MiR-142-3p Regulates ILC1s by Targeting HMGB1 via the NF-κB Pathway in a Mouse Model of Early Pregnancy Loss

Objective Innate lymphoid cells(ILCs)are a class of newly discovered immunocytes.Group 1 ILCs(ILC1s)are identified in the decidua of humans and mice.High mobility group box 1(HMGB1)is predicted to be one of the target genes of miR-142-3p,which is closely related to pregnancy-related diseases.Furthermore,miR-142-3p and HMGB1 are involved in regulating the NF-κB signaling pathway.This study aimed to examine the regulatory effect of miR-142-3p on ILC1s and the underlying mechanism involving HMGB1 and the NF-κB signaling pathway.Methods Mouse models of normal pregnancy and abortion were constructed,and the alterations of ILC1s,miR-142-3p,ILC1 transcription factor(T-bet),and pro-inflammatory cytokines of ILC1s(TNF-α,IFN-γand IL-2)were detected in mice from different groups.The targeting regulation of HMGB1 by miR-142-3p in ILC1s,and the expression of HMGB1 in normal pregnant mice and abortive mice were investigated.In addition,the regulatory effects of miR-142-3p and HMGB1 on ILC1s were detected in vitro by CCK-8,Annexin-V/PI,ELISA,and RT-PCR,respectively.Furthermore,changes of the NF-κB signaling pathway in ILC1s were examined in the different groups.For the in vivo studies,miR-142-3p-Agomir was injected in the uterus of abortive mice to evaluate the abortion rate and alterations of ILC1s at the maternal-fetal interface,and further detect the expression of HMGB1,pro-inflammatory cytokines,and the NF-κB signaling pathway.Results The number of ILC1s was significantly increased,the level of HMGB1 was significantly upregulated,and that of miR-142-3p was considerably downregulated in the abortive mice as compared with the normal pregnant mice(all P<0.05).In addition,miR-142-3p was found to drastically inhibit the activation of the NF-κB signaling pathway(P<0.05).The number of ILC1s and the levels of pro-inflammatory cytokines were significantly downregulated and the activation of the NF-κB signaling pathway was inhibited in the miR-142-3p Agomir group(all P<0.05).Conclusion miR-142-3p can regulate ILC1s by targeting HMGB1 via the NF-κB signaling pathway,and attenuate the inflammation at the maternal-fetal interface in abortive mice.

Anti-PD1 antibody and not anti-LAG-3 antibody improves the antitumor effect of photodynamic therapy for treating metastatic breast cancer

Photodynamic therapy(PDT)has limited effects in treating metastatic breast cancer.Immune checkpoints can deplete the function of immune cells;however,the expression of immune checkpoints after PDT is unclear.This study investigates whether the limited e±cacy of PDT is due to upregulated immune checkpoints and tries to combine the PDT and immune checkpoint inhibitor to observe the e±cacy.A metastatic breast cancer model was treated by PDT mediated by hematoporphyrin derivatives(HpD-PDT).The anti-tumor effect of HpD-PDT was observed,as well as CD4þT,CD8þT and calreticulin(CRT)by immunohistochemistry and immunofluorescence.Immune checkpoints on T cells were analyzed byflow cytometry after HpD-PDT.When combining PDT with immune checkpoint inhibitors,the antitumor effect and immune effect were assessed.For HpD-PDT at 100 mW/cm2 and 40,60 and 80 J/cm2,primary tumors were suppressed and CD4þT,CD8þT and CRT were elevated;however,distant tumors couldn't be inhibited and survival could not be prolonged.Immune checkpoints on T cells,especially PD1 and LAG-3 after HpD-PDT,were upregulated,which may explain the reason for the limited HpD-PDT effect.After PDT combined with anti-PD1 antibody,but not with anti-LAG-3 antibody,both the primary and distant tumors were signi-cantly inhibited and the survival time was prolonged,additionally,CD4þT,CD8þT,IFN-þCD4þT and TNF-þCD4þT cells were signi-cantly increased compared with HpD-PDT.HpD-PDT could not combat metastatic breast cancer.PD1 and LAG-3 were upregulated after HpD-PDT.Anti-PD1 antibody,but not anti-LAG-3 antibody,could augment the antitumor effect of HpD-PDT for treating metastatic breast cancer.

Exploring the relationship between red blood cell levels and emotional regulation through the miR191-Riok3-Mxi1 pathway

