Survivin shRNA Research Articles

Nucleolin-targeted cationic nanoparticle for delivery of survivin shRNA against colorectal cancer in vitro and in vivo

In this study, synthesis of Polylactic acid (PLA) polymer was carried out through ring-opening polymerization (ROP) method. Then, 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid as the CTA molecule was attached to the PLA, and subsequently, amphiphilic copolymer, with estimated structure of Poly(lactic acid)121-b-Poly(N-(3-aminopropyl)methacrylamide)103 (PLA121-b-PAPMA103) was obtained through the Reversible Addition Fragmentation chain Transfer (RAFT) reaction. The synthesized copolymer was self-assembled into spherical nanoparticles with size, Poly Dispersity Index (PDI) and zeta potential of 181.5 ± 21.3, 0.643 and of 44.9 ± 2.5 mV, respectively. The optimal copolymer/plasmid ratio was 4 with size, PDI and zeta potential of 161.7 ± 10.2, 0.277 and 36.5 ± 0.7, respectively for the delivery of short hairpin RNA (shRNA) plasmid against survivin. Additionally, the AS1411 aptamer was electrostatically decorated on the surface of the polyplex. In vitro studies on C26 cells revealed that the aptamer rendered nanoparticle transfer into the cell, dependent solely on the aptamer-receptor interactions. Lastly, in vivo studies involving tumor size and body weight change, survival and histological analysis indicated potential therapeutic index of the targeted nanoparticles with good safety profile.

Development of PNC-27 targeted codelivery system for survivin-shRNA and SN38 against colon adenocarcinoma in vitro and in vivo

Combination therapy, as a treatment modality with simultaneous use of two or more therapeutic agents, could provide more attention in the field of cancer therapy. In this regard, the combination of anti-cancer agents could increase the drug efficacy due to reduction of tumor growth and metastasis as well as apoptosis induction while reducing the drug resistance. In the current study, a single-walled carbon nanotube (SWCNT) nanoparticulate carrier, modified with polyethylene glycol and branched polyethylenimine (PEI 10 kDa), was synthesized for the simultaneous delivery of plasmid encoding survivin (psur) shRNA and SN38. The aforementioned nanoparticle was tagged with a targeting peptide, PNC27, for enhancing the internalization of therapeutic agents to cancerous cells. Competition assay using flow cytometry (FCM) experiment revealed the higher uptake of the targeted platform into C26 cells, treated with targeted carrier whereas cells treated with either untargeted NP or pretreated with an excess amount of free PNC27 peptide exhibited lower internalization efficiency. Furthermore, compared with either irinotecan at high dose or SWCNT-PEG10%PEI(10Br)/SN38/pEGFP at an equivalent dose, SWCNT-PEG10%PEI(10Br)/SN38/psur shRNA and SWCNT-PEG10%PEI(10Br)/SN38/psur shRNA/PNC27 significantly reduced the tumor volume in C26 tumorized female BALB/c mice, demonstrating the synergistic effects of SN38 and psur shRNA. In addition, the carrier conjugated with PNC27 facilitated the accumulation of psur shRNA and SN38 in cancer tissue causing a substantial inhibitory effect compared to that of untargeted groups. It could be concluded that the novel modified SWCNT loaded with SN38 and psur shRNA could enhance the therapeutic efficacy in vitro and in vivo, thereby providing an innovative approach for cancer treatment.

Survivin Regulates Bad Gene Expression by Binding to Its Promoter and Modulates Cell Cycle and Apoptosis in Esophageal Carcinoma Cell.

Esophageal cancer (EC) is the eighth most prevalent cancer and the sixth leading cause of cancer-related mortality worldwide. As an antiapoptotic and a proapoptotic protein, respectively, survivin and Bad play an important role in carcinogenesis of the most human cancers including EC. However, the regulatory relationships between them remain unclear. We sought to investigate the effects of survivin knockdown and overexpression on the expression of Bad gene, cell cycle progression, and apoptosis of esophageal carcinoma cell. The mRNA expression levels of survivin and Bad were determined in EC tissue samples. The knockdown and overexpression experiments were performed in ECA109 and KYSE450 cells via transfection with survivin overexpression and shRNA plasmids. A Bad overexpression experiment was conducted to confirm the biological effect on knockdown of survivin via modulating Bad expression. RT-qPCR and Western blot analysis were used to detect mRNA and protein expression, respectively. Cell cycle and apoptosis were analyzed by flow cytometry. The chromatin immunoprecipitation (ChIP) was conducted to determine the binding sites of survivin on the promoter of Bad gene. By analyzing the mRNA expression of survivin and Bad in 40 ESCC patient specimens, we found that the positive expression rate of survivin in tumor tissues (88%, 35/40) was remarkably high, compared with the distal nontumor tissues (48%, 19/40, p < 0.01). On the other hand, the positive expression rate of Bad in tumor tissues (70%, 28/40) was remarkably low, compared with the distal nontumor tissues (95%, 38/40, p < 0.01). Overexpression of survivin decreases Bad mRNA and protein expression and promotes transformation of cell cycle to S phase. Conversely, knockdown of survivin increases Bad mRNA and protein expression and induces cell cycle arrest and apoptosis. Bad overexpression inducing apoptosis of esophageal carcinoma cell shows the similar apoptotic effect with survivin knockdown. ChIP assays indicate that survivin directly binds to the Bad promoter region, diminishing the transcriptional activity of Bad. In conclusion, the result suggested that survivin regulates Bad gene expression by binding to its promoter and modulates cell cycle and apoptosis in esophageal carcinoma cell.

