Silencing essential meiotic endonuclease 1 inhibits the proliferation of liver cancer cells： A study of related mechanisms / 临床肝胆病杂志

ObjectiveTo investigate the expression of essential meiotic endonuclease 1 （EME1） in liver cancer tissue and its effect on the biological behavior of hepatoma cells. MethodsThe TCGA database was used to identify the differentially expressed genes between liver cancer tissue and paracancerous tissue. Immunohistochemistry and Western Blot were used to measure the expression abundance of EME1 in liver cancer tissue. A lentivirus was constructed by short hairpin RNA， and BEL-7404 cells were transfected with the lentivirus to interfere with the expression of the EME1 gene； the cells were divided into silencing group （shEME1 group） and control group （shCtrl group）. Quantitative real-time PCR and Western Blot were used to measure the mRNA and protein expression levels of EME1； Celigo Image Cytometer and MTT assay were used to measure cell proliferation rate； flow cytometry was used to observe cell cycle； Caspase 3/7 activity was used to measure cell apoptosis. The independent-samples t-test was used for comparison between two groups. ResultsTCGA results showed that the mRNA expression level of EME1 in liver cancer tissue was 18.9 times that in paracancerous tissue （t=5.00， P<0.001）， and the protein expression level of EME1 in liver cancer tissue was 7.0 times （based on immunohistochemistry： 8.4±2.6 vs 1.2±0.4， t=7.55， P<0.001） or 2.5 times （based on Western Blot： 249.0%±35.5% vs 100.0%±77.8%， t=3.02， P<0.05） that in paracancerous tissue. After lentivirus infection， compared with the shCtrl group， the shEME1 group had an mRNA expression level of EME1 reduced by 29.9% （29.9%±0.9% vs 100.0%±3.6%， t=32.82， P<0.001）， a protein expression level of EME1 reduced by 35.7% （35.7%±14.9% vs 100.0%±28.9%， t=3.42， P<0.05）， and a level of cell counting reduced by 45.1% （4 053±167 vs 8 988±477， t=16.91， P<0.001）， as well as a level of cell activity reduced to 66.9% （0.518±0.046 vs 0.774±0.022， t=8.74， P<0.001） and a level of colony forming ability reduced to 29.0% （75±6 vs 260±9， t=28.92， P<0.001）. Compared with the shCtrl group， the shEME1 group had a significant increase in the proportion of cells in G1 phase （49.9% vs 44.0%， t=8.96， P<0.001） and significant reductions in the proportion of cells in G2/M phase （15.9% vs 17.9%， t=9.13， P<0.001） and S phase （34.2% vs 38.1%， t=6.91， P<0.001）， while Caspase 3/7 activity was enhanced by 1.5 times （145.8%±5.9% vs 100.0%±2.3%， t=12.50， P<0.001）. ConclusionEME1 is highly expressed in liver cancer tissue， and silencing the EME1 gene can inhibit the proliferation of hepatoma cells and promote cell apoptosis.

Silencing of activating transcription factor 4 inhibits neuronal necroptosis after intracerebral hemorrhage in vitro / 中国组织工程研究

BACKGROUND:Neuronal necroptosis induced by intracerebral hemorrhage is an important cause of secondary brain injury.Activating transcription factor 4(ATF4)is a member of the transcription activator family,which plays an important role in secondary brain injury after intracerebral hemorrhage.However,the mechanism of ATF4 in neuronal necroptosis after intracerebral hemorrhage remains unclear. OBJECTIVE:To explore the effect of ATF4 silencing(ATF4 small interfering RNA,ATF4 siRNA)on neuronal necroptosis after intracerebral hemorrhage. METHODS:The HT-22 mouse hippocampal neuron cell line and the BV-2 mouse microglial cell line were co-cultured,and hemin was used to mimic an in vitro model of intracerebral hemorrhage.A gradient concentration of hemin was used to treat cells and was set in the interval of 0-100 μmol/L,and the cell viability was evaluated by MTT assay after 24 hours of administration of hemin.The cells were divided into four groups:the blank control group without any intervention;the control group was treated with hemin(50 μmol/L),and the other two groups were treated with negative control small interfering RNA(NC siRNA)and ATF4 small interfering RNA(ATF4 siRNA)48 hours before administration of hemin.After the cells were treated with hemin(50 μmol/L)for 24 hours,PI/Hoechst staining was used to detect neuronal necroptosis.Western blot assay was used to detect the protein expression of ATF4,receptor-interacting protein 3(RIP3),and mixed lineage kinase domain-like protein(MLKL),and double immunofluorescent staining was located in neurons to observe the level of neuronal necroptosis and the regulatory effect of ATF4 on it. RESULTS AND CONCLUSION:(1)50 μmol/L of hemin could induce neuronal necroptosis to a greater extent.(2)The number of PI+/Hoechst+ cells in the control group and NC siRNA group was higher than that in the blank control group(P<0.000 1).The number of PI+/Hoechst+ cells in the ATF4 siRNA group was lower than that in the control group(P<0.000 1).(3)Compared with the control group,the ATF4 siRNA group not only inhibited the expression of ATF4 protein(P<0.001),but also inhibited the expression of RIP3 and MLKL protein(P<0.001).(4)Through double immunofluorescent staining,compared with the control group,the protein expression of RIP3 and MLKL was significantly reduced in the ATF4 siRNA group(P<0.000 1).(5)The results show that the silencing of the ATF4 gene can directly or indirectly inhibit the expression of genes related to neuronal necroptosis after intracerebral hemorrhage,and play a vital role in alleviating secondary brain injury.