Objective:To assess emotional fluctuations,physical and mental health status,and indicators closely related to red blood cells,such as RIO kinase 3(Riok3),MAX interactor 1(Mxi1),and microRNA 191(miR191),in participants with different levels of red blood cells.Methods:Participants who underwent physical examinations at Dongfang Hospital between April and October 2019 were divided into healthy,blood deficiency,and anemia groups(30 individuals in the healthy and blood deficiency group respectively,and 13 in the anemia group).The physical and mental conditions of the participants were evaluated through questionnaires,and emotional fluctuations were assessed through an emotion-inducing experiment,in which participants watched video segments designed to induce specific emotions.Relative expression levels of miR191,Riok3,and Mxi1 from venous blood samples were also determined.Results:The main psychological factors identified in the anemia and blood deficiency groups were obsessive-compulsive symptoms,depression,anxiety,and other negative emotions.Relative gene expression levels indicated that miR191 was upregulated and Riok3 and Mxi1 were downregulated in both the blood deficiency and anemia groups.Regarding the emotional score of disgust on video stage,the main effect was significant(F=335.58,P<.001),which showed that watching the three videos caused participants to have a dominant emotion,and there is a difference on group(F=5.35,P=.01),with higher disgust scores in the anemia and blood deficiency groups.The symptoms of blood deficiency and anemia,such as weakness in limbs were significantly negatively correlated with Riok3 and Mxi1 expression(r=-0.38 and-0.31 respectively),but was significantly positively correlated with miR191 expression(r=0.29).Conclusion:We determined that a close relationship exists between red blood cell levels and emotional status.Our findings suggest that individuals with anemia and blood deficiency are more likely to experience psychological problems and negative emotions,particularly disgust.We also demonstrate that emotional regulation is related to mir191-Riok3-Mxi1 pathway activity,identifying these pathway components are potential targets for genetic therapies in combination with psychological therapy.

Exploring the mechanism of electroacupuncture at different acupoints on acute colitis rats based on JAK2/STAT3/SOCS1 signaling pathway

Objective:To investigate the mechanism of JAK2/STAT3/SOCS1 signaling pathway in electroacupuncture of different acupoints on acute colitis rats.Methods:36 SPF SD rats were randomly divided into 6 groups,with 6 rats in each group.The rat model of acute colitis was prepared by enema with glacial acetic acid solution.After the model was established,electroacupuncture was given to each acupoint group,with density wave,frequency 2Hz-50 Hz,intensity 2 mA,muscle tremor as the degree 20 min/time,1 time/day,for 3 consecutive days.Observe the general condition of rats;the pathological changes of colonic mucosa in rats were observed by HE method.The contents of serum interleukin-4(IL-4)and interleukin-8(IL-8)were detected by ELISA.Western blot and RT-PCR were used to detect the expression of JAK2,STAT3,SOCS1 protein and mRNA in rat colon tissue.Results:In contrast to the normal group,the overall condition of the model group was worse,the colonic mucosa was severely damaged,even necrotic,and the ulcer surface was obvious.The content of IL-4 in serum was obviously reduced,and the content of IL-8 was obviously go up(P<0.01).The protein content of JAK2,STAT3 and the expression of JAK2,STAT3 mRNA in colon tissue of rats were obviously go up,while the protein content of SOCS1 and the expression of SOCS1 mRNA were obviously reduced(P<0.01).In contrast to the model group,the general condition of rats in each acupoint group was significantly improved,the damage and necrosis of colonic mucosa and ulcer surface were obviously alleviated,the content of IL-4 in serum was obviously go up,and the content of IL-8 was significantly decreased(P<0.01).The protein content of JAK2,STAT3 and the expression of JAK2,STAT3 mRNA in colon tissue of rats were obviously reduced,while the protein content of SOCS1 and the expression of SOCS1 mRNA were obviously go up(P<0.05,P<0.01).Comparison of different acupoint groups,the colonic mucosal injury in the Zusanli group was significantly reduced,the content of serum IL-4 was significantly increased,and the content of IL-8 was significantly decreased(P<0.05,P<0.01).The protein content and mRNA expression of JAK2 and STAT3 in colon tissue were significantly down-regulated,while the protein content and mRNA expression of SOCS1 were significantly go up(P<0.05,P<0.01).Conclusion:Electroacupuncture at each acupoint can improve the damage of colonic mucosa and reduce the inflammatory response.The therapeutic effect of Zusanli(ST36)is better than that of Tianshu(ST25),Dachangshu(BL25)and Shangjuxu(ST37).The mechanism may be related to the regulation of JAK2/STAT3/SOCS1 signaling pathway related proteins and inflammatory cytokines IL-4 and IL-8.

METTL3介导的CCNE1 m^(6)A修饰调控乳腺癌细胞对哌柏西利敏感性的研究

目的:探讨RNA甲基转移酶METTL3介导的m^(6)A修饰对乳腺癌细胞哌柏西利敏感性的影响及作用机制。方法:将乳腺癌细胞MCF-7随机分为:si-NC组,si-METTL3组,si-CCNE1组,si-METTL3+OE-NC组和si-METTL3+OE-CCNE1组;qPCR和Western blot检测METTL3和CCNE1的mRNA和蛋白表达水平;CCK-8检测哌柏西利敏感性;RIP实验检测METTL3和CCNE1 mRNA的相互作用;MeRIP-qPCR检测CCNE1的m^(6)A修饰水平;加入放线菌素D检测CCNE1 mRNA稳定性。结果:在MCF-7细胞中,METTL3和CCNE1的mRNA水平显著升高(P<0.05);抑制METTL3或抑制CCNE1的表达显著提高MCF-7细胞对哌柏西利的敏感性(P<0.05),且抑制METTL3显著降低CCNE1的mRNA和蛋白表达水平(P<0.05);RIP实验证实METTL3和CCNE1 mRNA具有相互作用,且CCNE1发生了m^(6)A修饰;与si-NC组相比,si-METTL3组中CCNE1的m^(6)A修饰水平显著减少,CCNE1 mRNA稳定性显著降低;进一步实验表明,与si-METTL3+OE-NC组相比,si-METTL3+OE-CCNE1组中哌柏西利敏感性显著降低(P<0.05)。结论:在MCF-7细胞中,METTL3和CCNE1表达均上调;抑制METTL3能够提高MCF-7细胞对哌柏西利的敏感性,这与METTL3介导的m^(6)A修饰调控CCNE1 mRNA稳定性有关。