Novel Polymeric Hybrid Nanocarrier for Curcumin and Survivin shRNA Co-delivery Augments Tumor Penetration and Promotes Synergistic Tumor Suppression.

A major barrier for co-delivery of gene medicine with small molecular chemotherapeutic drugs in solid tumors is the inadequate tumor penetration and transfection. In this study, a novel polymeric nanocarrier with integrated properties of tumor penetration, nuclear targeting, and pH-responsive features was designed, and further used to achieve the synergistic anti-tumor effect of curcumin (CUR) and survivin shRNA (pSUR). The polymeric hybrid nanocarrier was constructed from the FDA-approved polymer PLGA and a novel conjugated triblock polymer W5R4K-PEG2K-PHIS (WPH). CUR and pSUR were simultaneously encapsulated in the dual-drug-loaded nanoparticles (CUR/pSUR-NPs) by a modified double-emulsion solvent evaporation (W/O/W) method. The obtained nanoparticles exhibited better pharmaceutical properties with a uniform spherical morphology and sustained release manners of CUR and pSUR. Excellent features including preferable cellular uptake, efficient endosomal escape, enhanced tumor penetration, and elevated transfection efficiency were further proven. Additionally, a markedly enhanced anti-tumor efficacy for CUR/shRNA-NPs was achieved on SKOV-3 and Hela cells. The synergistic anti-tumor effect involved the inhibition of tumor cell proliferation, induction of cell apoptosis, and the activation of caspase-3 pathways. This work sets up an innovative co-delivery nanosystem to suppress tumor growth, contributing to the development of a comprehensive nanoparticulate strategy for future clinical applications.

Targeted rod-shaped mesoporous silica nanoparticles for the co-delivery of camptothecin and survivin shRNA in to colon adenocarcinoma in vitro and in vivo

Simultaneous drug and gene delivery to cancer cells has been introduced to provide advantages of the synergistic effects of gene to sensitize the cancer cells to chemotherapeutic agent. In the current study, nucleolin-targeted co-delivery system, based on PEGylated rod-shaped mesoporous silica NPs was developed as a biocompatible nanocarrier for simultaneous delivery of camptothecin and survivin shRNA-expressing plasmid (iSur-DNA) to colon adenocarcinoma. The structural characterization including hydrodynamic radius and morphological characteristics of the prepared system demonstrated the mesoporous rod-shaped structure of the prepared system with 100–150 nm diameter. Camptothecin was loaded into the rod-shaped MSN NPs with encapsulation efficiency of 32%. At the next stage, the prepared camptothecin-loaded system was PEGylated and then iSur-DNA was condensed with C/P ratio of 6 to form PEG@MSNR-CPT/Sur. Then, the prepared camptothecin-iSur-DNA loaded PEGylated rod-shaped mesoporous silica NPs were tagged with AS1411 DNA aptamer (Apt-PEG@MSNR-CPT/Sur) in order to provide selective therapy against colorectal adenocarcinoma. The obtained results showed that the prepared platform controlled the release of anticancer drug, camptothecin. The experimental results indicated potent synergistic effect of iSur-pDNA and CPT in in vitro cytotoxicity, apoptosis induction and in vivo antitumor effect. In addition, tagging the system with AS1411 DNA aptamer facilitated drug uptake into nucleolin positive colorectal cancer cells leading to higher cellular toxicity and apoptosis induction in C26 cells compared to nucleolin-negative CHO cell line.Apt-PEG@MSNR-CPT/Sur system significantly supressed tumor growth rate in C26 tumor bearing mice while improving survival rate and pharmacokinetics of the platform in comparison with PEG@MSNR-CPT and PEG@MSNR-CPT/Sur. It could be concluded that the developed nucelolin targeted nanomedicine for co-delivery of camptothecin and iSur-DNA could serve as a versatile nanotherapeutic system against colorectal cancer.