Effect of interfering hsa_circ_0103232 on the biological behavior of uveal melanoma cells / 中华实验眼科杂志

Objective:To investigate the effects of interference with hsa_circ_0103232 on the proliferation, metastasis, cell cycle and apoptosis of melanoma cells C918 and MUM2B.Methods:C918 and MUM2B cells were cultured, and the interference efficiency of three small interfering RNA (siRNA) targeting hsa_circ_0103232 were detected by real-time fluorescent quantitative polymerase chain reaction (PCR).The siRNA with the highest interference efficiency was used for the following experiment.Both C918 and MUM2B cells were divided into negative control transfection (siCtrl) groups and interference (si-hsa_circ_0103232) groups.The proliferation of C918 and MUM2B cells was examined by cell counting kit-8 (CCK-8) assay and cell colony formation assay.The migration of C918 and MUM2B cells was determined by transwell assay.The cell cycle distribution and apoptosis rate of C918 and MUM2B cells were detected by flow cytometry.The localization of hsa_circ_0103232 in C918 and MUM2B cells was tested by the fluorescence in situ hybridization experiment (FISH).Results:The results of real-time quantitative PCR showed that among the three siRNAs targeting hsa_circ_0103232, si-hsa_circ_0103232#1 had the best effect, which reduced the expression level of gene in C918 and MUM2B cells to 0.263±0.016 and 0.469±0.028, significanthy lower than 1.013±0.008 and 1.004±0.108 of control groups (both at P<0.001).CCK-8 results showed that the proliferation activity of C918 and MUM2B cells was significantly lower in si-hsa_circ_0103232 group than in siCtrl group after transfection (all at P<0.05).The results of cell clone formation showed that the clone number of C918 and MUM2B cells in si-hsa_circ_0103232 group were 12±1 and 45±7, which were significantly lower than 28±4 and 83±3 in siCtrl group, and the differences were statistically significant ( t=4.93, 7.42; both at P<0.05).Transwell assay results showed that the number of migrating C918 and UM2B cells in si-hsa_circ_0103232 group were 4±1 and 24±2, respectively, which were significantly lower than 37±12 and 57±3 in siCtrl group, and the differences were statistically significant ( t=3.91, 10.80; both at P<0.05).The results of flow cytometry showed that compared with siCtrl group, the proportion of G1 phase cells in C918 and MUM2B cells in si-hsa_circ_0103232 group increased significantly, the proportion of G2/M phase cells decreased significantly, and the number of apoptotic cells increased significantly (all at P<0.05).FISH experiment showed that hsa_circ_0103232 was located in the nuclei of C918 and MUM2B cells. Conclusions:Interference with hsa_circ_0103232 can inhibit the proliferation, migration and cycle progression of C918 and MUM2B cells, and promote their apoptosis.hsa_circ_0103232 may be a new therapeutic target for uveal melanoma.

Effect of RhoC silencing on migration and invasion of oral squamous cell carcinoma / 中国病理生理杂志

AIM:To explore the expression of RhoC in oral squamous cell carcinoma(OSCC)and its effects on the malignant biological behavior of OSCC cells.METHODS:The UALCAN and K-M plotter databases,alongside tis-sue sample analyses,facilitated understanding RhoC expression in cancer and its links to clinicopathological traits.Two small interfering RNAs(RhoC-siRNA)were constructed according to the RhoC gene sequence.The mRNA and protein ex-pression levels of RhoC in OSCC cells were determined.The protein levels of FAK,p-FAK,MAPK,p-MAPK,matrix me-talloproteinase-2(MMP-2)and MMP-9 were also examined by Western blot.Furthermore,the invasion and migration of OSCC cells were analyzed by Transwell assay and scratch test.Finally,the pulmonary metastasis model of nude mice was established.RESULTS:The results of the databases showed that RhoC was highly expressed in OSCC tissues,which was closely related to pathological stage,pathological grade and lymph node metastasis,but not significantly related to the sur-vival rate of patients.Furthermore,compared with paracancer tissues,the mRNA and protein expression levels of RhoC were increased in OSCC tissues(P<0.01).Silencing of RhoC prominently reduced the migration and invasion of OSCC cells as well as the protein levels of p-FAK,p-MAPK,MMP2 and MMP9(P<0.05).The protein levels of MAPK and FAK were unchanged(P>0.05).The fluorescence intensity of the experimental group was significantly lower than that of the control group,and the results of HE staining showed that the number of lung nodules in the experimental group was sig-nificantly reduced(P<0.05).CONCLUSION:RhoC can effectively influence the migration and invasion of OSCC cells,and its potential mechanism may be related to FAK/MAPK/MMPs signaling pathway.

m 6A RNA Methylation Decreases Atherosclerotic Vulnerable Plaque Through Inducing T Cells