The flavonoid glycoside vaccarin inhibits adipogenesis and stimulates lipolysis via Hedgehog signaling in 3T3-L1 adipocytes

Vaccarin,a flavonoid glycoside isolated from Vaccaria segetalis,is non-toxic to 3T3-L1 cells up to concentrations of 200μM.Accordingly,we investigated the effects of this natural product on adipogenesis and lipolysis in 3T3-L1 adipocytes.Our results revealed that vaccarin significantly inhibited lipid accumulation by suppressing the adipogenesis-related transcription factors peroxisome proliferator-activated receptorγ(PPARγ)and the CCAAT/enhancer-binding proteinα(C/EBPα).Specifically,lipid accumulation decreased by up to 27.7±2.7%when 3T3-L1 adipocytes were treated with a 10μM concentration of vaccarin.Mechanistic studies showed that the compound inhibited adipogenesis through activation of the Hedgehog(Hh)signaling pathway and so restoring Smo and Gli1 expression at an early stage of differentiation.In mature 3T3-L1 cells,vaccarin significantly increased the secretion of glycerol into the surrounding medium and thus indicating that it accelerated the degradation of triglycerides.In addition,vaccarin,was shown to enhance lipolysis through stimulation of the transcription levels of lipoprotein lipase,monoglycerides lipase,adipose triacylglyceride lipase,hormone-sensitive lipase and adipose differentiated-related protein.All told,vaccarin suppressed lipid accumulation and enhanced lipolysis during adipocyte differentiation by restoring Hh signaling.As such,it is a phytochemical capable of halting adipocyte hyperplasia and,thereby,ameliorating the effects of obesity.

3-Dimensional Kinematic Comparison of Arm Movements between an Individual with NGLY1 Deficiency and a Neurotypical Individual

NGLY1 Deficiency is an ultra-rare autosomal recessively inherited disorder. Characteristic symptoms include among others, developmental delays, movement disorders, liver function abnormalities, seizures, and problems with tear formation. Movements are hyperkinetic and may include dysmetric, choreo-athetoid, myoclonic and dystonic movement elements. To date, there have been no quantitative reports describing arm movements of individuals with NGLY1 Deficiency. This report provides quantitative information about a series of arm movements performed by an individual with NGLY1 Deficiency and an aged-matched neurotypical participant. Three categories of arm movements were tested: 1) open ended reaches without specific end point targets;2) goal-directed reaches that included grasping an object;3) picking up small objects from a table placed in front of the participants. Arm movement kinematics were obtained with a camera-based motion analysis system and “initiation” and “maintenance” phases were identified for each movement. The combination of the two phases was labeled as a “complete” movement. Three-dimensional analysis techniques were used to quantify the movements and included hand trajectory pathlength, joint motion area, as well as hand trajectory and joint jerk cost. These techniques were required to fully characterize the movements because the NGLY1 individual was unable to perform movements only in the primary plane of progression instead producing motion across all three planes of movement. The individual with NGLY1 Deficiency was unable to pick up objects from a table or effectively complete movements requiring crossing the midline. The successfully completed movements were analyzed using the above techniques and the results of the two participants were compared statistically. Almost all comparisons revealed significant differences between the two participants, with a notable exception of the 3D initiation area as a percentage of the complete movement. The statistical tests of these measures revealed no significant differences between the two participants, possibly suggesting a common underlying motor control strategy. The 3D techniques used in this report effectively characterized arm movements of an individual with NGLY1 deficiency and can be used to provide information to evaluate the effectiveness of genetic, pharmacological, or physical rehabilitation therapies.

Prediction of the Photofading of Selected Derivatives of 5-(4-X-Phenylazo)-3-Cyano-1-(H or Ethyl)-6-Hydroxy-4-Methyl-2-Pyridone: Theoretical Studies, Comparison of AM1 and PM3 Methods

We analysed the photooxidation reaction in the electro-(1O2) and nucleophilic (O2•−) reaction of 2-pyridone azo derivatives. First, we calculated the energy (enthalpies) of tautomers formation, which is a measure of durability and the probability of their formation. We performed the light fastness calculations of the monoazopyridone dyes. Using the semi-empirical methods of quantum chemistry AM1 and PM3, the reactivity indicators of superdelocalisability (SrE(N)) and the electron density distribution in ground state on the highest occupied HOMO orbital and the lowest unoccupied excited state LUMO in 2-pyridone phenylazo derivatives were calculated. Superdelocalisability coefficients enable the stability to oxidising agents of various chemical molecules depending on the tautomeric forms in which they may occur. The results of the electron density calculations at the HOMO and LUMO boundary orbitals allow to determine the tendency to electrophilic attack with singlet oxygen 1O2 or nucleophilic attack of the superoxide anion O2•−on a specific atom in the molecule. The structure of the dyes was optimised with MM+, MD and AM1 or PM3 until a constant energy value was achieved with a convergence criterion of 0.01 kcal/mol.

