阻断PLCE基因对膀胱癌细胞侵袭能力的影响

目的构建PLCE基因的shRNA表达载体转染膀胱癌细胞T24株,观察其对T24细胞转移侵袭等恶性行为的影响。方法设计合成PLCE基因mRNA的寡核苷酸序列,插入绿色荧光蛋白质粒真核表达载体pGenesil中,构建重组载体pGenesil-PLCε。重组载体转染T24细胞后,RT-PCR和Western blot检测PLCE基因和蛋白表达;以侵袭小室、明胶酶谱分析及免疫组化检测细胞侵袭能力。结果成功构建了针对PLCE基因的shRNA表达载体。两个阳性重组质粒P1和P2转染膀胱癌细胞T24,P1和P2组目的基因/内参照吸光度比值较空载HK-A组明显降低(P<0.01);P1和P2组目的蛋白/内参照吸光度比值较空载HK-A组明显降低(P<0.01)。细胞侵袭实验中阳性质粒P1和P2转染组穿膜细胞数也明显低于空白对照组和阴性质粒HK-A转染组(P<0.01);转染P1、P2均使细胞分泌MMP-2、MMP-9明显低于空白对照和HK-A转染组(P<0.01)。结论阻断PLCE基因能有效抑制膀胱癌T24细胞系中PLCE基因和蛋白表达,并对T24细胞的侵袭转移能力具有一定的抑制作用。     

Influence of PLCE gene blockage on invasive power of human bladder cancer cells

目的 构建PLCE基因的shRNA表达载体转染膀胱癌细胞T24株,观察其对T24细胞转移侵袭等恶性行为的影响.方法 设计合成PLCE基因mRNA的寡核苷酸序列,插入绿色荧光蛋白质粒真核表达载体pGenesil中,构建重组载体pGenesil-PLCε.重组载体转染T24细胞后,RT-PCR和Western blot检测PLCE基因和蛋白表达;以侵袭小室、明胶酶谱分析及免疫组化检测细胞侵袭能力.结果 成功构建了针对PLCE基因的shRNA表达载体.两个阳性重组质粒P1和P2转染膀胱癌细胞T24,P1和P2组目的基因/内参照吸光度比值较空载HK-A组明显降低(P＜0.01);P1和P2组目的蛋白/内参照吸光度比值较空载HK-A组明显降低(P＜0.01).细胞侵袭实验中阳性质粒P1和P2转染组穿膜细胞数也明显低于空白对照组和阴性质粒HK-A转染组(P＜0.01);转染P1、P2均使细胞分泌MMP-2、MMP-9明显低于空白对照和HK-A转染组(P＜0.01).结论 阻断PLCE基因能有效抑制膀胱癌T24细胞系中PLCE基因和蛋白表达,并对T24细胞的侵袭转移能力具有一定的抑制作用.     

MTSS1基因在膀胱癌中表达下调促进T24细胞侵袭转移

目的筛查膀胱浸润性尿路上皮癌差异表达的基因,并探讨MTFSS1基因对膀胱癌T24细胞侵袭能力的影响。方法应用基因芯片结合生物信息学软件筛查膀胱浸润性尿路上皮癌差异表达的基因,并通过荧光定量PCR和Western Blot进行验证;通过转染技术,应用MTT和Transwell小室法分别检测MTSS1基因对膀胱癌T24细胞增殖和侵袭能力的影响。结果膀胱浸润性尿路上皮癌和其癌旁组织表达差异2倍以上的基因有1392个,荧光定量PCR和Westem Blot显示MTSS1在癌组织中较在癌旁组织中表达降低,两者差异35倍,P=0.0065,主要在趋化因子信号转导通路中发挥重要作用。转染结果显示MTSS1基因高表达列T24细胞增殖能力没有影响,而能明显抑制T24细胞的侵袭转移能力(P0.05)。结论 MTSS1基因在膀胱浸润性尿路上皮癌中表达下调是其促进该肿瘤侵袭转移的重要因素之一。     

