Growth inhibition induced by short hairpin RNA to silence survivin gene in human pancreatic cancer cells

BACKGROUND: Survivin is known to be overexpressed in various human malignancies, including pancreatic cancer, and mediates cancer cell proliferation and tumor growth, so the regulation of this molecule could be a new strategy for treating pancreatic cancer. In this study, short hairpin RNAs (shRNAs) specific to survivin were introduced into human pancreatic cancer Patu8988 cells to investigate the inhibitory effects on survivin expression and cell proliferation in vitro and in vivo. METHODS: Three kinds of shRNA specific to the survivin gene were designed and cloned into eukaryotic expression plasmid pGenesil-1 vector. Subsequently the recombinant plasmids were transfected into human pancreatic cancer Patu8988 cells with lipfectamine (TM) 2000 reagent. The mRNA and protein expressions of survivin in the transiently transfected Patu8988 cells were determined by RT-PCR, flow cytometry, and Western blotting analysis. The proliferation inhibition rates of stably transfected Patu8988 cells were determined by MTT assay. The antitumor activities of the three kinds of survivin-shRNA plasmids were evaluated in BALB/c nude mice inoculated with Patu8988 cells and bearing human pancreatic cancer. RESULTS: The three survivin-shRNA plasmids named pGenesil-1-survivin-1, pGenesil-1-survivin-2 and pGenesil-1-survivin-1+2 (with double interfering RNA sites) were successfully constructed, and were confirmed by restriction enzyme cutting and sequencing. At 48 hours after transfection, the expression of survivin mRNA and protein was inhibited in Patu8988 cells transfected with pGenesil-1-survivin-1, pGenesil-1-survivin-2, and pGenesil-1-survivin-1+2 when compared with that of either pGenesil-1-NC (with scrambled small interfering RNA) transfected cells or control cells (P<0.05). The MTT results showed that the proliferation rates of Patu8988 cells stably transfected with survivin-shRNA plasmids were reduced when compared with that of either pGenesil-1-NC transfected cells or control cells (P<0.01). Furthermore, when Patu8988 cells stably transfected with survivin-shRNA were injected into BALB/c nude mice, tumor growth was dramatically lower and the tumor was smaller than that of either pGenesil-1-NC transfected cells or control cells (P<0.01). The inhibitory effect of pGenesil-1-survivin-1 was the best among the three kinds of survivin-shRNA plasmids, but no combination of inhibitory effects was found in pGenesil-1-survivin-1+2. CONCLUSIONS: shRNAs specific to survivin have gene silencing effects and inhibit pancreatic cancer cell proliferation. shRNA activity against survivin could be of potential value in gene therapy for pancreatic cancer. However, shRNAs with double combining sites did not significantly enhance the interference compared with single site shRNAs, therefore further studies on this are needed.

Influence of Osteopontin Short Hairpin RNA on the Proliferation and Activity of Rat Vascular Smooth Muscle Cells

To investigate the influence of osteopontin （OPN） short hairpin RNA （shRNA） on the proliferation and activity of rat vascular smooth muscle cells （VSMCs）, the expressing vector of shRNA targeting OPN was constructed and transferred into the rat VSMCs. After amplification and purification, pGenesil-1/OPNshRNA1 （PG1）, pGenesil-1/OPNshRNA2 （PG2） and pGenesil-1/OPNshRNAHK （PGH） were transfected into the cultured rat VSMC by LipofectamineTM 2000. Transfected cells were visualized by using an inverted fluorescent microscope. VSMCs transfected by optimal recombined plasmid was selected by culturing in G418 48 h later. Nude cells and cells transfected by PGH were used as control. The expression levels of OPN mRNA and protein were assayed by RT-PCR and Western blotting. The OPN of VSMCs was suppressed by transfection of optimal recombined plasmid, and the changes in cell proliferation, adhesion and motility were evaluated by MTT, adhesion test and transwell chamber test. Levels of type I and Ⅲ collagen were measured with ELISA kit. Our results showed that VSMCs stably transfected by OPN shRNA accounted for over 50% of total cells. OPN mRNA and protein were reduced by 81% and 67% （P〈0.01） by PG1, 73% and 52% （P〈0.01） by PG2, respectively while no change was found in PGH and non-treated VSMCs. PG1 significantly suppressed the proliferation, adhesion, mobility of VSMCs and reduced the amount of type Ⅰ and Ⅲ collagen. It is concluded that recombinant plasmid can be success-fully transfected into VSMCs by LipofectamineTM 2000 and inhibit the expression of OPN. The proliferation, adhesion and mobility of VSMCs can be inhibited by knocking down OPN expression. Moreover, the transferring capability of cells is attenuated, and the secretion of type Ⅰ and Ⅲ collagen is inhibited aftter knocking-down of OPN expression. The study provides experimental evidence for clinical prevention of restenosis after percutaneous coronary intervention （PCI） by RNA interference （RNAi） technology.

