Abnormal β-catenin gene expression with invasiveness of primary hepatocellular carcinoma in China

AIM To study the abnormal expression of β-catenin gene and its relationship with invasiveness of primary hepatocellular carcinoma among Chinese people.METHODS Thirty-four hepatocellular carcinoma (HCC) specimens and adjacent para-cancerous tissues, 4 normal liver tissues were immunohistochemically stained to study subcellular distribution of β-catenin. Semiquantitive analysis of expression of β-catenin gene exon 3 mRNA was examined by RT-PCR and in situ hybridization. The relationship between expressions of both β-catenin protein, mRNA and clinicopathological characteristics of HCC was also analyzed.RESULTS Immunohistochemistry showed that all normal liver tissues and para-cancerous tissues examined displayed membranous type staining for β-catenin protein,occasionally with weak expression in the cytoplasm.While 21 cases (61.8%) of HCC examined showed accumulated type in cytoplasms or nuclei. The accumuled type Labling Index (LI) of cancer tissue and paracancarous tissue was (59.9 ± 26.3) and (18.3 ± 9.7)respectively (P＜0.01). Higher accumulated type LI was closely related with invasiveness of HCC. Results of RTPCR showed the β-catenin gene exon 3 mRNA Expression Index (El) of 34 HCCs was higher than that of paracancerous tissue and normal liver tissue. Using in situ hybridization, the signal corresponding to β-catenin gene exon 3 mRNA was particularly strong in cytoplasm of HCC when compared with those of para-cancerous and normal liver tissues. Over expression of β-catenin exon 3 was also found to be correlated with high metastatic potential of HCC.CONCLUSION Abnormal expression of β-catenin gene may contribute importantly to the invasiveness of HCC among Chinese people.

PIN1 Gene Overexpression and β-catenin Gene Mutation/Expression in Hepatocellular Carcinoma and Their Significance

The evolution of hepatocellular carcinoma （HCC） is a compound process which involves many kinds of genes and transductional pathways. The expression of the peptidyl-proplyl- isomerase PIN1 gene, the mutation in exon 3 of β-catenin and its correspondent abnormal expression and their roles in the hepatocellular carcinogeneisis were investigated. Among 29 pair cases of HCC and non-carcinoma tissues, the expression of PIN1 gene was detected by immunochemical staining. Mutations in exon 3 of β-catenin gene and differential expression of β-catenin gene were investigated by the methods of PCR-SSCP, direct sequencing and immunohistochemical technique as well. The results indicated： （1） 44.8% （13/29） cases of HCC presented higher level of PIN1 gene expression than non-cancerous tissues （X^2 =32.63, P〈0.05）, especially in cytoplasm and nucleus, while there was lower level of PIN1 expression in non-cancerous tissues; （2） 58.6% （17/29） HCC tissues showed β-catenin protein accumulation in cytoplasm and nucleus. 46.2% （6/13） HCC tissues indicated β-catenin protein accumulation with higher level of PIN1 expression, while 53.8% （7/13） HCC tissues indicated β-catenin protein accumulation with lower level or trace of PIN 1 expression （X^2 =0.00, P〉0.05）; （3） 24.1% （7/29） of primary tumor lesions carried gene mutations in exon 3 of β-catenin, and accompanied by β-catenin protein accumulation. There was no mutation in non-cancerous tissues All the mutation presented in tissues with low level of PIN1 expression. There was no mutation of β-catenin gene in tissues with high PIN1 expression level （X^2=58.12, P〈0.05）. So it was postulated that the increase of PIN1 gene expression could promote hepatocellular carcinogenesis via a way different from β- catenin gene mutation.

