Hsa-miR-494-3p attenuates gene HtrA3 transcription to increase inflammatory response in hypoxia/reoxygenation HK2 Cells

AbstractThe occurrence of cardiac surgery-associated acute kidney injury (CSA-AKI) increases hospital stay and mortality. MicroRNAs has a crucial role in AKI. This objective of the current study is to explore the function of hsa-miR-494-3p in inflammatory response in human kidney tubular epithelial (HK2) cells with hypoxia/reoxygenation. According to KDIGO standard, patients after cardiac surgery with cardiopulmonary bypass were divided into two groups: AKI (n = 10) and non-AKI patients (n = 8). HK2 were raised in the normal and hypoxia/reoxygenation circumstances and mainly treated by overexpression ofmiR-494-3p and HtrA3. The relationship between miR-494-3p and HtrA3 was determined by dual-luciferase reporter assay. Our result showed that Hsa-miR-494-3p was elevated in the serum of patients with CSA-AKI, and also induced in hypoxic reoxygenated HK2 cells. Hsa-miR-494-3p also increased a hypoxia-reoxygenation induced inflammatory response in HK2 cells. Moreover, as a target gene of miR-494-3p, overexpression of HtrA3 downregulated the hypoxia-reoxygenation induced inflammatory response in HK2 cells. Overexpression of hsa-miR-494-3p-induced inflammatory response was inhibited by overexpression of HtrA3. Collectively, we identified that hsa-miR-494-3p, a miRNA induced in both circulation of AKI patients and hypoxia-reoxygenation-treated HK2 cells, enhanced renal inflammation by targeting HtrA3, which may suggest a possible role as a new therapeutic target for CSA-AKI.

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Upregulation of miR-150-5p alleviates LPS-induced inflammatory response and apoptosis of RAW264.7 macrophages by targeting Notch1

Open Life Sciences ◽

10.1515/biol-2020-0058 ◽

2020 ◽

Vol 15 (1) ◽

pp. 544-552

Author(s):

Xiaoyan Deng ◽

Zhixing Lin ◽

Chao Zuo ◽

Yanjie Fu

Keyword(s):

Inflammatory Response ◽

Underlying Mechanism ◽

Notch Receptor ◽

Raw264.7 Cells ◽

Luciferase Reporter ◽

Raw264.7 Macrophages ◽

Factor Α ◽

Anti Inflammation ◽

Necrosis Factor ◽

Dual Luciferase Reporter Assay

AbstractCirculating miR-150-5p has been identified as a prognostic marker in patients with critical illness and sepsis. Herein, we aimed to further explore the role and underlying mechanism of miR-150-5p in sepsis. Quantitative real-time-PCR assay was performed to detect the expression of miR-150-5p upon stimulation with lipopolysaccharide (LPS) in RAW264.7 cells. The levels of tumor necrosis factor-α, interleukin (IL)-6 and IL-1β were measured by ELISA assay. Cell apoptosis was determined using flow cytometry. Western blot was used to assess notch receptor 1 (Notch1) expression in LPS-induced RAW264.7 cells. Dual-luciferase reporter assay was employed to validate the target of miR-150-5p. Our data showed that miR-150-5p was downregulated and Notch1 was upregulated in LPS-stimulated RAW264.7 cells. miR-150-5p overexpression or Notch1 silencing alleviated LPS-induced inflammatory response and apoptosis in RAW264.7 cells. Moreover, Notch1 was a direct target of miR-150-5p. Notch1 abated miR-150-5p-mediated anti-inflammation and anti-apoptosis in LPS-induced RAW264.7 cells. miR-150-5p alleviated LPS-induced inflammatory response and apoptosis at least partly by targeting Notch1 in RAW264.7 cells, highlighting miR-150-5p as a target in the development of anti-inflammation and anti-apoptosis drugs for sepsis treatment.

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MiR-196b Promotes the Invasion and Migration of Lung Adenocarcinoma Cells by Targeting AQP4

Technology in Cancer Research & Treatment ◽

10.1177/1533033820985868 ◽

2021 ◽

Vol 20 ◽

pp. 153303382098586

Author(s):

Xuhui Wu ◽

Gongzhi Wu ◽

Huaizhong Zhang ◽

Xuyang Peng ◽

Bin Huang ◽

...

