MiRNAs (miRs) are emerging as valuable biomarker for disease monitoring, diagnosis, and prognostic evaluation, particularly cancer. Herein, we have developed a highly sensitive and specific fluorescent biosensor for miRNA detection using a DNA nanoframework-confined well-organized catalytic hairpin assembly (DOCO-CHA) system. The biosensor leverages DNA dendrimers, self-assembled from Y-DNA1 and Y-DNA2, to provide abundant intercalation sites for immobilizing a large number of catalytic hairpin assembly (CHA) probes, thereby enhancing the reaction environment and improving detection efficiency. The DOCO-CHA system also demonstrated remarkable target recycling amplification, further enhancing sensitivity. Under optimal conditions, the biosensor achieved miR-21 detection with a detection limit of 16.4 pM. Moreover, the system exhibited excellent specificity and stability, with recoveries of 96.3 %-99.3 % in real serum sample analysis. These findings suggest that the developed DOCO-CHA system holds great potential for miRNA-based diagnostics.

Recurrence and metastasis of gastric cancer is a major therapeutic challenge for treatment. The presence of cancer stem cells (CSCs) is a major obstacle to the success of current cancer therapy, often leading to treatment resistance and tumor recurrence and metastasis. Therefore, it is important to develop effective strategies to eradicate CSCs. In this study, we developed a combined therapeutic strategy of photothermal therapy (PTT) and gastric cancer stem cells (GCSCs) inhibition by successfully synthesizing nanoliposomes loaded with IR780 (photosensitizer) and EN4 (c-Myc inhibitor). The nanocomposites are biocompatible and exhibit superior photoacoustic (PA) imaging properties. Under laser irradiation, IR780-mediated PTT effectively and rapidly killed tumor cells, while EN4 synergistically inhibited the self-renewal and stemness of GCSCs by suppressing the expression and activity of the pluripotent transcription factor c-Myc, preventing the tumor progression of gastric cancer. This Nano-EN-IR@Lip is expected to be a novel clinical nanomedicine for the integration of gastric cancer diagnosis, treatment and prevention.

Cancer-associated fibroblasts (CAFs) are the predominant stromal cells in the microenvironment and play important roles in tumor progression, including chemoresistance. However, the response of CAFs to chemotherapeutics and their effects on chemotherapeutic outcomes are largely unknown. In this study, we showed that epirubicin (EPI) treatment triggered ROS which initiated autophagy in CAFs, TCF12 inhibited autophagy flux and further promoted exosome secretion. Inhibition of EPI-induced reactive oxygen species (ROS) production with N-acetyl-L-cysteine (NAC) or suppression of autophagic initiation with short interfering RNA (siRNA) against ATG5 blunted exosome release from CAFs. Furthermore, exosome secreted from EPI-treated CAFs not only prevented ROS accumulation in CAFs but also upregulated the CXCR4 and c-Myc protein levels in recipient ER+ breast cancer cells, thus promoting EPI resistance of tumor cells. Together, the current study provides novel insights into the role of stressed CAFs in promoting tumor chemoresistance and reveal a new function of TCF12 in regulating autophagy impairment and exosome release.

目的 探讨血清可溶性E-钙黏蛋白(sE-cadherin)在慢性乙型肝炎(CHB)病毒感染中作用的分子机制.方法 2015年7月至2018年4月该院招募的研究对象中,检测包括51例乙型肝炎包膜抗原(HBeAg)阴性CHB、54例HBeAg阳性CHB和109例健康人血清中sE-cadherin、总抗氧化能力(TAC)、谷胱甘肽(GSH)、超氧化物歧化酶(SOD)、总氧化活性(TOA)、NADPH氧化酶2(NOX2)和丙二醛(MDA)等指标水平,并进行分析.结果 CHB患者的血清sE-cadherin水平显著高于健康人,差异有统计学意义(P<0.01),CHB患者血清sE-cadherin水平与TAC、抗氧化酶(GSH和SOD)呈显著负相关(P<0.05),血清sE-cadherin水平与肝酶标志物(谷丙转氨酶和谷草转氨酶)、氧化性标志物(TOA、NOX2和MDA)水平呈正相关(P<0.05).结论 血清sE-cadherin可能是反映CHB病毒感染发展过程中炎症和氧化应激状态的一个全新生物标志物.

