Mechanism Of circHIPK3 Research Articles

Circular RNA HIPK3 facilitates ferroptosis in gestational diabetes mellitus by regulating glutathione peroxidase 4 DNA methylation.

Gestational diabetes mellitus (GDM) is the most frequently occurring complication during pregnancy, with a high prevalence rate. Ferroptosis, a type of iron-dependent cell death, is closely associated with GDM nosogenesis. The present study aimed to examine the potential role and mechanism of circHIPK3 in GDM. Placental tissues, plasma samples, and HTR-8/SVneo cells were used. A receiver operating characteristic curve was used to analyze the diagnostic value of circHIPK3 in GDM. Actinomycin D and RnaseR were added to identify circHIPK3 characteristics. The expression of circHIPK3, miR-1278, and DNA methyltransferase 1 (DNMT1) was assessed using a quantitative reverse transcriptase-PCR. Cell counting kit-8 and terminal deoxynucleotidyl transferase dUTP nick end labeling assays and specific kits were employed to assess cell viability, apoptosis, reactive oxygen species (ROS), malondialdehyde, iron, glutathione, and glutathione peroxidase 4 (GPX4) levels. The interaction between miR-1278 and circHIPK3 or DNMT1 was validated via luciferase reporter and RNA pull-down assays. circHIPK3 expression was found to be high in GDM placental tissues, plasma, and cells, with a high diagnostic value. In high glucose (HG)-induced HTR-8/SVneo cells, the inhibition of circHIPK3 provoked cell viability and mitigated cell apoptosis, ROS, and iron levels, but it was rescued through the downregulation of miR-1278. Mechanism experiments showed that circHIPK3 bound with miR-1278 targeting DNMT1 in GDM. The elevation in DNMT1 expression abolished the effects of miR-1278 overexpression on ferroptosis in HG-cultured HTR-8/SVneo cells. Overall, circHIPK3 might facilitate ferroptosis via miR-1278/DNMT1 to regulate GPX4 DNA methylation in HG-cultured HTR-8/SVneo cells. CircHIPK3 could be a therapeutic agent for GDM treatment.

Circ_0001052 promotes cardiac hypertrophy via elevating Hipk3.

Cardiac hypertrophy (CH) is a crucial risk factor for sudden death. Circular RNAs (circRNAs) exert significant effects in various biological and pathological processes. Circ_0001052 is sourced from Hipk3 (homeodomain-interacting protein kinase 3) and is reported to aggravate myocardial fibrosis. The purpose of the current study was to clarify the role and mechanism of circ-Hipk3 in CH. Transverse aortic constriction (TAC) was used to create an in vivo CH model, and angiotensin II (Ang II) therapy was used to create an in vitro CH model in cardiomyocytes (CMs). It was uncovered that circ_0001052 exerted pro-hypertrophic effects in Ang II-treated CMs. Next, the circular characteristics of circ_0001052 were verified, and we identified that circ_0001052 positively regulated Hipk3. Hipk3 exerted the same functions as circ_0001052 did. It is significant to note that circ_0001052 acted as the ceRNA of Hipk3 by sponging miR-148a-3p and miR-124-3p. According to rescue assays, miR-148a-3p and miR-124-3p partially reversed the effects of circ_0001052. Further, we testified that circ_0001052 recruited Srsf1 to stabilize Hipk3. Finally, rescue assays demonstrated that circ_0001052 promoted CH via up-regulation of Hipk3. In conclusion, our work unveiled that circ_0001052 promoted hypertrophic effects through elevating Hipk3 via sponging miR-148a-3p and miR-124-3p and recruiting Srsf1.

Circular RNA_HIPK3-Targeting miR-93-5p Regulates KLF9 Expression Level to Control Acute Kidney Injury.

Acute kidney injury (AKI) is a clinical syndrome caused by various reasons that results in the rapid decline of renal function in a short period of time. Severe AKI can lead to multiple organ dysfunction syndrome. Circular RNA HIPK3 (circHIPK3) derived from the HIPK3 gene is involved in multiple inflammatory processes. The present research was performed to explore the function of circHIPK3 on AKI. The AKI model was established by ischemia/reperfusion (I/R) in C57BL/6 mice or hypoxia/reoxygenation (H/R) in HK-2 cells. The function and mechanism of circHIPK3 on AKI were explored via biochemical index measurement; hematoxylin and eosin (HE) staining; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT); flow cytometry; enzyme-linked immunosorbent assay (ELISA); western blot; quantitative real-time polymerase chain reaction (RT-qPCR); detection of reactive oxygen species (ROS) and adenosine triphosphate (ATP); and luciferase reporter assays. circHIPK3 was upregulated in kidney tissues of I/R-induced mice and in H/R-treated HK-2 cells, while the microRNA- (miR-) 93-5p level was decreased in H/R-stimulated HK-2 cells. Furthermore, circHIPK3 silencing or miR-93-5p overexpression could reduce the level of proinflammatory factors and oxidative stress and recover the cell viability in H/R-stimulated HK-2 cells. Meanwhile, the luciferase assay showed that Krüppel-like transcription factor 9 (KLF9) was the downstream target of miR-93-5p. Forced expression of KLF9 blocked the function of miR-93-5p on H/R-treated HK-2 cells. Knockdown of circHIPK3 improved the renal function and reduced the apoptosis level in vivo. In conclusion, circHIPK3 knockdown alleviated oxidative stress and apoptosis and inhibited inflammation in AKI via miR-93-5p-mediated downregulation of the KLF9 signal pathway.