ABSTRACT Introduction: Knockdown of fat mass and obesity-associated gene (FTO) can induce N6-methyladenosine (m 6A) ribonucleic acid (RNA) methylation. The objective of this study was to explore the effect of m 6A RNA methylation on atherosclerotic vulnerable plaque by FTO knockdown. Methods: A total of 50 New Zealand white rabbits were randomly divided into pure high-fat group, sham operation group, vulnerable plaque group, empty load group, and FTO knockdown group (10 rabbits/group). Results: Flow cytometry showed that helper T (Th) cells in the FTO knockdown group accounted for a significantly higher proportion of lymphocytes than in the vulnerable plaque group and empty load group (P<0.05). Th cells were screened by cell flow. The level of m 6A RNA methylation in the FTO knockdown group was significantly higher than in the vulnerable plaque group and empty load group (P<0.05). The levels of total cholesterol, triglyceride, and low-density lipoprotein C were higher at the 12th week than at the 1st week, but the high-density lipoprotein C level was lower at the 12th week than at the 1st week. At the 12th week, the interleukin-7 level was significantly lower in the adeno-associated virus-9 (AVV9)-FTO short hairpin RNA group than in the control and AVV9-green fluorescent protein groups (P<0.001). Conclusion: After successfully establishing a vascular parkinsonism rabbit model, m 6A RNA methylation can decrease Th cells and vulnerable atherosclerotic plaques.

Effects of ICMT gene silencing on the invasion and migration of human salivary adenoid cystic carcinoma cells in vitro / 口腔疾病防治

Objective @#To investigate the effect of isoprene cysteine carboxymethyltransferase (ICMT) gene on the migration and invasion of salivary adenoid cystic cancer cells (SACC) and the related mechanism, to provide experimental evidence for molecular targeted therapy of SACC.@*Methods@# Adenoid cystic cancer cells SACC-LM and SACC-83 were cultured in vitro, and siRNA was transfected into human SACC-LM and SACC-83 cells (experimental group) by transient transfection of a liposome vector. A blank control group and negative control group were set up respectively (transfected NC-siRNA). qRT-PCR was peformed to measure the mRNA expression of ICMT and RhoA in each group after transfection and to determine the silencing efficiency. The expression of ICMT, membrane RhoA, total RhoA, matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) and Rho associated with coiled helical binding protein kinase 1 (ROCK1) in each group was detected by Western blot. The proliferation abilityies of SACC cells was detected by CCK-8 assay. The migration and invasion ability of SACC cells were detected by comparing the relative healing area of cell scratch assay and the number of Transwell assay cells. @*Results@#After transfection of ICMT-siRNA into SACC-LM and SACC-83 cells, the expression of ICMT gene and protein in the experimental group was significantly decreased compared with the negative control group and blank control group (P<0.05), but there were no significant differences in the expression of RhoA gene and total protein among all groups (P>0.05). The expression of RhoA membrane proteins, ROCK1, MMP-2, MMP-9 in the experimental group was significantly decreased compared with that in the negative control group and blank control group (P<0.05). Cell proliferation ability was significantly decreased (P<0.05). The migration and invasion abilities were significantly decreased (P<0.05). @*Conclusion @#In vitro silencing of ICMT gene can effectively inhibit the migration and invasion of human SACC-LM and SACC-83 cells, and the mechanism may be related to RhoA-ROCK signaling pathway.

Research progress of RNA drugs delivery / 药学学报

Ribonucleic acid (RNA) medicines have strong therapeutic potential for numerous rare genetic illnesses and malignancies because of its exact programmability based on Watson-Crick base pairing principle and unique ability to regulate gene expression. However, RNA medicines still have limitations in many areas, including stability, half-life time, immunogenicity, organ selectivity, cellular uptake and endosomal escape efficiency despite their great therapeutic potentials. This review briefly introduced numerous RNA medications [mostly messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA) and antisense oligonucleotide (ASO)] that have intrigued of researchers in recent years, as well as their action mechanism in vivo. A number of delivery techniques, such as chemical modification, ligands coupling and nanocarriers have been proposed. The manufacture and applications of lipid nanoparticle, polymer nanoparticle and exosomes were discussed in depth. The goal of this work is to give a theoretical foundation and design concepts for the development of effective and safe RNA delivery technology, as well as to facilitate RNA therapeutic clinical translation.

Thyroxine promotes the progression of integrin α vβ 3-positive differentiated thyroid cancer through the ERK1/2 pathway / 中华核医学与分子影像杂志