Short Hairpin RNA-mediated MDR1 Gene Silencing Increases Apoptosis of Human Ovarian Cancer Cell Line A2780/Taxol

Objective： Recurrent ovarian cancer is often resistant to drugs such as paclitaxel. Short hairpin RNA （shRNA） targeting MDRI, a gene involved in the process of drug resistance, may be a promising strategy to overcome drug resistance. Methods： Construction and identification of eukaryotic expression plasmid was transiently transfected into human ovarian cancer ce plasmid of shRNA targeting on MDR1 gene. The ne A2780/Taxol. Apoptosis was determined by flow cytometry using annexin V-FITC/PI double labeling. Expression of MDRI mRNA was detected by quantitative polymerase chain reaction （qPCR） and P-glycoprotein expression was detected using Western blot. Results The IC50 of paclitaxel in MDR1 shRNA-transfected group was significantly reduced （1.986±0.153）μmol/ml as compared with that in negative control （5.246±0.107）μmol/ml and empty vector-transfected group （5.212±0.075）μmol/ml （P〈0.05）. The percent of the relative reverse sensitivity to paclitaxel on A2780/Taxol cells was 67.1%, and the apoptotic rate was significantly increased [（6.977±0.333）%] compared with control [（1.637±0.111）%] and empty vector-transfected group [（1.663±0.114）%] （P〈0.05）. Expressions of MDR1 mRNA and P-glycoprotein were significantly reduced compared with control （P〈0.05）. Conclusion： The present study demonstrated that the eukaryotic expression plasmid of shRNA targeting on MDRI inhibited the expression of MDRI effectively, thus enhance the sensitivity of A2780/Taxol cells to paclitaxel.

Lentivirus-mediated short hairpin RNA interference of CENPK inhibits growth of colorectal cancer cells with overexpression of Cullin 4A

BACKGROUND Colorectal cancer(CRC)is one of the most common malignant tumors worldwide.The identification of novel diagnostic and prognostic biomarkers for CRC is a key research imperative.Immunohistochemical analysis has revealed high expression of centromere protein K(CENPK)in CRC.However,the role of CENPK in the progression of CRC is not well characterized.AIM To evaluate the effects of knockdown of CENPK and overexpression of Cullin 4A(CUL4A)in RKO and HCT116 cells.METHODS Human colon cancer samples were collected and tested using a human gene expression chip.We identified CENPK as a potential oncogene for CRC based on bioinformatics analysis.In vitro experiments verified the function of this gene.We investigated the expression of CENPK in RKO and HCT116 cells using quantitative polymerase chain reaction(qPCR),western blot,and flow cytometry.The effect of short hairpin RNA(shRNA)virus-infected RKO cells on tumor growth was evaluated in vivo using quantitative analysis of fluorescence imaging.To evaluate the effects of knockdown of CENPK and overexpression of CUL4A in RKO and HCT116 cells,we performed a series of in vitro experiments,using qPCR,western blot,MTT assay,and flow cytometry.RESULTS We demonstrated overexpression of CENPK in human colon cancer samples.CENPK was an independent risk factor in patients with CRC.The downstream genes FBX32,CUL4A,and Yesassociated protein isoform 1 were examined to evaluate the regulatory action of CENPK in RKO cells.Significantly delayed xenograft tumor emergence,slower growth rate,and lower final tumor weight and volume were observed in the CENPK short hairpin RNA virus infected group compared with the CENPK negative control group.The CENPK gene interference inhibited the proliferation of RKO cells in vitro and in vivo.The lentivirus-mediated shRNA interference of CENPK inhibited the proliferation of RKO and HCT116 colon cancer cells,with overexpression of the CUL4A.CONCLUSION We indicated a potential role of CENPK in promoting tumor proliferation,and it may be a novel diagnostic and prognostic biomarker for CRC.