松脂醇二葡萄糖苷激活Wnt/β-catenin信号通路保护成骨细胞

背景:松脂醇二葡萄糖苷可以促进骨形成与骨基质的合成,加速骨组织修复,但其对成骨细胞的影响和作用机制仍需进一步探索。目的:基于Wnt/β-catenin信号通路探讨松脂醇二葡萄糖苷对地塞米松干预成骨细胞的影响和作用机制。方法:设置不同浓度的地塞米松组和松脂醇二葡萄糖苷组对成骨细胞干预24 h,筛选最佳干预浓度;使用地塞米松、松脂醇二葡萄糖苷和Wnt/β-catenin抑制剂XAV-939对成骨细胞进行干预,设置对照组、地塞米松组、抑制剂组、松脂醇二葡萄糖苷组、松脂醇二葡萄糖苷+抑制剂组。CCK-8法检测细胞活性,检测各组细胞碱性磷酸酶和半胱氨酸天冬氨酸蛋白酶3/7酶活性,Annexin V/PI染色与EdU法检测细胞凋亡与增殖情况,Real-Time qPCR检测Wnt3a、β-catenin、c-myc、骨钙素、Ⅰ型胶原蛋白的mRNA表达水平,Western Blot检测Wnt3a、β-catenin、c-myc、骨钙素、Ⅰ型胶原蛋白的蛋白表达水平。结果与结论:①地塞米松和松脂醇二葡萄糖苷干预成骨细胞24 h后,发现浓度为10μmol/L的地塞米松细胞抑制率为实验最佳干预浓度;松脂醇二葡萄糖苷浓度在100μmol/L时,细胞增殖最为明显;②与地塞米松组比较,松脂醇二葡萄糖苷组的碱性磷酸酶活性显著增强(P<0.05),半胱氨酸天冬氨酸蛋白酶3/7酶活性显著降低(P<0.05);Annexin V/PI染色和EdU法细胞增殖检测结果表明,松脂醇二葡萄糖苷可抑制地塞米松干预后成骨细胞的凋亡,促进成骨细胞增殖;③与地塞米松组和抑制剂组比较,松脂醇二葡萄糖苷组和松脂醇二葡萄糖苷+抑制剂组中Wnt3a、β-catenin、c-myc、骨钙素、Ⅰ型胶原蛋白mRNA和蛋白表达水平均显著升高(P<0.05);④结果表明,松脂醇二葡萄糖苷可以抑制地塞米松干预后的成骨细胞凋亡,通过激活Wnt/β-catenin信号通路来保护成骨细胞活性,促进成骨细胞增殖,可能发挥延缓激素性股骨头坏死的作用。

压应力激活SOST/Wnt/β-catenin 通路诱导软骨终板细胞退变

背景:在许多可以导致椎间盘退变的因素中(衰老、营养匮乏、机械因素等),力学负荷被认为是极其重要的因素,但其机制尚不清楚。目的:探讨硬骨素和Wnt/β-catenin信号通路在压应力诱导终板软骨退变中的作用。方法:提取4周龄雄性SD大鼠软骨终板细胞,体外利用力学加载仪器对终板软骨细胞施加压应力,于压缩细胞1,3,5,7 d,采用CCK-8法测定细胞活力;Western blot、RT-qPCR及细胞免疫荧光等检查终板软骨细胞内软骨标记物(聚集蛋白聚糖、Ⅱ型胶原)、钙化相关因子(Runx2、骨钙素)、细胞外基质降解酶及信号通路基因(硬骨素、β-catenin)等表达。结果与结论:①压应力作用下,终板软骨细胞活力会随着压应力时间、强度增加而降低;②终板软骨细胞的软骨标记物(聚集蛋白聚糖、Ⅱ型胶原)表达下降,而钙化相关因子(Runx2、骨钙素)表达上升;③压应力能促进终板软骨细胞的细胞外基质降解,基质金属蛋白酶3、基质金属蛋白酶13表达量增加;④细胞内Wnt/β-catenin信号通路表达出现异常,其特异性抑制因子硬骨素伴随着β-catenin的异常积累而表达下降。结果说明:在压应力作用下,终板软骨细胞内硬骨素的表达下降,导致Wnt/β-catenin信号通路激活,促进软骨终板的钙化、退变与细胞外基质的降解,进而促进椎间盘退变。

Peripheral blood RNA biomarkers can predict lesion severity in degenerative cervical myelopathy

