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.

Investigation of transcriptional gene silencing and mechanism induced by shRNAs targeted to RUNX3 in vitro

AIM:To investigate transcriptional gene silencing induced by short hairpin RNAs (shRNAs) that target gene prompter regions of RUNX3 gene, and whether shRNAs homologous to DNA sequences may serve as initiators for methylation. METHODS:According to the principle of RNAi design, pSilencer3.1-H1-shRNA/RUNX3 expression vector was constructed, The recombinant plasmid shRNA was transfected into human stomach carcinoma cell line SGC7901 with Lipofectamine 2000. Then, the positive cell clones were screened by G418. The mRNA and protein expression level of RUNX3 in the stable transfected cell line SGC7901 were determined by RT-PCR, Western blotting and immunocytochemistry. Characteristics of the cell lines including SGC7901, pSilencer3.1-H1/SGC7901 and pSilencer3.1-H1-shRNA/RUNX3/SGC7901 were analyzed with growth curves, clone formation rate and cell-cycle distribution. The activated level of RUNX3 was examined after treatment with the different density of 5’-aza-2’-deoxycytidine (5-Aza-CdR) by using semi- quantitative RT-PCR and Western blotting. RESULTS:In the cell line SGC7901 transfected with pSilencer3.1-H1-shRNA/RUNX3, mRNA and proteinexpression of the RUNX3 gene was lost identified by RT- PCR, Western blotting and immunocytochemistry assay. The growth of pSilencer3.1-H1-shRNA/ RUNX3/SGC7901 cells without expression of RUNX3 was the fastest (P < 0.05), its rate of clone formation was the highest (P < 0.01), and the cell distribution in G0/G1 and S/M phases was lowest and highest, respectively (P < 0.05), compared with that of the transfected pSilencer3.1-H1 and non-transfected cells. Through RT-PCR and Western blot assay, inactivated RUNX3 could not be reactivated by 5-Aza-CdR. CONCLUSION:We found that, although shRNAs targeted to gene prompter regions of RUNX3 could effectively induce transcriptional repression with chromatic changes characteristic of inaction promoters, this was independent of DNA methylation, and the presence of RNA-dependent transcriptional silencing showed that RNA-directed DNA methylation might be an existing gene regulatory mechanism relative to the methylated in humans.

AAV-mediated expression of p65shRNA and bone morphogenetic protein 4 synergistically enhances chondrocyte regeneration

BACKGROUND:Adeno-associated virus(AAV)gene therapy has been proven to be reliable and safe for the treatment of osteoarthritis in recent years.However,given the complexity of osteoarthritis pathogenesis,single gene manipulation for the treatment of osteoarthritis may not produce satisfactory results.Previous studies have shown that nuclear factorκB could promote the inflammatory pathway in osteoarthritic chondrocytes,and bone morphogenetic protein 4(BMP4)could promote cartilage regeneration.OBJECTIVE:To test whether combined application of AAV-p65shRNA and AAV-BMP4 will yield the synergistic effect on chondrocytes regeneration and osteoarthritis treatment.METHODS:Viral particles containing AAV-p65-shRNA and AAV-BMP4 were prepared.Their efficacy in inhibiting inflammation in chondrocytes and promoting chondrogenesis was assessed in vitro and in vivo by transfecting AAV-p65-shRNA or AAV-BMP4 into cells.The experiments were divided into five groups:PBS group;osteoarthritis group;AAV-BMP4 group;AAV-p65shRNA group;and BMP4-p65shRNA 1:1 group.Samples were collected at 4,12,and 24 weeks postoperatively.Tissue staining,including safranin O and Alcian blue,was applied after collecting articular tissue.Then,the optimal ratio between the two types of transfected viral particles was further investigated to improve the chondrogenic potential of mixed cells in vivo.RESULTS AND CONCLUSION:The combined application of AAV-p65shRNA and AAV-BMP4 together showed a synergistic effect on cartilage regeneration and osteoarthritis treatment.Mixed cells transfected with AAV-p65shRNA and AAV-BMP4 at a 1:1 ratio produced the most extracellular matrix synthesis(P<0.05).In vivo results also revealed that the combination of the two viruses had the highest regenerative potential for osteoarthritic cartilage(P<0.05).In the present study,we also discovered that the combined therapy had the maximum effect when the two viruses were administered in equal proportions.Decreasing either p65shRNA or BMP4 transfected cells resulted in less collagen II synthesis.This implies that inhibiting inflammation by p65shRNA and promoting regeneration by BMP4 are equally important for osteoarthritis treatment.These findings provide a new strategy for the treatment of early osteoarthritis by simultaneously inhibiting cartilage inflammation and promoting cartilage repair.

Effect of vector-expressed shRNAs on hTERT expression

AIM: To study the effect of short hairpin RNAs (shRNAs)expressed from DNA vector on hTERT expression.METHODS: Oligonucleotides coding for four shRNAs against hTERT were cloned into a mammalian shRNA expressionvector pUC18U6 to form pUC18U6ht1-4, which were thenintroduced into HepG2 cells by using liposome-mediated transfection. HepG2 cells transfected by pUC18U6 and pUC18U6GFPsir, which expressed shRNA against green fluorescent protein (GFP), were used as controls. hTERT mRNA in the transfected cells were quantified by using real-time fluorescent RT-PCR.RESULTS: Among the four shRNAs against hTERT, two decreased the hTERT mRNA level. Compared with the controls, pUC18U6ht which expressed the two shRNAs reduced hTERT mRNA by 39% and 49% (P＜0.05).CONCLUSION: hTERT expression is inhibited by the shRNAs expressed from the DNA vector.