The aberrant classical miRNAs are considered to play significant roles in tumor progression. However, it remains unclear for nonclassical miRNAs, a set of Drosha-independent miRNAs in the process of various biology. Here, we reveal that a nonclassical miR-4646-5p plays a pivotal role in gastric cancer (GC) metastasis. MiR-4646-5p, one of Drosha-independent mirtronic miRNA, is aberrant up-regulated in Drosha-low expressed GC and Drosha-knockdown gastric cancer cells. Mirtronic miR-4646-5p is a specific transcription splicing product of intron 3 of the host gene Abhd16a with the aid of SRSF2. The enhanced miR-4646-5p can stabilize HIF1A by targeting PHD3 to positive feedback regulate Abhd16a and miR-4646-5p itself expressions. ABHD16A, as an emerging phosphatidylserine-specific lipase, involves in lipid metabolism leading to lysophosphatidylserines (lyso-PSs) accumulation, which stimulates RhoA and downstream LIMK/cofilin cascade activity through GPR34/Gi subunit, thus causes metastasis of gastric cancer. In addition, miR-4646-5p/PHD3/HIF1A signaling can also up-regulate RhoA expression and synergistically promote gastric cancer cell invasion and metastasis. Our study provides new insights of nonclassical mirtronic miRNA on tumor progress and may serve as a new diagnostic biomarker for gastric cancer. MiR-4646-5p and its host gene Abhd16a mediated abnormal lipid metabolism may be a new target for clinical treatment of gastric cancer.

Objective. To demonstrate whether procalcitonin (PCT) combined with calcitonin (CT) could provide additional diagnostic value to other clinically available rheumatoid arthritis- (RA-) related biomarkers in the early diagnosis of RA. Method. The blood samples aseptically collected by venipuncture were centrifuged within 1 hour and frozen at -80°C. PCT and CT levels were measured using electrochemiluminescence immunoassay (ECLIA) in 260 subjects (48 patients with early RA, 34 patients with established RA, 37 patients with systemic lupus erythematosus, 30 with osteoarthritis, 31 with gouty arthritis, and 80 healthy participants). Anti-cyclic citrullinated peptide (Anti-CCP) and anti-RA33 antibodies (Anti-RA33) were analyzed by ELISA. RF was detected by transmission immunoturbidimetry. Mann–Whitney U tests and Kruskal-Wallis tests compared differences among groups. Spearman’s rank correlation analysis determined the relationship between biomarkers. Receiver-operator characteristic (ROC) curves were generated, and diagnostic performance was assessed by area under the curve (AUC), as well as specificity, sensitivity, likelihood ratios (LR). Results. Median serum PCT concentrations were significantly higher ( p < 0.0001 ) in patients with early RA (0.065 ng/ml) when compared with healthy controls (0.024 ng/ml), and patients with osteoarthritis (0.025 ng/ml). When compared with gouty arthritis (GA) controls (0.072 ng/ml) and systemic lupus erythematosus (SLE) controls (0.093 ng/ml), median serum PCT concentrations were not significant in patients with early RA (0.065 ng/ml). Median serum CT concentrations were significantly lower ( p < 0.0001 ) in patients with early RA (0.880 pg/ml) compared with healthy controls (3.159 pg/ml), patients with SLE (2.480 pg/ml), and patients with GA (2.550 pg/ml). When compared with osteoarthritis controls (0.586 pg/ml), median serum CT concentrations were not significant in patients with early RA (0.880 pg/ml). ROC curve analysis comparing early RA with healthy controls demonstrated that the AUC of RF, anti-CCP, and anti-RA33 were 0.66, 0.73, and 0.64, respectively; the additions of PCT and CT further improved the diagnostic ability of early RA with the AUC of 0.97, 0.98, and 0.97, respectively ( p < 0.01 ). The sensitivities of RF, anti-CCP, and anti-RA33 for early RA were 33.33%, 44.74%, and 58.33%, respectively, and the additions of PCT and CT showed very high sensitivities of 83.33%, 92.11%, and 87.50%. The high-value groups of PCT moderately correlated with the anti-RA33 levels ( r = 0.417 , p < 0.05 ). CT had no significant correlation with disease duration, radiographic progression, or clinical/serological variables, such as ESR levels, CRP levels, RF, anti-CCP, and anti-RA33 levels in early RA. Conclusions. Serum PCT and CT combined with clinically available RA-related biomarkers could further improve the diagnostic efficiency of early RA.

