Placenta-derived exosomal miR-135a-5p promotes gestational diabetes mellitus pathogenesis by activating PI3K/AKT signalling pathway via SIRT1

Most people are aware of gestational diabetes mellitus (GDM), a dangerous pregnancy complication in which pregnant women who have never been diagnosed with diabetes develop chronic hyperglycaemia. Exosomal microRNA (miRNA) dysregulation has been shown to be a key player in the pathophysiology of GDM. In this study, we looked into how placental exosomes and their miRNAs may contribute to GDM. When compared to exosomes from healthy pregnant women, it was discovered that miR-135a-5p was elevated in placenta-derived exosomes that were isolated from the maternal peripheral plasma of GDM women. Additionally, we discovered that miR-135a-5p encouraged HTR-8/SVneo cell growth, invasion and migration. Further research revealed that miR-135a-5p activates HTR-8/SVneo cells' proliferation, invasion and migration by promoting PI3K/AKT pathway activity via Sirtuin 1 (SIRT1). The transfer of exosomal miR-135a-5p generated from the placenta could be viewed as a promising agent for targeting genes and pertinent pathways involved in GDM, according to our findings.     

下调lncRNA MCF2L-AS1靶向miR-33b-5p抑制胃癌细胞增殖、迁移、侵袭并促进凋亡

摘要 目的：探讨lncRNA MCF2L-AS1对胃癌细胞恶性生物学行为的影响及分子机制。方法：选取45例胃癌患者的癌组织及癌旁正常组织,或培养胃黏膜上皮细胞GES-1、胃癌细胞HGC-27,采用RT-qPCR检测MCF2L-AS1和miR-33b-5p的表达水平。采用双荧光素酶报告实验检测MCF2L-AS1和miR-33b-5p的靶向关系。将HGC-27细胞分为si-NC组、si-MCF2L-AS1组、mimic NC组、miR-33b-5p mimic组、si-MCF2L-AS1+inhibitor NC组、si-MCF2L-AS1+miR-33b-5p inhibitor组,分别转染si-NC、si-MCF2L-AS1、mimic NC、miR-33b-5p mimic或共转染si-MCF2L-AS1+inhibitor NC、si-MCF2L-AS1+miR-33b-5p inhibitor。采用MTT实验检测细胞增殖情况,流式细胞术检测细胞凋亡率,克隆形成实验检测细胞克隆形成数,Transwell实验检测迁移和侵袭细胞数。结果：与癌旁正常组织或GES-1细胞相比,胃癌组织或HGC-27细胞中MCF2L-AS1表达水平升高、miR-33b-5p表达水平降低,差异均有统计学意义（P<0.05）。MCF2L-AS1可靶向调控miR-33b-5p。下调MCF2L-AS1或过表达miR-33b-5p,miR-33b-5p表达水平升高,HGC-27细胞凋亡率升高,但细胞增殖、克隆形成数、迁移和侵袭数均减少,差异均有统计学意义（P<0.05）。抑制miR-33b-5p可减弱下调MCF2L-AS1对HGC-27细胞的生物学作用。结论：下调MCF2L-AS1通过上调miR-33b-5p抑制胃癌细胞增殖、迁移、侵袭并促进凋亡;MCF2L-AS1通过靶向调控miR-33b-5p表达进而参与胃癌细胞的恶性生物学行为。     

Downregulation of microRNA-92b-3p suppresses proliferation,migration, and invasion of gastric cancer SGC-7901 cells by targeting Homeobox D10

To investigate the effect and mechanism of microRNA-92b-3p (miR-92b-3p) targeting Homeobox D10 (HOXD10) on proliferation, migration, and invasion of gastric cancer, we detected t he expression of miR-92b-3p and HOXD10 in SGC-7901 cells. The effects of miR-92b-3p or HOXD10 on proliferation, migration, invasion, and matrix metalloproteinase (MMP)-2/9 expression in SGC-7901 cells were measured by the Cell Counting Kit-8 assay, Transwell assay, and Western blot, respectively. The results showed that miR-92b-3p expression was increased, and HOXD10 expression was decreased in SGC-7901 cells, compared with human normal gastric epithelial cells GES-1. Functional experiments demonstrated that cell proliferation, migration, invasion, and expression of MMP-2/9 in SGC-7901 cells were significantly inhibited by miR-92b-3p silencing and HOXD10 overexpression. Moreover, HOXD10 was a potential target gene of miR-92b-3p as evidenced by the TargetScan software and double luciferase reporter assay. In the rescue experiment, knockdown of HOXD10, accompanied by higher expression of MMP-2/9, could significantly eliminate the inhibitory effects of miR-92b-3p silencing on cell proliferation, migration, and invasion. In conclusion, miR-92b-3p is highly expressed in gastric cancer SGC-7901 cells, and interfering with its expression might inhibit SGC-7901 cell proliferation, migration, and invasion via downregulating MMP-2/9 expression and targeting HOXD10.     