miR-126对不同类型膀胱癌细胞增殖、迁移与侵袭的影响

目的 miR-126对不同类型膀胱癌细胞增殖、迁移与侵袭的影响。方法膀胱癌细胞系(EJ、BIU-87、T24、5637)和正常膀胱组织细胞系(SV-HUC-1)培养48h后,采用qPCR检测miR-126在不同转移潜能膀胱癌细胞株EJ、BIU-87、T24、5637中的表达;使用Lipo-2000脂质体将miR-126 siRNA转染入膀胱癌细胞,采用CCK-8增殖实验、划痕愈伤实验、Transwell实验、细胞黏附实验进行检测。结果与对照组相比,miR-126在EJ、BIU-87、T24、5637细胞株中呈过量表达状态,差异有统计学意义(P0.05)。miR-126 siRNA转染入膀胱癌细胞后,EJ、BIU-87、T24、5637细胞存活率、迁移、侵袭、黏附能力明显降低,差异有统计学意义(P0.05)。结论 miR-126在不同膀胱癌细胞中呈异常高表达状态,降低细胞内miR-126可以有效抑制膀胱癌细胞的增殖、迁移和侵袭能力,为膀胱癌的靶向分子治疗提供了新靶点。     

PLCε通过PKCα/β/TBX3信号通路调控人膀胱癌细胞系T24的迁移和侵袭

目的体外实验研究PLCε基因调节人膀胱癌细胞迁移和侵袭的分子机制。方法设计并合成针对PLCε基因mRNA的寡核苷酸序列,构建重组腺病毒Ad-sh PLCε。T24细胞感染Ad-sh PLCε腺病毒后,用Western blot检测PKCα/β及TBX3、E-cadherin的表达;用划痕实验、Transwell迁移和侵袭实验检测膀胱癌细胞T24的迁移、侵袭能力。结果 Ad-sh PLCε重组腺病毒感染膀胱癌细胞T24,对其PLCεmRNA表达抑制率为75.6%,对其PLCε蛋白表达抑制率为67.4%。Ad-sh PLCε感染组T24细胞迁移能力较空载组和空白对照组明显减弱(P0.05);重组腺病毒Ad-sh PLCε感染组T24细胞穿膜细胞数较空载组和空白对照组明显减少(P0.05)。Ad-sh PLCε重组腺病毒感染膀胱癌T24细胞后下游PKCα/β的活化受到抑制(P0.05),同时,TBX3的表达减少,而E-cadherin的表达增高(P0.05)。结论通过以重组腺病毒干扰抑制PLCε基因,能有效抑制膀胱癌T24细胞系中PLCε下游PKCα/β的活化情况及TBX3,E-cadherin的表达变化,并且对T24细胞的迁移、侵袭能力具有一定的抑制作用。     

siRNA下调SNCG基因表达抑制膀胱癌细胞系5637的增殖和侵袭

目的探讨突触核蛋白γ(SNCG)基因对膀胱癌细胞5637增殖和侵袭能力的影响。方法设计并合成3对SNCG-siRNA序列,转染5637细胞后,用RT-q PCR和Western blot检测SNCG的表达。分别通过CCK-8增殖实验和侵袭实验评估5637细胞增殖和侵袭能力。结果与膀胱癌细胞系T24、EJ和UMUC-3相比,5637细胞中SNCG表达水平最高(P0.05);与空白和阴性对照组相比,3对SNCG-siRNA序列转染5637后均可抑制SNCG mRNA和SNCG蛋白的表达(P0.05),但SNCG-siRNA-244组抑制效果最明显;实验组5637细胞的增殖受到明显抑制(P0.05),且细胞的侵袭能力显著下降(P0.05)。结论通过特异性干扰SNCG基因的表达可抑制膀胱癌细胞5637的增殖和侵袭,SNCG的siRNA序列可能成为膀胱癌治疗的新靶点。     

miR-205调控YES1表达对人膀胱癌细胞增殖的影响

目的探讨miR-205对膀胱癌T24细胞增殖的影响。方法合成miR-205的模拟物并转染膀胱癌T24细胞,荧光显微镜下观察转染效率,实时荧光定量PCR法检测转染48 h后,T24细胞中miR-205的表达情况,MTT法检测转染24、48和72 h后,T24细胞的增殖情况,实时荧光定量PCR法和western blot检测转染48 h后膀胱癌细胞内源性YES1 mRNA及蛋白的表达变化。结果 miR-205的模拟物转染至T24细胞的效率为80%,转染miR-205模拟物48 h后,T24细胞内miR-205的表达水平显著升高(P0.05),T24细胞的增殖率显著低于阴性对照组(P0.05),且呈时间依赖性,YES1 mRNA及蛋白的表达水平明显低于阴性对照组(P0.05)。结论 miR-205抑制膀胱癌T24细胞增殖可能与靶向YES1基因相关。     