Degenerative cervical myelopathy is a common cause of spinal cord injury,with longer symptom duration and higher myelopathy severity indicating a worse prognosis.While numerous studies have investigated serological biomarkers for acute spinal cord injury,few studies have explored such biomarkers for diagnosing degenerative cervical myelopathy.This study involved 30 patients with degenerative cervical myelopathy(51.3±7.3 years old,12 women and 18 men),seven healthy controls(25.7±1.7 years old,one woman and six men),and nine patients with cervical spondylotic radiculopathy(51.9±8.6 years old,three women and six men).Analysis of blood samples from the three groups showed clear differences in transcriptomic characteristics.Enrichment analysis identified 128 differentially expressed genes that were enriched in patients with neurological disabilities.Using least absolute shrinkage and selection operator analysis,we constructed a five-gene model(TBCD,TPM2,PNKD,EIF4G2,and AP5Z1)to diagnose degenerative cervical myelopathy with an accuracy of 93.5%.One-gene models(TCAP and SDHA)identified mild and severe degenerative cervical myelopathy with accuracies of 83.3%and 76.7%,respectively.Signatures of two immune cell types(memory B cells and memory-activated CD4^(+)T cells)predicted levels of lesions in degenerative cervical myelopathy with 80%accuracy.Our results suggest that peripheral blood RNA biomarkers could be used to predict lesion severity in degenerative cervical myelopathy.

WNT/β-catenin-M2 macrophage interplay as a target for therapy against hepatocellular carcinoma:Role of Calculus bovis

Liver cancer,and in particular hepatocellular carcinoma(HCC)is a disease of rising prevalence and incidence.To date,definitive treatment options include either surgical excision or ablation of the affected area.With increasing research on several pathways that could be involved in the progression of HCC,new elements within these pathways emerge as potential targets for novel therapies.The WNT/β-catenin pathway favors the presence of M2 tumor-associated macrophages which in turn promote tumor growth and metastasis.The inhibition of this pathway is considered a good candidate for such targeted therapeutic interventions.Interestingly,as Huang et al show in their recently published article,Calculus bovis which is used in traditional Chinese medicine can exert an inhibitory effect on theβ-catenin pathway and become a potential candidate for targeted pharmacotherapy against liver cancer.

The autophagy-lysosome pathway:a potential target in the chemical and gene therapeutic strategies for Parkinson’s disease

Parkinson’s disease is a common neurodegenerative disease with movement disorders associated with the intracytoplasmic deposition of aggregate proteins such asα-synuclein in neurons.As one of the major intracellular degradation pathways,the autophagy-lysosome pathway plays an important role in eliminating these proteins.Accumulating evidence has shown that upregulation of the autophagy-lysosome pathway may contribute to the clearance ofα-synuclein aggregates and protect against degeneration of dopaminergic neurons in Parkinson’s disease.Moreover,multiple genes associated with the pathogenesis of Parkinson’s disease are intimately linked to alterations in the autophagy-lysosome pathway.Thus,this pathway appears to be a promising therapeutic target for treatment of Parkinson’s disease.In this review,we briefly introduce the machinery of autophagy.Then,we provide a description of the effects of Parkinson’s disease–related genes on the autophagy-lysosome pathway.Finally,we highlight the potential chemical and genetic therapeutic strategies targeting the autophagy–lysosome pathway and their applications in Parkinson’s disease.

Dysregulation of genes involved in the long-chain fatty acid transport in pancreatic ductal adenocarcinoma