miR-34a attenuates myocardial fibrosis in diabetic cardiomyopathy mice via targeting Pin-1

Diabetic cardiomyopathy (DCM) is characterized by myocardial hypertrophy and fibrosis. This study aimed to investigate the effects of microRNA (miR)-34a on myocardial fibrosis in DCM and its potential mechanism of targeting Pin-1 signaling. Vimentin and Pin-1 proteins in mouse cardiac tissues were detected by immunohistochemical staining. Locked nucleic acid in situ hybridization was used to measure miR-34a expression in cardiac tissues. Primary mouse cardiac fibroblasts (CFs) were transfected with a mimics control/miR-34a mimics or Pin-1 plasmid and cultured in high-glucose (HG) Dulbecco's modified Eagle's medium. The miR-34a levels were measured by quantitative polymerase chain reaction. The apoptosis and viability of transfected cells were detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling and Cell Counting Kit-8 assays respectively. A cell migration experiment and dual-luciferase reporter assay were also performed. The body weight and fasting blood glucose of DCM mice were significantly higher than those in the control (CTL) group. In addition, DCM mice had decreased serum insulin levels and impaired cardiac function. The number of CFs in the DCM group was higher than in the CTL group and Pin-1 expression was upregulated. The expression level of miR-34a in the cardiac tissue of mice in the DCM group was obviously downregulated compared with the CTL group. The HG stimulation of CFs for 48 h significantly downregulated the expression level of miR-34a and was associated with increased Type I collagen expression, cell viability, and migration and decreased apoptosis. However, these effects could be reversed by overexpressing miR-34a in HG-induced CFs. Furthermore, we found that Pin-1 was a direct target of miR-34a. Our results suggest that miR-34a can attenuate myocardial fibrosis in DCM by reducing Type I collagen production, cell viability, and migration and increasing the apoptosis of CFs by targeting Pin-1 signaling.     

miR-216b-5p regulates proliferation and apoptosis of ox-LDL-stimulated VSMCs and HUVECs via IGF2

Atherosclerosis (AS) is one of the principal causes of cardiovascular disorder. Reportedly, vascular smooth muscle cells (VSMCs) and human umbilical vein endothelial cells (HUVECs) play key roles in AS development, and microRNAs (miRNAs) regulate their functions. The function of miR-216b-5p in AS remains unknown. Human VSMCs and human HUVECs were treated with ox-LDL to establish the in vitro model of AS. MiR-216b-5p and IGF2 expressions in VSMCs and HUVECs were probed by qRT-PCR and western blot. The viability, cell cycle progression, and apoptosis of VSMCs and HUVECs were evaluated by Cell Counting Kit-8 (CCK-8), 5-ethynyl-2′-deoxyuridine, and flow cytometry assays, respectively. The binding sites between IGF2 3′UTR and miR-216b-5p were validated by dual-luciferase reporter assay. miR-216b-5p expression was declined in ox-LDL-induced VSMCs and HUVECs. In VSMCs, miR-216b-5p overexpression inhibited excessive proliferation and induced apoptosis. MiR-216b-5p could markedly restrain the viabiblity of VSMCs induced by ox-LDL and enhanced the viability of HUVECs. Additionally, IGF2 was confirmed as the direct target of miR-216b-5p and transfection of IGF2 overexpression plasmids rescued the effects of miR-216b-5p on VSMCs and HUVECs. miR-216b-5p alleviates the dysfunction of VSMCs and HUVECs caused by ox-LDL via repressing IGF2, and exerts protective functions to block the development of AS.     