Drosha-dependent canonical microRNAs (miRNAs) play a crucial role in the biological functions and development of cancer. However, the effects of Drosha-independent non-canonical miRNAs remain poorly understood. In our previous work, we found a set of aberrant miRNAs, including some upregulated miRNAs, called Drosha-independent noncanonical miRNAs, in Drosha-knockdown gastric cancer (GC) cells. Surprisingly, Drosha-silenced GC cells still retained strong malignant properties (e.g., proliferation ability and cancer stem cell (CSC) characteristics), indicating that aberrantly upregulated non-canonical miRNAs may play an important role in the maintenance of the malignant properties in GC cells that express low Drosha levels. Here, we report that miR6778-5p, a noncanonical miRNA, acts as a crucial regulator for maintenance of CSC stemness in Drosha-silenced GC cells. MiR-6778-5p belongs to the 5'-tail mirtron type of non-canonical miRNAs and is transcript splice-derived from intron 5 of SHMT1 (coding cytoplasmic serine hydroxymethyltransferase). It positively regulates expression of its host gene, SHMT1, via targeting YWHAE in Drosha-knockdown GC cells. Similar to its family member SHMT2, SHMT1 plays a crucial role in folate-dependent serine/glycine inter-conversion in one-carbon metabolism. In Drosha wild type GC cells, SHMT2 mediates a mitochondrial-carbon metabolic pathway, which is a major pathway of one-carbon metabolism in normal cells and most cancer cells. However, in Drosha-silenced or Drosha low-expressing GC cells, miR-6778-5p positively regulates SHMT1, instead of SHMT2, thus mediating a compensatory activation of cytoplasmic carbon metabolism that plays an essential role in the maintenance of CSCs in gastric cancer (GCSCs). Drosha wild type GCSCs with SHMT2 are sensitive to 5-fluorouracil; however, Drosha low-expressing GCSCs with SHMT1 are 5-FU-resistant. The loss of miR-6778-5p or SHMT1 notably mitigates GCSC sphere formation and increases sensitivity to 5-fluorouracil in Drosha-knockdown gastric cancer cells. Thus, our study reveals a novel function of Drosha-independent noncanonical miRNAs in maintaining the stemness of GCSCs.

Background: Distinguishing the natural course of chronic HBV infection (CHB) development is extremely desired. The pore-forming protein Gasdermin D (GSDMD) contributes to the development of HBV infection and might be used to distinguish different phases of CHB. We aim to investigate the serum expression of GSDMD in the natural course of CHB development and to evaluate its diagnostic efficacy.Methods: Levels of GSDMD were measured with ELISA kits that we developed. We evaluated the ability of serum GSDMD to differentiate clinical stages in 73 CHB patients. Besides, The HepG2-NTCP cells infected by virus from HepAD38-cell and CHB patients’ serum were applied to evaluate the GSDMD expression in vitro.Findings: The GSDMD elevated gradually with the development of HBV infection (P < 0.05) and the same phenomenon was observed in vitro. A cutoff value as ≤ 3·0 ng/mL identified healthy individuals from liver diseases patients with 96·479 % sensitivity (AUC, 0·954; P < 0.01). When the cutoff value as 3·0-104·5 ng/mL, the AUC and sensitivity of asymptomatic carriers patients were 0·694 and 91·603 %. In CHB patients, with the cutoff value as 104·5-160·0 ng/mL, the AUC and sensitivity were 0·701 and 56·897 %. When the cutoff value as 160-178 ng/mL, the active chronic hepatitis B patients, the AUC and sensitivity were 0·662 and 52·174 %. Additionally, HBV-related cirrhosis patients, GSDMD positive defined as > 178 ng/mL, the sensitivity was 52·174 %.Interpretation: Serum GSDMD, varied along with the development of HBV-related diseases, making it an early and efficacious serum biomarker for auxiliary definitions of the natural course of CHB.Funding Statement: This work was supported by the Outstanding Young Talents Plan of the Second Hospital of Chongqing Medical University (Pu Li).Declaration of Interests: There are no conflicts of interest.Ethics Approval Statement: The study was carried out based on the principles of the Declaration of Helsinki and was approved by the medical ethics committee of the Second Affiliated Hospital of Chongqing Medical University.