2型糖尿病合并骨质疏松症患者血清Chi3L1 NEFAs与糖脂代谢及骨密度的相关性

目的:探讨2型糖尿病(T2DM)合并骨质疏松患者血清壳多糖酶3样蛋白1(Chi3L1)、游离脂肪酸(NEFAs)与糖脂代谢及骨密度的相关性。方法:选取2020年1月至2023年4月在我院收治的110例T2DM患者作为研究对象,根据其是否发生骨质疏松,将T2DM患者分为骨质疏松组(38例)和非骨质疏松组(72例)。收集T2DM患者的临床资料。结果:骨质疏松组中Chi3L1水平显著低于非骨质疏松组,而NEFAs水平显著高于非骨质疏松组(P<0.001)。骨质疏松组患者的糖化血红蛋白(HbAlc)、胰岛素抵抗指数(HOMA-IR)均显著高于非骨质疏松组(P<0.05),骨密度显著低于非骨质疏松组(P<0.05)。血清Chi3L1水平与HbAlc及HOMA-IR均呈现负相关性,与骨密度呈现正相关性(P<0.05);NEFAs水平与HbAlc及HOMA-IR均呈现正相关性,与骨密度呈现负相关性(P<0.05)。与非骨质疏松组相比,骨质疏松组T2DM患者握力水平低患者比例升高(P<0.05)。NEFAs、Chi3L1均是T2DM患者发生骨质疏松的影响因素(P<0.05)。结论:血清Chi3L1水平降低、NEFAs水平升高与T2DM患者发生骨质疏松有关,且与糖脂代谢及骨密度存在显著相关性。

Adsorption mechanism of N-laurel-1,3-diaminopropane in a hematite-quartz flotation system

The strong collecting performance of N-laurel-1,3-diaminopropane（ND13） with respect to quartz encouraged us to study its separation of hematite and quartz mixtures in a laboratory cell flotation test The results show that the best separation results can be achieved when the pulp pH is 7.27 and 58.3 mg/L collector plus 6.67 mg/L depressant are added to the mixture.Products with 58.45%,62.78%and 63.72% iron grades can be achieved respectively when mass ratio of hematite to quartz is 2：3,1：1,and 3：2.The adsorption mechanism of ND13 on a quartz surface was investigated by zeta-potential and X-ray photoelectron spectroscopy measurements.The results reveal that electrostatic and hydrogen bonding adsorption take place between ND13 and the quartz surface,and that ND13 mainly interacts with the oxygen atoms on the quartz surface.

CsABF3-activated CsSUT1 pathway is implicated in pre-harvest water deficit inducing sucrose accumulation in citrus fruit

Pre-harvest water deficit(PHWD)plays an important role in sugar accumulation of citrus fruit.However,the mechanism is not known well.Here,it was confirmed that PHWD promoted sucrose accumulation of citrus fruit,but had limited effect on fructose,glucose and total acid.A sucrose transporter,Cs SUT1,which localizes to the plasma membrane,was demonstrated to function in sucrose transport induced by PHWD.Compared to wild-type,Cs SUT1 overexpression in citrus calli stimulated sucrose,fructose and glucose accumulation,while its silencing in juice sacs reduced sucrose accumulation.Increased sugar accumulation in transgenic lines enhanced plant drought tolerance,and resulted in decreased electrolyte leakage,malondialdehyde and hydrogen peroxide contents,as well as increased superoxide dismutase activity and proline contents.An abscisic acid(ABA)-responsive transcription factor,Cs ABF3,was found with a same expression pattern with Cs SUT1 under PHWD.Yeast one-hybrid,electrophoretic mobility shift assay and dual-luciferase assays all revealed that Cs ABF3 directly bound with the Cs SUT1 promoter by ABA responsive elements.When Cs ABF3 was overexpressed in citrus calli,the sucrose,fructose and glucose concentration increased correspondingly.Further,transgenic studies demonstrated that Cs ABF3 could affect sucrose accumulation by regulating Cs SUT1.Overall,this study revealed a regulation of Cs ABF3 promoting Cs SUT1 expression and sucrose accumulation in response to PHWD.Our results provide a detail insight into the quality formation of citrus fruit.