Downregulation of survivin by adenovirus-mediated shRNA promotes apoptosis in skin cancer cells.

BackgroundSurvivin, a member of the inhibitor of apoptosis protein family, is highly expressed in many cancers and has important roles in inhibiting apoptosis by blocking caspase activation. However, its antitumor effects remain largely unknown. Here we explore the function of survivin in skin cancer.MethodsWe used qPCR and Western blot to examine survivin expression in skin cancer patients and cell line. We generated several survivin shRNA constructs and tested the effects of survivin shRNA on cancer cell viability using MTT assay, flow cytometry, and TUNEL assay.ResultsWe found that survivin was upregulated in both skin cancer patients and skin cancer cell line A431. Knockdown survivin via shRNA inhibited cancer cell proliferation and promoted apoptosis in both A431 cell and in vivo xenograft tumor mouse model. The antitumor effect is comparable to resveratrol, a drug known to inhibit cancer progression. Moreover, we showed that inhibition of survivin was able to increase the expression of cleaved caspase 7/caspase 9 and activate the ataxia-telangiectasia mutated-NF-κB pathway in A431 cells.ConclusionSurvivin-shRNA possesses antitumor abilities in vitro and in vivo by inhibiting the proliferation and promoting apoptosis of A431 cells. It may serve as a potential anticancer target for skin cancer therapy in the future.

Effects of survivin shRNA-APC double gene co-expression stably transfected cell lines on the angiogenesis of HT-29 colon cancer cells subcutaneous xenografts in nude mice

Objective To investigate the effects of survivin shRNA-APC double gene co-expression stably transfected cell lines on the VEGF、COX-2 expressions and angiogenesis of subcutaneous exnotransplanted tumor tissues cell of HT-29 colon cancer in nude mice. Methods Forty nude mice were randomly divided into five groups, the negative control group, empty vector group, Survivin shRNA group, APC group, double-gene group.The stably transfected cell lines and HT-29 colon cancer cells were cultured, PBS suspension resulted in cell density of 2×107/ml, injected with respective stably transfected cell lines to establish an SXT model.All the mice were sacrificed after six weeks in order to separate the subcutaneous tumor, the expressions of the VEGF, COX-2mRNA and protein were detected by Real time PCR and immunohistochemistry, CD34 antibody was used to mark the vascular endothelial cells, and the MVD values were detected by immunohistochemistry. Results Tumors were formed in the nude mice of each group.The expressions of VEGF, COX-2 mRNA in Survivin shRNA group ((50.84±3.64)%, (50.11±3.91)%), APC group((74.28±6.87)%, (72.39±6.55)%)and Survivin shRNA-APC double-gene group ((21.78±4.00)%, (20.74±5.12)%)were significantly lower than those in the empty vector groups((100.00±0.00)%, (100.00±0.00)%) or negative control group ((98.22±0.38)%, (97.61 + 0.77)), the differences were statistically significant(P<0.05); the expressions of VEGF, COX-2 mRNA in Survivin shRNA-APC double-gene group were significantly lower than those in APC groups, Survivin shRNA group, the differences were statistically significant(P<0.05). The expressions of VEGF, COX-2 protein in Survivin shRNA group(5.15±1.02, 5.26±0.91), APC group(4.96±1.12, 4.93±1.18), and Survivin shRNA-APC double-gene groups(1.81±0.84, 1.80±0.81)were significantli lower than those in the negative control group (8.95±0.55, 8.77±0.60) and empty vector group(9.17±0.49, 9.01±0.80), the differences were statistically significant(P<0.05), the expressions of VEGF, COX-2 protein in the Survivin shRNA-APC double-gene group were significantly lower than those than in APC group, Survivin shRNA group(P<0.05); the expressions of MVD in APC group(12.14±3.45), Survivin shRNA group(11.39±2.94)and Survivin shRNA-APC double-gene group(3.96±2.20)were lower than those in the negative control group(25.09±5.59)and empty vector group(27.87±7.36), the differences were statistically significant(P<0.05), the MVD in the Survivin shRNA-APC double-gene group was even lower than that in APC group, Survivin shRNA group, the differences were statistically significant (P<0.05). Conclusion Survivin shRNA-APC double gene co-expression stably transfected cell lines can significantly reduce the expression of the VEGF, COX-2 mRNA and protein and then inhibit the angiogenesis of transplanted tumor tissue, and its inhibitory effect is more effective than that og Survivin shRNA and APC single gene stable strain. Key words: Colon cancer; Survivin shRNA-APC double gene; VEGF; COX-2; MVD

Transfection with Livin and Survivin shRNA inhibits the growth and proliferation of non‑small cell lung cancer cells.