Objective:To explore whether thyroxine (T 4) could promote differentiated thyroid cancer (DTC) progression by binding to integrin α vβ 3in vitro and its downstream mechanism. Methods:Papillary thyroid cancer cell lines TPC-1, K1 and follicular thyroid cancer (FTC) cell line FTC133 were cultured in vitro, and the expressions of integrin α vβ 3 in those 3 DTC cell lines were determined with immunofluorescence and flow cytometry analysis. After the treatment of T 4, tetraiodo thyroacetic acid (Tetrac) and Arg-Gly-Asp (RGD) peptide alone or in combination, the proliferation and metastatic potential of DTC cell lines were detected by cell counting kit-8 (CCK-8), Transwell migration and invasion assays. The small interfering RNA (siRNA) transfection was used to verify whether integrin α v or β 3 subunit knockdown could reverse the effect of T 4 on DTC cells. The expression levels of downstream signaling proteins phosphorylated extracellular signal-regulated kinase (p-ERK)1/2 and total extracellular signal-regulated kinase (ERK)1/2 were detected by Western blot. The effects of mitogen-activated protein kinase kinase (MEK)1/2 inhibitor (GSK1120212) on the proliferation, migration and invasion of T 4-treated cells were detected. One-way analysis of variance and Tukey test were used for data analysis. Results:The integrin α vβ 3 expressions in TPC-1, K1 and FTC133 cells were all positive, with the relative mean fluorescence intensity (MFI) of 61.93±18.61, 16.89±2.43 and 32.36±0.83, and the percentages of positive cells of (94.38±1.30)%, (74.11±3.87)% and (50.67±1.78)%, respectively ( F values: 13.36 and 217.30, P=0.006 and P<0.001). Compared with control group, the proliferation, migration and invasion in the three DTC cell lines treated with T 4 were significantly enhanced (96 h, F values: 62.67-297.50, q values: 13.15-20.73, all P<0.001). T 4-induced cell proliferation, migration and invasion were markedly reversed by Tetrac or RGD (96 h, q values: 8.61-17.54, all P<0.001). T 4-induced cell proliferation, migration and invasion were also significantly inhibited by the knockdown of integrin α v or β 3 subunit (72 h, F values: 7.75-70.98, q values: 4.77-15.21, all P<0.05). Western blot results showed that the phosphorylation levels of ERK1/2 in DTC cells were significantly increased by T 4 treatment, and the T 4-induced activation of ERK1/2 signaling pathway could be blocked by Tetrac, RGD, integrin α v or β 3 subunit knockdown. T 4-induced cell proliferation, migration and invasion were significantly reversed by GSK1120212 (96 h, F values: 47.53-151.40, q values: 10.32-16.65, all P<0.001). Conclusion:T 4 can promote cell proliferation and metastasis of DTC cells by binding to integrin α vβ 3 and activating the ERK1/2 pathway.

Analysis of differential expression profiles of Piwi-interacting RNA in diabetic nephropathy patients / 中华肾脏病杂志

Objective:To investigate the correlation between Piwi-interacting RNA (piRNA) and diabetic nephropathy (DN).Methods:The differential expression profiles of piRNAs in renal tissues of patients with DN (experimental group) and renal tissues adjacent to tumors of patients with renal tumors (control group) were detected by high-throughput sequencing. The biological function of differentially expressed piRNAs was described by gene ontology and Kyoto encyclopedia of genes and genomes enrichment analysis. Real-time fluorescence quantitative PCR was used to detect the serum expression level of target piRNAs in patients with DN. Spearman correlation analysis was used to analyze the correlation between serum target piRNAs and clinical indexes of patients with DN.Results:The results of high throughput sequencing showed that there were 127 differentially expressed piRNAs between DN group and control group, with screening condition of |log 2(fold changes)|≥2 and P<0.05. Among them, there were 99 up-regulated piRNAs and 28 down-regulated piRNAs. The top 5 up-regulated piRNAs were piRNA-hsa-161686, piRNA-hsa-349255, piRNA-hsa-355720, piRNA-hsa-151229 and piRNA-hsa-154959, respectively. The top 5 down-regulated piRNAs were piRNA-hsa-1929960, piRNA-hsa-174194, piRNA-hsa- 148658, piRNA-hsa-172594 and piRNA-hsa-172421, respectively. The PCR verification results of 3 up-regulated genes and 3 down-regulated genes with low P values and high expression levels showed that serum expression level of piRNA-hsa-77976 was significantly down-regulated in patients with DN ( P=0.028), which was consistent with that of sequencing, while the expression levels of other genes were inconsistent with the sequencing results or had no statistical significance. Bioinformatics analysis results predicted that significantly differentially expressed piRNAs might participate in the regulation of DN through Rap1, Ras, PI3K-Akt and axon guiding pathways. The results of correlation analysis showed that the expression level of piRNA-hsa-77976 was negatively correlated with blood urea nitrogen ( r=-0.584, P=0.028), serum creatinine ( r=-0.637, P=0.014), cystatin C ( r=-0.738, P=0.003) and β2 microglobulin ( r=-0.822, P<0.001), and positively correlated with estimated glomerular filtration rate ( r=0.661, P=0.010). Conclusion:The differential expression of piRNA is closely related to DN, and may be used as a new biomarker for the diagnosis and prognosis of DN.

