Effect of shRNA Inhibiting HIF1α Gene on TIMP1 Expression in RPE Cells

Small hairpin RNA （shRNA） was used to silence the HIF1α gene in human retinal pigment epithelial cells （RPE） under hypoxia in order to observe the effect of gene silencing on the expression of matrix metalloproteinase tissue inhibitor 1 （TIMP1）. By using chemical hypoxic inducer CoCl2 to mimic RPE hypoxic environment, shRNA against the targeting region of HIF1α mRNA sequence was synthesized by a method of in vitro transcription, and the HIF1α was interfered in RPE cultured under hypoxia （induced by 150 μmol/L CoCl2 ）. RT-PCR was employed to detect the expression of HIF1α and TIMP1. The expression levels of HIF1α and TIMP1 were measured by using Western blotting. The results showed that after the RPE were transfected with specific shRNA against HIF1α mRNA, RT PCR revealed that under hypoxia, the efficacy of HIF1α gene silencing in RPE was 83.4 %. Western blotting revealed that the expression levels of HIF1α protein was dramatically dropped. In addition, RT-PCR results demonstrated that the expression of TIMP1 mRNA was decreased by 28.9 %, and the expression levels of TIMP1 protein were also significantly reduced by Western blotting. It was suggested that shRNA targeted against HIF1α mRNA could effectively silence the HIF1α gene, subsequently effectively inhibit the hypoxia-induced up-regulation of TIMP1.

Effect of p120 Catenin Silencing on Biological Behaviors of PANC-1 Cells

This study examined the possible role of p120ctn in the pathogenesis and development of pan-creatic cancer.PANC-1 cells,a kind of human pancreatic carcinoma cell line,were cultured in this study.p120ctn was immunocytochemically detected in PANC-1 cells.The recombinant lentivirus vector was constructed to knock down the p120ctn expression of PANC-1 cells.Real-time quantitative PCR (RQ-PCR) and Western blotting were used to determine the expression of p120ctn and E-cadherin in PANC-1 cells after p120ctn knockdown.The adhesion,invasion and migration capacity of PANC-1 cells after p120ctn knockdown was detected by cell adhesion,invasion and migration assays.Cell growth was measured by the MTT method.Cell cycle and apoptosis were analyzed by fluorescence-activated cell sorting.The results showed that p120ctn knockdown led to significantly down-regulated E-cadherin and a reduced cell-to-cell adhesion ability in PANC-1 cells.shRNA-mediated knockdown of p120ctn reduced invasion and migration capacity of PANC-1 cells,inhibited cell growth,caused a significant decrease in the percentage of cells in G1,an increase in S,and promoted apoptosis of PANC-1 cells.It was concluded that p120ctn plays a pivotal role in the proliferation and metastasis of pancreatic carcinoma,suggesting that p120ctn is a novel target for pancreatic carcinoma treatment.

AT1a Receptor Has Interacted with Angiotensin-converting Enzymes 2 mRNA Expression in Mouse Brainstem

Objectives To examine in vivo interactions between angiotensin Ⅱ （Ang Ⅱ ） AT1 a receptor （AT1 aR）, angiotensin-converting enzymes （ACE） and ACE2 using small hairpin RNA （shRNA） gene-silencing methods in mice brainstem nucleus tractus solitarius （NTS）. Methods C57BL mice （n = 8 ） were used as animal model. Method of micro-injection in the nucleus of NTS was adopted. After ten days, mice were killed and their brain tissue were fixed and sectioned. The expression levels of AT1 aR, ACE and ACE2 mRNA at both sides of NTS were examined by in situ hybridization. Based on compared t-test, the changing for mRNA expression was examined. Results After the expression of ATlaR mRNA was significantly inhibited （61.6% ± 6.8% ） by ATlaR-shRNA, it was associated with decreases in ACE2 mRNA expression from （ 1.05 ± 0. 12） μCi/mg to （0. 74 ± 0.09 ） μCi/mg （ 29.0% ± 14. 5% , P 〈 0. 01 ） on the same side of the brainstem. ACE mRNA expression was consistent at both sides （ 0. 50 μCi/mg ± 0. 09μCi/mg and 0. 53 μCi/mg ± 0. 08 μCi/mg）, with insignificant difference （ P 〉 0. 05 ）. Conclusions The gene silencing result showed that there were interactions between brainstem AT1 aR and ACE2. ACE mRNA expression was not altered by RNA interference treatment at AT1 aR.

SNCG shRNA suppressed breast cancer cell xenograft formation and growth in nude mice

Background Overexpression of breast cancer-specific gene 1 （SNCG） is associated with poor prognosis in advanced breast cancer patients. This study aimed to determine the effects of SNCG knockdown in breast cancer cells by using small hairpin RNA （shRNA）.Methods Four different SNCG shRNA oligonucleotides were designed and chemically synthesized to construct mammalian expression vectors. These vectors were then stably transfected into a breast cancer MCF-7 cell line to knockdown SNCG expression. After SNCG knockdown was confirmed, the stable cell lines were inoculated into nude mice. SNCG mRNA and protein expressions were analyzed by semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR） and immunohistochemistry, respectively in both the stable cell lines and xenografts.Results All four SNCG shRNA constructs significantly reduced SNCG mRNA and protein levels in MCF-7 cells, as compared to the unrelated sequence control shRNA and the liposome control mice （P〈0.05）. SNCG-knockdown MCF-7cells formed significantly smaller tumor masses than cells expressing the unrelated sequence control or the liposome control mice （P〈0.05）.Conclusion SNCG shRNA effectively suppressed breast cancer cell formation in vivo and may be a useful clinical strategy to control breast cancer

Gene silencing efficiency of shRNA expression vectors targeting Cx43 in vitro

Our previous studies showed that there were close relationships between connexin 43(Cx43)and acupoints and meridians.In order to further investigate the effect of Cx43 in acupuncture treatment,RNA interference technique was used to construct small hairpin RNA(shRNA)expression vectors targeting Cx43 and identify the efficiency of RNA interference in NIH/3T3 cell lines for further use in vivo.Aiming directly at the two targets of Cx43 mRNA sequence of the rat and mouse homology region,we synthesized two pairs of comple-mentary oligonucleotide strands in vitro.Double strands were formed after annealing,and then inserted into Pgenesil-1 plasmid expression vector.After identification by enzyme cutting and sequencing,the recombinant plasmids named P-Cx43-shRNA(1),P-Cx43-shRNA(2)and P-con-shRNAwere transfected into the NIH/3T3 cells.Immunofluorescence and Western blot assays were used to detect the protein level of Cx43 after being screened by G418.The results of enzyme cutting and sequencing showed that we successfully constructed two shRNA expression vectors targeting Cx43,and a control expres-sion vector for rat and mouse.Also,the Cx43 protein level was decreased by 73.5%(P<0.01)and 10.8%,accord-ingly.The Cx43 protein level was not influenced by the transfection of P-con-shRNA.The outcomes demonstrate that the plasmid P-Cx43-shRNA(1)can specifically silence better the expression of Cx43 in NIH/3T3 cells,which offers an experimental evidence for further in vivo investigation.