Keyword(s):

Lung Adenocarcinoma ◽

Cell Invasion ◽

Target Gene ◽

Luciferase Reporter Assay ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Reporter Assay ◽

Invasion And Migration ◽

And Migration ◽

Dual Luciferase Reporter Assay

Objective: We aimed to investigate the mechanism of the regulatory axis of miR-196b/AQP4 underlying the invasion and migration of lung adenocarcinoma (LUAD) cells. Methods: LUAD miRNA and mRNA expression profiles were downloaded from TCGA database and then differential analysis was used to identify the target miRNA. Target gene for the miRNA was obtained via prediction using 3 bioinformatics databases and intersection with the differentially expressed mRNAs searched from TCGA-LUAD. Then, qRT-PCR and western blot were used to validate the expression of miR-196b and AQP4. Dual-luciferase reporter assay was performed to confirm the targeting relationship between miR-196b and AQP4. Transwell assay was used to investigate the migration and invasion of LUAD cells. Results: MiR-196b was screened out by differential and survival analyses, and the downstream target gene AQP4 was identified. In LUAD, miR-196b was highly expressed while AQP4 was poorly expressed. Besides, overexpression of miR-196b promoted cell invasion and migration, while overexpression of AQP4 had negative effects. Moreover, the results of the dual-luciferase reporter assay suggested that AQP4 was a direct target of miR-196b. In addition, we also found that overexpressing AQP4 could suppress the promotive effect of miR-196b on cancer cell invasion and migration. Conclusion: MiR-196b promotes the invasion and migration of LUAD cells by down-regulating AQP4, which helps us find new molecular targeted therapies for LUAD.

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MiR-940 promotes malignant progression of breast cancer by regulating FOXO3

Bioscience Reports ◽

10.1042/bsr20201337 ◽

2020 ◽

Vol 40 (9) ◽

Author(s):

Huayao Zhang ◽

Jingwen Peng ◽

Jianguo Lai ◽

Haiping Liu ◽

Zhiyuan Zhang ◽

...

Keyword(s):

Breast Cancer ◽

Colony Formation ◽

Target Gene ◽

Luciferase Reporter Assay ◽

Luciferase Reporter ◽

Reporter Assay ◽

Poor Survival ◽

High Level ◽

The Relationship ◽

Proliferation And Invasion

Abstract Breast cancer (BC) is a common cancer with poor survival. The present study aimed to explore the effect of miR-940 on the process of BC cells and its target gene FOXO3. The expression of miR-940 was assessed in BC tissues and cells using qRT-PCR. Furthermore, the correlation between miR-940 and prognosis of BC patients from the TCGA database was analyzed. CCK8 assays and colony formation assays were used to explore the effect of miR-940 on BC cell proliferation. The invasion abilities were detected by transwell assays. Luciferase reporter assay was performed to scrutinize the relationship between miR-940 and FOXO3. Finally, rescue experiments were performed through FOXO3 down-regulation and miR-940 inhibitors by using CCK8 assays, colony formation assays and transwell assays. miR-940 was significantly up-regulated in BC cells and tissues. In addition, the high level of miR-940 correlated with poor survival of BC patients (P=0.023). CCK8 assays, colony formation assays and transwell assays indicated that miR-940 promoted the proliferation and invasion abilities of BC cells. The luciferase reporter assay suggested that miR-940 directly targeted FOXO3. Moreover, we found that the effect of si-FOXO3 was rescued by miR-940 inhibitors in BC cells. miR-940 may promote the proliferation and invasion abilities of BC cells by targeting FOXO3. Our study suggested that miR-940 could be a novel molecular target for therapies against BC.

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MiR-22-3p suppresses sepsis-induced acute kidney injury by targeting PTEN

Bioscience Reports ◽

10.1042/bsr20200527 ◽

2020 ◽

Vol 40 (6) ◽

Author(s):

Xudong Wang ◽

Yali Wang ◽

Mingjian Kong ◽

Jianping Yang

Keyword(s):