Lung cancer is reported to be a major public health issue worldwide and the overall prognosis of patients remains poor. The expression levels of Livin and Survivin, of the inhibitors of apoptosis (IAP) family, are associated with prognostic significance in the majority of solid tumors. Therefore, in the presents study, short hairpin (sh)RNA expression vectors inhibiting the Livin and Survivin genes were constructed to examine the effects of the transfection of Livin shRNA and/or Survivin shRNA on the biological functions of tumor cells. The transfection efficiency was measured using fluorescence reverse transcription‑quantitative polymerase chain reaction and western blot analyses. The cell growth inhibition ratio was measured using a CCK assay. Cell apoptosis following transfection and in tumor tissues were measured using a TUNEL assay, and a cancer xenograft model was used to investigate the effect of Livin shRNA and/or Survivin shRNA on tumor growth. The results indicated that the mRNA and protein expression levels were suppressed following the transfection of Livin and Survivin shRNA into tumor cells (P<0.05, compared with control group). The growth of tumor cells invivo and invitro was significantly inhibited following transfection with Livin and Survivin shRNA, compared with that in the other groups (P<0.05). Taken together, the transfection of cells with Livin and Survivin inhibited tumor growth invivo and invitro, with the co‑transfection of Livin and Survivin shRNA showing increased efficiency, compared with transfection of either the Livin vector or Survivin vector alone. The combined inhibition of Livin and Survivin may be a promising multitargeted gene therapeutic strategy in cancer treatment.

Lentivirus‑mediated knockdown of chondroitin polymerizing factor inhibits glioma cell growth invitro.

Glioma is the most common primary tumor in the central nervous system, characterized by rapid progression, aggressive behavior, frequent recurrence and poor prognosis. In the present study we demonstrated that chondroitin polymerizing factor(CHPF) is highly expressed in human glioma tissues and 4glioma cell lines. To explore the role of CHPF in glioma, a lentiviral vector expressing CHPF shRNA was constructed and transfected into the glioma U251 cells, which stably downregulated the expression levels of the CHPF gene in U251 cells invitro. U251 cell proliferation inhibition rates were determined by MTT assay. The effect of survivin shRNA on U251 cell cycle distribution and cell apoptosis was determined by flow cytometry. Compared to the shRNA‑Ctrl group of cells, the shRNA-CHPF group of cells exhibited decreased proliferation and a significant increase in the proportion of cells in the G0/G1phase. In addition, we found that knockdown of the expression of CHPF increased apoptosis in glioma U251 cells. Therefore, our results confirmed that CHPF promotes growth and inhibits apoptosis in glioma U251 cells. Thus, by invivo and invitro data, the present study suggests that CHPF could be a new potential therapeutic target for glioma.

Effects of monomethoxypolyethylene glycol-chitosan nanoparticle-mediated dual silencing of livin and survivin genes in prostate cancer PC-3M cells.

Monomethoxypolyethylene glycol-chitosan (mPEG-CS) nanoparticles were used as interfering RNA carriers to transfect human prostate cancer PC-3M cells to evaluate the effects of livin and survivin gene silencing on the proliferation and apoptosis. mPEG-CS nanoparticles with sizes of approximately 60 nm were first synthesized by ionic crosslinking. Through electrostatic adsorption, mPEG-CS-livin short hairpin RNA (shRNA), mPEG-CS-survivin shRNA, and mPEG-CS-(livin shRNA + survivin shRNA) nanoparticles were then prepared to transfect PC-3M cells. The mRNA and protein expression levels of livin and survivin were measured by reverse transcription-PCR and western blotting, respectively. The inhibitory effects of down-regulated livin and survivin gene expression on the cell proliferation were evaluated by MTT assay. Cell apoptosis was assessed visually using Hoechst staining. Livin and survivin expression levels in all shRNA interference groups were effectively down-regulated at both the mRNA and protein levels. Dual silencing of livin and survivin genes markedly inhibited cell proliferation and facilitated apoptosis, with better outcomes than those of individual shRNA treatments. mPEG-CS nanoparticle-mediated dual shRNA interference of livin and survivin genes significantly reduced the expression levels in PC-3M cells, inhibited proliferation, and promoted apoptosis. As these effects were superior to single interference, this method may have synergistic effects.