Influence of Osteopontin Short Hairpin RNA on the Proliferation and Invasion of Human Renal Cancer Cells

The influence of short hairpin RNA(shRNA)-mediated osteopontin(OPN)gene silencing on the proliferation and invasion of human renal cancer ACHN cells was investigated.Four types of OPN shRNA recombinant plasmids were constructed and RT-PCR assays were used to screen the most highly functional shRNA recombinant plasmids,which were transferred into the cultured ACHN cells by LipofectamineTM 2000.The cells transfected by shRNA expression vectors(ACHN/OPN)were visualized under an inverted microscope and screened...

Construction of Short Hairpin RNA Vector with σNS & σC Genes of Avian Reovirus and Determination of Interference Effect

[ Objective] The aim was to explore novel method for treatment of Avian Reovirus. [ Method] According to the design principle of siRNA target sequences, siRNA templates were designed and synthesized and then cloned into the shRNA expression vector, namely, pSilencer-CMV 4.1 neo. Short hairpin RNA vector C1, C2, C3, which contain σC gene, and shRNA vector NS1, NS2, NS3, which contain aNS gene, were constructed separately. The constructed shRNA vectors and negative control were co-transfected into DF-1 cells with the eukaryotic expression vector pEG- FP-σC and pEGFP-σNS, respectively. [ Result] Observation through fluorescence microscope indicated that the constructed 6 shRNA could inhibit the expression of fusion protein to different degrees. In addition, results of Real-time PCR suggested that C3 and NS1 have the best interference effect to the viral duplication in vitro. [ Conclusionl Construction and selection of specific shRNA expression vectors inhibiting Avian Reovirus are significant for researching effects of σC and oNS proteins in infection and duplication of ARV, providing new idea for ARV antiviral therapy.

Knockdown of polypyrimidine tract binding protein facilitates motor function recovery after spinal cord injury

After spinal cord injury(SCI),a fibroblast-and microglia-mediated fibrotic scar is formed in the lesion core,and a glial scar is formed around the fibrotic scar as a res ult of the activation and proliferation of astrocytes.Simultaneously,a large number of neuro ns are lost in the injured area.Regulating the dense glial scar and re plenishing neurons in the injured area are essential for SCI repair.Polypyrimidine tra ct binding protein(PTB),known as an RNA-binding protein,plays a key role in neurogenesis.Here,we utilized short hairpin RNAs(shRNAs)and antisense oligonucleotides(ASOs)to knock down PTB expression.We found that reactive spinal astrocytes from mice were directly reprogrammed into motoneuron-like cells by PTB downregulation in vitro.In a mouse model of compressioninduced SCI,adeno-associated viral shRNA-mediated PTB knockdown replenished motoneuron-like cells around the injured area.Basso Mouse Scale scores and forced swim,inclined plate,cold allodynia,and hot plate tests showed that PTB knockdown promoted motor function recovery in mice but did not improve sensory perception after SCI.Furthermore,ASO-mediated PTB knockdown improved motor function resto ration by not only replenishing motoneuron-like cells around the injured area but also by modestly reducing the density of the glial scar without disrupting its overall structure.Together,these findings suggest that PTB knockdown may be a promising therapeutic strategy to promote motor function recovery during spinal cord repair.