MiR-29b-3p aggravates cardiac hypoxia/reoxygenation injury via targeting PTX3

Our current research aimed to decipher the role and underlying mechanism with regard to miR-29b-3p involving in myocardial ischemia/reperfusion (I/R) injury. In the present study, cardiomyocyte H9c2 cell was used, and hypoxia/reoxygenation (H/R) model was established to mimic the myocardial I/R injury. The expressions of miR-29b-3p and pentraxin 3 (PTX3) were quantified deploying qRT-PCR and Western blot, respectively. The levels of LDH, TNF-α, IL-1β and IL-6 were detected to evaluate cardiomyocyte apoptosis and inflammatory response. Cardiomyocyte viability and apoptosis were examined employing CCK-8 assay and flow cytometry, respectively. Verification of the targeting relationship between miR-29b-3p and PTX3 was conducted using a dual-luciferase reporter gene assay. It was found that miR-29b-3p expression in H9c2 cells was up-regulated by H/R, and a remarkable down-regulation of PTX3 expression was demonstrated. MiR-29b-3p significantly promoted of release of inflammatory cytokines of H9c2 cells, and it also constrained the proliferation and promoted the apoptosis of H9c2 cells. Additionally, PTX3 was inhibited by miR-29b-3p at both mRNA and protein levels, and it was identified as a direct target of miR-29b-3p. PTX3 overexpression could reduce the inflammatory response, increase the viability of H9c2 cells, and inhibit apoptosis. Additionally, PTX3 counteracted the function of miR-29b-3p during the injury of H9c2 cells induced by H/R. In summary, miR-29b-3p was capable of aggravating the H/R injury of H9c2 cells by repressing the expression of PTX3.     

miR-142通过靶向HMGB1对宫颈癌细胞生物学行为影响的实验研究

摘要 目的：通过实验探究miR-142靶向高迁移率族蛋白1（high-mobility group box 1 protein,HMGB1）对宫颈癌（cervical cancer,CC）细胞生物学行为的影响及其潜在的作用机制。方法：采用实时荧光定量PCR（RT-PCR）和蛋白质免疫印迹法（Western Blot）检测CC组织和正常组织中miR-142和HMGB1 mRNA及蛋白表达水平,采用荧光素酶报告实验分析miR-142与HMGB1的靶向关系,CCK-8法检测CC细胞生存能力,克隆形成实验检测CC细胞增殖能力,划痕修复实验检测CC细胞迁移能力,基质胶侵袭实验检测CC细胞侵袭能力。结果：CC组miR-142 mRNA和蛋白表达水平显著低于正常组（P<0.05）,HMGB1 mRNA和蛋白表达水平显著高于正常组（P<0.05）,且CC癌组织中miR-142和HMGB1 mRNA和蛋白表达水平均呈显著负相关（r=-0.399,P=0.002;r=-0.429,P=0.001）;miR-142与HMGB1存在靶向关系;CCK-8法实验、克隆形成实验、划痕修复实验和基质胶侵袭实验结果显示,miR-142 mimic组细胞生存、增殖、迁移和侵袭能力显著低于miR-NC组（P<0.05）,miR-142 inhibitor组细胞生存、增殖、迁移和侵袭能力显著高于miR-NC组;Western Blot实验结果显示,HMGB1过表达时miR-142 mimic+plasmid组HMGB1蛋白表达水平显著高于miR-142 mimic+control plasmid组（P<0.05）,显著低于miR-NC+plasmid组（P<0.05）;CCK-8法实验、克隆形成实验、划痕修复实验和基质胶侵袭实验结果显示,HMGB1过表达时miR-142 mimic+plasmid组细胞生存、增殖、迁移和侵袭能力显著高于miR-142 mimic+control plasmid组（P<0.05）,显著低于miR-NC+plasmid组（P<0.05）。结论：miR-142可通过靶向负调控HMGB1表达,进而抑制CC细胞生存、增殖、迁移和侵袭。     

The lncRNA ENST00000608794 acts as a competing endogenous RNA to regulate PDK4 expression by sponging miR-15b-5p in dexamethasone induced steatosis