The novel circular RNA HIPK3 accelerates the proliferation and invasion of hepatocellular carcinoma cells by sponging the micro RNA-124 or micro RNA-506/pyruvate dehydrogenase kinase 2 axis

ABSTRACT Circular RNAs (circRNAs) have been confirmed to be associated with the progression of various cancers, including hepatocellular carcinoma (HCC). However, the role and mechanism of circHIPK3 in HCC are still unclear. To investigate its function, circHIPK3 expression was first determined by RT–qPCR in HCC tissues or cells. Functionally, cell proliferation and invasion were investigated by CCK-8, EdU, or Transwell assays. In terms of understanding the mechanism, the interaction of the circRNA HIPK3/micro RNA 124 (miRNA 124) or micro RNA 506 (miRNA506) /PDK2 regulatory loop was verified by dual-luciferase reporter gene assay. In addition, a xenograft tumor model was established to confirm the impact of circHIPK3 on the growth of HCC cells in vivo. We found that circHIPK3 was upregulated in HCC patients and associated with clinical characteristics, while miR-124 and miR-506 were downregulated in HCC patients. Additionally, we proved that knock down of circHIPK3 remarkably suppressed the proliferation and invasion of HCC cells. Mechanistically, circHIPK3 directly bound to miR-124 or miR-506 and inhibited their expression, and PDK2 was a target gene of miR-124 or miR-506. Moreover, circHIPK3 overexpression reversed the inhibitory effect of miR-124 or miR-506 on HCC progression. miR-124 or miR-506 could also suppress tumorigenesis of HCC cells by PDK2. Furthermore, in vivo evidence confirmed that knock down of circHIPK3 inhibited tumor formation. We suggest that circHIPK3 can accelerate the proliferation and invasion of HCC cells by sponging miR-124 or miR-506 to upregulate PDK2, which is the underlying mechanism of circHIPK3-induced HCC progression.

CircHIPK3 Alleviates High Glucose Toxicity to Human Renal Tubular Epithelial HK-2 Cells Through Regulation of miR-326/miR-487a-3p/SIRT1.

BackgroundThe intervention of circular RNA HIPK3 (circHIPK3) in diabetes has drawn increasing attention in recent years. However, the underlying mechanism of circHIPK3 in diabetic nephropathy (DN) has not been fully elucidated. Thus, the current study aims to investigate the role of circHIPK3 in high glucose (HG)-induced toxicity to human renal tubular epithelial HK-2 cells.MethodsThe expression of circHIPK3 in HK-2 cells induced by HG was determined by qRT-PCR and Western blot. The regulatory effects of circHIPK3 and miR-326/miR-487a-3p on cells proliferative and apoptosis were evaluated by CCK-8 and flow cytometry. Dual-luciferase reporter assay was applied to predict the target genes of miR-326 or miR-487a-3p.ResultsExpression level of circHIPK3 in HK-2 cells was remarkably decreased after the treatment of HG. The overexpression of circHIPK3 effectively reversed the HG-induced HK-2 cell proliferation inhibition and apoptosis. Furthermore, SIRT1 was confirmed to be the target gene of miR-326 and miR-487a-3p, which were showed to be the downstream genes of circHIPK3. The silencing of miR-326 or miR-487a-3p was also proved to induce proliferation and reduce apoptosis in HG-induced HK-2 cells.ConclusionOur data suggest that overexpression of circHIPK3 can attenuate the proliferation inhibition of HK-2 induced by HG and inhibit apoptosis through sponging miR-326 or miR-487a-3p to regulate SIRT1.

Circular RNA circHIPK3 as a novel circRNA regulator of autophagy and endothelial cell dysfunction in atherosclerosis.

The aim of the study was to explore the role and mechanism of circHIPK3 in atherosclerosis. AS model was constructed in vivo and in vitro for high fat-fed and ox-LDL treatment. RT-PCR was used to assess the level of circHIPK3. The autophagy level of HUVECs was detected by Western blot, transmission electron microscopy, and LC3II fluorescence intensity. HUVECs lipid accumulation was assessed by oil red staining. Luciferase assay was performed to verify the relationship of circRNA and miRNA, miRNA, and target gene. The expression of circHIPK3 was downregulated in HFD mice, and ox-LDL treated HUVECs. The level of autophagy was decreased in AS, which was reversed by overexpression of circHIPK3. Meanwhile, forced expression of circHIPK3 would reduce the accumulation of lipid in HUVECs. CircHIPK3 could inhibit lipid content in ox-LD-treated HUVECs via activating autophagy. This progression mechanism may target the miR-190b/ATG7 signal pathway, which indicates a suitable role in the pathogenesis of atherosclerosis.