Mechanism for regulation of trophoblast invasion through piR-3127964 via PIWIL-3/sialophorin pathway / 中华围产医学杂志

Objective:To investigate the molecular mechanism for regulation of trophoblast invasion by piR-3127964, which is differentially expressed in placental tissues of preeclamptic and healthy pregnant women.Methods:Placenta samples of healthy (control group, n=12) and preeclamptic pregnant (PE group, n=10) women who delivered by caesarean section and chorionic villi specimens of patients undergoing artificial abortion were collected in the Department of Obstetrics of the First Affiliated Hospital of Chongqing Medical University during November 2020 to August 2021. Total RNA was extracted from placenta samples and sequenced and the expression of piR-3127964 in different tissues was determined by real-time quantitative-polymerase chain reaction (qRT-PCR). The expressions of PIWI proteins including PIWIL-1, PIWIL-2 and PIWIL-3 in different tissues were detected by Western blot. The expressions of two candidate targets, guanine nucleotide-binding protein-like 3-like (GNL3L) mRNA and sialophorin (SPN) mRNA were evaluated by qRT-PCR after exogenous treating HTR-8/SVneo cells with mimics, inhibitor or negative control of piR-3127964, respectively. qRT-PCR was also used to detect the relative expression of GNL3L and SPN at mRNA level in placentas of all women. The interactions between GNL3L/SPN and piR-3127964 were analyzed by double luciferase reporter gene detection. The localization of piR-3127964 and SPN in chorionic villi was detected by fluorescence in situ hybridization and immunofluorescence. Transwell assay was performed to analyze the influence of piR-3127964 on the invasion of HTR-8/SVneo cells and the possible mechanism. Independent sample t-test, analysis of variance, and LSD post test were used for analysis Results:(1) Enrichment pathways of candidate targets predicted by differentially expressed piR-3127964 were associated with cell motility. There were statistically significant differences in piR-3127964 expression in villi, healthy and preeclamptic placentas (2.950±0.853 vs 1.036±0.303 vs 0.254±0.155, F=27.35, P<0.05), and piR-3127964 was predominantly expressed in extravillous cytotrophoblasts (EVTs). (2) The expression of PIWIL-3 protein in placentas of preeclamptic patients was significantly lower than that in healthy placentas and villi (0.810±0.400 vs 3.175±0.429 and 6.843±1.379, F=49.36, P<0.05). (3) Compared with the control group, exogenous piR-3127964 mimics (piR-mimics) and inhibitors (piR-inhibitor) significantly affected the expression of SPN mRNA (0.971±0.045 vs 0.732±0.010, F=6.50; 1.076±0.073 vs 1.293±0.092, F=7.58; both P<0.05), while the expression of GNL3L mRNA had no significant correlation with piR-3127964 level. (4) The luciferase activity of wild-type SPN (SPN-WT) plasmids was significantly affected by piR-mimics (1.010±0.049 vs 0.645±0.047, t=9.34, P<0.05) and piR-inhibitor (1.035±0.058 vs 1.397±0.015, t=－10.60, P<0.05). (5) SPN mRNA was significantly upregulated in placentas of preeclamptic patients than in healthy placentas (2.097±0.239 vs 1.305±0.290, t=－4.22, P<0.05), but no significant difference in the expression of GNL3L mRNA was observed. Immunofluorescence experiment showed that SPN was expressed in EVTs. (6) The invasive potential of HTR8/SVneo cells treated with piR-inhibitor was significantly inhibited, but this effect could be reversed by SPN knockdown (160.714±53.860 vs 371.667±103.061 and 344.333±120.267, F=9.76, both P<0.05). Conclusions:piR-3127964 expression is abnormally downregulated in placentas of preeclamptic patients, resulting in inhibition of trophoblasts invasion through upregulation of SPN expression, which may be related to the pathogenesis of preeclampsia.

Research progress of ionizable lipid nanoparticles for siRNA delivery / 药学学报

Small interfering RNA (siRNA) is the initiator of RNA interference and inhibits gene expression by targeted degradation of specific messenger RNA. siRNA-mediated gene regulation has high efficiency and specificity and exhibits great significance in the treatment of diseases. However, the naked or unmodified siRNA has poor stability, easy to degrade by nuclease, short half-life, and low intracellular delivery. As an emerging non-viral nucleic acid delivery system, ionizable lipid nanoparticles play an important role in improving the druggability of siRNA. At present, one siRNA drug based on ionizable lipid nanoparticles has been approved for the treatment of rare disease. This review introduces the research progress in ionizable lipid nanoparticles for siRNA delivery, focusing on the effect of each component of lipid nanoparticles on the efficiency of siRNA-mediated gene silencing, which provides new references for the studies on ionizable lipid nanocarriers for siRNA delivery.

Progress on RNA-based therapeutics for genetic diseases / 浙江大学学报·医学版

RNA therapeutics inhibit the expression of specific proteins/RNAs by targeting complementary sequences of corresponding genes or encode proteins for the synthesis desired genes to treat genetic diseases. RNA-based therapeutics are categorized as oligonucleotide drugs (antisense oligonucleotides, small interfering RNA, RNA aptamers), and mRNA drugs. The antisense oligonucleotides and small interfering RNA for treatment of genetic diseases have been approved by the FDA in the United States, while RNA aptamers and mRNA drugs are still in clinical trials. Chemical modifications can be applied to RNA drugs, such as pseudouridine modification of mRNA, to reduce immunogenicity and improve the efficacy. The secure and effective delivery systems such as lipid-based nanoparticles, extracellular vesicles, and virus-like particles are under development to address stability, specificity, and safety issues of RNA drugs. This article provides an overview of the specific molecular mechanisms of eleven RNA drugs currently used for treating genetic diseases, and discusses the research progress of chemical modifications and delivery systems of RNA drugs.

