Yonsei Med J.  2019 Apr;60(4):381-388. 10.3349/ymj.2019.60.4.381.

Knockdown of LncRNA H19 Relieves LPS-Induced Damage by Modulating miR-130a in Osteoarthritis

• Hu Y
• Li S
• Zou Y

• KMID: 2450194
• DOI: http://doi.org/10.3349/ymj.2019.60.4.381

Abstract

PURPOSE
Osteoarthritis (OA) is a commonly occurring illness without a definitive cure, at present. Long non-coding RNAs (lncRNAs) have been widely confirmed to be involved in the modulation of OA progression. This study aimed to investigate the role and mechanism of lncRNA H19 in OA.
MATERIALS AND METHODS
Abundances of H19 and microRNA-130a (miR-130a) in lipopolysaccharide (LPS)-treated C28/I2 cells were measured by reverse-transcription quantitative PCR (RT-qPCR). CCK-8 and flow cytometry analyses were carried out to assess cell viability and apoptosis. Starbase online software was used to predict the putative binding sites between H19 and miR-130a. Luciferase reporter, RNA pull down, and RT-qPCR were performed to analyze the true interaction between H19 and miR-130a.
RESULTS
A notably dose-dependent elevation of H19 levels was observed in LPS-treated C28/I2 cells. Knockdown of H19 ameliorated the injury of LPS-induced C28/I2 cells, reflected by induced viability, decreased apoptosis, and reduced inflammatory factor secretions. Moreover, H19 negatively regulated the expression of miR-130a via acting as a molecular sponge for miR-130a. The stimulatory effects of H19 on cell damage were abolished following the restoration of miR-130a.
CONCLUSION
LncRNA H19 aggravated the injury of LPS-induced C28/I2 cells by sponging miR-130a, hinting a novel regulatory mechanism and a potential therapeutic target for OA.

Keyword

Osteoarthritis; lipopolysaccharide; lncRNA H19; miR-130a

MeSH Terms

Apoptosis
Binding Sites
Cell Survival
Flow Cytometry
Luciferases
Osteoarthritis*
Polymerase Chain Reaction
Porifera
RNA
RNA, Long Noncoding*
Sincalide
Luciferases
RNA
RNA, Long Noncoding
Sincalide

Figure

• 

  Fig. 1 H19 is upregulated in LPS-treated C28/I2 cells in a dose-dependent manner. (A) C28/I2 cells were treated with 0, 1, 5, and 10 µg/mL of LPS for 12 h, and then relative expression of H19 was measured by RT-qPCR. (B) LPS-exposed C28/I2 cells were transfected with si-NC or si-H19, followed by the detection of H19 expression by RT-qPCR. *p<0.01, †p<0.001. LPS, lipopolysaccharide; RT-qPCR, reverse-transcription quantitative PCR.

• 

  Fig. 2 LncRNA H19 deficiency remits LPS-induced cell damage in C28/I2 cells. After treatment with 5 µl of LPS, along with si-NC or si-H19, cell viability (A), apoptosis (B), and the release of inflammatory cytokines (IL-1β, IL-6, TNF-α) (C-E) in C28/I2 cells were determined by CCK-8, flow cytometry, and ELISA assays. *p<0.01, †p<0.001. LPS, lipopolysaccharide.

• 

  Fig. 3 LncRNA H19 identified as a molecular sponge for miR-130a. (A) Bioinformatics software was used to predict putative binding sites between miR-130a and H19, as well as mutant sites in the H19-MUT reporter. (B and C) 293T cells were co-transfected with H19-WT or H19-MUT and miR-130a or anti-miR-130a for 48 h, and then the activity of luciferase constructs focused on H19-WT and H19-MUT were determined by Dual-Luciferase reporter assay. (D) The mutant sites in the miR-130a-MUT reporter. (E and F) RNA pull down analysis was performed to verify the interplay between H19 and miR-130a. †p<0.001.

• 

  Fig. 4 The negative regulation between H19 and miR-130a in LPS-treated C28/I2 cells. (A) RT-qPCR assay was employed for the detection of H19 expression in H19 or si-H19-transfected cells. (B) The expression of miR-130a was negatively regulated by H19 in LPS-exposed C28/I2 cells. (C) RT-qPCR assay was employed for the detection of miR-130a expression in miR-130a or anti-miR-130a-transfected cells. (D) The expression of H19 was negatively regulated by miR-130a in LPS-exposed C28/I2 cells. *p<0.01, †p<0.001. LPS, lipopolysaccharide; RT-qPCR, reverse-transcription quantitative PCR.

• 

  Fig. 5 Effects of H19 are abrogated by miR-130a overexpression. (A) C28/I2 cells were treated with 0, 1, 5, and 10 µg/mL of LPS for 12 h, and then relative expression of miR-130a was determined by RT-qPCR. LPS-induced C28/I2 cells were transfected with pcDNA3.1, H19, H19+miR-NC, or H19+miR-130a. At about 48 h post-transfection, cell viability (B), apoptosis (C), and the levels of inflammatory cytokines (IL-1β, IL-6, TNF-α) (D-F) were measured by CCK-8, flow cytometry, and ELISA assays. *p<0.01. LPS, lipopolysaccharide; RT-qPCR, reverse-transcription quantitative PCR.

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