目的:探究肝硬化相关血清学指标与肝硬化Child-Pugh分级之间的关系和临床意义.方法:收集重庆医科大学附属第二医院感染科2016年7月至2017年4月间肝硬化患者血清235例,其中Child-Pugh A组55例,B组90例,C组90例,以及同期健康体检人群35例,分别检测肝功能、肝纤维化、血小板计数等指标并分析其特征.结果:透明质酸(hyaluronic acid,HA)、层粘连蛋白(laminin,LN)、Ⅲ型胶原蛋白(typeⅢcollagen,PC-Ⅲ)、Ⅳ型胶原蛋白(typeⅣcollagen,Ⅳ-C)、天门冬氨酸氨基转移酶(aspartate aminotransferase,AST)、丙氨酰氨基转移酶(alanyl aminotransferase,ALT)及天门冬氨酸氨基转移酶与血小板计数比(aspartate aminotransferase to platelet ratio index,APRI)指数之间差异均具有统计学意义(P＜0.05).HA、PC-Ⅲ、Ⅳ-C、LN和AST指标对预测肝硬化的曲线下面积(AUC)分别为0.910、0.804、0.833、0.753、0.730.HA、PC-Ⅲ、Ⅳ-C与Child-Pugh分级呈高度正相关(r＞0.7,P＜0.0l),APRI与分级呈显著性正相关(0.4＜r＜0.7,P＜0.05).结论:HA、PC-Ⅲ、Ⅳ-C、APRI可作为Child-Pugh分级的潜在临床指标,这将有助于临床更好地制定诊疗方案及预后评估.