Effects of Foxp3 gene silencing on the expression of inflammatory cytokines and the proliferation and migration of human periodontal ligament fibroblasts in an inflammatory environment / 华西口腔医学杂志

OBJECTIVES@#This study aimed to clarify the effects of Foxp3 silencing on the expression of inflammatory cytokines in human periodontal ligament cells (hPDLFs) in an inflammatory environment and on cell proliferation and invasiveness, as well as to explore the role of Foxp3 gene in the development of periodontitis.@*METHODS@#An small interfering RNA (siRNA) construct specific for Foxp3 was transfected into hPDLFs. Foxp3 silencing efficiency was verified by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, and the siRNA with the optimum silencing effect of Foxp3 gene was screened. Using lipopolysaccharide to simulate an inflammatory environment in vitro, CCK-8 detected the effect of silencing Foxp3 on hPDLFs proliferation under inflammatory conditions. Wound-healing experiments and transwell assays were conducted to detect the effect of silencing Foxp3 on hPDLF migration under inflammatory conditions. The expression of the inflammatory cytokines interleukin (IL)-6 and IL-8 was detected by RT-PCR and Western blotting under inflammatory conditions.@*RESULTS@#After siRNA transfection, RT-PCR and Western blotting analyses showed that the expression of Foxp3 mRNA in the Foxp3-si3 group decreased significantly (t=21.03, P<0.000 1), and the protein expression of Foxp3 also decreased significantly (t=12.8, P<0.001). In the inflammatory environment, Foxp3 gene silencing had no significant effect on hPDLFs proliferation (P>0.05), and Foxp3 gene silencing promoted hPDLFs migration (P<0.05). Moreover, the expression of IL-6 and IL-8 increased (P<0.05).@*CONCLUSIONS@#In an inflammatory environment, Foxp3 gene silencing promoted hPDLFs migration but had no significant effect on hPDLFs proliferation. The expression of inflammatory factors expressed in hPDLFs increased after Foxp3 gene silencing, indicating that Foxp3 gene inhibited inflammation in periodontitis.

Lipid nanoparticle delivery of siRNA targeting Cyp2e1 gene attenuates subacute alcoholic liver injury in mice / 浙江大学学报·医学版

OBJECTIVES@#To investigate the effect and mechanism of lipid nanoparticle (LNP) delivery of small interfering RNA (siRNA) targeting Cyp2e1 gene on subacute alcoholic liver injury in mice.@*METHODS@#siRNA targeting Cyp2e1 gene was encapsulated in LNP (si-Cyp2e1 LNP) by microfluidic technique and the resulting LNPs were characterized. The optimal dose of si-Cyp2e1 LNP administration was screened. Forty female C57BL/6N mice were randomly divided into blank control group, model control group, si-Cyp2e1 LNP group, LNP control group and metadoxine group. The subacute alcoholic liver injury mouse model was induced by ethanol feeding for 10 d plus ethanol gavage for the last 3 d. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, and the superoxide dismutase (SOD) activity as well as malondialdehyde, reactive oxygen species, glutathione, triacylglycerol, total cholesterol contents in liver tissue were measured in each group, and liver index was calculated. The expression of genes related to oxidative stress, lipid synthesis and inflammation in each group of mice were measured by realtime RT-PCR.@*RESULTS@#Compared with the model control group, the levels of liver index, serum ALT, AST activities, malondialdehyde, reactive oxygen species, triacylglycerol, total cholesterol contents in liver tissue decreased, but the SOD activity as well as glutathione increased in the si-Cyp2e1 LNP group (all P<0.01). Hematoxylin-eosin staining result showed disorganized hepatocytes with sparse cytoplasm and a large number of fat vacuoles and necrosis in the model control group, while the si-Cyp2e1 LNP group had uniformly sized and arranged hepatocytes with normal liver tissue morphology and structure. Oil red O staining result showed si-Cyp2e1 LNP group had lower fat content of the liver compared to the model control group (P<0.01), and no fat droplets accumulated. Anti-F4/80 monoclonal antibody fluorescence immunohistochemistry showed that the si-Cyp2e1 LNP group had lower cumulative optical density values compared to the model control group (P<0.01) and no significant inflammatory reaction. Compared with the model control group, the expression of catalytic genes P47phox, P67phox and Gp91phox were reduced (all P<0.01), while the expression of the antioxidant enzyme genes Sod1, Gsh-rd and Gsh-px were increased (all P<0.01). The mRNA expression of the lipid metabolism genes Pgc-1α and Cpt1 were increased (all P<0.01) and the lipid synthesis-related genes Srebp1c, Acc and Fasn were decreased (all P<0.01); the expression of liver inflammation-related genes Tgf-β, Tnf-α and Il-6 were decreased (all P<0.01).@*CONCLUSIONS@#The si-Cyp2e1 LNP may attenuate subacute alcoholic liver injury in mice mainly by reducing reactive oxygen levels, increasing antioxidant activity, blocking oxidative stress pathways and reducing ethanol-induced steatosis and inflammation.