YBX1 inhibits mitochondrial-mediated apoptosis in ischemic heart through the PI3K/AKT signaling pathway

Background:Myocardial infarction(MI)is associated with higher morbidity and mortality in the world,especially in cold weather.YBX1 is an RNA-binding protein that is required for pathological growth of cardiomyocyte by regulating cell growth and protein synthesis.But YBX1,as an individual RNA-binding protein,regulates cardiomyocytes through signaling cascades during myocardial infarction remain largely unexplored.Methods:In vivo,the mouse MI model was induced by ligating the left anterior descending coronary artery(LAD),and randomly divided into sham operation group,MI group,MI+YBX1 knockdown/overexpression group and MI+negative control(NC)group.The protective effect of YBX1 was verified by echocardiography and triphenyltetrazolium chloride staining.In vitro,mitochondrial-dependent apoptosis was investigated by using CCK8,TUNEL staining,reactive oxygen species(ROS)staining and JC-1 staining in hypoxic neonatal mouse cardiomyocytes(NMCMs).Results:YBX1 expression of cardiomyocytes was downregulated in a mouse model and a cellular model on the ischemic condition.Compared to mice induced by MI,YBX1 overexpression mediated by adeno-associated virus serotype 9(AAV9)vector reduced the infarcted size and improved cardiac function.Knockdown of endogenous YBX1 by shRNA partially aggravated ischemia-induced cardiac dysfunction.In hypoxic cardiomyocytes,YBX1 overexpression decreased lactic dehydrogenase(LDH)release,increased cell viability,and inhibited apoptosis by affecting the expression of apoptosis related proteins,while knockdown of endogenous YBX1 by siRNA had the opposite effect.Overexpression of YBX1 restored mitochondrial dysfunction in hypoxic NMCMs by increasing mitochondrial membrane potential and ATP content and decreasing ROS.In hypoxic NMCMs,YBX1 overexpression increased the expression of phosphorylated phosphatidylinositol 3 kinase(PI3K)/AKT,and the anti-apoptosis effect of YBX1 was eliminated t by LY294002,PI3K/AKT inhibitor.Conclusion:YBX1 protected the heart from ischemic damage by inhibiting the mitochondrial-dependent apoptosis through PI3K/AKT pathway.It is anticipated that YBX1 may serve as a novel therapeutic target for MI.

circDNMT1 调节 miR-377-3p/PUM1 轴对三阴性乳腺癌细胞增殖、凋亡和上皮-间质转化的影响

目的探讨circDNMT1调节miR-377-3p/PUM1轴对三阴性乳腺癌(TNBC)细胞增殖、凋亡和上皮-间质转化(EMT)的影响。方法收集儋州市人民医院2018年至2021年收治的24例TNBC患者的TNBC组织和其邻近正常组织,采用qRT-PCR、Western blot法分别检测组织及小鼠正常乳腺上皮细胞系HC11和TNBC细胞系4T1、Eph41424、JC中circDNMT1表达、miR-377-3p表达、PUM1蛋白表达。将4T1细胞分为4T1组(未转染)、si-NC组(转染si-NC)、si-DNMT1组(转染si-DNMT1)、si-DNMT1+anti-NC组(同时转染si-DNMT1和anti-NC)、si-DNMT1+anti-miR-377-3p组(同时转染si-DNMT1和anti-miR-377-3p),qRT-PCR检测各组4T1细胞中circDNMT1、miR-377-3p表达;CCK-8检测各组4T1细胞增殖情况;流式细胞术检测各组4T1细胞凋亡情况;Western blot检测各组4T1细胞PUM1、EMT相关蛋白及凋亡相关蛋白表达;TargetScan网站预测miR-377-3p与circDNMT1、PUM1的结合位点,双荧光素酶报告验证miR-377-3p与circDNMT1、PUM1的靶向关系。BALB/c小鼠接种4T1细胞后随机分为空白对照组(注射等量生理盐水)、阴性对照组(尾静脉注射si-NC)、DNMT1沉默组(尾静脉注射si-DNMT1)、联合对照组(尾静脉注射si-DNMT1和anti-NC)、联合沉默组(尾静脉注射si-DNMT1和anti-miR-377-3p),记录小鼠肿瘤质量,采用苏木精-伊红染色观察肿瘤形态变化。结果在TNBC组织和细胞中circDNMT1、PUM1上调,miR-377-3p下调,且在4T1细胞中表达差异最显著,因此本研究选择4T1细胞进行后续实验。与4T1组、si-NC组比较,si-DNMT1组4T1细胞中miR-377-3p表达、4T1细胞凋亡率及Bax、cleaved caspase-3、E-cadherin蛋白表达水平显著升高(P<0.05),circDNMT1水平、PUM1蛋白表达水平、24 h和48 h OD值、Bcl-2蛋白表达水平、N-cadherin蛋白表达水平、Vimentin蛋白表达水平均显著减少/降低(P<0.05);与si-DNMT1组、si-DNMT1+anti-NC组比较,si-DNMT1+anti-miR-377-3p组4T1细胞中miR-377-3p表达、4T1细胞凋亡率、Bax蛋白表达水平、cleaved caspase-3蛋白表达水平、E-cadherin蛋白表达水平显著降低(P<0.05),PUM1蛋白表达水平、24 h和48 h OD值、Bcl-2蛋白表达水平、N-cadherin蛋白表达水平、Vimentin蛋白表达水平均显著增加/升高(P<0.05)。与miR-NC+WT-circDNMT1组比较,miR-377-3p mimic+WT-circDNMT1组细胞荧光素酶活性显著降低(P<0.05);与miR-NC+WT-PUM1组比较,miR-377-3p mimic+WT-PUM1组细胞荧光素酶活性显著降低(P<0.05)。空白对照组、阴性对照组小鼠肿瘤细胞排列密集,边界清晰;DNMT1沉默组、联合对照组肿瘤细胞排列疏松,胞核固缩,细胞碎片增多;联合沉默组小鼠肿瘤细胞排列比较密集,边界趋于清晰。与空白对照组、阴性对照组比较,DNMT1沉默组小鼠肿瘤质量显著减少(P<0.05);与DNMT1沉默组、联合对照组比较,联合沉默组小鼠肿瘤质量显著增加(P<0.05)。结论沉默circDNMT1可能通过上调miR-377-3p来抑制PUM1表达,进而抑制TNBC细胞增殖和EMT,促进细胞凋亡。

