Suppression of non-small cell lung tumor development by the let-7 microRNA family

Many microRNAs (miRNAs) target mRNAs involved in processes aberrant in tumorigenesis, such as proliferation, survival, and differentiation. In particular, the let-7 miRNA family has been proposed to function in tumor suppression, because reduced expression of let-7 family members is common in non-small cell lung cancer (NSCLC). Here, we show that let-7 functionally inhibits non-small cell tumor development. Ectopic expression of let-7g in K-Ras(G12D)-expressing murine lung cancer cells induced both cell cycle arrest and cell death. In tumor xenografts, we observed significant growth reduction of both murine and human non-small cell lung tumors when overexpression of let-7g was induced from lentiviral vectors. In let-7g expressing tumors, reductions in Ras family and HMGA2 protein levels were detected. Importantly, let-7g-mediated tumor suppression was more potent in lung cancer cell lines harboring oncogenic K-Ras mutations than in lines with other mutations. Ectopic expression of K-Ras(G12D) largely rescued let-7g mediated tumor suppression, whereas ectopic expression of HMGA2 was less effective. Finally, in an autochthonous model of NSCLC in the mouse, let-7g expression substantially reduced lung tumor burden.     

Effects of HMGA2 on malignant degree,invasion,metastasis,proliferation and cellular morphology of ovarian cancer cells

Objective:To analyze effects of high mobility group AT-hook 2(HMGA2)on malignant degree,invasion,metastasis,proliferation and cellular morphology of ovarian cancer cells.Methods:Three methods were applied to observe the effect on HMGA2 expression in ovarian cancer cells and ovarian epithelial cells.Results:After the application of siRNA-HMGA2,number of T29A2-cell clones was decreased,there was significant difference compared with the negative control Block-iT.After application of let-7c,number of T29A2+cell clones was decreased significantly,however,after the application of Anti-let-7,the number of clones restored,and there was no significant difference compared with the negative control group.After interference,the number of T29A2-cells which passed through Matrigel polycarbonate membrane were significantly lower than the negative control group.After the treatment of siRNA-HMGA2,let-7c and sh-HMGA2respectively,growth and proliferation of T29A2-,T29A2+and SKOV3 were slower,and the phenomenon was most obvious in SKOV3.Stable interference of HMGA2 induced mesenchymalepithelial changes in the morphology of SKOV3-sh-HMGA2.Conclusions:HMGA2 can promote malignant transformation of ovarian cancer cells,enhance cell invasion and metastasis,and promote cell growth and proliferation of ovarian cancer cells,which can cause ovarian cancer to progress rapidly and affect the quality of life.     

Epithelial mesenchymal transition of non-small-cell lung cancer cells A549 induced by SPHK1

Objective:To explore the effect and molecular mechanism of SPHK1 in the invasion and metastasis process of non-small-cell lung cancer cells(A549).Methods:Recombinant retrovirus was used to mediate the production of A549/vector,A549/SPHK1,A549/scramble,and A549/SPHK1/RNAi that stably expressed or silenced SPHK1.The invasion and migration capacities of A549 cells overexpressing or silencing SPHK1 were determined using Transwell invasion assay and scratch wound repair experiment.The protein and mRNA expression levels of E-cadherin,fibronectin,vimentin in A549/vector,A549/SPHK1,A549/scramble,A549/SPHK1/RNAi were detected with Western blot(WB) and quantitative PCR(QPCR) methods,respectively.Results:Transwell invasion assay and scratch wound repair experiments showed that over-expression of SPHK1 obviously enhanced the invasion and migration capacities of A549 cells.WB and QPCR detection results showed that,the expression of E-cadherin(a molecular marker of epithelial cells) and fibronectin,vimentin(molecular markers of mesenchymal cells) in A549 cells was upregulated after overexpression of SPHK1;while SPHK1 silencing significantly reduced the invasion and metastasis capacities of A549 cells,upregulated the expression of molecular marker of epithelial cells,and downregulated the expression of molecular marker of mesenchymal cells.Conclusions:SPHK1 promotes epithelial mesenchymal transition of non-small-cell lung cancer cells and affects the invasion and metastasis capacities of these cells.     

Fibroblast growth factors 7 and 10 are expressed in the human embryonic pancreatic mesenchyme and promote the proliferation of embryonic pancreatic epithelial cells