Progresses on RNA-based therapeutics for genetic diseases / 浙江大学学报·医学版

RNA therapeutics inhibit the expression of specific proteins/RNAs by targeting complementary sequences of corresponding genes, or synthesize proteins encoded by the desired genes to treat genetic diseases. RNA-based therapeutics are categorized as oligonucleotide drugs (antisense oligonucleotides, small interfering RNA, RNA aptamers), and mRNA drugs. The antisense oligonucleotides and small interfering RNA for treatment of genetic diseases have been approved by the FDA in the United State, while RNA aptamers and mRNA drugs are still in clinical trials. Chemical modifications are applied to RNA drugs, such as pseudouridine modification of mRNA, to reduce immunogenicity and improve the efficacy. The secure and effective delivery systems like lipid-based nanoparticles, extracellular vesicles, and virus-like particles are under development to address stability, specificity, and safety issues of RNA drugs. This article provides an overview of the specific molecular mechanisms of 11 RNA drugs currently used for treating genetic diseases, and discusses the research progress of chemical modifications and delivery systems of RNA drugs.

Application progress on contrast-enhanced ultrasound in acute rejection after kidney transplantation / 器官移植

Early diagnosis of acute rejection is of significance for the protection of renal allograft function. Pathological puncture biopsy is the gold standard for the diagnosis of acute rejection of renal allografts. Nevertheless, it may provoke multiple complications, such as bleeding, infection and renal parenchymal injury, which limit its widespread application. In recent years, the sensitivity of contrast-enhanced ultrasound in the diagnosis of acute rejection has been constantly improved. Ultrasound-targeted microbubble technique has further enhanced the diagnostic specificity of contrast-enhanced ultrasound, making it possible to replace pathological puncture biopsy. Besides, in the field of acute rejection treatment, microbubble ultrasonic cavitation may promote local delivery of immunosuppressants by inducing sonoporation and exhibit anti-rejection effect. In this article, the application of contrast-enhanced ultrasound in the diagnosis and treatment of acute rejection after kidney transplantation was reviewed, aiming to provide reference for widespread application of contrast-enhanced ultrasound in kidney transplantation.

Advances in approved nucleic acid drugs and lipid nanoparticle system / 药学学报

Nucleic acids, as a next generation of biotechnology drugs, not only can fundamentally treat diseases, but also own significant platform characteristics in view of technology and production. Therefore, nucleic acid-based drugs have broad clinical applications in biomedical fields. However, nucleic acids are degradable and unstable, and have very low intracellular delivery efficiency in vitro and in vivo, which greatly limits their applications. In recent years, ionizable lipid-based lipid nanoparticles have shown promising application potentials and have been successfully applied to COVID-19 (Coronavirus Disease 2019) vaccines in clinic. Lipid nanoparticles demonstrate high in vivo delivery efficiency and good safety profile due to their unique structural and physicochemical properties, which provides many possibilities for their clinical applications for nucleic acid delivery in the future. This review focused on the characteristics of nucleic acid drugs and their delivery barriers, and discussed the approved nucleic acid drugs to illustrate the key aspects of the success of their delivery carrier system. In addition, problems to be solved in the field were highlighted.

Targeted therapy of Apt-siRNA combining with docetaxel for PSMA-positive prostate cancer and monitoring imaging of 99Tc m-Apt-siRNA / 中华核医学与分子影像杂志

Objective:Apt-A10-3.2 (aptamer of prostate specific membrane antigen (PSMA)) can be used as a specific ligand for early diagnosis and targeted treatment of prostate cancer. Mouse double minute 2 homolog (MDM2) is closely related to the malignancy of prostate cancer, and MDM2 small interfering RNA (siRNA) can silence MDM2 gene through RNA interference. To design a novel chimera of PSMA Apt-MDM2 siRNA and combine it with docetaxel (DTX) to explore a new diagnosis and treatment model combining targeted therapy of PSMA-positive prostate cancer with 99Tc m-chimera imaging monitoring. Methods:Apt-siRNA were obtained by covalent connection of PSMA Apt-A10-3.2 and MDM2 siRNA, which was combined with DTX to treat PSMA-positive prostate cancer cell lines (22RV1 and LNCaP). Cell lines were treated with Apt-siRNA alone or in combination with DTX. The levels of MDM2 and apoptosis-related proteins (B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X (Bax), poly ADP-ribose polymerase (PARP), caspase-3) were detected by Western blot, which were used to evaluate the therapeutic effect. Fifteen BALB/c mice bearing 22RV1 xenografts were treated with PBS, DTX+ Apt-siRNA (200 pmol) and DTX+ Apt-siRNA (400 pmol), respectively. Tumor volume and MDM2 level were observed, and 99Tc m-Apt-siRNA SPECT imaging was performed to obtain the tumor/muscle (T/M) ratio. One-way analysis of variance, Tukey′s test and linear regression analysis were used for data analysis. Results:The levels of MDM2 protein were significantly decreased by Apt-siRNA (0.25±0.02, F=183.40, P<0.001; 0.56±0.03, F=37.15, P<0.001) in 22RV1 and LNCaP cells. After the treatment of Apt-siRNA+ DTX, the levels of Bcl-2 were significantly decreased, and the levels of Bax, PARP and caspase-3 were significantly increased. MDM2 protein level (400 pmol: 0.59±0.12; F=49.99, P=0.023) and tumor volume (400 pmol: (0.22±0.07) cm 3;F=71.30, P=0.039) were significantly inhibited by Apt-siRNA+ DTX in mice bearing 22RV1 xenografts. As for 99Tc m-Apt-siRNA SPECT imaging in vivo, T/M ratio of treatment group was significantly decreased (400 pmol: 2.07±0.22; F=34.99, P=0.022), and there was a linear regression relationship between T/M ratio and the expression level of MDM2 ( R2=0.875, P<0.001). Conclusion:Apt-siRNA combined with DTX can effectively inhibit the progression of prostate cancer, and realize visual targeted diagnosis and treatment of PSMA-positive prostate cancer by coupling radionuclide technetium.