Characteristics of short double stranded RNA against hepatitis C virus: a literature-based analysis

Objective： To describe the characteristics of short interfering double stranded RNA （dsRNA） against hepatitis C virus （HCV） and to fred out the determining factors in design for desirable inhibitory efficacy. Methods： The data were collected and analyzed by retrieval of 229 published short dsRNAs designed for degradation ofHCV RNA. Results： Statistical analyses showed that the most frequently involved short dsRNAs were directing against 5＇NTR/core and genotype lb, accounting for 64.2% and 69.9%, respectively. Inhibitory efficacy varied with the structural characteristics of short dsRNAs, of which the most potential were those directed against HCV core region with inhibitory efficacy of 70.2%. Moreover, the mean inhibitory efficacy of short dsRNAs with GC contents from 30% to 52% was higher than that of those with GC contents out of this range. Conclusion： Based on this pooled data in a relatively large sample, the present results provided clues to design for short dsRNAs with more potent inhibitory efficacy.

Effects of p27^(Kip1)- and p53- shRNAs on kanamycin damaged mouse cochlea

AIM： To study the effects of adeno-associated virus （AAV） delivered short hairpin RNAs （shRNAs） on adult CD-1 mouse cochlea damaged by aminoglycoside anti-biotic kanamycin. METHODS： Three different shRNAs were designed （p27^Kip1, p53 and p27^Kip1＋p53） and tested in COS cells. A total of 20 adult CD-1 mice were used in the experiment. Mice were divided into fve different groups （four animals/group） depending on the AAV-shRNA construct they received and whether they received kanamycin or not. Saline and AAV-EGFP injected animals were used as controls. All constructs were injected through the round window membrane （RWM） into the cochlea. Cochleae were harvested after 1 mo. Apoptosis was detected with Tunel labeling from paraffin-embedded cochlear tissue sections.RESULTS： AAV2/2-p27^Kip1-shRNA and AAV2/2-p53-shRNA were tested in COS cells. Western blotting analysis confirmed that both constructs silenced their target genes effectively in the cell culture. AAV2/2-shRNA constructs were injected into the cochlea of CD-1 mice through the intact RWM. Cotransductionof Cotransduction of individual AAV2/2-shRNAs with AAV2/2-EGFP resulted in EGFP expression in the organ of Corti.Kanamycin Kanamycin treatment had no effect on the expression pattern of the EGFP. AAV2/2-shRNA treated mice （either with p53 or p27Kip1and p53 together） showed fewer apop-totic hair cells in the cochlea than the control group （P 〈 0.05; AAV2/2-p53-shRNA vs saline P = 0.00014; AAV2/2-p27＋p53-shRNA vs saline P = 0.0011）. AAV2/2-p27-shRNA injected cochleae showed no significant difference in the number of apoptotic cells when compared to the saline injected cochleae.CONCLUSION： Silencing of p53 protein in the kana-mycin treated ears may decrease cell death in the organ of Corti.

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.

RNA interference and antiviral therapy

RNA interference (RNAi) is an evolutionally conserved gene silencing mechanism present in a variety of eukaryotic species. RNAi uses short double-stranded RNA (dsRNA) to trigger degradation or translation repression of homologous RNA targets in a sequence-specific manner. This system can be induced effectively in vitro and in vivo by direct application of small interfering RNAs (siRNAs), or by expression of short hairpin RNA (shRNA) with non-viral and viral vectors. To date, RNAi has been extensively used as a novel and effective tool for functional genomic studies, and has displayed great potential in treating human diseases, including human genetic and acquired disorders such as cancer and viral infections. In the present review, we focus on the recent development in the use of RNAi in the prevention and treatment of viral infections. The mechanisms, strategies, hurdles and prospects of employing RNAi in the pharmaceutical industry are also discussed.

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.

STAT3-specific nanocarrier for shRNA/drug dual delivery and tumor synergistic therapy

Non-small cell lung cancer(NSCLC)is a major disease with high incidence,low survival rate and prone to develop drug resistance to chemotherapy.The mechanism of secondary drug resistance in NSCLC chemotherapy is very complex,and studies have shown that the abnormal activation of STAT3(Signal Transducer and Activator of Transcription 3)plays an important role in it.In this study,the pGPU6/GFP/Neo STAT3-shRNA recombinant plasmid was constructed with STAT3 as the precise target.By modifying hydrophilic and hydrophobic blocks onto chitosan,a multifunctional vitamin E succinate-chitosan-polyethylene glycol monomethyl ether histidine(VES-CTS-mPEG-His)micelles were synthesized.The micelles could encapsulate hydrophobic drug doxorubicin through self-assembly,and load the recombinant pGPU6/GFP/Neo STAT3-shRNA(pDNA)through positive and negative charges to form dual-loaded nanoparticles DOX/VCPH/pDNA.The co-delivery and synergistic effect of DOX and pDNA could up-regulate the expression of PTEN(Phosphatase and Tensin Homolog),down-regulate the expression of CD31,and induce apoptosis of tumor cells.The results of precision targeted therapy showed that DOX/VCPH/pDNA could significantly down-regulate the expression level of STAT3 protein,further enhancing the efficacy of chemotherapy.Through this study,precision personalized treatment of NSCLC could be effectively achieved,reversing its resistance to chemotherapy drugs,and providing new strategies for the treatment of drug-resistant NSCLC.