The contribution of ncRNAs, especially long non-coding RNAs (lncRNAs) to drug induced steatosis remains largely unknown. The aim of this study was to investigate the role of lncRNA ENST00000608794 in dexamethasone induced steatosis. We found that ENST00000608794 is expressed at higher levels in dexamethasone treated HepG2 cell, and ENST00000608794 can bind and be regulated by miR-15b-5p. Ectopic expression of ENST00000608794 enhanced steatosis and the protein expression of PDK4 which is a critical gene in lipid metabolism and also is a target of miR-15b-5p. However, the differentiated PDK4 expression between control and ectopic expression of ENST00000608794 was absence in the presence of miR-15b-5p inhibitor. Moreover, in dexamethasone treated HepG2 cell lines, ENST00000608794 increased whether with miR-15b-5p inhibitor treatment or not, while increase of PDK4 expression by dexamethasone was greatly compromised in the presence of miR-15b-5p mimic. Meanwhile, dexamethasone induced steatosis could be ameliorated by silencing ENST00000608794 or expressing miR-15b-5p. Taken together, the results suggested that ENST00000608794 plays an important role in dexamethasone induced steatosis, which was partly mediated by derepressing of PDK4 through competitively binding to miR-15b-5p.     

MiR-26b-3p regulates osteoblast differentiation via targeting estrogen receptor α

BackgroundUnderstanding of the molecular mechanisms of miRNAs involved in osteoblast differentiation is important for the treatment of bone-related diseases.MethodsMC3T3-E1 cells were induced to osteogenic differentiation by culturing with bone morphogenetic protein 2 (BMP2). After transfected with miR-26b-3p mimics or inhibitors, the osteogenic differentiation of MC3T3-E1 cells was detected by ALP and ARS staining. Cell viability was analyzed by MTT. The expressions of miR-26b-3p and osteogenic related markers and signaling were examined by qPCR and western blot. Direct binding of miR-26b-3p and ER-α were determined by dual luciferase assay.ResultsmiR-26b-3p was significantly down-regulated during osteoblast differentiation. Overexpression of miR-26b-3p inhibited osteoblast differentiation, while inhibition of miR-26b-3p enhanced osteoblast differentiation. Further studies demonstrated miR-26b-3p inhibited the expression of estrogen receptor α (ER-α) by directly targeting to the CDS region of ER-α mRNA. Overexpression of ER-α rescued the suppression effects of miR-26b-3p on osteoblast differentiation, while knockdown of ER-α reversed the upregulation of osteoblast differentiation induced by knockdown of miR-26b-3p.ConclusionOur study demonstrates that miR-26b-3p suppresses osteoblast differentiation of MC3T3-E1 cells via directly targeting ER-α.     

Long non-coding RNA ZFAS1 alleviates sepsis-induced myocardial injury via target miR-34b-5p/SIRT1

Long non-coding RNA ZFAS1 is down-regulated in sepsis. However, whether ZFAS1 participates in sepsis-induced cardiomyopathy (SIC) remains largely unknown. LPS injection to rats was used to establish an in vivo sepsis model, while LPS stimulation with H9C2 cell was used to mimic an in vitro sepsis-induced myocardial injury model. Western blots and quantitative RT-PCR were performed to evaluate protein and mRNA levels, respectively. ELISA was conducted to determine cytokine levels in supernatant. Flow cytometry was used to test apoptosis. Dual-luciferase assay was performed to validate binding between ZFAS1 and miR-34b-5p, miR-34b-5p and SIRT1. Our data revealed that ZFAS1 and SIRT1 were down-regulated, while miR-34b-5p was up-regulated in LPS-induced H9C2 cells. Inhibition of miR-34b-5p or overexpression of ZFAS1 alleviated inflammatory response and cell apoptosis in LPS-stimulated H9C2 cells. A mechanism study revealed that ZFAS1 sponged miR-34b-5p and thus elevated expression of SIRT1, which was prohibited by miR-34b-5p. ZFAS1 alleviated inflammatory response and cell apoptosis in LPS-stimulated H9C2 cells via the miR-34b-5p/SIRT1 axis, providing novel potential therapeutic targets for SIC.     

miRNA-301b-3p accelerates migration and invasion of high-grade ovarian serous tumor via targeting CPEB3/EGFR axis