Acute Kidney Injury ◽

Inflammatory Response ◽

Periodic Acid ◽

Kidney Injury ◽

Luciferase Reporter ◽

Tunel Staining ◽

Tnf Α

Abstract Background: Septic acute kidney injury is considered as a severe and frequent complication that occurs during sepsis. The present study was performed to understand the role of miR-22-3p and its underlying mechanism in sepsis-induced acute kidney injury. Methods: Rats were injected with adenovirus carrying miR-22-3p or miR-NC in the caudal vein before cecal ligation. Meanwhile, HK-2 cells were transfected with the above adenovirus following LPS stimulation. We measured the markers of renal injury (blood urea nitrogen (BUN), serum creatinine (SCR)). Histological changes in kidney tissues were examined by hematoxylin and eosin (H&E), Masson staining, periodic acid Schiff staining and TUNEL staining. The levels of IL-1β, IL-6, TNF-α and NO were determined by ELISA assay. Using TargetScan prediction and luciferase reporter assay, we predicted and validated the association between PTEN and miR-22-3p. Results: Our data showed that miR-22-3p was significantly down-regulated in a rat model of sepsis-induced acute kidney injury, in vivo and LPS-induced sepsis model in HK-2 cells, in vitro. Overexpression of miR-22-3p remarkably suppressed the inflammatory response and apoptosis via down-regulating HMGB1, p-p65, TLR4 and pro-inflammatory factors (IL-1β, IL-6, TNF-α and NO), both in vivo and in vitro. Moreover, PTEN was identified as a target of miR-22-3p. Furthermore, PTEN knockdown augmented, while overexpression reversed the suppressive role of miR-22-3p in LPS-induced inflammatory response. Conclusions: Our results showed that miR-22-3p induced protective role in sepsis-induced acute kidney injury may rely on the repression of PTEN.

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MicroRNA-384 Inhibits the Progression of Papillary Thyroid Cancer by Targeting PRKACB

BioMed Research International ◽

10.1155/2020/4983420 ◽

2020 ◽

Vol 2020 ◽

pp. 1-11 ◽

Cited By ~ 2

Author(s):

Yongxia Wang ◽

Beixi Wang ◽

Hong Zhou ◽

Xiangnan Zhang ◽

Xinlai Qian ◽

...

Keyword(s):

Thyroid Cancer ◽

Papillary Thyroid Cancer ◽

Target Gene ◽

Underlying Mechanism ◽

Soft Agar ◽

Luciferase Reporter ◽

Papillary Thyroid ◽

Soft Agar Assay ◽

Spearman’S Correlation ◽

Dual Luciferase Reporter Assay

Background. Growing evidence shows that dysregulation of miRNAs plays a significant role in papillary thyroid cancer (PTC) tumorigenesis and development. The abnormal expression of miR-384 has been acknowledged in the proliferation or metastasis of some cancers. However, the function and the underlying mechanism of miR-384 in PTC progression remain largely unknown. Methods. Real-time PCR was conducted to detect miR-384 expression in 58 cases of PTC and their adjacent noncancerous tissues. MTT, soft agar assay Transwell assay, and wound-healing assay were carried out to explore the biological function of miR-384 in PTC cell lines of BCPAP and K1. Bioinformatics analysis, dual-luciferase reporter assay, western blot, and functional complementation analysis were conducted to explore the target gene of miR-384. Moreover, Spearman’s correlation analysis was conducted to reveal the correlation between miR-384 and PRKACB mRNA in PTC. Results. The expression of miR-384 decreased obviously in PTC, especially in the tumors with lymph node metastasis or larger tumor size. The ectopic upregulation of miR-384 significantly suppressed PTC progression, and the inhibition of miR-384 had the opposite effects. Moreover, PRKACB gene was confirmed as the target of miR-384. Conclusion. The study suggests that miR-384 serves as a tumor suppressor in PTC progression by directly targeting the 3′-UTR of PRKACB gene.

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Regulation of Connective Tissue Growth Factor Expression by miR-133b for the Treatment of Renal Interstitial Fibrosis in Aged Mice with Unilateral Ureteral Obstruction

10.21203/rs.3.rs-55357/v2 ◽

2020 ◽

Author(s):

Dan Cao ◽

Yuan Wang ◽

Yingjie Zhang ◽

Yinping Zhang ◽

Qi Huang ◽

...

Keyword(s):

