枸杞多糖对大鼠角膜缘干细胞体外增殖的影响研究

目的通过体外分离和培养大鼠角膜缘干细胞(Limbal Stem Cells, LSCs)，观察枸杞多糖(Lyciumbarbarum Polysaccharide, LBP)对LSCs体外增殖的影响，为中药体外培养LSCs的研究提供实验资料。 方法体外分离和培养LSCs，显微镜观察LSCs的细胞形态特征，通过免疫荧光检测转录因子p63初步鉴定大鼠LSCs；分对照组和枸杞多糖处理组，每组20例，免疫印迹检测增殖细胞核抗原(Proliferating Cell Nuclear Antigen, PCNA)，初步探讨枸杞多糖对LSCs的增殖影响。 结果镜下观察LSCs体积小，呈多边形或椭圆形，胞核较大，核质比例大，免疫荧光结果显示体外分离的LSCs可表达p63(阳性率为76.41±1.66%)；枸杞多糖处理后LSCs的PCNA蛋白的表达水平高于对照组，并且随着枸杞多糖浓度的增加，PCNA蛋白的表达升高(P<0.05)。 结论枸杞多糖对LSCs的增殖和生长具有促进和维持的作用。     

Human papillomavirus and urinary bladder cancer revisited

This review aims to present data on the association between human papillomavirus (HPV) and urinary bladder cancer (BC), especially of the subtype squamous cell carcinoma (SCC). Furthermore, the current data on the relation between p16, HPV, and BC are reviewed. PubMed was searched for ‘Humans’ [MESH] AND ‘Papillomaviridae’ [MESH] AND ‘Urinary Bladder Neoplasms’ [MESH], resulting in 157 potential articles. After profound reviewing, 18 articles were included in this review. Only original articles in English were included. A variable number of HPV genotypes in a small number of cases have been investigated in several studies with various methodology. HPV was present in 0–100% of cases depending on inclusion and exclusion criteria. SCC studies are mostly hampered by low number of cases whereas the few studies with a high number show a slightly higher prevalence of different HPV genotypes compared to pure urothelial carcinoma. Studies on p16 status in HPV positive cases are even more scarcely reported and show conflicting results. Most studies fail to prove clear-cut relevance of HPV in BC irrespectively of histological subtype. Negative p16 staining cannot rule out positive HPV status.     

miR-513a-3p靶向鼠双微体基因2对胃癌细胞增殖迁移侵袭的影响

目的探讨miR-513a-3p靶向鼠双微体基因2(MDM2)对胃癌细胞的增殖、迁移和侵袭的影响及其作用机制。方法采用脂质体法将miR-NC、miR-513a-3p、anti-miR-NC、anti-miR-513a-3p、si-NC、si-MDM2、miR-513a-3p+pcDNA3.1和miR-513a-3p+pcDNA3.1-MDM2转染至BGC-823细胞中,采用实时荧光定量聚合酶链反应(qRT-PCR)检测miR-513a-3p的表达水平,采用Western blot检测cyclin D1、MMP-2、p21、E-cadherin和MDM2蛋白的表达水平,四甲基偶氮唑蓝法检测各组胃癌细胞BGC-823的活性,Transwell法检测各组胃癌细胞BGC-823的迁移和侵袭能力,双荧光素酶报告基因检测实验检测miR-513a-3p与MDM2的靶向关系。结果胃癌细胞BGC-823、MGC-803中miR-513a-3p的表达水平分别为0.21±0.02和0.34±0.03,与胃上皮细胞GES-1(0.76±0.08)比较,差异均有统计学意义(均P<0.05)。培养24、48和72 h后,miR-NC组细胞的吸光度(A)值分别为0.57±0.05、1.03±0.10和1.43±0.14,miR-513a-3p组细胞的A值分别为0.36±0.03、0.48±0.05和0.63±0.06,差异均有统计学意义(均P<0.05);miR-NC组细胞的迁移和侵袭数分别为(130±11.80)个和(117±10.60)个,miR-513a-3p组细胞分别为(58±5.64)个和(50±5.13)个,差异均有统计学意义(均P<0.05)。培养24、48和72 h后,si-NC组细胞的A值分别为0.53±0.05、0.95±0.10和1.36±0.14,si-MDM2组细胞的A值分别为0.39±0.04、0.57±0.06和0.80±0.08;si-NC组细胞的迁移和侵袭数分别为(141±12.02)个和(109±10.60)个,si-MDM2组的迁移和侵袭数分别为(66±6.67)个和(61±6.18)个,差异均有统计学意义(均P<0.05)。培养24、48和72 h后,miR-513a-3p+pcDNA3.1组细胞的A值分别为0.34±0.03、0.46±0.05和0.61±0.06,miR-513a-3p+pcDNA3.1-MDM2组细胞的A值分别为0.48±0.05、0.82±0.08和1.17±0.12,差异均有统计学意义(均P<0.05);miR-513a-3p+pcDNA3.1组细胞的迁移和侵袭数分别为(56±5.71)个和(51±5.16)个,miR-513a-3p+pcDNA3.1-MDM2组分别为(113±10.28)个和(104±10.02)个,差异均有统计学意义(均P<0.05)。结论miR-513a-3p可抑制胃癌细胞的增殖、迁移和侵袭能力,其机制可能与靶向调控MDM2的表达有关,可为胃癌的预防和治疗提供新靶点。     