High-grade ovarian serous carcinoma (HGS-OvCa), a type of ovarian cancer with poor prognosis due to distant metastasis, is urgently in need of new therapeutic targets. microRNAs (miRNAs), a class of small noncoding RNAs, perform significant roles in tumor progression. Mounting evidence has revealed the aberrant expression of miRNA in various cancers, one of which is HGS-OvCa. Present study planned to investigate that miRNA-301b-3p accelerates migration and invasion of high-grade ovarian serous tumor via targeting CPEB3/EGFR axis. Upregulation of miR-301b-3p was uncovered in HGS-OvCa tissues and cell lines, and was identified to be associated with metastasis. The Kaplan-Meier analysis confirmed the association of miR-301b-3p with poor prognosis of HGS-OvCa patients. Transwell assay validated the oncogenic effect of miR-301b-3p on migration and invasion of HGS-OvCa cells. Cytoplasmic polyadenylation element binding protein 3 (CPEB3) was then identified as a target of miR-301b-3p. It was also discovered that CPEB3 was downregulated in HGS-OvCa tissues and cell lines. The Spearman correlation curve presented the negative correlation of CPEB3 expression with miR-301b-3p. Furthermore, rescue assays proved that miRNA-301b-3p regulated the invasion and migration through CPEB3. Western blot and qRT-PCR analysis showed that miRNA-301b-3p induced epidermal growth factor receptor and downstream metastasis-related proteins, p38, and extracellular signal-regulated kinase 1/2 (ERK1/2), through CPEB3. To be concluded, these results indicated that miRNA-301b-3p accelerated migration and invasion of high-grade ovarian serous tumor via targeting CPEB3/EGFR axis.     

miR-193b-5p regulates chondrocytes metabolism by directly targeting histone deacetylase 7 in interleukin-1β-induced osteoarthritis

There is increasing evidence regarding the pivotal roles of microRNAs (miRNAs) and histone deacetylases (HDACs) in the development of osteoarthritis (OA). This study aimed to determine whether miR-193b-5p regulates HDAC7 expression directly to affect cartilage degeneration. Expression levels of miR-193b-5p, HDAC7, matrix metalloproteinase 3 (MMP3), and MMP13 were determined in normal and OA cartilage and primary human chondrocytes (PHCs) stimulated with interleukin-1β (IL-1β). PHCs were transfected with a miR-193b-5p mimic or inhibitor to verify whether miR-193b-5p influences the expression of HDAC7 and MMPs. A luciferase reporter assay was performed to demonstrate the binding between miR-193b-5p and the 3′-untranslated region (UTR) of HDAC7. Expression of miR-193b-5p was reduced in IL-1β-stimulated PHCs and in OA cartilage compared to that in normal cartilage. Luciferase reporter assay exhibited the repressed activity of the reporter construct containing the 3′UTR of HDAC7. Both miR-193b-5p overexpression and HDAC7 inhibition decreased the expression of MMP3 and MMP13, whereas the inhibition of miR-193b-5p enhanced HDAC7, MMP3, and MMP13 expression. miR-193b-5p downregulates HDAC7 directly and, as a result, inhibits MMP3 and MMP13 expression, which suggests that miR-193b-5p has a protective role in OA.     

Retracted: Propranolol suppresses HUVEC viability,migration, VEGF expression,and promotes apoptosis by downregulation of miR-4295

Infantile hemangioma (IH) is a common benign tumor. Human umbilical vein endothelial cells (HUVECs) have the potential of stem cells, which has been widely used in vascular endothelial cell experiments. Oral propranolol was first reported to treat hemangioma in 2008. However, the role of propranolol in IH remains unclear. Therefore, in this study, we investigated the effects of propranolol on HUVECs in vitro, to explore the underlying mechanism of propranolol in IH. HUVECs were treated with 0.15, 1.5, and 15 μM of propranolol, and transfected with microRNA-4295 (miR-4295) mimic. Cell viability, migration, and apoptosis were examined using Cell Counting Kit-8, transwell assay, and flow cytometry analysis, respectively. In addition, the expressions and concentrations of miR-4295, vascular endothelial growth factor (VEGF), VEGF-A, FLT1, FLT2, and FOXF1 were assessed using real-time polymerase chain reaction, Western blot assay, and enzyme-linked immunosorbent assay. We found that 15 μM of propranolol decreased HUVEC viability the most. Then, cell migration and the concentrations of VEGF and VEGF-A were reduced, and apoptosis was increased when treated with propranolol. Meanwhile, the expressions of VEGF, VEGF-A, FLT1, FLT2, and FOXF1 were downregulated by propranolol exposure. Further study showed that miR-4295 expression was upregulated in IH tissues, and propranolol treatment downregulated miR-4295 expression in HUVECs. MiR-4295 overexpression alleviated the reductions of viability, migration, and factors expression, as well as the increase of apoptosis. Propranolol suppressed HUVEC viability, migration, the expression of VEGF, VEGF-A, FLT1/2, FOXF1, and promoted apoptosis via downregulation of miR-4295. This study lays a foundation for further study of the effect of propranolol on IH.     

