Cyclin-dependent kinase 4 and 6 inhibitors (CDK4/6 inhibitors) can significantly extend tumor response in patients with metastatic luminal A breast cancer, yet intrinsic and acquired resistance remains a prevalent issue. Understanding the molecular features of CDK4/6 inhibitor sensitivity and the potential efficacy of their combination with novel targeted cell death inducers may lead to improved patient outcomes. Herein, we demonstrate that ferroptosis, a form of regulated cell death driven by iron-dependent phospholipid peroxidation, partly underpins the efficacy of CDK4/6 inhibitors. Mechanistically, CDK4/6 inhibitors downregulate the cystine transporter SLC7A11 by inhibiting SP1 binding to the SLC7A11 promoter region. Furthermore, SLC7A11 is identified as critical for the intrinsic sensitivity of luminal A breast cancer to CDK4/6 inhibitors. Both genetic and pharmacological inhibition of SP1 or SLC7A11 enhances cell sensitivity to CDK4/6 inhibitors and synergistically inhibits luminal A breast cancer growth when combined with CDK4/6 inhibitors in vitro and in vivo. Our data highlight the potential of targeting SLC7A11 in combination with CDK4/6 inhibitors, supporting further investigation of combination therapy in luminal A breast cancer.

The emergence of XBB.1.16 has gained rapid global prominence. Previous studies have elucidated that the infection of SARS-CoV-2 induces alterations in the mitochondrial integrity of host cells, subsequently influencing the cellular response to infection. In this study, we compared the differences in infectivity and pathogenicity between XBB.1.16 and the parental Omicron sublineages BA.1 and BA.2 and assessed their impact on host mitochondria. Our findings suggest that, in comparison with BA.1 and BA.2, XBB.1.16 exhibits more efficient spike protein cleavage, more efficient mediating syncytia formation, mild mitochondriopathy, and less pathogenicity. Altogether, our investigations suggest that, based on the mutation of key sites, XBB.1.16 exhibited enhanced infectivity but lower pathogenicity. This will help us to further investigate the biological functions of key mutation sites.

Non-alcoholic fatty liver disease (NAFLD), characterized by excessive lipid accumulation in hepatocytes, is an increasing global healthcare burden. Sirtuin 2 (SIRT2) functions as a preventive molecule for NAFLD with incompletely clarified regulatory mechanisms. Metabolic changes and gut microbiota imbalance are critical to the pathogenesis of NAFLD. However, their association with SIRT2 in NAFLD progression is still unknown. Here, we report that SIRT2 knockout (KO) mice are susceptible to HFCS (high-fat/high-cholesterol/high-sucrose)-induced obesity and hepatic steatosis accompanied with an aggravated metabolic profile, which indicates SIRT2 deficiency promotes NAFLD-NASH (nonalcoholic steatohepatitis) progression. Under palmitic acid (PA), cholesterol (CHO), and high glucose (Glu) conditions, SIRT2 deficiency promotes lipid deposition and inflammation in cultured cells. Mechanically, SIRT2 deficiency induces serum metabolites alteration including upregulation of L-proline and downregulation of phosphatidylcholines (PC), lysophosphatidylcholine (LPC), and epinephrine. Furthermore, SIRT2 deficiency promotes gut microbiota dysbiosis. The microbiota composition clustered distinctly in SIRT2 KO mice with decreased Bacteroides and Eubacterium, and increased Acetatifactor. In clinical patients, SIRT2 is downregulated in the NALFD patients compared with healthy controls, and is associated with exacerbated progression of normal liver status to NAFLD to NASH in clinical patients. In conclusion, SIRT2 deficiency accelerates HFCS-induced NAFLD-NASH progression by inducing alteration of gut microbiota and changes of metabolites.