Growth Factor ◽

Target Genes ◽

Interstitial Fibrosis ◽

Tissue Growth ◽

Luciferase Reporter ◽

Renal Interstitial Fibrosis ◽

Mrna And Protein Expression ◽

Hk2 Cells ◽

Tgf Β1 ◽

Dual Luciferase Reporter Assay

Abstract Introduction: Renal interstitial fibrosis, an important pathological feature of kidney aging and chronic renal failure, is regulated by mesenchymal stem cells (MSCs). We have previously demonstrated low expression of miR-133b in MSC-derived extracellular vesicles (MSC-EVs) in aged rats. However, miR-133b can mediate the inhibition of epithelial-mesenchymal transition (EMT) of renal tubules induced by transforming growth factor-β1 (TGF-β1). We investigated the effect of miR-133b for the treatment of geriatric renal interstitial fibrosis and evaluated its target genes.Methods: We performed real-time polymerase chain reaction to detect miR-133b expression induced during EMT of HK2 cells by TGF-β1 at different concentrations (0, 6, 8, and 10 ng/mL) and at different time points (0, 24, 48, and 72 h). The target genes of miR-133b were validated using the dual-luciferase reporter assay. In vitro experiments were performed to evaluate mRNA and protein expression of miR-133b targets, E-cadherin, α-smooth muscle actin (SMA), fibronectin, and collagen 3A1 (Col3A1), in HK2 cells transfected with miR-133b under TGF-β1 stimulation. A 24-month-old unilateral ureteral obstruction (UUO) mouse model was established and injected with transfection reagent and miR-133b into the caudal vein. The target gene of miR-133b and other parameters mentioned above such as mRNA and protein expression levels and renal interstitial fibrosis were detected at 7 and 14 days.Results: miR-133b expression gradually decreased with an increase in TGF-β1 concentration and treatment time, and the miR-133b mimic downregulated connective tissue growth factor (CTGF) expression. The dual-luciferase reporter assay confirmed CTGF as a direct target of miR-133b. Transfection of the miR-133b mimic inhibited TGF-β1-induced EMT of HK2 cells; this effect was reversed by CTGF overexpression. miRNA-133b expression significantly increased (approximately 70-100 times) in mice kidney tissues after injection of the miRNA-133b overexpression complex, which significantly alleviated renal interstitial fibrosis in mice with UUO.Conclusion: miR-133b exerted targeted inhibitory effects on CTGF expression, which consequently reduced TGF-β1-induced EMT of HK2 cells and renal interstitial fibrosis in aged mice with UUO.

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LncRNA NKILA Promotes Epithelial-Mesenchymal Transition of Liver Cancer Cells by Targeting miR-485-5p

Journal of Oncology ◽

10.1155/2021/1281031 ◽

2021 ◽

Vol 2021 ◽

pp. 1-9

Author(s):

Yuxu Wang ◽

Chao Li ◽

Yuyi Shi ◽

Jing Kuai

Keyword(s):

Liver Cancer ◽

Normal Liver ◽

Liver Cells ◽

Luciferase Reporter Assay ◽

Biological Behavior ◽

Luciferase Reporter ◽

Reporter Assay ◽

Mesenchymal Transition ◽

The Relationship ◽

Dual Luciferase Reporter Assay

Objective. Liver cancer (LC), one of the familiar malignancies, has a very high morbidity all over the world. The onset of the disease is hidden, and the patients usually do not express any special symptoms. Most of them will have been developed to the middle and later stage when they are diagnosed. This is one of the main reasons why the prognosis of LC is extremely pessimistic all the year round. Recently, researchers have focused mainly on molecular studies, among which LncRNA is a hot spot. This research aims to explore the biological behaviors of LncRNA NKILA and miR-485-5p in LC cells and verify the relationship between them, thereby providing a new theoretical basis for future prevention and treatment. Methods. Ninety-four early LC patients admitted to our hospital from January 2015 to January 2017 were regarded as the research objects. In addition, human LC cells SMMC-7721, HepG2, and normal liver cells HL-7702 were purchased. The LncRNA NKILA and miR-485-5p level in cancer and adjacent tissues, LC, and normal liver cells of patients was tested by PCR. Patients were followed up for 3 years. Then, LncRNA NKILA and miR-485-5p’s effects on prognosis and cell biological behavior were analyzed. At last, the relationship between LncRNA NKILA and miR-485-5p was assessed by a dual-luciferase reporter assay. Results. The LncRNA NKILA expression was high in LC tissues and cells ( P < 0.050 ), while miR-485-5p was low compared with the normal adjacent tissues ( P < 0.050 ). Prognostic follow-up manifested that high LncRNA NKILA or low miR-485-5p could predict the poor prognosis and high mortality risk of the patients ( P < 0.050 ). LC cells with downregulated LncRNA NKILA documented inhibited proliferation, invasion, and EMT, while the apoptosis level of the cells increased ( P < 0.050 ). The proliferation, invasion, and EMT were inhibited by miR-485-5p increase, while the apoptosis of the cells decreased after upregulating miR-485-5p ( P < 0.050 ). Online websites predicted that LncRNA NKILA had a binding site with miR-485-5p, and dual-luciferase reporter assay confirmed that LncRNA NKILA could directly target with miR-485-5p ( P < 0.050 ). The miR-485-5p in LC cells increased after LncRNA NKILA was silenced ( P < 0.050 ). The rescue experiment documented that LncRNA NKILA inhibition on LC cells was reversed by inhibiting miR-485-5p ( P < 0.050 ). Conclusion. The LncRNA NKILA with high expression advances LC cell proliferation, invasion, and EMT by targeting miR-485-5p.