胃窦癌组织中LAG-3 FGL1 MHC-Ⅱ的表达与预后的关系

目的:探索新型免疫检查点淋巴细胞激活基因3(lymphocyte-activation gene 3,LAG-3)、纤维蛋白原样蛋白1(fibrinogenlike protein 1,FGL1)、主要组织相容性复合体Ⅱ类分子(major histocompatibility complex classⅡ,MHC-Ⅱ)在胃窦癌(gastric antral cancer,GAC)中的表达情况与预后的相关性。方法:收集2012年1月至2014年12月于天津医科大学肿瘤医院诊断为GAC的67例患者病理标本,分别进行石蜡切片制作,采用免疫组织化学法检测LAG-3、FGL1、MHC-Ⅱ三个指标的表达情况,并用统计学方法分析组间差异。采用Kaplan-Meier法评估LAG-3、FGL1、MHC-Ⅱ的表达水平与GAC患者预后之间的关系并绘制生存曲线。结果:GAC患者中,肿瘤大小<4 cm的患者和无淋巴结转移的患者LAG-3免疫细胞阳性率更高(P<0.05);女性患者MHC-Ⅱ免疫细胞阳性率更高(P<0.05)。免疫细胞中LAG-3、MHC-Ⅱ高表达的患者总生存期(overall survival,OS)较好(P<0.05);肿瘤细胞中MHC-Ⅱ高表达的患者OS、无病生存期(disease-free survival,DFS)较差(P<0.05);而FGL1在免疫细胞和肿瘤细胞中的表达与OS、DFS无显著相关性(P>0.05)。结论:GAC患者LAG-3、MHC-Ⅱ在不同区域的表达量存在差异,GAC患者LAG-3及其配体在免疫细胞的表达对预后产生积极影响,提示免疫细胞中LAG-3/MHC-Ⅱ可以作为GAC患者预后标志物,为临床个体化免疫治疗提供新的依据。

METTL3介导的PDK1 mRNA m^(6)A修饰通过Akt/mTOR信号通路促进肺上皮细胞增殖

腺苷N6-位点甲基化(m^(6)A)在细胞增殖过程中发挥重要作用。RNA甲基转移酶3(METTL3)作为催化m^(6)A关键酶,其介导m^(6)A修饰在肺上皮细胞增殖中的作用机制尚不明确。本研究旨在探讨METTL3介导m^(6)A修饰调控肺上皮细胞增殖的效应及机制。结果显示,在肺上皮细胞中敲低METTL 3显著抑制细胞生长,而过表达METTL3则促进了细胞增殖(P<0.05)。进一步的蛋白质免疫印迹结果显示,细胞生长和增殖的关键蛋白质PCNA在METTL 3敲降的肺上皮细胞中蛋白质水平的表达显著下调,并且Akt以及mTOR的磷酸化水平显著降低(P<0.05)。细胞免疫荧光结果发现,METTL 3敲降的肺上皮细胞中m^(6)A修饰水平显著降低(P<0.05)。实时荧光定量PCR及蛋白质免疫印迹结果表明,Akt-mTOR信号通路上游调控分子PDK1的mRNA和蛋白质表达水平在METTL 3敲降的肺上皮细胞中显著下降(P<0.05)。机制上,m^(6)A-IP-qPCR和RIP-qPCR结果进一步表明,METTL3催化PDK 1 mRNA的3′UTR区域m^(6)A修饰,进而被YTH N6-甲基腺苷RNA结合蛋白1(YTHDF1)识别,增强其mRNA的稳定性。总之,本研究揭示了METTL3通过增强PDK1 m^(6)A修饰,进而激活Akt-mTOR信号通路,促进细胞增殖。本研究为METTL3在上皮细胞增殖中的新角色提供了证据,同时为治疗肺上皮细胞损伤修复提供了新的治疗靶点。

DNA Methylation of KLRC1 and KLRC3 in Autoimmune Thyroiditis:Perspective of Different Water Iodine Exposure