RON基因在膀胱癌组织中的表达及对T24细胞增殖的影响

目的探讨RON基因对T24细胞增殖与迁移的影响。方法收集膀胱癌组织标本,通过RT-q PCR检测RON在膀胱癌组织中的表达,其次用siRNA技术干扰T24细胞RON,用RT-q PCR、Western blot检测干扰效果,CCK-8、Transwell实验及流式细胞术分别检测细胞的增殖、迁移及细胞凋亡。结果膀胱癌组织中RON表达与TNM分期呈正相关(P0.05)。在T24细胞的RON-siRNA组,细胞增殖能力减弱(P0.05),迁移与侵袭能力降低(P0.05),凋亡细胞数增加(P0.05),同时P-ERK蛋白表达减少,但Total-ERK表达不变。结论 RON基因促进膀胱癌细胞的增殖与迁移,有望成为膀胱癌治疗的新靶点。     

PIK3CA基因对人非小细胞肺癌A549细胞侵袭及迁移能力的影响

目的探讨PIK3CA基因对非小细胞肺癌侵袭及迁移能力的影响及可能机制。方法实时荧光定量PCR检测非小细胞肺癌组织、癌旁组织、非小细胞肺癌A549细胞与人支气管上皮细胞PIK3CA mRNA的表达,构建靶向PIK3CA基因的si RNA质粒,并转染至非小细胞肺癌A549细胞,实时荧光定量PCR技术与Westem blot方法分别检测PIK3CA mRNA与蛋白表达的变化,利用Transwell实验检测转染后细胞侵袭和转移能力的变化,Westem blot方法检测转染后A549细胞p-Akt蛋白表达变化。结果与癌旁正常组织比较,PIK3CA mRNA和蛋白表达水平在非小细胞肺癌组织显著上升(P0.05),A549细胞中PIK3CA mRNA和蛋白表达水平明显高于人支气管上皮细胞(P0.05)。PIK3CA基因沉默6h,A549细胞PIK3CA mRNA和蛋白表达水明显下降(P0.05);PIK3CA基因沉默48h,A549细胞侵袭和转移能力显著降低,p-Akt蛋白表达显著降低(P0.05)。结论 PIK3CA基因能够降低非小细胞肺癌侵袭及迁移能力,其作用机制可能与调控p-Akt蛋白表达有关。     

Notch信号通路调控上皮-间质转化影响膀胱癌侵袭性和耐药性

目的体外探讨Notch信号通路对膀胱癌细胞侵袭性与耐药性的影响及分子机制。方法采用Notch信号通路受体完全阻断剂(γ分泌酶抑制剂)处理膀胱癌T24、5637和J82细胞48 h后,倒置显微镜观察膀胱癌细胞增生及形态;用RT-PCR和Western blot在mRNA和蛋白水平检测上皮-间质转化(EMT)分子标志物E-cadherin、N-cadherin、vimentin和Alpha-smooth muscle actin的表达;MTT、Transwell检测膀胱癌细胞耐药性及侵袭能力。结果完全阻断Notch信号通路后,镜下显示膀胱癌细胞形态变小,细胞分散;EMT分子标志物E-cadherin mRNA和蛋白水平表达上调(P0.05),N-cadherin、vimentin、Alpha-smooth muscle actin mRNA和蛋白水平表达下调(P0.05);膀胱癌细胞T24、5637和J82增殖明显被抑制(P0.05);膀胱癌细胞T24、5637和J82穿过微孔膜的细胞数明显减少(P0.05)。结论 Notch信号通路可通过调控EMT的变化而改变膀胱癌的侵袭性与耐药性。     

Influence of the microenvironment on invasiveness of human bladder carcinoma cell lines