MicroRNA‐375‐3p enhances chemosensitivity to 5‐fluorouracil by targeting thymidylate synthase in colorectal cancer

Resistance to chemotherapy is a major challenge for the treatment of patients with colorectal cancer (CRC). Previous studies have found that microRNAs (miRNAs) play key roles in drug resistance; however, the role of miRNA‐373‐3p (miR‐375‐3p) in CRC remains unclear. The current study aimed to explore the potential function of miR‐375‐3p in 5‐fluorouracil (5‐FU) resistance. MicroRNA‐375‐3p was found to be widely downregulated in human CRC cell lines and tissues and to promote the sensitivity of CRC cells to 5‐FU by inducing colon cancer cell apoptosis and cycle arrest and by inhibiting cell growth, migration, and invasion in vitro. Thymidylate synthase (TYMS) was found to be a direct target of miR‐375‐3p, and TYMS knockdown exerted similar effects as miR‐375‐3p overexpression on the CRC cellular response to 5‐FU. Lipid‐coated calcium carbonate nanoparticles (NPs) were designed to cotransport 5‐FU and miR‐375‐3p into cells efficiently and rapidly and to release the drugs in a weakly acidic tumor microenvironment. The therapeutic effect of combined miR‐375 + 5‐FU/NPs was significantly higher than that of the individual treatments in mouse s.c. xenografts derived from HCT116 cells. Our results suggest that restoring miR‐375‐3p levels could be a future novel therapeutic strategy to enhance chemosensitivity to 5‐FU.     

Knockdown of PVT1 inhibits IL-1β-induced injury in chondrocytes by regulating miR-27b-3p/TRAF3 axis

Long noncoding RNA plasmacytoma variant translocation 1 (PVT1) has been identified to implicate in the progression of osteoarthritis (OA). However, the mechanism underlying PVT1 in OA development remains largely unknown. This study aimed to investigate the effect of PVT1 on interleukin-1 beta (IL-1β)-induced injury in chondrocytes and explore potential mechanism. The cartilage tissues from 25 OA patients and normal controls were collected. Human transformed chondrocytes C28/I2 were stimulated by IL-1β. The levels of PVT1, microRNA (miR)-27b-3p, and tumor necrosis factor receptor-associated factor 3 (TRAF3) were detected by quantitative real-time polymerase chain reaction or western blot. IL-1β-induced injury was investigated by cell viability, apoptosis, autophagy and inflammatory response using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, flow cytometry, western blot and enzyme linked immunosorbent assay, respectively. The target association between miR-27b-3p and PVT1 or TRAF3 was explored by luciferase reporter, RNA immunoprecipitation and RNA pull-down assays. We found that PVT1 expression was enhanced in OA patients and IL-1β-treated C28/I2 cells. Silence of PVT1 promoted cell viability and autophagy but suppressed apoptosis and inflammatory response in IL-1β-treated C28/I2 cells. miR-27b-3p was confirmed as a target of PVT1 and its deficiency reversed the suppressive effect of PVT1 knockdown on IL-1β-induced injury. TRAF3 was a target of miR-27b-3p and attenuated the effect of miR-27b-3p on IL-1β-induced injury in C28/I2 cells. Moreover, TRAF3 expression was positively regulated by PVT1 via sponging miR-27b-3p. Collectively, knockdown of PVT1 increased cell viability and autophagy but inhibited apoptosis and inflammatory response in chondrocytes treated by IL-1β via up-regulating miR-27b-3p and down-regulating TRAF3.     

Combined use of cytology,p16 immunostaining and genotyping for triage of women positive for high-risk human papillomavirus at primary screening

Human papillomavirus (HPV) testing is very sensitive for primary cervical screening but has low specificity. Triage tests that improve specificity but maintain high sensitivity are needed. Women enrolled in the experimental arm of Phase 2 of the New Technologies for Cervical Cancer randomized controlled cervical screening trial were tested for high-risk HPV (hrHPV) and referred to colposcopy if positive. hrHPV-positive women also had HPV genotyping (by polymerase chain reaction with GP5+/GP6+ primers and reverse line blotting), immunostaining for p16 overexpression and cytology. We computed sensitivity, specificity and positive predictive value (PPV) for different combinations of tests and determined potential hierarchical ordering of triage tests. A number of 1,091 HPV-positive women had valid tests for cytology, p16 and genotyping. Ninety-two of them had cervical intraepithelial neoplasia grade 2+ (CIN2+) histology and 40 of them had CIN grade 3+ (CIN3+) histology. The PPV for CIN2+ was >10% in hrHPV-positive women with positive high-grade squamous intraepithelial lesion (61.3%), positive low-grade squamous intraepithelial lesion (LSIL+) (18.3%) and positive atypical squamous cells of undetermined significance (14.8%) cytology, p16 positive (16.7%) and, hierarchically, for infections by HPV33, 16, 35, 59, 31 and 52 (in decreasing order). Referral of women positive for either p16 or LSIL+ cytology had 97.8% sensitivity for CIN2+ and women negative for both of these had a 3-year CIN3+ risk of 0.2%. Similar results were seen for women being either p16 or HPV16/33 positive. hrHPV-positive women who were negative for p16 and cytology (LSIL threshold) had a very low CIN3+ rate in the following 3 years. Recalling them after that interval and referring those positive for either test to immediate colposcopy seem to be an efficient triage strategy. The same applies to p16 and HPV16.