ABSTRACT
BACKGROUND/AIMS: Acute myocardial infarction (AMI) occurs when blood supply to the heart is diminished (ischemia) for long time; ischemia is primarily caused due to hypoxia. The present study evaluated the effects of long non-coding RNA H19 on hypoxic rat H9c2 cells and mouse HL-1 cells. METHODS: Hypoxic injury was confirmed by measuring cell viability, migration and invasion, and apoptosis using MTT, Transwell and flow cytometry assays, respectively. H19 expression after hypoxia was estimated by qRT-PCR. We then measured the effects of non-physiologically expressed H19, knockdown of miR-139 with or without H19 silence, and abnormally expressed Sox8 on hypoxia-induced H9c2 cells. Moreover, the interacted miRNA for H19 and downstream target gene were virtually screened and verified. The involved signaling pathways and the effects of abnormally expressed H19 on contractility of HL-1 cells were explored via Western blot analysis. RESULTS: Hypoxia induced decreases of cell viability, migration and invasion, increase of cell apoptosis and up-regulation of H19. Knockdown of H19 increased hypoxia-induced injury in H9c2 cells. H19 acted as a sponge for miR-139 and H19 knockdown aggravated hypoxia-induced injury by up-regulating miR-139. Sox8 was identified as a target of miR-139, and its expression was negatively regulated by miR-139. The mechanistic studies revealed that overexpression of Sox8 might decrease hypoxia-induced cell injury by activating the PI3K/AKT/mTOR pathway and MAPK. Besides, H19 promoted contractility of HL-1 cells. CONCLUSION: These findings suggest that H19 alleviates hypoxia-induced myocardial cell injury by miR-139-mediated up-regulation of Sox8, along with activation of the PI3K/AKT/mTOR pathway and MAPK.
Subject(s)
Cell Hypoxia , MicroRNAs/metabolism , RNA, Long Noncoding/metabolism , 3' Untranslated Regions , Animals , Antagomirs/metabolism , Apoptosis , Base Sequence , Cell Line , Cell Movement , Cell Survival , Humans , MicroRNAs/antagonists & inhibitors , MicroRNAs/genetics , Mitogen-Activated Protein Kinases/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , RNA Interference , RNA, Long Noncoding/antagonists & inhibitors , RNA, Long Noncoding/genetics , RNA, Small Interfering/metabolism , Rats , Real-Time Polymerase Chain Reaction , SOXE Transcription Factors/antagonists & inhibitors , SOXE Transcription Factors/genetics , SOXE Transcription Factors/metabolism , Sarcoplasmic Reticulum/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Sequence Alignment , Signal Transduction , TOR Serine-Threonine Kinases/metabolism , Up-RegulationABSTRACT
This study was aimed to analyze the efficiency and influence factors of PBSC collection by an automatic (AutoPBSC procedure) and a semiautomatic apheresis procedure ( MNC procedure) of COBE Spectra cell separators. According to the different objects, A total of 109 apheresis cases were divided into autologous cohort (patient) and allogeneic cohort (donor). The quantity and quality of the collections and the characteristics of apheresis procedure were compared, the yields and influence factors of two cohorts with two kinds of procedures were analyzed respectively. The results showed that the collections of two procedure in patients and donors which processed the similar blood volumes were insignificantly different in MNC%, CD34⁺ %, CD34⁺ cell counts and Hb concentration (P > 0.05) ; the collections by AutoPBSC procedure had got fewer platelets, less product volumes whereas more ACD-A used, longer apheresis time in comparison with MNC procedure (P < 0.05). Correlation analysis indicated that MNC (r = 0.314,P = 0.015) , CD34⁺ cell counts (r = 0.922, P = 0.000) in collections were positively correlated with preahperesis in the autologus cohort by two procedures, CD34⁺ cell counts were correlated with WBC (r = 0.369, P = 0.004) and MNC (r = 0.495,P = 0.000) in collections; MNC (r = 0.896, P = 0.000) was positive correlated with preahperesis by AutoPBSC procedures and CD34⁺ cell counts also (r = 0.666,P = 0.000) by MNC procedure in the allogeneic cohort. Male had got more MNC and CD34⁺ cell counts than female (P < 0.05), age ≤ 40 had got more MNC and CD34⁺ cell counts than age>40 (P < 0.05) in patients by AutoPBSC procedure; age > 40 had got more CD34⁺ cell counts than age ≤ 40 by MNC procedure(P < 0.05). Only male had got more MNC and CD34⁺ cell counts than female (P < 0.05) by MNC procedure in donors. It is concluded that with same amount of blood processing, the PBSC collections from autologous patients and allogeneic donors had got a high degree of uniformly in purity of MNC and purity and concentration of CD34(+) cell counts by two procedure, whereas sex and age imposed more influence on PBSC collection in autologous.