Apoptosis induced by short hairpin RNA-mediated STAT6 gene silencing in human colon cancer cells

Background The relationship between signal transduction and tumors has become one of the loci in cancer research. Signal transducer and activator of the transcription 6 （STAT6） signaling pathway is found to be activated in some cancer cells. But the function of the pathway in cancer cells is unknown. This study was undertaken to investigate the effect of the Stat6 signaling pathway on apoptosis in human colon cancer cells （HT-29 cells） and the possible mechanism of Stat6 by RNA interference techniques.

Shuanghuanglian injection downregulates nuclear factor-kappa B expression in mice with viral encephalitis

A mouse model of viral encephalitis was induced by intracranial injection of a Coxsackie virus B3 suspension. Quantitative real-time reverse transcription-PCR and western blot assay were applied to detect mRNA and protein expression of intelectin-2 and nuclear factor-kappa B in the viral encephalitis and control groups. Nuclear factor-kappa B and intelectin-2 mRNA and protein expression were significantly increased in mice with viral encephalitis. After intraperitoneal injection of Shuanghuanglian at a dose of 1.5 mg/kg for 5 successive days, intelectin-2 and nuclear factor-kappa B protein and mRNA expression were significantly decreased. To elucidate the relationship between intelectin-2 and nuclear factor-kappa B, mice with viral encephalitis were administered an intracerebral injection of 107 pfu recombinant lentivirus expressing intelectin shRNA. Both protein and mRNA levels of intelectin and nuclear factor-kappa B in brain tissue of mice were significantly decreased. Experimental findings suggest that Shuanghuanglian injection may downregulate nuclear factor-kappa B production via suppression of intelectin production, thus inhibiting inflammation associated with viral encephalitis.

Reversal of multidrug resistance in renal cell carcinoma by short hairpin RNA targeting MDR1 gene

Background Over-expression of P-glycoprotein （P-gp）, encoded by the MDR1 gene, confers multidrug resistance （MDR） in renal cell carcinoma （RCC） and is a major reason for unsuccessful chemotherapy. This study aimed to determine the effct of RNA interference （RNAi） on the reversal of MDR in human RCC. Methods We designed and selected one short hairpin RNA （shRNA） targeting MDR1 gene, which is stably expressed from integrated plasmid and transfected by lentivirus fluid in human RCC A498 cell. Results The MDRl-targeted RNAi resulted in decreased MDR1 gene mRNA level （P 〈0.001）, almost abolished P-gp expression and reversed MDR to different chemotherapy drugs in the RCC A498 cell line. Conclusion MDR could be reversed by RNAi in human RCC A498 cell line, which may be used for clinical application

Recombinant adenovirus-mediated shRNA silencing of midkine gene in BxPC-3 cells

Objective：To investigate the silencing effects of recombinant adenovirus Ad-shRNA-MK on midkine（MK） gene in pancreatic cancer cells. Methods：Ad-shRNA-MK was used to infect pancreatic cancer BxPC-3 cells. Assays were conducted for knockdown of the MK gene on the day of infection and on the 1 ^st, 3^rd, 5^th, 7^th, and 9^th days post-infection by using immunocytochemistry, real-time RT-PCR, and Western blot analysis. Results：The adenoviral Ad-shRNA-PTN was constructed successfully, and infection was confirmed by electron microscopic observation. By using real-time RT-PCR, the inhibition rates of MK mRNA expression in the BxPC-3 cells were 20%, 80%, 55%, and 23% on the 1st, 3^th, 5^th, and 7^th days post-infection. Immunocytochemistry and Western blot analysis confirmed this effect at the gene product level. Conclusion：Efficient and specific knockdown of MK in pancreatic cancer cells by adenoviral Ad-shRNA-PTN is a potentially powerful tool for the study of gene therapy of pancreatic cancer nerve infiltration.