目的:探讨稳定干扰癌相关成纤维细胞(cancer-associated fibroblast,CAF)中Yes相关蛋白1(Yes-associated protein 1,YAP1)表达对乳腺癌MDA-MB-231细胞迁移和侵袭的影响.方法:用生物信息学方法分析乳腺原代CAF与对应正常成纤维细胞(normal fibroblast,NF)的mRNA芯片数据中YAP1的表达,并采用实时荧光定量PCR和蛋白质印迹法检测乳腺原代及永生化CAF与NF细胞中YAP1 mRNA和蛋白的表达水平,以验证芯片结果的真实性和准确性.用NF与CAF条件培养液分别培养乳腺癌MDA-MB-231细胞,然后采用Transwell小室法比较NF与CAF对乳腺癌细胞迁移和侵袭的影响.构建靶向YAP1基因的shRNA重组质粒并转染入CAF,采用实时荧光定量PCR和蛋白质印迹法检测CAF细胞中YAP1 mRNA和蛋白的表达水平,然后采用划痕愈合实验和Transwell小室法分别检测干扰YAP1表达后的CAF条件培养液对乳腺癌MDA-MB-231细胞迁移和侵袭能力的影响.用Hippo通路抑制剂XMU-MP-1处理CAF,采用蛋白质印迹法检测CAF细胞中YAP1总蛋白和磷酸化蛋白水平,Transwell小室法检测XMU-MP-1处理后的CAF条件培养液对MDA-MB-231细胞侵袭能力的影响.采用实时荧光定量PCR法检测干扰YAP1表达后的CAF细胞中YAP1下游与迁移和侵袭密切相关的转化因子β1(transforming growth factor-beta 1,TGF-β1)和白细胞介素6 (interleukin 6,IL6)的表达,并采用Collagen Ⅰ胶原凝胶收缩实验检测干扰YAP1表达对细胞外基质重塑的影响.结果:mRNA芯片检测发现,与正常对照NF相比,乳腺原代CAF中YAP1高表达(P＜0.05).实时荧光定量PCR和蛋白质印迹法检测证实原代及永生化CAF中YAP1 mRNA (P＜0.05)和蛋白(P＜0.01)水平均明显高于NF.与NF相比较,CAF条件培养液可明显促进乳腺癌MDA-MB-231细胞侵袭(P＜0.01).成功构建YAP1 shRNA稳定转染的CAF细胞株,其中YAP1 mRNA和蛋白表达水平均明显下调(P值均＜ 0.01).与未干扰YAP1表达的CAF对照组相比,经稳定干扰YAP1的CAF条件培养液处理后,乳腺癌MDA-MB-231细胞的迁移(P＜0.05)和侵袭(P＜0.01)能力均明显减弱.用Hippo通路抑制剂XMU-MP-1处理后,CAF细胞中YAP1磷酸化蛋白水平明显降低(P＜0.01),经该CAF条件培养液处理后MDA-MB-231细胞的侵袭能力明显增强(P＜0.05).干扰YAP1表达后的CAF细胞中TGF-β1和IL6 mRNA表达水平均明显下调(P值均＜ 0.05),且细胞外基质的胶原凝胶收缩能力明显减弱(P＜0.05).结论:稳定干扰CAF细胞中YAP1表达可能通过下调细胞因子TGF-β1和IL6的表达,以及重塑细胞外基质,抑制乳腺癌MDA-MB-231细胞的迁移和侵袭.

目的：探讨S100A9在乙型肝炎病毒X（HBx）介导的HepG2细胞增殖及迁移中的作用。方法：用表达HBx蛋白的重组腺病毒AdHBx感染HepG2细胞后，用CCK-8实验检测细胞增殖能力及划痕愈合实验检测细胞迁移能力；在HepG2/AdHBx细胞中转染S100A9-siRNA及其对照siRNA后，检测HepG2细胞增殖及迁移能力；在HepG2/AdHBx和对照组HepG2/AdGFP细胞中，采用Real- time PCR及Western Blot检测S100A9基因及蛋白的表达情况；在HepG2/AdHBx细胞中，加入不同剂量的NF-κB抑制剂BAY11-7082后，检测各组中S100A9的基因及蛋白表达情况。结果：HBx促进HepG2细胞的增殖与迁移；S100A9-siRNA抑制S100A9的表达后，HBx促进HepG2细胞的增殖与迁移的作用降低，HBx介导的HepG2细胞的增殖与迁移部分依赖于S100A9；S100A9基因及蛋白表达在HepG2/AdHBx中较对照组HepG2/AdGFP显著升高，HBx可致S100A9表达增加；抑制NF-κB转录活性后，AdHBx+BAY11-7082组S100A9基因及蛋白表达较对照组显著降低，阻断NF-κB转录活性可部分抑制HBx调控的S100A9表达。结论：HBx可调控S100A9的表达且与NF-κB活化有关， S100A9参与 HBx介导的HepG2细胞的增殖与迁移。

探讨miR-200c在癌相关成纤维细胞(cancer-associated fibroblasts,CAF)活化中的作用,研究其对人乳腺癌细胞MDA-MB-231侵袭能力的影响.通过质粒构建的方法分别获得miR-200c稳定干扰的NF(normal fibroblast)及其对照细胞株、miR-200c稳定过表达的CAF及其对照细胞株;采用Western blotting法检测miR-200c对CAF活化标志物α-SMA和FAP表达的影响;采用细胞划痕愈合实验检测miR-200c对CAF迁移能力的影响;采用胶收缩实验和侵袭实验分别检测miR-200c对CAF细胞外基质和对癌细胞侵袭能力的影响.成功构建了miR-200c稳定干扰的NF及其对照细胞株、miR-200c稳定过表达的CAF及其对照细胞株;与低表达miR-200c的细胞株相比,高表达miR-200c细胞株的α-SMA和FAP表达水平及迁移能力明显降低,其胶收缩能力和癌细胞的侵袭能力也明显减弱.miR-200c能够明显抑制CAF细胞的活化并通过细胞外基质重塑(extracellular matrix remodeling,ECM)的方式抑制癌细胞的侵袭.