BACKGROUND Pancreatic ductal adenocarcinoma(PDAC)is an aggressive lethal malignancy with limited options for treatment and a 5-year survival rate of 11%in the United States.As for other types of tumors,such as colorectal cancer,aberrant de novo lipid synthesis and reprogrammed lipid metabolism have been suggested to be associated with PDAC development and progression.AIM To identify the possible involvement of lipid metabolism in PDAC by analyzing in tumoral and non-tumoral tissues the expression level of the most relevant genes involved in the long-chain fatty acid(FA)import into cell.METHODS A gene expression analysis of FASN,CD36,SLC27A1,SLC27A2,SLC27A3,SLC27A4,SLC27A5,ACSL1,and ACSL3 was performed by qRT-PCR in 24 tumoral PDAC tissues and 11 samples from non-tumoral pancreatic tissues obtained via fine needle aspiration or via surgical resection.The genes were considered significantly dysregulated between the groups when the p value was<0.05 and the fold change(FC)was≤0.5 and≥2.RESULTS We found that three FA transporters and two long-chain acyl-CoA synthetases genes were significantly upregulated in the PDAC tissue compared to the non-tumoral tissue:SLC27A2(FC=5.66;P=0.033),SLC27A3(FC=2.68;P=0.040),SLC27A4(FC=3.13;P=0.033),ACSL1(FC=4.10;P<0.001),and ACSL3(FC=2.67;P=0.012).We further investigated any possible association between the levels of the analyzed mRNAs and the specific characteristics of the tumors,including the anatomic location,the lymph node involvement,and the presence of metastasis.A significant difference in the expression of SLC27A3(FC=3.28;P=0.040)was found comparing patients with and without lymph nodes involvement with an overexpression of this transcript in 17 patients presenting tumoral cells in the lymph nodes.CONCLUSION Despite the low number of patients analyzed,these preliminary results seem to be promising.Addressing lipid metabolism through a broad strategy could be a beneficial way to treat this malignancy.Future in vitro and in vivo studies on these genes may offer important insights into the mechanisms linking PDAC with the long-chain FA import pathway.

Recovery of the injured neural system through gene delivery to surviving neurons in Parkinson’s disease

A critical unaddressed problem in Parkinson’s disease is the lack of therapy that slows or hampers neurodegeneration.While medications effectively manage symptoms,they offer no long-term benefit because they fail to address the underlying neuronal loss.This highlights that the elusive goals of halting progression and restoring damaged neurons limit the long-term impact of current approaches.Recent clinical trials using gene therapy have demonstrated the safety of various vector delivery systems,dosages,and transgenes expressed in the central nervous system,signifying tangible and substantial progress in applying gene therapy as a promising Parkinson’s disease treatment.Intriguingly,at diagnosis,many dopamine neurons remain in the substantia nigra,offering a potential window for recovery and survival.We propose that modulating these surviving dopamine neurons and axons in the substantia nigra and striatum using gene therapy offers a potentially more impactful therapeutic approach for future research.Moreover,innovative gene therapies that focus on preserving the remaining elements may have significant potential for enhancing long-term outcomes and the quality of life for patients with Parkinson’s disease.In this review,we provide a perspective on how gene therapy can protect vulnerable elements in the substantia nigra and striatum,offering a novel approach to addressing Parkinson’s disease at its core.

槲皮素联合木犀草素对大肠癌细胞凋亡和β-catenin及其靶基因的作用

目的:研究槲皮素和木犀草素及其联合使用对CT26大肠癌细胞增殖和细胞凋亡的作用及机制。方法:实验分为对照组、槲皮素组(QCT)、木犀草素组(LTL)和联合组(QCT+LTL),分别给予等体积DMSO、槲皮素、木犀草素及槲皮素联合木犀草素处理CT26细胞。CCK-8(Cell Counting Kit-8)法检测细胞增殖;Annexin-FITC/PI双染,流式细胞仪检测细胞凋亡;酶底物试剂盒检测Caspases活性;Western blot法检测β-catenin及其下游c-Myc、Cyclin D1和Survivin的蛋白表达水平。结果:与对照组比较,槲皮素和木犀草素治疗可抑制CT26细胞增殖,诱导细胞凋亡,伴随Caspase-3、Caspase-8和Caspase-9活性增强,同时还可抑制β-catenin、c-Myc、Cyclin D1及Survivin蛋白表达(P<0.05,P<0.01)。槲皮素和木犀草素联合可增强对CT26细胞的作用。结论:槲皮素和木犀草素可抑制CT26大肠癌生长,诱导细胞凋亡,两药联合作用显著优于单药,其机制与下调β-catenin、c-Myc、Cyclin D1及Survivin表达,活化Caspase-3、Caspase-8和Caspase-9相关。

AAV2-PDE6B restores retinal structure and function in the retinal degeneration 10 mouse model of retinitis pigmentosa by promoting phototransduction and inhibiting apoptosis