Aims/hypothesis The fibroblast growth factor (FGF) family consists of 22 members. In rodents, several FGFs are expressed in the pancreas, where they participate in epithelial–mesenchymal interactions. Our objective was to describe the pattern of expression of FGFs in the human embryonic pancreas and to analyse their effect on pancreas development.Methods The expression of FGFs was analysed by RT-PCR. To investigate the cell types expressing FGF7 and FGF10, we separated epithelial from mesenchymal cells using immunomagnetic beads linked to E-cadherin antibodies and performed real-time PCR. The effect of FGF7 and FGF10 on proliferation of human embryonic pancreatic epithelial cells was evaluated in vitro by measuring BrdU incorporation.Results We found that different FGFs are expressed in the human embryonic pancreas, and we focused on FGF7 and FGF10. We defined a new approach to separating epithelial cells (containing the pancreatic progenitor cells) from mesenchymal cells. This allowed us to demonstrate that human embryonic pancreatic mesenchymal cells express both FGF7 and FGF10. We next demonstrated that FGF7 and FGF10 were able to induce the proliferation of the epithelial cells in vitro.Conclusion/interpretation These findings indicate that it is now possible to efficiently separate human embryonic pancreatic epithelial from mesenchymal cells, an important step to characterize and expand progenitor cells. This method allowed us to demonstrate that human embryonic pancreatic mesenchyme expresses FGF7 and FGF10 that act on epithelial cells to activate their proliferation. Such growth factors could thus be used to expand human embryonic pancreatic epithelial cells.     

p38 MAPK和JNK信号通道在TGF-β1诱导的肺泡上皮细胞上皮-间质转化中的意义

目的 探讨p38丝裂原活化蛋白激酶（p38 MAPK）和c-Jun氨基端激酶（JNK）信号通道在转化生长因子β1 （TGF-β1）诱导的人肺泡上皮细胞上皮-间质转化（epithelial to mesenchymal transition,EMT）中的意义,从而进一步揭示肺纤维化的发病机制.方法 使用TGF-β1诱导A549细胞EMT,分别在蛋白水平（使用p38 MAPK抑制剂SB203580和JNK抑制剂SP600125）和RNA水平（RNA干扰）抑制p38 MAPK和JNK信号通道,检测抑制后A549细胞的p38 MAPK和JNK的蛋白和mRNA,以及间质细胞表型蛋白包括结蛋白、波形蛋白、α-平滑肌肌动蛋白和上皮细胞表型蛋白包括E-钙黏素、紧密连接蛋白-1、水通道蛋白-5的表达.结果 TGF-β1可以诱导A549细胞EMT,表现为间质细胞表型蛋白表达的增加和上皮细胞表型蛋白表达的减少,这一过程p38 MAPK和JNK通道表达也增加,无论蛋白水平抑制或基因沉默p38 MAPK和JNK均可减轻A549细胞的EMT.结论 p38 MAPK和JNK信号通道在TGF-β1诱导的A549细胞EMT过程中起重要作用.     

活化血管内皮生长因子受体-1诱导肝癌细胞MHCC97-H上皮-间叶表型转化的实验研究

目的 探讨血管内皮生长因子受体1(VEGFR-1)促肝细胞癌(HCC)侵袭转移的作用机制.方法 用VEGFR-1的特异性配体血管内皮生长因子-B(VEGF-B)诱导活化肝癌细胞MHCC97-H,观察MHCC97-H细胞形态学改变,RT-PCR和Western blot检测MHCC97 H细胞上皮标志物钙黏蛋白(E-cad)、连环蛋白-a(a-cat)和间叶标志物波形蛋白、神经钙黏蛋白(N-cad)的mRNA和蛋白质表达改变,细胞荧光免疫组织化学法检测E-cad、a-cat和波形蛋白、N-cad表达部位改变,细胞侵袭和迁移试验检测MHCC97-H细胞侵袭和运动能力的改变.组间比较采用单因素方差分析.结果 VEGFR-1活化后MHCC97-H细胞变成梭形、纺锤状,细胞间隙增宽,有的伸出伪足;活化前上皮标志物E-cad、a-cat的mRNA吸光度值(A值)分别为12.55±2.98、14.23±1.36,活化后E-cad、a-cat的A值分别为6.78±3.66、6.18±0.92,活化后上皮标志物的mRNA表达显著下调,F=17.21,P＜0.05.活化前上皮标志物E cad、a-cat蛋白的A值分别为20 878±11.54、7520.45±8.66,活化后E cad、a-cat的A值分别为8031.23±10.44、5425.15±7.37,活化后上皮标志物的蛋白表达显著下调,F=30.49,P＜0.05.波形蛋白、Ncad mRNA的A值分别为0.72±1.77、4.46±6.50,活化后的分别为26.58±7.97、26.98±10.79,活化后间叶标志物的mRNA表达显著上调,F=26.24,P＜0.05.活化前波形蛋白、Ncad蛋白的A值分别为6100.22±12.73、1244.64±10.27,活化后的分别为12836.99±9.67、4586.70±8.58,活化后间叶标志物的蛋白表达显著上调,F=19.16,P＜0.05.上皮标志物E-cad和a-cat在胞膜表达减少,胞质中的表达增加,波形蛋白和N-cad在胞质中表达显著增加;MHCC97-H细胞运动和侵袭能力显著增强,与活化前相比,F=20.13,P＜0.05,差异有统计学意义.结论 VEGFR-1促进肝细胞癌侵袭和转移是通过诱导肝癌细胞发生上皮-间叶表型转化实现的.     