Drosha is an RNA III-like enzyme that has an aberrant expression in some tumors. Our previous studies showed the aberrant Drosha in gastric tumors. However, the roles of nuclear Drosha, the main regulator of microRNA (miRNA) biogenesis, in gastric cancer (GC) progression remain poorly understood. In this study, we demonstrated that nuclear Drosha is significantly associated with cell invasion of GC and that Drosha silence impedes the tumor invasion. Knockdown of Drosha led to a set of dysregulated miRNAs in GC cells. Multiple targets of these miRNAs were the members in cell migration, invasion and metastasis-associated signaling (e.g. ECM-receptor interaction, focal adhesion, p53 signaling and MAPK signaling pathway) revealed by bioinformatics analysis. LAMC2 (a key element of ECM-receptor signaling) and CD82 (a suppressor of p53 signaling) are the targets of miR-622 and miR-197, respectively. High levels of LAMC2 and low levels of CD82 were significantly related to the worse outcome for GC patients. Furthermore, overexpression of LAMC2 and knockdown of CD82 markedly promoted GC cell invasion and activated EGFR/ERK1/2-MMP7 signaling via upregulation of the expression of phosphorylated (p)-EGFR, p-ERK1/2 and MMP7. Our findings suggest that nuclear Drosha potentially has a role in the development of GC.

Tumor microenvironment contributes to tumor angiogenesis. However, the role of the activated cancer associated-fibroblasts (CAFs) in angiogenesis is still unclear. Here we report that miR-205/YAP1 signaling in the activated stromal fibroblasts plays a critical role in VEGF-independent angiogenesis in breast tumor. Methods: miR-205 expression was assessed by quantitative real-time polymerase chain reaction (qRT-PCR); YAP1 expression by qRT-PCR, western blotting and immunohistochemistry; IL11 and IL15 expression by qRT-PCR, western blotting and ELISA. Tube formation and three-dimensioned sprouting assays in vitro, and orthotopic Xenografts in vivo were conducted as angiogenesis experiments. The mechanism of miR-205/YAP1-mediated tumor angiogenesis was analyzed via overexpression and shRNA, siRNA, or antibody neutralization experiments in combination with anti-VEGF antibody or Axitinib. Results: miR-205/YAP1 signaling axis activates breast normal fibroblasts (NFs) into CAFs, promotes tubule formation and sprouting of Human Umbilical Vein Endothelial Cells (HUVECs). Rescue of miR-205 in CAFs blunts angiogenesis processes. YAP1, a target of miR-205, does not regulate VEGF expression but specifically enhances IL11 and IL15 expressions, maintaining tumor angiogenesis even in the presence of Axitinib or after exhaustion of VEGF by neutralizing VEGF antibody. IL11 and IL15 released from CAFs activate STAT3 signaling in HUVECs. Blockage of IL11 and IL15 expression in CAFs results in the inactivation of STAT3-signaling in HUVECs and repression of the CAF-induced angiogenesis. The blunt angiogenesis halts the invasion and metastasis of breast cancer cells in vivo. Conclusions: These results provide a novel insight into breast CAF-induced tumor angiogenesis in a VEGF-independent manner.