目的 将pCMV-Tag2B-Flag标签连接到人线粒体溶质载体蛋白25A37(SLC25A37)的基因上构建真核表达载体,检测其对人肺癌细胞H460功能的影响.方法 采用PCR技术扩增人乳腺文库中SLC25A37的编码区片段,用限制性内切酶EcoR I和Xho I双酶作为该片段和Flag的载体,经连接后形成重组质粒.通过Western Blot(WB)技术鉴定目的基因在细胞中的表达,利用CCK-8和克隆形成方法检测SLC25A37对肺癌H460细胞增殖的影响,划痕试验检测SLC25A37对H460细胞迁移能力的影响.结果 将构建好的Flag-SLC25A37真核表达质粒转染H460细胞后成功表达融合蛋白;过表达SLC25A37后能够增强H460细胞的增殖和迁移能力.结论 携带Flag标签的人SLC25A37基因的真核表达载体能在人肺癌H460细胞中表达,该基因能够增强H460细胞的增殖和迁移能力.SLC25A37可能是促癌基因,并可能为肺癌的治疗提供重要的靶标.

Background Limited response to programmed death ligand-1 (PD-L1)/programmed death 1 (PD-1) immunotherapy is a major hindrance of checkpoint immunotherapy in non-small cell lung cancer (NSCLC). The abundance of PD-L1 on the tumor cell surface is crucial for the responsiveness of PD-1/PD-L1 immunotherapy. However, the negative control of PD-L1 expression and the physiological significance of the PD-L1 inhibition in NSCLC immunotherapy remain obscure.Methods Bioinformatics analysis was performed to profile and investigate the long non-coding RNAs that negatively correlated with PD-L1 expression and positively correlated with CD8+T cell infiltration in NSCLC. Immunofluorescence, in vitro PD-1 binding assay, T cell-induced apoptosis assays and in vivo syngeneic mouse models were used to investigate the functional roles of LINC02418 and mmu-4930573I07Rik in regulating anti-PD-L1 therapeutic efficacy in NSCLC. The molecular mechanism of LINC02418-enhanced PD-L1 downregulation was explored by immunoprecipitation, RNA immunoprecipitation (RIP), and ubiquitination assays. RIP, luciferase reporter, and messenger RNA degradation assays were used to investigate the m6A modification of LINC02418 or mmu-4930573I07Rik expression. Bioinformatics analysis and immunohistochemistry (IHC) verification were performed to determine the significance of LINC02418, PD-L1 expression and CD8+T cell infiltration.Results LINC02418 is a negative regulator of PD-L1 expression that positively correlated with CD8+T cell infiltration, predicting favorable clinical outcomes for patients with NSCLC. LINC02418 downregulates PD-L1 expression by enhancing PD-L1 ubiquitination mediated by E3 ligase Trim21. Both hsa-LINC02418 and mmu-4930573I07Rik (its homologous RNA in mice) regulate PD-L1 therapeutic efficacy in NSCLC via Trim21, inducing T cell-induced apoptosis in vitro and in vivo. Furthermore, METTL3 inhibition via N6-methyladenosine (m6A) modification mediated by YTHDF2 reader upregulates hsa-LINC02418 and mmu-4930573I07Rik. In patients with NSCLC, LINC02418 expression is inversely correlated with PD-L1 expression and positively correlated with CD8+T infiltration.Conclusion LINC02418 functions as a negative regulator of PD-L1 expression in NSCLC cells by promoting the degradation of PD-L1 through the ubiquitin-proteasome pathway. The expression of LINC02418 is regulated by METTL3/YTHDF2-mediated m6A modification. This study illuminates the underlying mechanisms of PD-L1 negative regulation and presents a promising target for improving the effectiveness of anti-PD-L1 therapy in NSCLC.

Abstract Radiotherapy has shown measurable efficacy with objective response rate and prolonged progression-free survival (PFS) in patients with breast cancer (BC). However, radioresistance is still a major challenge in BC radiotherapy, leading to limited clinical application and treatment failure. Therefore, elucidating the underlying mechanisms and developing effective strategies to enhance the radiosensitivity is of clinical importance. In the present study, we demonstratedCCAAT enhancer-binding protein alpha (CEBPA) as a specific marker for predicting the efficacy of radiotherapy in BC patients. CEBPA enhanced radiotherapeutic sensitivity of BC by increasing the apoptosis both in vitro and in vivo. Mechanistically, CEBPA executed radio-sensitizing function in BC by promoting ferroptosis through negatively regulating the nuclear factor E2-related factor 2 (Nrf2) pathway genes. Furthermore, high expression of CEBPA was positively correlated with the infiltration of cytotoxic immune cells in BC microenvironment. In summary, our work revealed CEBPA as a critical regulator to enhance radiosensitivity, induce ferroptosis, as well as modulate tumor immune microenvironment in BC. Induction of CEBPA or ferroptosis with specific inducers or modulation of tumor immune microenvironment would be potential sensitizing therapeutic strategies in BC.