Distinct populations of tumor-initiating cells derived from a tumor generated by rat mammary cancer stem cells

Tumors derived from rat LA7 cancer stem cells (CSCs) contain a hierarchy of cells with different capacities to generate self-renewing spheres and tubules serially ex vivo and to evoke tumors in vivo. We isolated two morphologically distinct cell types with distinct tumorigenic potential from LA7-evoked tumors: cells with polygonal morphology that are characterized by expression of p21/^WAF1 and p63 and display hallmarks of CSCs and elongated epithelial cells, which generate tumors with far less heterogeneity than LA7 CSCs. Serial transplantation of elongated epithelial cells results in progressive loss of tumorigenic potential; tumor heterogeneity; CD44, E-cadherin, and epithelial cytokeratin expression and increased α-smooth muscle actin I and vimentin expression. In contrast, serial transplantation of LA7 CSCs can be performed indefinitely and results in tumors that maintain their heterogeneity, consistent with self-renewal and multilineage differentiation potential. Collectively, our data show that polygonal cells are CSCs, whereas epithelial elongated cells are lineage-committed progenitors with tumorigenic potential, and suggest that tumor progenitors, although lacking indefinite self-renewal potential, nevertheless may make a substantial contribution to tumor development. Because LA7 cells can switch between conditions that favor maintenance of pure CSCs vs. differentiation into other tumor cell types, this cell system provides the opportunity to study factors that influence CSC self-renewal and differentiation. One factor, p63, was identified as a key gene regulating the transition between CSCs and early progenitor cells.     

食管鳞癌中微小核糖核酸let-7的表达及病理生物学意义

目的 探讨微小RNA(miRNA)1et-7对食管鳞癌细胞增殖的影响及食管鳞癌组织中let-7表达水平与临床病理特征间的关系.方法 利用RNA干扰(RNAi)和细胞转染技术将食管鳞癌细胞Eca109分别转染入let-7、let-7抑制剂及随机序列.以正常培养的Eca109细胞为阴性对照组.噻唑蓝(MTT)比色法检测各组Eca109细胞的增殖情况.实时荧光定量聚合酶链反应(qRTPCR)检测各组细胞、45例食管鳞癌组织及其癌旁组织中let-7的表达水平,并分析其与食管鳞癌临床病理特征间的关系.结果 转染后72 h,转染let-7组吸光度(A)值较阴性对照组明显降低(P=0.005),转染let-7抑制剂组A值较阴性对照组明显升高(P=0.029).与阴性对照组let-7表达量比较,转染let-7组表达增加33%(1.33比1.00,P=0.039),转染let-7抑制剂组表达降低50%(0.50比1.00,P=0.014).食管鳞癌组织和癌旁组织中let-7相对表达量的比值为0.66±0.47,差异有统计学意义(P=0.001).汉族患者食管鳞癌组织中let-7相对表达量(0.48±0.43)低于哈萨克族(0.88±0.51,P=0.019).低分化食管鳞癌组织中let-7相对表达量(0.42±0.30)低于高分化食管鳞癌组织(0.84±0.38,P=0.015).有淋巴结转移者食管鳞癌组织中let-7相对表达量(0.50±0.35)低于无淋巴结转移者(0.80±0.52,P=0.032).结论 let-7对食管鳞癌的发生、发展起抑制作用,其表达水平与组织分化程度、淋巴结转移及民族相关.

Abstract：

Objective To estimate the effect of microRNA (miRNA) let-7 expression on human esophageal squamous cell carcinoma(ESCC) and the relationship between let-7 level and clinicopathological parameters. Methods ESCC cell line (Eca109) was transfected with let-7 or its inhibitor by RNAi and cell transfection techniques. Normal cultured Eca109 cell was served as negative control. The proliferation of Eca109 cell was detected by MTT. The expression of let-7 in Eca109 cells and 45 paired ESCC tissues and corresponding para-cancerous tissues were measured using real-time quantitative polymerase chain reaction (qRT-PCR). The relationship between let-7 level and clinicopathological parameters in patients with ESCC was analyzed. Results The A value of let-7 in Eca109 cells transfected with let-7 was lower than negative control (P=0.005), while it was higher in Eca109 cells transfected inhibitor than that in negative control 72 hours after transfection. In comparison with negative control, the expression of let-7 in Eca109 cells transfected with let-7 was increased 33% (1.33 vs 1.00,P=0. 039) and it was decreased 50% in Eca109 cells transfected with inhibitor (0.50 vs 1.00,P=0. 014). The ratio of let-7 expression in ESCC tissue and para-cancerous tissue was 0.66 ± 0.47 with significant differece (P= 0.001). Moreover, The level of let-7 expression in Han patients with ESCC was lower than Kazakh patients with ESCC (0.48±0.43 vs 0. 88±0.51,P=0. 019). The level of let-7 expression in poorly differentiated ESCC tissue was lower than well differentiated ESCC tissue (0.42±0.30 vs 0.84±0.38,P=0. 015). The level of let-7 expression in patients with lymph node metastasis was lower than those without lymph node metastasis (0.50±0.35vs 0. 80±0.52,P=0. 032) . Conclusion It is demonstrated that let-7 can inhibit the carcinogenesis and development of ESCC. The level of let-7 expression is associated with cell differentiation,lymph node metastasis and nationalities.