Retinitis pigmentosa is a group of inherited diseases that lead to retinal degeneration and photoreceptor cell death.However,there is no effective treatment for retinitis pigmentosa caused by PDE6B mutation.Adeno-associated virus(AAV)-mediated gene therapy is a promising strategy for treating retinitis pigmentosa.The aim of this study was to explore the molecular mechanisms by which AAV2-PDE6B rescues retinal function.To do this,we injected retinal degeneration 10(rd10)mice subretinally with AAV2-PDE6B and assessed the therapeutic effects on retinal function and structure using dark-and light-adapted electroretinogram,optical coherence tomography,and immunofluorescence.Data-independent acquisition-mass spectrometry-based proteomic analysis was conducted to investigate protein expression levels and pathway enrichment,and the results from this analysis were verified by real-time polymerase chain reaction and western blotting.AAV2-PDE6B injection significantly upregulated PDE6βexpression,preserved electroretinogram responses,and preserved outer nuclear layer thickness in rd10 mice.Differentially expressed proteins between wild-type and rd10 mice were closely related to visual perception,and treating rd10 mice with AAV2-PDE6B restored differentially expressed protein expression to levels similar to those seen in wild-type mice.Kyoto Encyclopedia of Genes and Genome analysis showed that the differentially expressed proteins whose expression was most significantly altered by AAV2-PDE6B injection were enriched in phototransduction pathways.Furthermore,the phototransductionrelated proteins Pde6α,Rom1,Rho,Aldh1a1,and Rbp1 exhibited opposite expression patterns in rd10 mice with or without AAV2-PDE6B treatment.Finally,Bax/Bcl-2,p-ERK/ERK,and p-c-Fos/c-Fos expression levels decreased in rd10 mice following AAV2-PDE6B treatment.Our data suggest that AAV2-PDE6B-mediated gene therapy promotes phototransduction and inhibits apoptosis by inhibiting the ERK signaling pathway and upregulating Bcl-2/Bax expression in retinitis pigmentosa.

Heterogeneity of mature oligodendrocytes in the central nervous system

Mature oligodendrocytes form myelin sheaths that are crucial for the insulation of axons and efficient signal transmission in the central nervous system.Recent evidence has challenged the classical view of the functionally static mature oligodendrocyte and revealed a gamut of dynamic functions such as the ability to modulate neuronal circuitry and provide metabolic support to axons.Despite the recognition of potential heterogeneity in mature oligodendrocyte function,a comprehensive summary of mature oligodendrocyte diversity is lacking.We delve into early 20th-century studies by Robertson and Río-Hortega that laid the foundation for the modern identification of regional and morphological heterogeneity in mature oligodendrocytes.Indeed,recent morphologic and functional studies call into question the long-assumed homogeneity of mature oligodendrocyte function through the identification of distinct subtypes with varying myelination preferences.Furthermore,modern molecular investigations,employing techniques such as single cell/nucleus RNA sequencing,consistently unveil at least six mature oligodendrocyte subpopulations in the human central nervous system that are highly transcriptomically diverse and vary with central nervous system region.Age and disease related mature oligodendrocyte variation denotes the impact of pathological conditions such as multiple sclerosis,Alzheimer's disease,and psychiatric disorders.Nevertheless,caution is warranted when subclassifying mature oligodendrocytes because of the simplification needed to make conclusions about cell identity from temporally confined investigations.Future studies leveraging advanced techniques like spatial transcriptomics and single-cell proteomics promise a more nuanced understanding of mature oligodendrocyte heterogeneity.Such research avenues that precisely evaluate mature oligodendrocyte heterogeneity with care to understand the mitigating influence of species,sex,central nervous system region,age,and disease,hold promise for the development of therapeutic interventions targeting varied central nervous system pathology.

Pan-TRK positive uterine sarcoma in immunohistochemistry without neurotrophic tyrosine receptor kinase gene fusions:A case report