目的 构建肌动蛋白结合Rho激活C末端样(ABRACL)基因启动子不同截短片段重组质粒,并检测其转录活性.方法 以含ABRACL启动子全长质粒为模板,利用PCR方法分别扩增ABRACL启动子不同截短片段;将扩增片段分别插入pGL3.0-basic载体,构建ABRACL启动子不同截短片段重组质粒;经双酶切及序列鉴定正确后,将重组质粒转染ZR75-1和A549细胞,采用双荧光素酶报告基因检测系统测定ABRACL启动子不同截短片段的双荧光素酶活性;检测转录因子MYB原癌基因样2(B-MYB)对ABRACL启动子不同截短片段转录活性的影响.结果 成功构建含ABRACL基因启动子不同截短片段重组质粒,双荧光素酶活性测定发现ABRACL基因启动子-400 bp片段活性较高,转录因子B-MYB可以增强ABRACL启动子-600 bp和-1000 bp片段的活性.结论 发现ABRACL基因启动子转录活性较高区域,为进一步研究ABRACL基因上游调控机制奠定基础.

目的 构建带Myc标签的三磷酸腺苷结合盒亚家族G成员2(ABCG2)基因的真核表达载体,并研究其生物学功能.方法 以人卵巢文库为模板,通过PCR方法扩增人的ABCG2基因,将其插入pXJ-40-myc载体上,经双酶切和基因测序确定后转染人ZR75-1乳腺癌细胞,通过Western印迹验证ABCG2蛋白表达;采用免疫荧光法检测细胞中ABCG2蛋白的定位;采用CCK-8法和划痕实验检测ABCG2基因对乳腺癌细胞增殖、迁移能力的影响;通过流式细胞术检测ABCG2基因对化疗药物米托蒽醌外排的影响.结果 在人卵巢文库中成功获取长约2000 bp的ABCG2基因,并构建在pXJ-40-myc载体上,测序结果与目的序列一致;质粒提取后转染人ZR75-1乳腺癌细胞;免疫荧光法结果表明,ABCG2蛋白主要在ZR75-1细胞的细胞膜和细胞质中表达;CCK-8法与划痕试验结果表明,转染pXJ-40-myc-ABCG2的乳腺癌细胞,与转染空载体质粒的细胞相比,增殖能力与迁移能力均提高;流式细胞术检测提示,ABCG2诱导细胞对米托蒽醌药物外排增多.结论 成功构建了pXJ-40-myc-ABCG2真核表达载体,并验证ABCG2在细胞内的定位和介导耐药作用,为今后探讨ABCG2在乳腺癌细胞发生发展中的作用奠定基础.

Sorafenib (SRF) is one of the most effective and common multi-kinase targeted therapeutic inhibitors used in advanced hepatocellular carcinoma (HCC). However, the occurrence of SRF resistance remains a major challenge. SLC7A11 is a critical ferroptosis-associated gene, the upregulation of which inhibits ferroptosis in various cancers. However, the mechanisms of SLC7A11 upregulation and the physiological significance of SLC7A11 activation in controlling SRF sensitivity are unclear. Here, we identified a novel lncRNA LINC00654 positively correlates with SLC7A11 expression and predicts poor clinical outcomes in HCC. LINC00654 inhibits HCC cell ferroptosis by altering the levels of Fe2+, total ROS, MDA and GSH/GSSG ratio and confers SRF resistance via SLC7A11 expression in HCC cells. Inhibition of LINC00654 or treatment of ferroptosis inducer RSL3 can partly reverse the sensitivity of SRF-resistant cells. Mechanistically, LINC00654 activates SLC7A11 expression by increasing the recruitment of the transcription factor STAT3 onto the SLC7A11 promoter. Furthermore, for the xenograft assay in nude mice, LINC00654/SLC7A11 axis confers SRF resistance, the effect of which can be reversed by LINC00654 knockdown or treatment of RSL3. Clinically, in HCC patients, LINC00654 and STAT3 expression were positively correlated with SLC7A11 expression. Thus, our work established LINC00654/STAT3/SLC7A11 as a vital axis to regulate HCC cell ferroptosis and SRF resistance. Evaluation of the expression of LINC00654 or SLC7A11 in HCC tissue would be an effective way in predicting the efficacy of SRF. Inhibition of LINC00654 or treatment of ferroptosis inducer would be a promising strategy to overcome SRF resistance.