Silencing Endothelin-3 Expression Attenuates the Malignant Behaviors of Human Melanoma Cells by Regulating SPARC Levels

Summary： Endothelin-3 （ET-3） is aberrantly expressed in both metastatic melanoma tissues and cul tured melanoma cells. Our previous work showed that ET-3 could promote survival of metastatic mela noma cells via its altered expression. In this study, we investigated the mechanisms responsible for these gene-induced phenotypes in melanoma cells. An ET-3 gene sequence-specific shRNA vector pLVTHM-ET3-RNAi was constructed and transfected into human malignant melanoma cells A375 and MMRU, and the resultant molecular events and cellular changes were examined. As compared with the empty-vector group, cell proliferation was slowed down, and the growth inhibition rates were 38.9% in A375 cells and 38.4% in MMRU cells after transfection. In addition, cell invasion capability was also inhibited, with a reduction of 62.2% in A375 cells and 54.3% in MMRU cells. The percentage of apoptotic cells was found to increase. Meanwhile, in both cell lines, secreted protein acidic and rich in cy teine （SPARC） levels were down-regulated together with inhibition of its upstream signaling molecule, NF-kB. Thus, the current results suggested that down-regulated expression of ET3 attenuates the ma lignant behaviors of human melanoma ceils partially by decreasing the expression of SPARC and NF-kB.

Construction of Controllable Expression Vector of Mus musculus Growth Hormone (GH) shRNA and Its Gene Si- lencing Efficiency Detection

Growth hormone （GH） is a polypeptide hormone secreted by the pituitary, which promotes animal growth, muscle development, metabolism regulation and other important physiological functions. In this study, a pair of mGH short hairpin RNA （shRNA） was designed according to mouse （ Mus musculus） GH mRNA sequence; pSingle-tTS-mGH shRNA-RFP, an integrated controllable expression vector of mGH shRNA, was constructed successfully. The recombinant vector was transfected into mouse pituitary tumor cell line AtT-20. After addition of doxycyelin （ DOX）, the expression of red fluorescent protein （RFP） was observed un- der a fluorescent microscope. The expression level of mGH in cells was detected by quantitative Realtime PCR （qRT-PCR） and Western blot. After DOX induction, the relative expression level of GH mRNA in cells transfected with GH shRNA was reduced by about 70% compared with that in DOX-free group and other control groups, exhibiting extremely significant differences （P 〈 0.01 ） ; moreover, the relative expression level of GH protein was reduced by about 90% ; the expression level of GH mRNA and GH protein exhibited no significant difference among other groups （P 〉 0.05）. In this study, a controllable expression vector of GH shRNA with high gene silencing efficiency was constructed successfully, which could be used to reveal GH autocfine / paracrine interactions and analyze functions of GH gene in growth, development and disease occurrence of animals by regulating GH expression levels.

Inhibition of Nogo expression to promote repair after spinal cord injury

Background One of the reasons for poor neuroregeneration after central nervous system injury is the presence of inhibitory factors such as Nogo. Here, we tested the inhibition of Nogo by RNA interference both in vitro and in vivo, using recombinant adenovirus-mediated transfection of short hairpin RNAs, to explore a new method of treatment for spinal cord injury. Methods We designed and cloned two Nogo-specific short hairpin RNAs and an unrelated short hairpin RNA, packaged the clones into adenovirus, and amplified the recombinant virus in 293 cells. We then tested the inhibition of Nogo expression both in vitro in adenovirus-transfected oligodendrocytes and in vivo in spinal cord tissue from adenovirus-transfected spinal cord injury model rats. We tested Nogo expression at the mRNA level by reverse-transcription PCR and at the protein level by Western blotting and immunohistochemistry. Results In vitro, the two specific Nogo short hairpin RNAs decreased Nogo mRNA expression by 51% and 49%, respectively, compared with Nogo expression in cells transfected with the unrelated control small hairpin RNA （P 〈0.005）. Similarly, Nogo protein expression decreased by 50% and 48%, respectively （P 〈0.005）. In vivo, in spinal cord injury model rats, the two specific Nogo short hairpin RNAs decreased Nogo mRNA expression by 45% and 40%, respectively, compared with Nogo expression in spinal cord injury model rats transfected with the unrelated control short hairpin RNA （P 〈0.005）. The Nogo protein level was similarly decreased. Conclusions We were successful in specifically downregulating Nogo at the mRNA and protein levels by adenovirus-mediated delivery of short hairpin RNAs, both in vitro and in vivo. This confirms the effectiveness of RNA interference for the inhibition of Nogo gene expression and the efficiency of using adenovirus for delivery. Thus gene therapy may be an effective treatment for spinal cord injury.

