HAb18G/CD147, a transmembrane glycoprotein highly expressed in various types of malignant cells, mainly functions as an inducer of matrix metalloproteinases to promote tumor growth, invasion and metastasis. However, whether there are other mechanisms underlying the role of HAb18G/CD147 in tumor progression remains to be elucidated. In this study, we investigated the functional effects of HAb18G/CD147 on autophagy in hepatoma cell line SMMC7721 using immunofluorescence staining, Western blot and transmission electronmicroscopy. Our data showed that specific small interference RNA (siRNA) considerably down-regulated the expression of HAb18G/CD147 in SMMC7721 cells at both messenger RNA (mRNA) and protein levels. The down-regulation of HAb18G/CD147 significantly promoted starvation-induced autophagy in a dose-dependent manner. Using trypan blue exclusion assay, we found that HAb18G/CD147 notably enhanced the survival of SMMC7721 cells through inhibiting starvation-induced autophagy. In addition, we demonstrated that HAb18G/CD147 down-regulated the expression of autophagy-regulating protein Beclin 1 in SMMC7721 cells. Furthermore, our data indicated that HAb18G siRNA-transfected SMMC7721 cells had a significantly decreased level of phosphorylated serine/threonine protein kinase B (pAkt) and the expression of Beclin 1 was inversely associated with the level of pAkt, suggesting that the Class I phosphatidylinositol 3 kinase-Akt pathway may be involved in the down-regulation of Beclin 1 by HAb18G/CD147. Overall, we provide the first experimental evidence to show that HAb18G/CD147 may play an important role in the inhibitory regulation of autophagy. Therefore, our data suggest a new molecular mechanism for HAb18G-mediated hepatoma progression.
HAb18G/CD147, a new hepatoma-associated antigen cloned and screened from human hepatocellular carcinoma cDNA library, is closely correlated with metastasis process in human hepatoma cells. In the present study we aimed to identify the pivotal molecules of the HAb18G/CD147 signal transduction pathway. The investigation showed that betaig-h3, a secretory extracellular matrix (ECM) protein, was upregulated in HAb18G/CD147-expressing human hepatoma T7721 cells and was downregulated by depressing HAb18G/CD147 expression. The expression of betaig-h3, upregulated in human hepatoma cells, was positively relative to the expression of HAb18G/CD147 in different human hepatoma cell lines. By overexpressing betaig-h3 in human SMMC-7721 hepatoma cells, we discovered that betaig-h3 promoted cell adhesion, invasion, and matrix metalloproteinase (MMP) secretion potential. HAb18G/CD147-induced invasion and metastasis potential of human hepatoma cells can be attenuated by antibodies specific for betaig-h3, and no significant differences on inhibitory effects were observed among T7721 cells incubated with antibodies for betaig-h3 or HAb18G/CD147 or both types together. Taken together, our study suggests that betaig-h3, regulated by the expression of HAb18G/CD147, is involved in the HAb18G/CD147 signal transduction pathway and mediates the HAb18G/CD147-induced invasion and metastasis process of human hepatoma cells.
Objective To investigate the inhibitory effects of siRNA targeting BCSG1 gene expression in tumor transplants of human breast cancer cell line in nude mice. Methods Four-pairs of small interfering RNA sequences of BCSG1 were chemically synthesized and inserted into the plasmid expression vectors, and were then transfected into human breast carcinoma cell line MCF7 by liposome method. Plasmid vector with unrelated sequence was used as the vector control. Cells transfected with 4siRNA sequences, control vector and naive FCF7 cells were transplanted into the nude mice. The tumor inhibition was analysised. Immunohistochemical SP method and semi-quantitative RT-PCR were adopted to detect the BCSG1 mRNA and protein expression, respectively. Breast tissue samples of human infiltrating ductal carcinoma, ductal hyperplasia and fibroadenoma were also used as the controls. Results The inhibition rates of tumor growth in four BCSG1-siRNA transfected groups were remarkably higher than those of the vector control group and naive MCF7 cells (P ＜0. 01 ). Compared with that of the vector control and na(i)ve MCF7 cell group, there was a significant decrease of BCSG-1 protein expression in the four experimental groups by immuno-histochemistry staining (P ＜0. 01 ). In addition, BCSG1 mRNA expression in the four groups transfected with BCSG1-siRNA were significantly less than that of the control vector group,naive MCF7 cell control group and human breast IDC (P ＜ 0. 01 ). Conclusion BCSG1-siRNA downregulates the expression of BCSG1 and inhibits effectively growth of the transplaned human breast cancer cell line in nude mice.