Objective This study aimed to identify differentially methylated genes(DMGs) associated with natural killer cells in patients with autoimmune thyroiditis(AIT), focusing on the influence of varying water iodine exposure levels.Methods Participants were divided into categories based on median water iodine(MWI)concentrations: iodine-fortified areas(IFA, MWI < 10 μg/L), iodine-adequate areas(IAA, 40 ≤ MWI ≤ 100μg/L), and iodine-excessive areas(IEA, MWI > 300 μg/L). A total of 176 matched AIT cases and controls were recruited and divided into 89, 40, and 47 pairs for IFA, IAA, and IEA, respectively. DMGs were identified using 850K Bead Chip analysis for 10/10 paired samples. Validation of DNA methylation and m RNA expression levels of the DMGs was conducted using Methyl Target^(TM) and QRT-PCR for 176/176paired samples.Results KLRC1, KLRC3, and SH2D1B were identified as significant DMGs. Validation revealed that KLRC1 was hypomethylated and highly expressed, whereas KLRC3 was hypermethylated and highly expressed in individuals with AIT. Furthermore, KLRC1 was hypomethylated and highly expressed in both IFA and IEA.Conclusion The DNA methylation status of KLRC1 and KLRC3 may play crucial roles in AIT pathogenesis. Additionally, DNA methylation of KLRC1 seems to be influenced by different iodine concentrations in water.

Knockdown of RCN1 contributes to the apoptosis of colorectal cancer via regulating IP3R1

Background:The incidence of colorectal cancer(CRC)has been increasing in recent years.Thus,the discovery of factors that can assist in alleviating CRC is urgently warranted.Methods:To identify a potential factor involved in the development of CRC,we screened the upregulated genes in tumor tissues through four datasets from an online database.The expression of reticulocalbin 1(RCN1),a Ca2+-binding protein,was upregulated in the four datasets.Based on loss-offunction experiments,the effect of RCN1 on cell viability was assessed by Cell Counting Kit-8(CCK-8)assay.The regulatory effect of RCN1 on apoptosis was evaluated through Annexin V-fluorescein 5-isothiocyanate(FITC)/propidium iodide(PI)staining assay and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL)assay in RKO and SW480 cells.Activation of endoplasmic reticulum(ER)stress signaling pathways was confirmed by estimating the phosphorylation and expression of PRKR-like ER kinase(PERK),inositol-requiring kinase-1(IRE1),transcription factor 6(ACT6),and CCAAT/enhancer-binding protein-homologous protein(CHOP).The intracellular Ca2+homeostasis regulated by RCN1 was determined through the detection of Ca2+concentration and mitochondrial membrane potential(MMP)measurement.Moreover,whether inositol 1,4,5-trisphosphate receptor type 1(IP3R1)was involved in the regulation of RCN1 in CRC was verified through the depletion of IP3R1 in RKO cells.Results:Knockdown of RCN1 reduced cell viability and facilitated apoptosis in RKO and SW480 cells.Phosphorylation of PERK and IRE1,activation of ATF6,and upregulation of CHOP were induced by the absence of RCN1,suggesting that the unfolded protein response(UPR)was activated in CRC cells.The concentration of Ca2+in mitochondria was increased after RCN1 depletion,followed by reduction in the MMP and release of cytochrome c from mitochondria to the cytoplasm in RKO and SW480 cells.Moreover,it was demonstrated that IP3R1 mediates the effect of RCN1 on apoptosis induced by ER stress in CRC cells.The downregulation of IP3R1 restored the RCN1 loss-induced apoptosis and the increased Ca2+concentration.Conclusion:Taken together,our results confirmed that silencing of RCN1 disrupted intracellular Ca2+homeostasis and promoted cell apoptosis caused by TG-induced ER stress by regulating IP3R1 and activating the UPR signaling pathways.

CircR-ZC3HC1 mediates MiR-384-5p/SIRT1 axis to promote neuronal autophagy and relieves ischemic stroke

Objective:Circular RNAs(circRNAs)have been shown to involve in pathological processes of ischemic stroke(IS),including autophagy.This study was designed to explore the effect of circR-ZC3HC1 on neuronal autophagy in IS and the related mechanisms.Methods:Expression of circR-ZC3HC1 in blood samples of IS patients and healthy controls was detected.Hippocampal neurons were treated with oxygen and glucose deprivation(OGD)to establish IS in vitro model.The expression of LC3 and p62 and the number of autophagosomes were examined to evaluate the autophagy level of OGD induced neurons using western blotting and transmission electron microscope.Cell apoptosis rate and the expression of cleaved caspase-3,Bax,and Bcl-2 were assessed byflow cytometry and western blotting.The binding relationships among circR-ZC3HC1,miR-384-5p,and SIRT1 were predicted and verified.Results:Low expression of circR-ZC3HC1 was found in blood samples of IS patients and OGD-treated neurons.Overexpressed circR-ZC3HC1 or inhibited miR-384-5p expression promoted autophagy and inhibited apoptosis of OGD-treated neurons,which could be reversed by further 3-MA treatment.Mechanistically,circR-ZC3HC1 targeted miR-384-5p to mediate SIRT1 expression.miR-384-5p overexpression or SIRT1 knockdown in the presence of circR-ZC3HC1 overexpression in OGD-treated neurons lead to reduced autophagy and enhanced apoptosis.Conclusion:Collectively,circR-ZC3HC1 promoted neuronal autophagy to attenuate IS via miR-384-5p/SIRT1 axis.