Tumor necrosis factor alpha affect hydrocortisone expression in mice adrenal cortex cells mainly through tumor necrosis factor alpha-receptor 1

Background Tumor necrosis factor alpha （TNF-α） is important in promoting relative adrenal insufficiency （RAI） due to systemic inflammatory response syndrome （SIRS）. We identified the TNF-α receptor involved in the inhibition of adrenal corticotrophin （ACTH）-stimulated hydrocortisone release by studying the expression of TNF-α receptors in adrenal cortex Y1 cells and the effect of downregulating TNF receptors on ACTH-stimulated hydrocortisone release. Methods We used real-time PCR and immunocytochemistry to evaluate the expression of TNF receptors on Y1 cells. TNF-receptor 1 （TNF-R1） DNA fragments corresponding to the short hairpin RNA （shRNA）-sequences were synthesized and cloned into pcDNATM 6.2-GW/EmGFP expression vector. Knockdown efficiency of TNF-R1 expression was evaluated in miRNA transfected and mock-miRNA transfected Y1 cells by quantitative real-time PCR （Q-PCR）. Hydro- cortisone expression levels were determined in TNF-Rl-knockdown and control Y1 cells treated with TNF-α and ACTH. Results Mouse adrenal cortex Y1 cells were positive for type I TNF-R1, but not type II TNF-receptor （TNF-R2）. Blocking TNF-R1 expression resulted in loss of TNF-a-mediated inhibition of ACTH-stimulated hydrocortisone expression suggesting a role for the TNF-R1 related signaling pathway in ACTH-stimulated hydrocortisone synthesis. Conclusion The inhibitory effect of TNF-α on ACTH-stimulated hydrocortisone synthesis was mediated via TNF-R1 in adrenal cortex.

shRNA-triggered RNAi inhibits expression of NDV NP gene in chicken embryo fibroblast

RNA interference(RNAi)technology is a powerful tool for identifying gene functions.Chicken embryo fibroblast(CEF)is an ideal model for studying the interaction between avian viruses and their hosts.To establish a methodological platform for RNAi studies in CEF,three plasmid vectors expressing short hairpin RNAs(shRNAs)targeted against the Newcastle disease virus(NDV)NP gene were constructed.One of them,ndv1,was proven effective on blocking viral replication in CEF and chicken embryos.Four hours prior to infection with NDV,the CEF was transfected with the plasmids by Silent-fect.An unrelated shRNA sequence(HK)was used in mock transfection.The expression of a potent shRNA resulted in up to 2.3,21.1 and 9.8 fold decreases in NP gene expression at 3,6 and 9 h post infection in CEF,respectively.The ndv1 was able to completely inhibit the replication of the virus in CEF within 48 post infection.Furthermore,the pathological changes in CEF caused by NDV were delayed,and the degree of pathological changes was lighter compared with the mock transfection in the presence of ndv1.When the complex of shRNASilent-fect and NDV was co-injected into the allantoic cavity of 10-day-old embryonated eggs with 10^(5) or 10^(6) ELD50 NDV,NDV replication was decreased by 94.14% and 62.15% after 17 h,respectively.These findings suggest that the newly synthesized NP protein is critical for NDV transcription and replication and provide a basis for identifying the functions of viral genes and screening for effective siRNAs against viruses in CEF and chicken embryo by RNAi.