cell line (16HBE).</p

Perinatal testosterone exposure potentiates vascular dysfunction by ERβ suppression in endothelial progenitor cells

<div><p>Recent clinical cohort study shows that testosterone therapy increases cardiovascular diseases in men with low testosterone levels, excessive circulating androgen levels may play a detrimental role in the vascular system, while the potential mechanism and effect of testosterone exposure on the vascular function in offspring is still unknown. Our preliminary results showed that perinatal testosterone exposure in mice induces estrogen receptor β (ERβ) suppression in endothelial progenitor cells (EPCs) in offspring but not mothers, while estradiol (E2) had no effect. Further investigation showed that ERβ suppression is due to perinatal testosterone exposure-induced epigenetic changes with altered DNA methylation on the ERβ promoter. During aging, EPCs with ERβ suppression mobilize to the vascular wall, differentiate into ERβ-suppressed mouse endothelial cells (MECs) with downregulated expression of SOD2 (mitochondrial superoxide dismutase) and ERRα (estrogen-related receptor α). This results in reactive oxygen species (ROS) generation and DNA damage, and the dysfunction of mitochondria and fatty acid metabolism, subsequently potentiating vascular dysfunction. Bone marrow transplantation of EPCs that overexpressed with either ERβ or a SIRT1 single mutant SIRT1-C152(D) that could modulate SIRT1 phosphorylation significantly ameliorated vascular dysfunction, while ERβ knockdown worsened the problem. We conclude that perinatal testosterone exposure potentiates vascular dysfunction through ERβ suppression in EPCs.</p></div

Perinatal testosterone exposure potentiates vascular dysfunction in old male offspring (20 months old).

<p>(a-c) The treated male offspring were given a bolus dose of 2mCi of ¹⁴C-OA through oral gavage, and the blood and tissues, including the heart, aorta and liver, were dissected for analysis of total radioactivity. (a) The in vivo ¹⁴C-OA uptake from the heart and aorta in 2h, n = 8. (b) The in vivo ¹⁴C-OA uptake from liver in 2h, n = 7. (c) The in vivo ¹⁴C-OA uptake in plasma in 1h, n = 6. (d-g) The plasma was collected from treated male offspring for analysis of total cholesterol, n = 10 (d); triglyceride, n = 10 (e); LDL cholesterol, n = 12 (f); and HDL cholesterol, n = 11 (g). (h,i) The aortas were dissected from treated mice for vessel tension analysis. The rings were pre-constricted with phenylephrine, and the acetylcholine (Ach, 10⁻¹⁰–10⁻⁴ mol/l) was injected at the plateau of the phenylephrine-induced contraction. (h) The 10⁻⁴ mol/l Ach-induced aorta ring relaxation, n = 9–12; (i) The Ach-induced aorta ring relaxation curves. (j) The treated mice were used to measure the mean of systolic blood pressure, n = 11. *, <i>P</i><0.05, vs CTL group. Results are expressed as mean ± SEM.</p

Bone marrow transplantation with ERβ overexpression in EPCs restores perinatal testosterone exposure-induced vascular dysfunction in male old offspring, while ERβ knockdown in EPCs worsens the problem.

<p>(a-c) The treated mice were given a bolus dose of 2mCi of ¹⁴C-OA through oral gavage, and the blood and tissues, including the heart, aorta and liver, were dissected for analysis of total radioactivity. (a) The in vivo ¹⁴C-OA uptake from the heart and aorta in 2h, n = 6. (b) The in vivo ¹⁴C-OA uptake from liver in 2h, n = 6. (c) The in vivo ¹⁴C-OA uptake in plasma in 1h, n = 7. (c) (d-g) The plasma was collected from treated mice for analysis of total cholesterol, n = 9 (d); triglyceride, n = 8 (e); LDL cholesterol, n = 11 (f); and HDL cholesterol, n = 12 (g). (h,i) The aortas were dissected from treated mice for vessel tension analysis. The rings were pre-constricted with phenylephrine, and the acetylcholine (Ach, 10⁻¹⁰–10⁻⁴ mol/l) was injected at the plateau of the phenylephrine-induced contraction. (h) The 10⁻⁴ mol/l Ach-induced aorta ring relaxation, n = 8–11; (i) The Ach-induced aorta ring relaxation curves. (j) The treated mice were used to measure the mean of systolic blood pressure, n = 10. *, <i>P</i><0.05, vs CTL group; ¶, <i>P</i><0.05, vs DHT group. Results are expressed as mean ± SEM.</p

Perinatal testosterone exposure induces ERβ suppression in EPCs through increased methylation on ERβ promoter, while E2 has no effect.

<p>The EPCs were isolated from 2-month old male offspring for in vitro cell culture analysis. (a) The representative bands for ERβ methylation in EPCs from male offspring. (b) DNA methylation on ERβ by real-time PCR based methylation specific PCR (MSP) analysis in EPCs, n = 4. (c) ChIP analysis on ERβ promoter in BM-derived EPCs, n = 5. (d) ChIP analysis on ERβ promoter in Circulating EPCs, n = 5. *, <i>P</i><0.05, vs CTL group; ¶, <i>P</i><0.05, vs DHT group. Results are expressed as mean ± SEM.</p

Perinatal testosterone exposure induces ROS generation and DNA damage, and dysfunction of mitochondria and fatty acid metabolism in both circulating EPCs and MECs in old male offspring (20 months old).

<p>The MECs from treated mice were isolated from the hearts during 20 months of age for in vitro culture analysis. (a) ROS formation, n = 6. (b) Quantitation of 3-nitrotyrosine (3-NT) formation, n = 4. (c) Representative γH2AX western blotting band. (d) Quantitation of γH2AX formation for (c), n = 5. (e) Mitochondrial DNA copies, n = 4. (f) Intracellular ATP levels, n = 5. (g) Representative western blotting band for OXPHOS proteins. (h) Quantitation of OXPHOS proteins for (g), n = 5. (i) In vitro ¹⁴C-OA fatty acid uptake, n = 4. (j) The in vitro palmitate oxidation rate, n = 4. *, <i>P</i><0.05, vs CTL group; ¶, <i>P</i><0.05, vs DHT group. Results are expressed as mean ± SEM.</p