To investigate the importance of the microenvironment in bladder cancer invasion, a panel of six bladder carcinoma cell lines (SD, RT112, JON, 1207, T24, and J82) was tested in both in vitro and in vivo invasion assays. Furthermore, invasiveness was correlated with the expression of components of the E-cadherin-catenin complex. The E-cadherin-negative cell lines, T24 and J82, displayed a high in vitro invasive capacity, whereas the E-cadherin-positive cell lines, SD and JON, completely lacked in vitro invasive capacity. In contrast, in vivo invasion was noted for all cell lines, with the exception of cell line JON. Most notably, SD formed highly invasive tumors in vivo. The in vivo invasiveness of the E-cadherin-positive bladder carcinoma cell lines was associated with a heterogeneous expression of the E-cadherin-catenin complex. The discrepancy between in vitro and in vivo invasive behavior implies that, in vivo, the microenvironment plays an important role in the establishment of the invasive phenotype. In addition, it was found that orthotopic xenografting of 1207 and T24 bladder carcinoma cells resulted in site-specific tumor take and an enhanced tumor outgrowth and invasiveness, respectively, compared with heterotopic (i.e., subcutaneous) inoculation. We conclude that the site-specific growth and invasion of the bladder carcinoma cell lines in vivo and the observed assay specific invasion (in vitro vs in vivo) points to an effect of the local (bladder) microenvironment on tumor cell behavior.     

Increased expression of hepatoma-derived growth factor correlates with poor prognosis in human nasopharyngeal carcinoma

Wang S & Fang W
(2011) Histopathology 58 , 217–224
Increased expression of hepatoma‐derived growth factor correlates with poor prognosis in human nasopharyngeal carcinoma Aims: To examine the correlation between hepatoma‐derived growth factor (HDGF) expression and clinicopathological data in nasopharyngeal carcinoma (NPC), including patient survival. Methods and results: Using real‐time polymerase chain reaction (PCR) and Western blot, mRNA and protein expression of HDGF was detected in normal nasopharyngeal tissues, NPC tissues and cell lines. HDGF levels were determined further by an immunohistochemical analysis in a retrospective series consisting of 160 primary NPC tissues and 71 non‐cancerous nasopharynx tissues. Overexpressed mRNA and HDGF protein was present in NPC. By immunohistochemical analysis, we found that 53.8% (86 of 160) and 19.4% (32 of 160) of NPC biopsy specimens showed higher HDGF expression of the nucleus and cytoplasm, respectively. Statistical analysis showed that the higher expression of nuclear HDGF was associated significantly with T stage (P = 0.005) and clinical stage (P = 0.038), but there was no association with lymph node (P = 0.059) or distant metastasis (P = 0.563). Patients with increased HDGF expression levels had poorer overall survival rates than those with low expression of HDGF levels (P = 0.006). Multivariate analysis revealed that high expression of nuclear HDGF was an independent prognostic indicator of patient survival. Conclusions: Increased nuclear expression of HDGF is a potential unfavourable prognostic factor for patients with NPC.     

High expression of Notch ligand Jagged2 is associated with the metastasis and recurrence in urothelial carcinoma of bladder

Background: The Notch signaling pathway is closely related with human organ development and tumorgenisis. Jagged2 is among the most popular topic in Notch studies currently. Recent studies found its vital role in tumor metastasis in breast cancer; however, its expression profile and its prognostic value in urothelial carcinoma of bladder have not been investigated. Methods: Immunohistochemistry was used to detect the expression of Jagged2 in 120 bladder urothelial carcinoma. Moreover, the expression of Jagged2 was analyzed by Western blot in 60 bladder urothelial carcinoma and 20 normal epithelial tissues. MTT assay and flow cytometry and transwell assay were used to examine the proliferative and invasive ability of bladder cancer cells with the treatment of GSIXX (the inhibitor of Jagged2). Prognostic value of Jagged2 expression and its correlation with tumor metastasis and recurrence were evaluated, and the proliferative and invasive ability and cell cycle process of the bladder cancer cells were detected as well. Results: There was a significantly higher Jagged2 expressions in bladder urothelial carcinoma and highly invasive bladder T24 cells than those in bladder normal tissues and the superficial bladder BIU-87 cells. Jagged2 expression was positively correlated with histological grade, p T stage, recurrence, and metastasis. With the increasing concentration of GSIXX, we found that not only the cell proliferation and invasion activity decreased significantly, but also the cell cycle was blocked at G₂/M stage. Conclusions: Jagged2 expression status was closely correlated with important histopathologic characteristics (grades and stages) and the recurrence and metastasis of bladder urothelial carcinomas. Furthermore, Jagged2 played an important function on the bladder cancer cells’ proliferation by regulating the cancer cell cycle from G₁/S to G₂/M and probably promoted the invasion and metastasis of bladder cancer.     