Key words:
Breast neoplasms; Proto-oncogenes; RNA interference; Tumor cells,cultured
Abstract Insulin is a protein hormone that controls the metabolism of sugar, fat and protein via signal transduction in cells, influencing growth and developmental processes such as reproduction and ageing. From nematodes to fruit flies, rodents and other animals, glucose signalling mechanisms are highly conserved. Reproductive termites (queens and kings) exhibit an extraordinarily long lifespan relative to non-reproductive individuals such as workers, despite being generated from the same genome, thus providing a unique model for the investigation of longevity. The key reason for this molecular mechanism, however, remains unclear. To clarify the molecular mechanism underlying this phenomenon, we sequenced the transcriptomes of the primary kings (PKs), primary queens (PQs), male (WMs) and female (WFs) workers of the lower subterranean termite Reticulitermes chinensis . We performed RNA sequencing and identified 33 insulin signalling pathway-related genes in R. chinensis . RT-qPCR analyses revealed that EIF4E and RPS6 genes were highly expressed in WMs and WFs workers, while mTOR expression was lower in PKs and PQs than in WMs and WFs. PQs and PKs exhibited lower expression of akt2-a than female workers. As the highly conserved insulin signalling pathway can significantly prolong the healthspan and lifespan, so we infer that the insulin signalling pathway regulates ageing in the subterranean termite R. chinensis . Further studies are recommended to reveal the biological function of insulin signalling pathway-related genes in the survival of termites to provide new insights into biomolecular homeostasis maintenance and its relationship to remarkable longevity.
Objective: To explore the function and molecular mechanism of Timeless in promoting hepatocellular carcinoma (HCC) growth. Methods: The expression of Timeless in HCC and paracancer tissues were analyzed by using the public data of HCC. Timeless was overexpressed in MHCC97L cells and silenced in MHCC97H cells, respectively, and the expression of Timeless and its downstream molecules were detected by real-time PCR and western blot. The effects of Timeless on cell glycolysis, oxidative phosphorylation and proliferation were detected by the glucose uptake experiment, lactic acid detection experiment, the extracellular fluid pH detection experiment, cell oxygen consumption test and cell viability assay, respectively. Results: The level of Timeless in HCC tissue was significantly higher than that of paracancer tissue (P<0.05). The relative cellular glucose uptake levels in the groups of Timeless knockdown, including siTimeless-1 and siTimeless-2 group were 0.510±0.119 and 0.508±0.099, respectively, significantly different from that of control group (P<0.05); The relative cellular uptake level of Timeless overexpressed group was 1.953±0.324, significantly different from that of vector transfected group (P<0.05). The relative levels of lactic acid production in the siTimeless-1 and siTimeless-2 group were 0.579±0.096 and 0.550±0.120, respectively, significantly different from that of control group (P<0.05); The relative production level of lactic acid in the Timeless overexpressed group was 1.463±0.179, significantly different that of vector transfected group (P<0.05). The extracellular pH values of siTimeless-1 and siTimeless-2 group were 7.390±0.035 and 7.370±0.060, respectively, significantly different from that of control group (P<0.05); the extracellular pH value of Timeless overexpressed group was 7.130±0.031, significantly different than vector transfected group (P<0.05). Oxygen consumption rate of siTimeless-1 and siTimeless-2 group were 3.686±0.389 and 3.955±0.431, respectively, significantly higher than 1.690±0.297 of control group (P<0.05); Oxygen consumption rate of Timeless overexpressed group was 1.302±0.336, significantly lower than 3.185±0.262 of vector transfected group (P<0.05) Timeless inhibited the expression of p53. The cell glucose uptake, lactic acid production, the pH of extracellular culture medium and cell oxygen consumption of control group were not significantly different from that of Timeless and p53 co-silenced group [(si-Timeless+sip53) group] (P>0.05); the glucose uptake, the production of lactic acid, the pH of the extracellular culture medium and the oxygen consumption of Timeless co-transfected with p53 (Timeless+p53) group were not significantly different from those of vector transfected group (P>0.05). Timeless promoted the proliferation of HCC cells through inhibiting the expression of p53. Conclusion: Timeless promotes reprogramming of glucose metabolism and proliferation of HCC cells by inhibiting the p53-dependent signaling pathway.