Abstract Purpose: Cyclin-dependent kinase 4 and 6 inhibitors (CDK4/6is) can significantly extend tumor response in patients with metastatic estrogen receptor–positive (ER+) breast cancer, but intrinsic and acquired resistance is common. Elucidation of the molecular features of CDK4/6i sensitivity and the efficiency of their combination with novel targeted cell death inducers may pave the way toward improving patient outcomes. Experimental Design: Ferroptosis-related characteristics were observed following treatment with the CDK4/6 inhibitor palbociclib. Transcriptomic analyses and functional assays were performed to determine the targets and regulatory mechanism of palbociclib in the ferroptosis pathway in ER+ breast cancer cell lines. A tumor xenograft model was used to study the synergistic antitumor effects of CDK4/6is in combination with ferroptosis inducers. Results: Ferroptosis, a form of regulated cell death driven by iron-dependent phospholipid peroxidation, is partly responsible for the efficacy of the CDK4/6 inhibitor palbociclib. Mechanistically, palbociclib downregulates cystine transporter SLC7A11 by inhibiting SP1 binding to the promoter region of SLC7A11. Furthermore, genetic or pharmacological inhibitors of SP1 or SLC7A11 can enhance cell sensitivity to palbociclib and synergistically inhibit ER+ breast cancer cell growth in combination with palbociclib. A syngeneic ER+ mouse mammary tumor model was used to verify that combined inhibition of SLC7A11 or SP1 and CDK4/6 resulted in marked suppression of tumor growth in vivo. Conclusions: Experimentally, ferroptosis represents some of the CDK4/6i-induced cell death response. This study illustrates the potential for targeting SLC7A11 in combination with CDK4/6 inhibitors and supports the investigation of combination therapy in ER+ breast cancer.

目的 构建带Flag标签的RING结构泛素连接酶26(Ring finger protein 26,RNF26)真核表达载体,表达Flag-RNF26的融合蛋白,通过细胞免疫荧光实验,观察其在人肝癌细胞HepG2中的定位及与带绿色荧光蛋白标签的死骨片1(sequestosome 1,SQSTM1)是否存在共定位.方法 以人乳腺文库为模板,应用PCR技术扩增出RNF26的编码区序列,双酶切后插入带Flag标签的pCMV-Tag2B载体中,构建pCMV-Tag2B-RNF26重组质粒;将测序正确的重组质粒和pCMV-Tag2B空载体分别转染人胚肾293T细胞中,Western印迹检测融合蛋白表达情况,将成功表达的质粒pCMV-Tag2B-RNF26与pEGFP-SQSTM1质粒转染至人肝癌细胞HepG2中,进行免疫荧光染色,荧光显微镜观察SQSTM1和RNF26的共定位情况.结果 插入片段的DNA序列测序结果与目的基因序列相同,Western印迹检测提示,融合蛋白成功表达;免疫荧光结果表明,RNF26与SQSTM1蛋白在细胞质中存在共定位.结论 pCMV-Tag2B-RNF26真核表达载体构建成功.带Flag标签的人RNF26蛋白在HepG2细胞内成功表达,且与SQSTM1蛋白存在共定位,为进一步研究RNF26和SQSTM1的相互作用关系建立了实验基础.