雌激素处理的hBMSC对高糖诱导血管内皮细胞损伤的保护作用*

目的: 探讨雌激素处理人骨髓间充质干细胞(hBMSC)对高糖诱导的人脐静脉血管内皮细胞(HUVEC)损伤的保护作用及机制。方法: 采用30 mmol/L葡萄糖刺激hBMSC细胞建立高糖模型并分组:以无刺激者为高糖对照组(HG组)、以20 μmol/L雌激素处理者为高糖雌激素组(HG+E2组)、以5 μmo/L蛋白激酶B(PKB/Akt)抑制剂Triciribine预处理45 min后,再以20 μmol/L雌激素处理者为高糖Akt抑制剂组(HG+E2+Triciribine组)和正常条件培养的hBMSC为正常对照组(NG组)。分别于处理12 h后,采用CCK8法检测各组hBMSC的细胞活力,硝酸还原酶法和ELISA法检测各组培养基上清中NO、VEGF和IL-8的含量(n=6),48 h后采用Western blot检测内皮型一氧化氮合酶(eNOS)和磷酸化eNOS(p-eNOS)蛋白表达水平(n=3)。此外,提取各组hBMSC的培养基上清作为条件培养基(CM)培养人脐静脉血管内皮细胞(HUVEC)并分组为:HG-CM组(HG组条件培养基处理)、HG+E2-CM组(HG+E2组条件培养液处理)、HG+E2+Triciribine-CM组(HG+E2+Triciribine组条件培养基处理)和HG-H组(高糖对照组,30 mmol/L葡萄糖终浓度处理),分别于12 h后,采用CCK8法检测各组HUVEC的细胞活力(n=6),24 h后采用流式细胞术检测各组HUVEC细胞的凋亡率(n=3);48 h后采用划痕实验观察各组HUVEC细胞的迁移率(n=3)。结果: 与NG组相比,HG组中hBMSC细胞活力和细胞内eNOS蛋白磷酸化水平降低(P<0.05),细胞培养液上清中NO、VEGF和IL-8含量减少(P<0.05);与HG组相比,HG+E2组中hBMSC的细胞活力和细胞中eNOS蛋白磷酸化水平显著增加(P<0.05),细胞培养基上清中NO、VEGF和IL-8含量增加(P<0.05),而当hBMSC细胞中Akt蛋白活性被抑制后,HG+E2+Triciribine组中上述结果指标呈反向变化(P<0.05)。此外,与HG-CM组相比,HG+E2-CM组中HUVECs的细胞活力和迁移能力显著增加(P<0.05),细胞凋亡比例降低(P<0.05),而与HG+E2-CM组相比,HG+E2+Triciribine-CM组中HUVECs的细胞活力和迁移能力降低(P<0.05),细胞凋亡比例增加(P< 0.05)。结论: 雌激素可能通过激活hBMSC细胞Akt/eNOS信号通路,促进NO、VEGF和IL-8的分泌,进而增加HUVECs的细胞活力和迁移能力,并抑制细胞凋亡的发生,对高糖诱导的HUVECs细胞损伤发挥保护作用。     