Role of biomimetic nanomaterials made from glioma cell- derived extracellular vesicles in targeted delivery of STAT3-siRNA / 中南大学学报(医学版)

OBJECTIVES@#Glioma is the most common primary intracranial tumor and there is still no ideal treatment at present. Gene therapy, as one of the new methods for treating glioma, has attracted attention in recent years. But its application in treating glioma is very limited due to lack of effective delivery vectors. This study aims to investigate the feasibility of biomimetic nanomaterials made from glioma cells-derived extracellular vesicles (EV) for targeted delivery of signal transducers and activators of transcription 3 (STAT3)-small interfering RNA (siRNA) in treating glioma.@*METHODS@#First, U251 glioma cells-derived extracellular vessel (EVU251) was extracted by ultra-centrifugal method. Nanoparticle tracking analysis was used to characterize the particle size distribution, the transmission electron microscope was used to analyze the morphology, and Western blotting was used to verify the expression of srface characteristic protein. The homing ability was verified by cell uptake assay after labeling EVU251 with membrane dye kit PKH67; the EVU251 contents were removed by a low permeability method and then EVMU251 was prepared through a microporous membrane. Finally, the biomimetic nanomaterials EVMU251@STAT3-siRNA were prepared by loading STAT3-SiRNA with electro-dyeing method. The real-time quantitative PCR was used to quantify the successful encapsulation of siRNA, and the encapsulation and drug loading rate was calculated; then Cy5-labeled siRNA was used to evaluate the ability of biomimetic nanomaterials (EVMU251@CY5-siRNA) to target U251 cells. Lysosomal escape ability of the biomimetic nanomaterial was evaluated by lysosomal dye lyso-tracker green. At last, the ability of EVMU251@STAT3-siRNA to knock down STAT3 gene and selective killing of U251 cells was detected by cell experiments in vitro.@*RESULTS@#The size of EVU251 ranged from 50 nm to 200 nm with a natural disc shape. The expression of extracellular vesicle marker proteins could be detected on the membrane of EVU251. The cell uptake assay demonstrated that it had homing ability to target U251 cells. After EVU251 was prepared as EVMU251@STAT3-siRNA, the particle size was (177.9±5.0) nm, the siRNA loading rate was (33.5±2.2)% and the drug loading rate was (3.24±0.21)%. The biomimetic nanomaterial EVMU251@STAT3-siRNA still had the ability to target U251 cells and successfully deliver siRNA to the cytoplasm without lysosomal degradation. The EVMU251@STAT3-siRNA can effectively knock down the expression of STAT3 gene and produce selective killing ability in U251 cells.@*CONCLUSIONS@#The biomimetic nanomaterials EVMU251@STAT3-siRNA made from glioma U251 cells-derived extracellular vesicles can knock down STAT3 gene of U251 cells and produce selective killing effect, which can provide a new idea for the treatment of glioma.

Tumor microenvironment responsive liposomes blocking CXCL12/CXCR4 pathway and synergistically enhancing immune efficacy of anti-PD-L1 / 药学学报

Blocking immune checkpoint programmed cell death receptor 1 (PD-1) or programmed death receptor-ligand 1 (PD-L1) can enhance anti-tumor activity of effector T cells. However, the lack of response in many patients to PD-1/PD-L1 therapy remains a question. Improving the immunosuppressive tumor microenvironment (TME) to enhance the efficacy of immune checkpoint inhibitors has become a promising cancer treatment strategy. We constructed a liposome system (PD-L1/siCXCL12-Lp) of CXCL12 siRNA and anti-PD-L1 peptide with matrix metalloproteinases (MMPs) responsiveness, which combined the TME regulation of siCXCL12 and the immune regulation of anti-PD-L1 peptide. All animal experiments were approved by the Biomedical Ethics Committee of Peking University. The authors found that PD-L1/siCXCL12-Lp directly down-regulated the expression of CXCL12 in vitro (33.8%) and in vivo (15.5%). It also effectively increased the ratio of CD8+/Treg by 20.0%, which helped the anti-PD-L1 peptide to better exert its immune effect. The combination therapy significantly inhibited tumor growth (52.08%) with great safety, which explored a new idea for cancer immunotherapy.