BACKGROUND The classification of uterine sarcomas is based on distinctive morphological and immunophenotypic characteristics,increasingly supported by molecular genetic diagnostics.Data on neurotrophic tyrosine receptor kinase(NTRK)gene fusionpositive uterine sarcoma,potentially aggressive and morphologically similar to fibrosarcoma,are limited due to its recent recognition.Pan-TRK immunohistochemistry(IHC)analysis serves as an effective screening tool with high sensitivity and specificity for NTRK-fusion malignancies.CASE SUMMARY We report a case of a malignant mesenchymal tumor originating from the uterine cervix,which was pan-TRK IHC-positive but lacked NTRK gene fusions,accompanied by a brief literature review.A 55-year-old woman presented to the emergency department with abdominal pain and distension,exhibiting significant ascites and multiple solid pelvic masses.Pelvic examination revealed a tumor encompassing the uterine cervix,extending to the vagina and uterine corpus.A punch biopsy of the cervix indicated NTRK sarcoma with positive immunochemical pan-TRK stain.However,subsequent next generation sequencing revealed no NTRK gene fusion,leading to a diagnosis of poorly differentiated,advanced-stage sarcoma.CONCLUSION The clinical significance of NTRK gene fusion lies in potential treatment with TRK inhibitors for positive sarcomas.Identifying such rare tumors is crucial due to the potential applicability of tropomyosin receptor kinase inhibitor treatment.

Genetic and epigenetic alterations associated with gestational diabetes mellitus and adverse neonatal outcomes

Gestational diabetes mellitus(GDM)is a metabolic disorder,recognised during 24-28 weeks of pregnancy.GDM is linked with adverse newborn outcomes such as macrosomia,premature delivery,metabolic disorder,cardiovascular,and neurological disorders.Recent investigations have focused on the correlation of genetic factors such asβ-cell function and insulin secretary genes(transcription factor 7 like 2,potassium voltage-gated channel subfamily q member 1,adipo-nectin etc.)on maternal metabolism during gestation leading to GDM.Epigenetic alterations like DNA methylation,histone modification,and miRNA expression can influence gene expression and play a dominant role in feto-maternal meta-bolic pathways.Interactions between genes and environment,resulting in differ-ential gene expression patterns may lead to GDM.Researchers suggested that GDM women are more susceptible to insulin resistance,which alters intrauterine surroundings,resulting hyperglycemia and hyperinsulinemia.Epigenetic modi-fications in genes affecting neuroendocrine activities,and metabolism,increase the risk of obesity and type 2 diabetes in offspring.There is currently no treatment or effective preventive method for GDM,since the molecular processes of insulin resistance are not well understood.The present review was undertaken to un-derstand the pathophysiology of GDM and its effects on adverse neonatal out-comes.In addition,the study of genetic and epigenetic alterations will provide lead to researchers in the search for predictive molecular biomarkers.

Autophagy-targeting modulation to promote peripheral nerve regeneration

Nerve regeneration following traumatic peripheral nerve injuries and neuropathies is a complex process modulated by diverse factors and intricate molecular mechanisms.Past studies have focused on factors that stimulate axonal outgrowth and myelin regeneration.However,recent studies have highlighted the pivotal role of autophagy in peripheral nerve regeneration,particularly in the context of traumatic injuries.Consequently,autophagy-targeting modulation has emerged as a promising therapeutic approach to enhancing peripheral nerve regeneration.Our current understanding suggests that activating autophagy facilitates the rapid clearance of damaged axons and myelin sheaths,thereby enhancing neuronal survival and mitigating injury-induced oxidative stress and inflammation.These actions collectively contribute to creating a favorable microenvironment for structural and functional nerve regeneration.A range of autophagyinducing drugs and interventions have demonstrated beneficial effects in alleviating peripheral neuropathy and promoting nerve regeneration in preclinical models of traumatic peripheral nerve injuries.This review delves into the regulation of autophagy in cell types involved in peripheral nerve regeneration,summarizing the potential drugs and interventions that can be harnessed to promote this process.We hope that our review will offer novel insights and perspectives on the exploitation of autophagy pathways in the treatment of peripheral nerve injuries and neuropathies.