目的： 探讨节律分子Timeless对肝癌细胞生长的影响及其分子机制。 方法： 采用肝癌公共数据信息分析Timeless在肝癌及癌旁组织中的表达；在高表达Timeless的肝癌细胞株MHCC97H和低表达Timeless的肝癌细胞株MHCC97L中，分别用RNA干扰和真核表达质粒方法沉默或上调肝癌细胞中Timeless的表达后，采用实时荧光定量PCR和Western blot检测干扰和过表达Timeless的效果以及Timeless下游分子的表达。采用葡萄糖摄取实验、乳酸检测实验、细胞外液pH检测实验、细胞氧耗实验和MTS实验，分析Timeless对肝癌细胞糖酵解、氧化磷酸化和细胞生长的影响。 结果： 肝癌组织中Timeless的表达高于癌旁组织(P<0.05)。敲低Timeless后，Timeless干扰组1和Timeless干扰组2细胞葡萄糖相对摄取水平分别为0.510±0.119和0.508±0.099，与对照组比较，差异均有统计学意义(均P<0.05)；Timeless过表达组细胞葡萄糖相对摄取水平为1.953±0.324，与空质粒组比较，差异有统计学意义(P<0.05)。Timeless干扰组1和Timeless干扰组2细胞乳酸产生相对水平分别为0.579±0.096和0.550±0.120，与对照组比较，差异有统计学意义(P<0.05)；Timeless过表达组细胞乳酸产生相对水平为1.463±0.179，与空质粒组比较，差异有统计学意义(P<0.05)。Timeless干扰组1和Timeless干扰组2细胞外pH值为7.390±0.035和7.370±0.060，与对照组比较，差异有统计学意义(P<0.05)；Timeless过表达组细胞外pH值为7.130±0.031，与空质粒组比较，差异有统计学意义(P<0.05)。Timeless干扰组1和Timeless干扰组2细胞耗氧率分别为3.686±0.389和3.955±0.431，与对照组(1.690±0.297)比较，差异有统计学意义(P<0.05)；Timeless过表达组细胞耗氧率为1.302±0.336，与空质粒组(3.185±0.262)比较，差异有统计学意义(P<0.05)。Timeless抑制p53的表达。与Timeless+p53对照组比较，Timeless+p53干扰组细胞葡萄糖相对摄取水平、乳酸产生相对水平、细胞外培养液pH和细胞氧耗率差异均无统计学意义(均P>0.05)；与Timeless+p53空质粒组比较，Timeless+p53过表达组细胞葡萄糖相对摄取水平、乳酸产生相对水平、细胞外培养液pH和细胞氧耗率差异均无统计学意义(均P>0.05)。Timeless通过抑制p53的表达促进肝癌细胞生长。 结论： Timeless通过抑制p53的表达促进细胞糖酵解和抑制氧化磷酸化，进而促进肝癌细胞生长。.
Objective To investigate the morphological features of a diseased inferior vena cava (IVC) diaphragm associated with Budd-Chiari syndrome,and to explore the pathogenic mechanisms.Methods Ten patients with a diseased IVC diaphragm associated with Budd-Chiari syndrome underwent radical resection in our hospital frome October 2007 to December 2010.We assessed the gross morphological features of these diaphragms.The structure of the diseased diaphragms was compared with that of the IVC retrohepatic segment using light and electron microscopy.Results The diseased diaphragms were located within 2.0 cm from the junction of the IVC and the right atrium.The diaphragms had thick edges and relatively thin centers; their surface connected with the inner membrane of the IVC.A mural thrombus was observed in the IVC below the diaphragm in nine patients,with partial calcification of the diaphragm in five patients.Light microscopy revealed that the diaphragm was composed of massive homogencous collagen fibers and fibroblasts.The collagen was highly disorganized and some collagen fibers showed hyaline degeneration.The edges of the diaphragms showed proliferation of fibrous connective tissue,together with some pathological changes,including the presence of some granulation tissue and neovessels.The structures of the normal inner,middle and outer membranes of the IVC remained intact.Electron microscopy revealed the diaphragms contained a relatively high number of fibroeytes,and a small number of endotheliocytes on their surface.Most of the cristae inside the endotheliocytes were disconnected from the inner membranes and fused,and were therefore blurred or missing,The rough endoplasmic reticulum was also slightly dilated.Apoptotic cells were infrequent,and were generally smaller than expected.The endotheliocytes on the normal IVC walls were well organized,showing a striped distribution.Mitochondria,rough endoplasmic reticolum and pinocytotic vesicles were found inside the endotheliocytes.Collageo fibers and elastic fibers below the endotheliocytes were sparse.Conclusion The diseased IVC diaphragm associated with Budd Chiari syndrome mainly develops as a result of fibrocyte proliferation,although IVC injury and inflammatory reactions may also be involved.
Key words:
Budd-Chiari syndrome; Inferior vena cava; Pathological membrane