CircTIAM1 overexpression promotes the progression of papillary thyroid cancer by regulating the miR-338-3p/LASP1 axis

Background:Papillary thyroid cancer(PTC)is the most prevalent histological type of differentiated thyroid malignancy.Circular RNAs(circRNAs)have been implicated in the pathogenesis and progression of various cancers.circTIAM1(hsa_circ_0061406)is a novel circRNA with aberrant expression in PTC.However,its functional roles in PTC progression remain to be investigated.Methods:The expression levels of circTIAM1 in the PTC and the matched para-cancerous tissues were detected by quantitative real-time reverse-transcription PCR(qRT-PCR).The subcellular localization of circTIAM1 was examined by fluorescence in-situ hybridization(FISH).Kaplan-Meier plot was used to analyze the association of clinicopathological features with circTIAM1 expression.Bioinformatics databases were utilized to predict the target miRNAs of circTIAM1 and the downstream target mRNAs.RNA pulldown,RIP assay,and dual-luciferase reporter assay were used to confirm the interactions.Functional experiments,such as CCK-8,EDU staining,and apoptosis assays,as well as in vivo xenograft model were employed to explore the impacts of circTIAM1,miR-338-3p,and LIM/SH3 protein 1(LASP1)on the malignant phenotype of the PTC cells.Results:CircTIAM1 was highly expressed in PTC cells.Moreover,circTIAM1 silencing suppressed the proliferation and invasion of PTC cells in vitro and impaired tumorigenesis in vivo.Furthermore,miR-338-3p was verified as a miRNA target of circTIAM1.LASP1 was also identified as a downstream target of miR-338-3p.The anti-tumorigenic effect of miR-338-3p overexpression and the pro-tumorigenic effect of LASP1 was further explored by functional assays,which demonstrated that circTIAM1 modulated the PTC progression through targeting miR-338-3p/LASP1 axis.Conclusion:The overexpression of circTIAM1 is associated with the malignant progression of PTC.A high level of circTIAM1 promotes the malignancy of PTC cells via the miR-338-3p/LASP1 axis.

Prognostic significance and relationship of SMAD3 phosphoisoforms and VEGFR-1 in gastric cancer:A clinicopathological study

BACKGROUND The TGF-β/SMAD3 and VEGFR-1 signaling pathways play important roles in gastric cancer metastasis.SMAD3 phosphorylation is a crucial prognostic marker in gastric cancer.AIM To determine the prognostic value and relationship of SMAD3 phospho-isoforms and VEGFR-1 in gastric cancer.METHODS This was a single-center observational study which enrolled 98 gastric cancer patients and 82 adjacent normal gastric tissues from patients aged 32-84 years(median age 65)between July 2006 and April 2007.Patients were followed up until death or the study ended(median follow-up duration of 28.5 mo).The samples were used to generate tissue microarrays(TMAs)for immunohistochemical(IHC)staining.The expressions of TGF-β1,pSMAD3C(S423/425),pSMAD3L(S204),and VEGFR-1 in gastric cancer(GC)tumor tissue and normal tissue were measured by IHC staining using TMAs obtained from 98 GC patients.Prognosis and survival information of the patients was recorded by Outdo Biotech from May 2007 to July 2015.The relationship between TGF-β1,pSMAD3C(S423/425),pSMAD3L(S204),and VEGFR-1 protein expression levels was analyzed using Pearson's correlation coefficient.The relationship between protein expression levels and clinicopathological parameters was analyzed using the Chi-squared test.A survival curve was generated using the Kaplan-Meier survival analysis.RESULTS TGFβ-1 and VEGFR-1 expression was significantly upregulated in gastric cancer tissue compared to adjacent noncancerous tissue.The positive expression of phosphorylated isoforms of Smad3 varied depending on the phosphorylation site[pSMAD3C(S423/425):51.0%and pSMAD3L(S204):31.6%].High expression of pSMAD-3L(S204)was significantly correlated with larger tumors(P=0.038)and later N stages(P=0.035).Additionally,high expression of VEGFR-1 was closely correlated with tumor size(P=0.015)and pathological grading(P=0.013).High expression of both pSMAD3L(S204)and VEGFR-1 was associated with unfavorable outcomes in terms of overall survival(OS).Multivariate analysis indicated that high expression of pSMAD3L(S204)and VEGFR-1 were independent risk factors for prognosis in GC patients.VEGFR-1 protein expression was correlated with TGF-β1(r=0.220,P=0.029),pSMAD3C(S423/425)(r=0.302,P=0.002),and pSMAD3L(S204)(r=0.201,P=0.047),respectively.Simultaneous overexpression of pSMAD3L(S204)and VEGFR-1 was associated with poor OS in gastric cancer patients.CONCLUSION Co-upregulation of pSMAD3L(S204)and VEGFR-1 can serve as a predictive marker for poor gastric cancer prognosis,and pSMAD3L(204)may be involved in enhanced gastric cancer metastasis in a VEGFR-1-dependent manner.