AAV-mediated expression of p65shRNA and bone morphogenetic protein 4 synergistically enhances chondrocyte regeneration

BACKGROUND:Adeno-associated virus(AAV)gene therapy has been proven to be reliable and safe for the treatment of osteoarthritis in recent years.However,given the complexity of osteoarthritis pathogenesis,single gene manipulation for the treatment of osteoarthritis may not produce satisfactory results.Previous studies have shown that nuclear factorκB could promote the inflammatory pathway in osteoarthritic chondrocytes,and bone morphogenetic protein 4(BMP4)could promote cartilage regeneration.OBJECTIVE:To test whether combined application of AAV-p65shRNA and AAV-BMP4 will yield the synergistic effect on chondrocytes regeneration and osteoarthritis treatment.METHODS:Viral particles containing AAV-p65-shRNA and AAV-BMP4 were prepared.Their efficacy in inhibiting inflammation in chondrocytes and promoting chondrogenesis was assessed in vitro and in vivo by transfecting AAV-p65-shRNA or AAV-BMP4 into cells.The experiments were divided into five groups:PBS group;osteoarthritis group;AAV-BMP4 group;AAV-p65shRNA group;and BMP4-p65shRNA 1:1 group.Samples were collected at 4,12,and 24 weeks postoperatively.Tissue staining,including safranin O and Alcian blue,was applied after collecting articular tissue.Then,the optimal ratio between the two types of transfected viral particles was further investigated to improve the chondrogenic potential of mixed cells in vivo.RESULTS AND CONCLUSION:The combined application of AAV-p65shRNA and AAV-BMP4 together showed a synergistic effect on cartilage regeneration and osteoarthritis treatment.Mixed cells transfected with AAV-p65shRNA and AAV-BMP4 at a 1:1 ratio produced the most extracellular matrix synthesis(P<0.05).In vivo results also revealed that the combination of the two viruses had the highest regenerative potential for osteoarthritic cartilage(P<0.05).In the present study,we also discovered that the combined therapy had the maximum effect when the two viruses were administered in equal proportions.Decreasing either p65shRNA or BMP4 transfected cells resulted in less collagen II synthesis.This implies that inhibiting inflammation by p65shRNA and promoting regeneration by BMP4 are equally important for osteoarthritis treatment.These findings provide a new strategy for the treatment of early osteoarthritis by simultaneously inhibiting cartilage inflammation and promoting cartilage repair.

补中益气汤协同Siβ-catenin调控Wnt/β-catenin信号通路干预肺腺癌顺铂耐药性的分子机制研究

目的:探讨补中益气汤含药血清协同Siβ-catenin调控Wnt/β-catenin信号通路干预A549/DDP细胞顺铂耐药性的分子机制研究。方法:采用siRNA干扰沉默β-catenin的表达,Western blot检测正常血清组、顺铂组、siCTNNB1-235组、siCTNNB1-510组、siCTNNB1-547组A549/DDP细胞的β-catenin蛋白的相对表达量;干扰β-catenin后,Western blot检测各组A549/DDP细胞β-catenin和Survivin的蛋白表达量,MTT法检测各组A549/DDP细胞对顺铂的IC50,Annexin V/PI染色法检测各组A549/DDP细胞顺铂诱导的凋亡率。结果:相较于正常血清组(1.00),siCTNNB1-235组(0.323±0.021)对β-catenin表达抑制率达67%(P=0.000)。RNAi技术沉默β-catenin后,与正常血清组(1.00)相比,补中益气汤联合顺铂组的Survivin(0.247±0.015)和β-catenin(0.257±0.015)蛋白的表达下调更为明显(P=0.000,P=0.000)。IC50和正常血清组(28.330±1.029)µmol/L相比,单独补中益气汤含药血清(20.350±1.155)µmol/L或单独瞬时转染siCTNNB1-235组(15.577±1.535)µmol/L均可降低A549/DDP细胞顺铂的IC50(P=0.000,P=0.000),2者联合(10.453±0.999)µmol/L使用可进一步降低顺铂的IC50(P=0.000)。与正常血清组(9.130±0.384)%相比,瞬时转染siCTNNB1-235联合顺铂组(64.393±0.244)%和补中益气汤联合顺铂组(70.120±0.400)%的总凋亡率均升高(P=0.000,P=0.000)。结论:补中益气汤含药血清和瞬时转染siβ-catenin在抑制Wnt/β-catenin信号通路改善肺腺癌顺铂耐药性方面具有协同效应,且补中益气汤具有siβ-catenin瞬时转染样效应。