miR-135b-5p抑制脓毒症引起的小鼠急性肺损伤(ALI)的作用及机制*

目的: 探究miR-135b-5p在小鼠脓毒症(sepsis)引起的急性肺损伤(ALI)模型中的表达水平及其对小鼠肺部炎症反应和细胞焦亡的影响。方法: 将C57BL/6小鼠随机分为6组,每组8只,通过盲肠结扎穿刺法(CLP)手术构建CLP诱导的脓毒症小鼠模型:腹腔注射0.1 mg/kg的巴比妥麻醉,腹部纵向切开暴露盲肠,结扎盲肠并用注射器针头进行穿孔,挤出部分肠道内容物后缝合伤口。假手术组(Sham组)开腹后不做任何处理缝合伤口,无CLP手术处理。治疗组分为CLP+NC mimic组,CLP+miR-135b-5p mimic组,CLP+NC mimic+empty vector组,CLP+消皮素D (GSDMD)组,CLP+miR-135b-5p mimic+GSDMD组。治疗组小鼠在CLP手术前一周皮下注射200 μl溶解于生理盐水的NC mimic(200 nmol/L),miR-135b-5p mimic(200 nmol/L),empty vector(100 nmol/L),GSDMD vector(100 nmol/L),每天注射1次,连续一周。术后24 h采用二氧化碳窒息法实施安乐死。采用qRT-PCR检测小鼠肺组织样本中miR-135b-5p和GSDMD mRNA的表达水平;苏木精-伊红(HE)染色检测小鼠肺组织形态和损伤状态;采用5 ml生理盐水冲洗小鼠右肺3次,每次持续约3~5 min,收集肺泡灌洗液(BALF),酶联免疫吸附实验(ELISA)检测小鼠肺泡灌洗液(BALF)中GSDMD、白介素1β(IL-1β)和白介素18(IL-18)的表达水平;蛋白免疫印迹法检测小鼠肺组织内含NLR家族PYRIN域蛋白3(NLRP3),半胱氨酸天冬氨酸蛋白水解酶1(caspase 1)以及切割后的N-端GSDMD端蛋白结构域(cleaved-GSDMD-N)的表达水平。双荧光素酶报告基因检测系统验证miR-135b-5p与GSDMD的靶向结合关系。结果: 与对照组相比,CLP组小鼠肺组织中有大量的炎症细胞浸润,肺泡损伤,细胞间质水肿及肺泡塌陷等病理特征,小鼠肺组织内细胞焦亡相关蛋白(NLRP3,caspase-1和GSDMD)的表达水平明显增加(P<0.01),但miR-135b-5p的表达水平明显下调(P<0.01);与CLP组相比,超表达miR-135b-5p能够明显抑制CLP诱导的小鼠肺组织内细胞焦亡(P<0.01),靶向抑制GSDMD的表达水平(P<0.01);超表达GSDMD能够逆转超表达miR-135b-5p对肺组织细胞焦亡的抑制作用(P<0.01),超表达miR-135b-5p能够通过靶向GSDMD抑制小鼠BALF中IL-1β及IL-18的表达水平(P<0.01)。结论: miR-135b-5p靶向下调GSDMD抑制细胞焦亡,改善脓毒症引起的ALI,为脓毒症诱导的ALI治疗提供了潜在的治疗靶点和理论依据。     

MicroRNA-450b-3p inhibits cell growth by targeting phosphoglycerate kinase 1 in hepatocellular carcinoma

Dysregulation of microRNAs frequently contributes to the occurrence and progression of human diseases, including hepatocellular carcinoma (HCC). In this study, the role of miR-450b-3p in HCC was investigated. Gene Expression Omnibus database and HCC specimens were used to evaluate the expression level of miR-450b-3p and the patient's prognosis. Cell functional analyses and tumor xenograft model were used to assess the role of miR-450b-3p in HCC. Bioinformatics was used to predict the downstream target gene of miR-450b-3p, which was verified by dual-luciferase reporter assay. MiR-450b-3p was found to be downregulated in HCC cell lines and tissues, compared with nontransformed immortal hepatic cells and adjacent normal liver tissues, respectively. Lower expression of miR-450b-3p was associated with poor overall survival and disease-free survival in patients with HCC. Ectopic expression of miR-450b-3p inhibited HCC cell viability, colony formation, and cell-cycle progression in vitro, and suppressed the growth of HCC xenograft tumors in vivo. Interestingly, a negative correlation between miR-450b-3p and phosphoglycerate kinase 1 (PGK1) protein was observed among HCC specimens. Additionally, miR-450b-3p inhibited PGK1 expression and phosphorylation of protein kinase B in HCC cell lines. Further experiments confirmed that PGK1 was a direct target of miR-450b-3p. Moreover, restoration of PGK1 abrogated the inhibitory effect of miR-450b-3p on HCC proliferation and cell division. In conclusion, miR-450b-3p is downregulated in human HCC and exerts tumor suppressive effects at least in part by inhibiting PGK1.