Wnts are powerful regulators of cell proliferation and differentiation. Activation of Wnt signalling in many tissues has also been associated with cancer. RNAi is a process of posttranscriptional gene silencing, by wh...Wnts are powerful regulators of cell proliferation and differentiation. Activation of Wnt signalling in many tissues has also been associated with cancer. RNAi is a process of posttranscriptional gene silencing, by which dsRNA induces sequence-specific degradation of homologous gene transcripts. In many eukaryotes, expression of nuclear-encoded mRNA can be strongly inhibited by the presence of a small double-stranded RNA corresponding to exon sequences in the mRNA. In this study the pAVU6＋27 vector, which has Sail and Xbal clone sites, was used to construct the siRNA expression vectors for mouse wnt9a. Four kinds of small interfering RNA inserts were designed, synthesized and visually tested for efficacy by in situ hybridization, the results demonstrated that dramatically reduced wnt9a signals were observed in the cells transfected with U6＋27 cassettes with anti-wnt9a hairpin siRNA inserts compared with the untransfected. The results of flow cytometry analysis showed that the cell proliferation was promoted after lowering expression of the mouse wnt9a in MA891 cells by RNAi. All that suggest the expression level of mouse wnt9a in breast cancer MA891 cells may play a role in adjusting the rate of proliferation.展开更多
The paper aimed to study the effect of lysyl oxidase-like 2 （LOXL2） on hepatocellular carcinoma （HCC） and explore the biological mechanisms of tumorigenicity and progression in HCC. The authors used four HCC cell ...The paper aimed to study the effect of lysyl oxidase-like 2 （LOXL2） on hepatocellular carcinoma （HCC） and explore the biological mechanisms of tumorigenicity and progression in HCC. The authors used four HCC cell lines to identify LOXL2. A lentiviral vector containing LOXL2-siRNA was constructed to silence the LOXL2 gene in SMMC-7721 cell line, and mRNA of the target gene was detected by real-time polymerase chain reaction （RT-PCR）. The effect of LOXL2 silencing on the growth of SMMC-7721 cells was explored with flow cytometry profiling and BrdU labeling. Downstream genes of LOXL2 were selected by microarray and verified by Western Blotting. In the results, LOXL2 expression was significantly up-regulated in four types of HCC cell lines, therefore, SMMC-7721 cell line was selected for further exploration. When SMMC-7721 cell line was infected with LOXL2-siRNA, the expression of LOXL2 mRNA decreased. The silencing of LOXL2 resulted in the cell cycle arrest at the G 1-phase, the increased apoptosis and the decreased growth of SMMC-7721 cells on the indicated days by BrdU. Moreover, the MDM2, BIRC3, CDC42, FOS and TGFBR2 genes were selected and verified to be the downstream genes of LOXL2. In conclusion, LOXL2 contributes to the genesis and progression of HCC cells and works by regulating downstream genes of LOXL2 in certain pathways. Therefore, LOXL2 may play an important role in the progression and prognosis of HCC.展开更多
文摘Wnts are powerful regulators of cell proliferation and differentiation. Activation of Wnt signalling in many tissues has also been associated with cancer. RNAi is a process of posttranscriptional gene silencing, by which dsRNA induces sequence-specific degradation of homologous gene transcripts. In many eukaryotes, expression of nuclear-encoded mRNA can be strongly inhibited by the presence of a small double-stranded RNA corresponding to exon sequences in the mRNA. In this study the pAVU6＋27 vector, which has Sail and Xbal clone sites, was used to construct the siRNA expression vectors for mouse wnt9a. Four kinds of small interfering RNA inserts were designed, synthesized and visually tested for efficacy by in situ hybridization, the results demonstrated that dramatically reduced wnt9a signals were observed in the cells transfected with U6＋27 cassettes with anti-wnt9a hairpin siRNA inserts compared with the untransfected. The results of flow cytometry analysis showed that the cell proliferation was promoted after lowering expression of the mouse wnt9a in MA891 cells by RNAi. All that suggest the expression level of mouse wnt9a in breast cancer MA891 cells may play a role in adjusting the rate of proliferation.
文摘The paper aimed to study the effect of lysyl oxidase-like 2 （LOXL2） on hepatocellular carcinoma （HCC） and explore the biological mechanisms of tumorigenicity and progression in HCC. The authors used four HCC cell lines to identify LOXL2. A lentiviral vector containing LOXL2-siRNA was constructed to silence the LOXL2 gene in SMMC-7721 cell line, and mRNA of the target gene was detected by real-time polymerase chain reaction （RT-PCR）. The effect of LOXL2 silencing on the growth of SMMC-7721 cells was explored with flow cytometry profiling and BrdU labeling. Downstream genes of LOXL2 were selected by microarray and verified by Western Blotting. In the results, LOXL2 expression was significantly up-regulated in four types of HCC cell lines, therefore, SMMC-7721 cell line was selected for further exploration. When SMMC-7721 cell line was infected with LOXL2-siRNA, the expression of LOXL2 mRNA decreased. The silencing of LOXL2 resulted in the cell cycle arrest at the G 1-phase, the increased apoptosis and the decreased growth of SMMC-7721 cells on the indicated days by BrdU. Moreover, the MDM2, BIRC3, CDC42, FOS and TGFBR2 genes were selected and verified to be the downstream genes of LOXL2. In conclusion, LOXL2 contributes to the genesis and progression of HCC cells and works by regulating downstream genes of LOXL2 in certain pathways. Therefore, LOXL2 may play an important role in the progression and prognosis of HCC.