CTNND1通过Wnt/β-catenin信号通路调控胰腺癌细胞增殖、迁移和侵袭

目的:研究CTNND1调控胰腺癌细胞增殖、迁移和侵袭的机制,为胰腺癌精准治疗提供新理论依据。方法:通过生信分析验证CTNND1在胰腺癌和正常组织中的表达,免疫组织化学(IHC)和qPCR进一步验证。Transwell、划痕实验和细胞增殖试验用于研究CTNND1对胰腺癌细胞增殖、迁移和侵袭的影响。裸鼠皮下成瘤实验检测CTNND1对人胰腺癌细胞成瘤能力的影响。结果:在胰腺癌细胞中敲低CTNND1可以抑制胰腺癌细胞的Wnt/β-catenin信号通路以及增殖、迁移和侵袭能力。加入LiCl(Wnt/β-catenin特异性激活剂)能部分恢复CTNND1敲低胰腺癌细胞的增殖、迁移和侵袭能力。裸鼠皮下成瘤显示,敲低CTNND1抑制了肿瘤裸鼠皮下成瘤。结论:敲低CTNND1能从体内外通过Wnt/β-catenin信号通路调控胰腺癌的增殖、迁移和侵袭及皮下成瘤能力。

低氧微环境通过TGFBI调控Wnt/β-catenin通路介导胰腺癌化疗耐药及机制研究

目的:研究低氧微环境通过TGFBI调控胰腺癌耐药的作用及分子机制。方法:以CCK-8方法检测细胞增殖;以Western blotting技术检测蛋白表达水平;以ImageJ软件分析蛋白灰度值;RNAi技术用于敲减TGFBI基因。结果:TGFBI在Panc-1细胞中表达比正常细胞增强;低氧促进胰腺癌细胞Panc-1增殖并减弱顺铂对Panc-1的抑制作用,而高氧抑制Panc-1细胞增殖并加强顺铂的杀伤作用;低氧促进TGFBI表达及EMT行为;低氧通过TGFBI调控Panc-1细胞增殖及顺铂的杀伤作用;低氧通过TGFBI调控Wnt/β-catenin信号,进而促进EMT行为。结论:低氧微环境通过增强TGFBI表达促进胰腺癌细胞增殖及耐药;低氧微环境通过TGFBI激活Wnt/β-catenin信号通路,促进EMT标志分子表达。

lncRNA ENST00000452996.1通过ERK/GSK-3β/β-catenin信号通路调控肝癌细胞增殖、迁移及侵袭

目的研究一种新的长链非编码RNA ENST00000452996.1对肝癌细胞增殖、迁移及侵袭等的影响及其调控机制。方法TCGA数据集分析肝癌组织ENST00000452996.1表达;构建ENST00000452996.1的shRNA载体,包装病毒感染Huh-7细胞(shENST00000452996.1组),CCK-8法、克隆形成法、流式细胞术、划痕法和Transwell小室法分别检测细胞增殖、凋亡、迁移和侵袭能力;Western blot法检测shENST00000452996.1组、ERK激动剂EGF处理组和GSK-3β抑制剂TDZD-8处理组的ERK/GSK-3β/β-catenin信号分子的表达。结果TCGA分析发现,肝癌组织ENST00000452996.1表达水平明显高于癌旁组织,且与Edmondson-Steiner分级呈正相关,与总生存时间呈负相关;与对照组相比,shENST00000452996.1组细胞增殖、迁移及侵袭能力明显下降,凋亡能力明显增强,p-ERK1/2、p-GSK-3β^(Ser9)和β-catenin表达均下调,GSK-3β和p-β-catenin表达上调,核β-catenin表达下降;与shENST00000452996.1组相比,EGF处理组p-ERK1/2表达升高,EGF处理组和TDZD-8处理组p-GSK-3β^(Ser9)和β-catenin及核β-catenin表达均升高,GSK-3β和p-β-catenin表达均降低。结论干扰ENST00000452996.1表达抑制ERK1/2磷酸化,阻止GSK3β^(Ser9)磷酸化,导致β-catenin磷酸化,抑制β-catenin核转位,推断ENST00000452996.1通过ERK/GSK-3β/β-catenin信号通路增强肝癌细胞增殖、迁移及侵袭能力,抑制细胞凋亡,从而发挥促进肝细胞癌的作用。