CircHIPK3 regulates melanoma cell behaviors by binding with miR-215–5p to upregulate YY1

ObjectTo investigate the role of circHIPK3 in melanoma. MethodsBioinformatics analysis and experiments including RT-qPCR, Pearson's correlation analysis, luciferase reporter, Western blot, and RIP assays were applied to explore the function and mechanism of circHIPK3 in melanoma. ResultsCircHIPK3 expression was strikingly upregulated while miR-215–5p was downregulated in melanoma tissues and cell lines. Pearson's correlation analysis unveiled circHIPK3 expression was positively correlated with Ki-67 (a marker of proliferation), which implied that circHIPK3 may play a vital role in the progression of melanoma. In mechanism, luciferase reporter and RIP assays validated that circHIPK3 was able to bind with miR-215–5p. Moreover, we confirmed that overexpression of circHIPK3 could facilitate cell proliferation and depress cell apoptosis in melanoma while overexpression of miR-215–5p exerted opposite effects. Besides, our findings indicated that miR-215–5p overexpression significantly reversed the circHIPK3 overexpressing-mediated promotive effect on cell proliferation and inhibitory effect on cell apoptosis. Furthermore, we found that miR-215–5p could directly target YY1. Upregulation of YY1 could notably offset the inhibitory effect of circHIPK3 downregulation on cell proliferation and the promotive effect on cell apoptosis. ConclusionOur study corroborated that circHIPK3 regulated melanoma cell behaviors via the miR-215–5p/YY1 axis, which might provide a novel insight for the treatment of melanoma patients.

RETRACTION: The circular RNA HIPK3 reduces hydrogen peroxide-induced injury of PC-12 cells by upregulation of miR-455.

Craniocerebral trauma is a serious disease accompanied with mechanical damage, oxidative damage, and neuronal apoptosis. Circular RNA HIPK3 (circHIPK3), an abundant circRNA, exerts the function in promoting cell growth. Therefore, we aimed to explore the biological roles and mechanism of circHIPK3 on cell damage in craniocerebral trauma. PC-12 cells were cotransfected with PLCDH-cirHIPK3 and microRNA-455 (miR-455) inhibitor and then were treated with H2 O2 for 24 hours. Cell viability, apoptosis and mean fluorescence intensity (MFI) were tested through trypan blue staining, flow cytometry and reactive oxygen species assay, respectively. Expression of circHIPK3 and miR-455 were tested through quantitative reverse transcription polymerase chain reaction. Expression of pro-PARP, cleaved-PARP, pro-caspase-3, cleaved-caspase-3, pro-caspase-9, cleaved-caspase-9, β-actin, beclin-1, p62, light chain 3-I (LC3-I), LC3-II, nuclear factor erythroid 2-related factor 2 (Nrf2), t-extracellular-regulated kinase (t-ERK) and p-ERK were tested via Western blot experiments. The 100 μM H2 O2 -induced cell damage presented as reducing cell viability, increasing apoptosis, autophagy, and MFI. Besides, H2 O2 could downregulate circHIPK3. However, overexpression of circHIPK3 attenuated cell damage caused by H2 O2 that it increased cell viability, decreased apoptosis, autophagy and MFI through upregulating miR-455. Additionally, circHIPK3 overexpression promoted the activation of Nrf2 and ERK signal pathways by upregulating miR-455. Our study demonstrated that circHIPK3 attenuated H2 O2 -induced cell damage and activated Nrf2 and ERK signal pathways by upregulation of miR-455.

Contributors

To investigate the role of circHIPK3 in melanoma.Bioinformatics analysis and experiments including RT-qPCR, Pearson's correlation analysis, luciferase reporter, Western blot, and RIP assays were applied to explore the function and mechanism of circHIPK3 in melanoma.CircHIPK3 expression was strikingly upregulated while miR-215–5p was downregulated in melanoma tissues and cell lines. Pearson's correlation analysis unveiled circHIPK3 expression was positively correlated with Ki-67 (a marker of proliferation), which implied that circHIPK3 may play a vital role in the progression of melanoma. In mechanism, luciferase reporter and RIP assays validated that circHIPK3 was able to bind with miR-215–5p. Moreover, we confirmed that overexpression of circHIPK3 could facilitate cell proliferation and depress cell apoptosis in melanoma while overexpression of miR-215–5p exerted opposite effects. Besides, our findings indicated that miR-215–5p overexpression significantly reversed the circHIPK3 overexpressing-mediated promotive effect on cell proliferation and inhibitory effect on cell apoptosis. Furthermore, we found that miR-215–5p could directly target YY1. Upregulation of YY1 could notably offset the inhibitory effect of circHIPK3 downregulation on cell proliferation and the promotive effect on cell apoptosis.Our study corroborated that circHIPK3 regulated melanoma cell behaviors via the miR-215–5p/YY1 axis, which might provide a novel insight for the treatment of melanoma patients.