Pro-inflammatory type-1 and anti-inflammatory type-2 macrophages differentially modulate cell survival and invasion of human bladder carcinoma T24 cells

Findings from numerous studies suggest that inflammation is likely to have an important role in bladder carcinogenesis and cancer disease progression. While macrophages (M?s) constitute a major inflammatory component of the stroma of human bladder carcinoma, the regulatory role of such inflammatory leukocytes in tumor cell survival and invasion remains elusive. Human urothelial bladder cancer (UBC) T24 cells and monocyte-derived macrophages were used to study the relative contribution of pro-inflammatory type-1 (M?-1) and anti-inflammatory type-2 (M?-2) macrophages in the regulation of UBC cell behaviour. Cell-to-cell studies indicated that the number of viable cells were considerable higher in T24 cell/M?-2 cocultures but lower in T24 cell/M?-1 cocultures when compared to cultures of T24 cells alone. M?-1-derived factors inhibit T24 cell growth but fail to induce caspase-3-mediated apoptosis. M?-2-derived factors have the ability to suppress the inhibitory effect of M?-1-derived factors on T24 cell growth. Exogenous interleukin (IL)-10 reverse M?-1-mediated arrest growth in T24 cell/M?-1 cell cocultures. Further analyses showed that M?-1-derived factors induced tumor necrosis factor (TNF)-α gene expression, promoted cellular invasiveness and increased phosphoinositide 3-kinase (PI 3-K)/Akt signaling pathway activity in T24 cells. Inhibition of PI 3-K activation in T24 cells or blockade of TNFα receptor in T24 cell/M?-1 cell cocultures decreased cellular invasiveness but did not affect T24 cell viability. Based on these observations, we propose that similar functional interactions between UBC cells and infiltrating macrophages can take place in vivo and influence tumor cell survival and invasion during bladder cancer progression.     

The relationship of BRMS1 and RhoGDI2 gene expression to metastatic potential in lineage related human bladder cancer cell lines

We have recently characterized a human bladder cancer cell line T24 and a more aggressive lineage related variant of it, T24T. To gain further insights, we have studied their metastatic ability in an in vivo model system. Results show that T24 forms significantly fewer [4/12 (1/11) mice had metastases with 1-2 lesions/mouse] metastasis in SCID/bg mice than T24T [14/14 (6/6) mice had metastases with a mean of 24-28 lesions/mouse]. To begin exploring the mechanisms underlying this difference, we evaluated the mRNA and protein expression levels of metastasis-suppressor genes, known to be important in the progression of other cancers, in our model of bladder cancer progression. A higher mRNA expression of BRMS1, a metastasis suppressor in breast cancer, was observed in T24 cells. In addition, RhoGDI2 mRNA expression was only observed in T24 when compared to T24T, suggesting that Rho activation might play a significant role in the metastatic cascade. However, a basal level mRNA expression of KISS1, described as metastasis suppressor in melanoma and breast, was observed in both the lines and had slightly higher expression in T24T. No difference of Nm23-H1, KAI1, MKK4/SEK1 and E-Cadherin protein levels were noted between these two lines. In summary, it appears that the T24/T24T paired cell lines constitute a useful model for the study of human bladder cancer metastasis that will allow both the discovery and mechanistic evaluation of genes potentially involved in this process. This revised version was published online in July 2006 with corrections to the Cover Date.     