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Abstract 12622: Pro-inflammatory Urinary CXCL10 and VCAM Are Elevated After Infant Cardiac Surgery-associated Acute Kidney Injury (AKI)

Circulation ◽

10.1161/circ.142.suppl_3.12622 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

Daniella Levy- Erez ◽

Lokesh Shah ◽

Kathryn D Howarth ◽

Sherin Meloni ◽

Benjamin Laskin ◽

...

Keyword(s):

Acute Kidney Injury ◽

Cardiac Surgery ◽

Therapeutic Targets ◽

Kidney Injury ◽

Urine Samples ◽

Inflammatory Biomarkers ◽

Rank Test ◽

Immune Biomarkers ◽

The Relationship ◽

Complex Surgery

Introduction: Acute kidney injury (AKI) occurs frequently after infant cardiac surgery and is associated with mortality. AKI mechanisms are unknown, limiting therapeutic targets. Emerging data implicates unregulated immune activation and AKI development. Hypothesis: Urinary immune biomarkers will be elevated in the urine of infants developing AKI. Methods: One hundred and twenty six infants were enrolled (median age 87 days, 74% male). Urine samples were collected pre-bypass and 6, 24, 48, and 72 hours after surgery. Urine samples underwent multiplex Luminex assays to detect six immune biomarkers: VCAM, CXCL10, MCP, IL-18, TWEAK, and C5-C9. Greater than 150% increase in serum creatinine defined AKI. The Kruskal-Wallis rank test determined the relationship between AKI and biomarker levels. Results: Thirty-five infants (27%) developed AKI. AKI subjects were younger (median 6 days (4-98) vs no AKI 107 days (7-164), p<0.01). The AKI group had more complex surgery (STAT 4-5) (60% AKI vs 19% no AKI, p<0.01). Bypass time was longer among the AKI group ((81 min vs 69 min (p<0.01)).AKI infants had higher urinary CXCL10 levels at 24 hours (14.3 pg/ml vs 5.3 pg/ml p=0.04), 48 hours (3.4 pg/ml vs 0.75 pg/ml p=0.01), and 72 hours (1.15 pg/ml vs 0.22 pg/ml p=0.05) (Figure 1). Six-hour VCAM levels were higher among AKI infants (Median 491 pg/ml vs 0 pg/ml p=0.04). Other biomarkers showed no significant differences between groups. (Table1). Conclusions: Urinary CXCL10 and VCAM are promising pro-inflammatory biomarkers for early AKI detection and may indicate eventual AKI therapeutic targets.

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Inhibition of TRAF1 protects renal tubular epithelial cells against hypoxia/reoxygenation injury

10.31083/jomh.2021.060 ◽

2021 ◽

Keyword(s):

Cell Viability ◽

Western Blotting ◽

Mtt Assay ◽

Cell Apoptosis ◽

Mapk Pathway ◽

Kidney Injury ◽

Control Group ◽

Injury Model ◽

Hk2 Cells ◽

Hypoxia Reoxygenation

Background and objective: This study aimed to explore the expression of TRAF1 in vitro kidney injury model, and the function mechanism of TRAF1 in the model growth and apoptosis. Methods: After transfecting HK2 cells with short hair RNA (shRNA), shTRAF1 gene silencing model was established. The cells were divided into shRNA group and shNC group. For kidney injury model, we used hypoxia/reoxygenation to establish H/R cell lines. MTT assay was used to determine cell viability. PI/FITC staining was used to determine cell apoptosis. The genes expressions were determined by RT-qPCR and western blotting, respectively. The concentration of MDA, SOD, iNOS and LDH was determined by ELISA. Results: The results of RT-qPCR and western blotting assay revealed that TRAF1 upregulated expression in AKI model cells. The results of MTT assay revealed that shRNA group exhibited signiﬁcantly higher cell viability and lower cell apoptosis compared with the control group in H/R HK2 cells. In addition, TRAF1 downregulated expression inhibits oxidative stress response in H/R treated HK2 cell. Mechanically, TRAF1 deﬁciency protects HK2 cell via inhibiting p38-MAPK pathway. Conclusions: Our study suggests that TRAF1 could be a target in kidney injury treatment.

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