Drosha is an RNA III-like enzyme that has an aberrant expression in some tumors. Our previous studies showed the aberrant Drosha in gastric tumors. However, the roles of nuclear Drosha , the main regulator of microRNA (miRNA) biogenesis, in gastric cancer (GC) progression remain poorly understood. In this study, we demonstrated that nuclear Drosha is significantly associated with cell invasion of GC and that Drosha silence impedes the tumor invasion. Knockdown of Drosha led to a set of dysregulated miRNAs in GC cells. Multiple targets of these miRNAs were the members in cell migration, invasion and metastasis-associated signaling (e.g. ECM-receptor interaction, focal adhesion, p53 signaling and MAPK signaling pathway) revealed by bioinformatics analysis. LAMC2 (a key element of ECM-receptor signaling) and CD82 (a suppressor of p53 signaling) are the targets of miR-622 and miR-197, respectively. High levels of LAMC2 and low levels of CD82 were significantly related to the worse outcome for GC patients. Furthermore, overexpression of LAMC2 and knockdown of CD82 markedly promoted GC cell invasion and activated EGFR/ERK1/2-MMP7 signaling via upregulation of the expression of phosphorylated (p)-EGFR, p-ERK1/2 and MMP7. Our findings suggest that nuclear Drosha potentially has a role in the development of GC.

目的:探讨稳定干扰整合素β3(integrin β3,ITGB3)基因对人乳腺癌BT549细胞增殖的影响.方法:采用实时荧光定量PCR法和蛋白质印迹法检测乳腺癌BT549、MCF-7和MDA-MB-453细胞中ITGB3 mRNA和蛋白的表达.将干扰ITGB3表达的LV-ITGB3-shRNA慢病毒感染BT549细胞后,应用实时荧光定量PCR法和蛋白质印迹法检测BT549细胞中ITGB3 mRNA和蛋白的表达水平,MTT法和FCM法检测BT549细胞的增殖能力和细胞周期,蛋白质印迹法检测BT549细胞中c-Myc和cyclin D1蛋白的表达情况.结果:乳腺癌BT549细胞中ITGB3 mRNA和蛋白的表达水平高于MCF-7和MDA-MB-453细胞(P值均＜0.05).LV-ITGB3-shRNA慢病毒感染后,BT549细胞中ITGB3 mRNA和蛋白的表达水平低于阴性对照组(LV-NC-shRNA感染BT549细胞)和空白对照组(BT549细胞未进行感染)(P值均＜ 0.01),细胞增殖能力增强(P＜0.01),S期细胞所占百分比上升(P＜0.01),c-Myc和cyclin D1蛋白的表达水平上调(P值均＜0.05).结论:稳定干扰BT549细胞中ITGB3表达后,可能通过上调c-Myc和cyclin D1蛋白的表达而促进细胞增殖.

Background The nuclear localization of Drosha is critical for its function as a microRNA maturation regulator. Dephosphorylation of Drosha at serine 300 and serine 302 disrupts its nuclear localization, and aberrant distribution of Drosha has been detected in some tumors. Aims The purpose of the present study was to assess cytoplasmic/nuclear Drosha expression in gastric cancer carcinogenesis and progression. Methods Drosha expression and its subcellular location was investigated by immunohistochemical staining of a set of tissue microarrays composed of normal adjacent tissues (374), chronic gastritis (137), precancerous lesions (94), and gastric adenocarcinoma (829) samples, and in gastric cancer cell lines with varying differentiation by immunofluorescence and western blot assay. Results Gradual loss of cytoplasmic Drosha was accompanied by tumor progression in both gastric cancer tissues and cell lines, and was inversely associated with tumor volume ( P = 0.002), tumor grade ( P < 0.001), tumor stage ( P = 0.018), and distant metastasis ( P = 0.026). Aberrant high levels of cytoplasmic Drosha were apparent in intestinal metaplasia and dysplasia tissues. The levels of nuclear Drosha were sharply decreased in chronic gastritis and maintained through precancerous lesions to gastric cancer. High levels of cytoplasmic Drosha predicted longer survival (LR = 7.088, P = 0.008) in gastric cancer patients. Conclusions Our data provide novel insights into gastric cancer that cytoplasmic Drosha potentially plays a role in preventing carcinogenesis and tumor progression, and may be an independent predictor of patient outcome.