Over the past decades, the incidence of thyroid cancer (TC) rapidly increased all over the world, with the papillary thyroid cancer (PTC) accounting for the vast majority of TC cases.It is crucial to investigate novel diagnostic and therapeutic targets for PTC and explore more detailed molecular mechanisms in the carcinogenesis and progression of PTC.Based on the TCGA and GEO databases, FAM111B is downregulated in PTC tissues and predicts better prognosis in PTC patients.FAM111B suppresses the growth, migration, invasion and glycolysis of PTC both in vitro and in vivo.Furthermore, estrogen inhibits FAM111B expression by DNMT3B methylation via enhancing the recruitment of DNMT3B to FAM111B promoter.DNMT3B-mediated FAM111B methylation accelerates the growth, migration, invasion and glycolysis of PTC cells.In clinical TC patient specimens, the expression of FAM111B is inversely correlated with the expressions of DNMT3B and the glycolytic gene PGK1.Besides, the expression of FAM111B is inversely correlated while DNMT3B is positively correlated with glucose uptake in PTC patients.Our work established E2/DNMT3B/FAM111B as a crucial axis in regulating the growth and progression of PTC.Suppression of DNMT3B or promotion of FAM111B will be potential promising strategies in the estrogen induced PTC.

线粒体是持续进行分裂和融合的动态细胞器.近年来,除了线粒体代谢作用相关的研究之外,线粒体动力学也开始逐渐引起研究的关注.越来越多的研究表明,线粒体动力学与肿瘤细胞生物学行为具有相关性.线粒体分裂蛋白1(mitochondrial fission protein 1,FIS1)介导线粒体分裂复合物的组装,参与线粒体分裂,是线粒体融合分裂过程中重要的蛋白质.然而,鲜有研究揭示FIS1在人宫颈癌中的表达及其作用.本研究对比了宫颈癌组织以及癌旁组织的转录物组数据,结果显示,与癌旁组织相比,人宫颈癌组织中的FIS1 mRNA水平明显降低(P＜0.01).进一步进行宫颈癌组织FIS1高表达组与低表达组的差异基因分析,发现差异基因主要与线粒体功能相关.随后,进行FIS1过表达后HeLa细胞增殖、迁移、线粒体裂变以及ROS水平的相关分析.结果显示,过表达FIS1基因,HeLa细胞增殖及迁移能力显著降低,细胞内线粒体裂变程度加剧并且细胞内ROS水平升高.综合以上结果,FIS1在人宫颈癌细胞中表达水平较低,而过表达FIS1可促使宫颈癌细胞因线粒体动力学失衡而发生一系列生物学功能异常.因此,本研究为进一步研究FIS1在宫颈癌治疗中的作用奠定了重要基础.

目的 构建带Flag标签的人溶质载体家族成员蛋白SLC25A39基因的真核表达载体,在获得Flag?SLC25A39融合蛋白表达后,研究SLC25A39表达对人乳腺癌细胞线粒体稳态的影响.方法 以人乳腺文库为模板采用PCR技术扩增出人SLC25A39的编码区序列,双酶切后将其插入到带Flag标签的pcDNA3.0载体上,转化大肠杆菌DH5α感受态细胞,菌液PCR鉴定阳性的克隆再进行质粒提取,测序正确后将重组质粒与空载体分别转染人乳腺癌细胞ZR75?1.Western印迹鉴定SLC25A39目的基因的表达;Mitotracker对乳腺癌细胞的线粒体进行染色,共聚焦显微镜对线粒体进行观察;Western印迹检测线粒体融合蛋白1(Mfn1)、线粒体分裂蛋白1(Fis1)的表达变化.结果 双酶切鉴定表明,Flag?SLC25A39真核表达载体构建成功;Western印迹验证SLC25A39基因表达成功;激光共聚焦显微镜下观察发现SLC25A39过表达的乳腺癌ZR75?1细胞分裂的线粒体较多;Western印迹证实SLC25A39基因过表达后线粒体分裂蛋白Fis1表达增高,线粒体融合蛋白Mfn1表达减弱.结论 成功构建带Flag标签的SLC25A39基因真核表达载体,该基因可促进人乳腺癌细胞的线粒体分裂,有望成为靶向线粒体治疗乳腺癌的潜在新靶点.