In vitro invasiveness of human bladder cancer from cell lines and biopsy specimens

Aggregates prepared from cell lines established from a human transitional cell carcinoma of the urothelium (Hu 456) or from apparently normal urothelium before (Hu 609) and after phenotypic transformation (Hu 609T) were confronted with fragments of embryonic chick cardiac muscle in organ culture. In this assay a correlation was found between in vitro invasiveness of animal cell lines and their capacity to produce invasive tumours in syngeneic animals [1, 10, 11]. The invasiveness of cells from established human urothelial lines was compared to the invasiveness of cells from fresh biopsy specimens of a normal urothelium, a non-invasive papilloma, and a metastasizing transitional cell carcinoma. Cells from all established lines (Hu 609, Hu 609T and HU 456) and from the biopsy specimens of the transitional cell carcinoma occupied and eventually replaced the cardiac muscle by contrast with cells from the normal urothelium or from the non-invasive papilloma. We concluded that the organ culture assay for invasiveness might be used to define malignancy of human bladder cell lines and to follow the various steps during the acquisition of invasiveness in vitro.     

Expression of Robo protein in bladder cancer tissues and its effect on the growth of cancer cells by blocking Robo protein

This study aimed to detect the expression of Slit signaling protein ligand Robo protein in human bladder cancer and para-carcinoma tissue, and observe the tumor cell survival and growth by inoculating the bladder cancer cells with the blocked signaling protein into the subcutaneous tissue of nude mice. The expression of Robo protein was detected in T24 cells in human bladder uroepithelium carcinoma and cultivated human bladder uroepithelium carcinoma confirmed by pathological diagnosis. The cultivated T24 cells were coated by the protein antibody and human bladder uroepithelium carcinoma T24 tumor-bearing mice model was established. The tumor cell survival and growth were observed in the antibody coating group and non-coating group. The tumor body size was measured. The immunohistochemical detection showed that Robo protein isoforms Robo1 and Robo 4 were expressed in T24 cells of cancer tissues, paracarcinoma tissues and cultured human uroepithelium carcinoma. The expression of Robo1 was significantly higher than that of Robo4 (P<0.05). The cancer cells could be detected in nodular tumor of mice in each group. The volume of the tumor-bearing mice in the nodular tumor of the non-coating group was larger than that of anti-Robol antibody coating group and the difference was statistically significant (P<0.01). There was no significant difference in tumor volume between anti-Robo4 antibody coating group and non-coating group (P>0.05); The difference was statistically significant compared with the anti-Robo1 antibody coating group (P<0.01). In conclusion, Robo protein isoforms Robo1 and Robo4 were expressed in human bladder cancer T24 cells. To block Robo4 signal protein had little effect on the survival and growth of the transplantation tumor and to block Robo1 signal protein would seriously affect the survival and growth of the transplantation tumor, suggesting that Robo1 might play an important role in the growth and metastasis of bladder cancer, and might become a new target for the treatment of human bladder cancer and drug research.     

Genetic and phenotypic changes associated with the acquisition of tumorigenicity in human bladder cancer

There has been a general lack of human paired cell lines that both reproduce the in vivo spectrum of tumor progression of bladder cancer and have some of the genetic changes associated with progression in human tumor tissue. T24, a cell line established from an invasive human transitional cell carcinoma (TCC) of the bladder, has been used extensively in bladder cancer research. However, a significant limitation of this cell line is its lack of tumorigenicity when injected into immunocompromised mice. This characteristic was used to our advantage as we sought to characterize T24T, a highly tumorigenic variant that could then be used to elucidate the genes responsible for human bladder tumor progression. In culture, T24T has a faster doubling time, reaches a higher cell density in monolayer culture, and is more motile than T24 at higher cell densities. T24T is able to form colonies in soft agar, whereas T24 is not, and expresses HRAS, a gene associated with increased aggressiveness in human TCC, at higher levels than T24. Most importantly, T24T forms solid tumors when injected subcutaneously in SCID mice both with and without Matrigel (Sigma, St. Louis, MO), whereas T24 does not. Cytogenetically, the 2 cell lines contain at least 5 shared structural anomalies, as determined by detailed karyotyping. Interestingly, T24T has acquired 4 new structural changes, 3 of which [add(10)(p12), i(10)(q10), -15] have been observed in loss of heterozygosity (LOH) studies of tumor progression in human TCC. It appears that the T24/T24T model may be an excellent tool for the study of human TCC progression because of its relationship with known karyotypic changes associated with human bladder cancer progression. We are currently taking advantage of these paired cell lines to identify genes involved in human TCC progression. Genes Chromosomes Cancer 27:252-263, 2000.