PurposeRecently, long noncoding RNA LINC01134 has been shown to reduce cell viability and apoptosis via the antioxidant stress pathway, thereby enhancing OXA resistance in hepatocellular carcinoma. However, the association of LINC01134 with ferroptosis and the underlying molecular mechanisms remain to be elucidated.MethodsBioinformatics analysis was employed to screen lncRNAs positively correlated with GPX4 and poor clinical prognosis. And Western blot and RT-PCR analysis in HCC cells confirmed the effect of LINC01134 on GPX4 expression. In addition, LINC01134 siRNA was transfected in HCC cells to detect the changes in cell viability, ROS, lipid peroxidation, MDA levels and GSH/GSSG levels. CCK-8, colony formation and apoptosis assays were performed to determine the effect of LINC01134 on cell death. The effect of LINC01134 and OXA on Nrf2 transcriptional binding to GPX4 was analyzed using dual luciferase reporter assay and CHIP. The expression of GPX4 and Nrf2 in HCC tissues was detected by FISH and IHC.ResultsLINC01134 is a novel lncRNA positively correlated with GPx4 and associated with poor clinical prognosis. Silenced LINC01134 conferred OXA sensitivity by enhancing total ROS, lipid ROS, MDA levels and decreasing GSH/GSSG ratio. Mechanistically, LINC01134 and OXA could promote Nrf2 recruitment to the GPX4 promoter region to exert transcriptional regulation of GPX4. Clinically, LINC01134 was positively correlated with GPX4 or Nrf2, demonstrating the clinical significance of LINC01134, Nrf2 and GPX4 in OXA resistance of HCC.ConclusionsWe identified LINC01134/Nrf2/GPX4 as a novel and critical axis to regulate HCC growth and progression. Targeting GPX4, knocking down LINC01134 or Nrf2 could be a potential therapeutic strategy for HCC.

Phosphoglycerate kinase 1 (PGK1), a critical component of the glycolytic pathway, relates to the development of various cancers. However, the mechanisms of PGK1 inhibition and physiological significance of PGK1 inhibitors in cancer cells are unclear. Long non-coding RNAs (lncRNAs) play a vital role in tumor growth and progression. Here, we identify a lncRNA LINC00926 that negatively regulates PGK1 expression and predicts good clinical outcome of breast cancer. LINC00926 downregulates PGK1 expression through the enhancement of PGK1 ubiquitination mediated by E3 ligase STUB1. Moreover, hypoxia inhibits LINC00926 expression and activates PGK1 expression largely through FOXO3A. FOXO3A/LINC00926/PGK1 axis regulates breast cancer glycolysis, tumor growth, and lung metastasis both in vitro and in vivo. In breast cancer patients, LINC00926 expression is negatively correlated with PGK1 and positively correlated with FOXO3A expression. Our work established FOXO3A/LINC00926/PGK1 as a critical axis to regulate breast cancer growth and progression. Targeting PGK1 or supplement of LINC00926 or FOXO3A could be potential therapeutic strategies in breast cancer.

BACKGROUND AND AIMS:Oxaliplatin (OXA) is one of the most common chemotherapeutics in advanced hepatocellular carcinoma (HCC), the resistance of which poses a big challenge. Long noncoding RNAs (lncRNAs) play vital roles in chemoresistance. Therefore, elucidating the underlying mechanisms and identifying predictive lncRNAs for OXA resistance is needed urgently.METHODS:RNA sequencing (RNA-seq) and fluorescence in situ hybridization (FISH) were used to investigate the OXA-resistant (OXA-R) lncRNAs. Survival analysis was performed to determine the clinical significance of homo sapiens long intergenic non-protein-coding RNA 1134 (LINC01134) and p62 expression. Luciferase, RNA immunoprecipitation (RIP), chromatin immunoprecipitation (ChIP), and chromatin isolation by RNA purification (ChIRP) assays were used to explore the mechanisms by which LINC01134 regulates p62 expression. The effects of LINC01134/SP1/p62 axis on OXA resistance were evaluated using cell viability, apoptosis, and mitochondrial function and morphology analysis. Xenografts were used to estimate the in vivo regulation of OXA resistance by LINC01134/SP1/p62 axis. ChIP, cell viability, and xenograft assays were used to identify the demethylase for LINC01134 up-regulation in OXA resistance.RESULTS:LINC01134 was identified as one of the most up-regulated lncRNAs in OXA-R cells. Higher LINC01134 expression predicted poorer OXA therapeutic efficacy. LINC01134 activates anti-oxidative pathway through p62 by recruiting transcription factor SP1 to the p62 promoter. The LINC01134/SP1/p62 axis regulates OXA resistance by altering cell viability, apoptosis, and mitochondrial homeostasis both in vitro and in vivo. Furthermore, the demethylase, lysine specific demethylase 1 (LSD1) was responsible for LINC01134 up-regulation in OXA-R cells. In patients with HCC, LINC01134 expression was positively correlated with p62 and LSD1 expressions, whereas SP1 expression positively correlated with p62 expression.CONCLUSIONS:LSD1/LINC01134/SP1/p62 axis is critical for OXA resistance in HCC. Evaluating LINC01134 expression in HCC will be effective in predicting OXA efficacy. In treatment-naive patients, targeting the LINC01134/SP1/p62 axis may be a promising strategy to overcome OXA chemoresistance.

Lactate dehydrogenase A (LDHA), a critical component of the glycolytic pathway, relates to the development of various cancers, including thyroid cancer. However, the regulatory mechanism of LDHA inhibition and the physiological significance of the LDHA inhibitors in papillary thyroid cancer (PTC) are unknown. Long non-coding RNA (lncRNA) plays a vital role in tumor growth and progression. Here, we identified a novel lncRNA LINC00671 negatively correlated with LDHA, downregulating LDHA expression and predicting good clinical outcome in thyroid cancer. Moreover, hypoxia inhibits LINC00671 expression and activates LDHA expression largely through transcriptional factor STAT3. STAT3/LINC00671/LDHA axis regulates thyroid cancer glycolysis, growth, and lung metastasis both in vitro and in vivo. In thyroid cancer patients, LINC00671 expression is negatively correlated with LDHA and STAT3 expression. Our work established STAT3/LINC00671/LDHA as a critical axis to regulate PTC growth and progression. Inhibition of LDHA or STAT3 or supplement of LINC00671 could be potential therapeutic strategies in thyroid cancer.

目的 构建带Flag标签的肾小管间质性肾炎抗原样蛋白1(Tinagl1)基因真核表达载体,检测其对肝癌细胞HepG2 ERK/AKT磷酸化、生长和迁移的影响.方法 以人乳腺cDNA文库为模板,PCR扩增Tinagl1基因片段,将其插入pcDNA3.0载体,经双酶切和测序验证后,将重组质粒转染到肝癌HepG2细胞中,采用Western印迹检测重组蛋白对ERK和AKT磷酸化水平的影响,生长曲线和划痕实验检测其对肝癌细胞HepG2生长和迁移的影响.结果 双酶切和Western印迹结果表明,pcDNA3.0-Flag-Tinagl1真核表达质粒构建成功,Tinagl1可降低ERK和AKT的磷酸化水平;生长曲线和划痕实验表明Tinagl1抑制肝癌细胞HepG2生长和迁移.结论 构建了带Flag标签的Tinagl1真核表达载体,并能抑制肝癌细胞HepG2中ERK/AKT磷酸化、生长和迁移,为进一步研究Tinagl1在肝癌细胞中的功能奠定了基础.

目的 构建SLC25A5基因的真核表达载体,同时验证该基因与肝癌细胞生长之间的关系.方法 以人乳腺文库为模板,采用PCR技术扩增出SLC25A5的CDS区编码序列,应用双酶切将PCR产物插入到带Flag标签的pCMV?Tag2B载体上,酶切和测序验证后转染到人肝癌HepG2细胞中,通过Western印迹检测其表达情况,CCK?8法、克隆形成实验测定其对肝癌HepG2细胞生长的影响.结果 双酶切结果显示,pCMV?Tag2B?Flag?SLC25A5真核表达载体构建成功;提取质粒转染人肝癌HepG2细胞;Western印迹技术验证该基因蛋白成功表达;CCK?8法和克隆形成实验证明过表达pCMV?Tag2B?Flag?SLC25A5促进肝癌HepG2细胞增殖.结论 pCMV?Tag2B?Flag?SLC25A5真核表达载体构建成功,并证明其可促进肝癌细胞增殖,为进一步研究SLC25A5在肝癌中的发生发展作用奠定了基础.