Targeting survivin using a combination of miR-494 and survivin shRNA has synergistic effects on the suppression of prostate cancer growth

Castration-resistant prostate cancer (CRPC) remains an obstacle in the current treatment provided for prostate cancer (PCa). Survivin, an apoptosis inhibitor, has been found to be involved in the progression of PCa, and is a promising candidate target for CRPC therapy. Micro (mi) RNAs are involved in the progression of PCa through the regulation of multiple genes. One of the objectives of the present study was to investigate the effect of miRNA (miR)-494 on the expression of survivin, as well as on PCa growth. The present study also aimed to assess whether co-transfecting miR-494 with survivin short hairpin (sh)RNA has synergistic effects on suppressing PCa proliferation or the expression of survivin. Gene Expression Omnibus datasets with clinical PCa miRNA expression profiles were utilized to analysis the expression of miR-494 in Ca, compared with normal prostate samples. PC3 cells, a CRPC cell line, were transfected with either an miR-494 expression adenovius, a survivin shRNA adenovirus or the two together, to examine their effect on PCa growth and the expression of survivin in vitro and in vivo. miR-494 was downregulated in PCa tissue samples and in the PC-3 cell line. miR-494 targeted survivin at the translational level in PCa. Overexpression of miR-494 and silencing survivin RNA through the use of survivin shRNA inhibited the expression of survivin and attenuated PC-3 cell growth in vitro and in vivo. Notably, co-transfecting miR-494 with survivin shRNA had synergistic effects on suppressing prostate cancer proliferation via further suppression of the expression of survivin. These results suggested that using multiple methods to inhibit the function of survivin may have improved efficacy for treating PCa.

Effects of RNA interference-mediated knockdown of livin and survivin using monomethoxypolyethylene glycol-chitosan nanoparticles in MG-63 osteosarcoma cells

MG-63 human osteosarcoma cells were transfected with short hairpin RNA (shRNA) against livin and survivin using monomethoxypolyethylene glycol‑chitosan (mPEG‑CS) nanoparticles (NPs) as carriers, with the aim of evaluating the effect on cell proliferation and apoptosis. mPEG‑CS NPs sized ~100 nm were prepared by ionic crosslinking. mPEG‑CS‑livin shRNA, mPEG‑CS‑survivin shRNA and mPEG‑CS‑(livin shRNA + survivin shRNA) NPs were constructed by electrostatic adsorption at NP suspension/gene solution ratios of 3:1 to transfect MG‑63 cells. The expression levels of livin and survivin mRNA and protein were measured by reverse transcription‑polymerase chain reaction and western blotting, respectively. The inhibitory effects of downregulated livin and survivin expression on cell proliferation were measured using an MTT assay. The apoptosis‑inducing effects of livin and surivin knockdown were investigated using a Hoechst staining kit. All shRNA groups resulted in reduced expression of livin and survivin mRNA and protein in MG‑63 cells. The MTT assay and Hoechst staining indicated that simultaneous knockdown of livin and survivin genes inhibited the proliferation of MG‑63 cells and promoted their apoptosis, to a greater extent than knocking down either gene individually. The simultaneous interference mediated by mPEG‑CS NPs significantly reduced livin and survivin expression in MG‑63 cells, suppressed proliferation and facilitated apoptosis, to a greater extent than knockdown of either livin or survivin alone were. Thus the results indicate a synergistic effect of livin and survivin.

Survivin silencing and TRAIL expression using oncolytic adenovirus increase anti-tumorigenic activity in gemcitabine-resistant pancreatic cancer cells.

In this study, we demonstrated that survivin downregulation with TRAIL expression greatly enhanced the cytotoxic death of pancreatic cancer cells after gemcitabine treatment. Using real-time RT-PCR, we analyzed five survivin shRNAs to identify the best target sequence for suppression of human survivin, with the goal of treating gemcitabine-resistant pancreatic cancer cells. Survivin shRNA 5, corresponding to target 5, showed the greatest reduction in survivin mRNA levels. Furthermore, combined treatment with survivin shRNA-expressing adenovirus with gemcitabine plus TRAIL decreased uncleaved PARP and increased consequent PARP cleavage, which was correlated with the greatest levels of survivin downregulation and cell death. These results indicate that survivin functions as a common mediator of gemcitabine- and TRAIL-induced cell death. Using a nude mouse model implanted with MiaPaCa-2 pancreatic cancer cells, we observed tumor regression induced by an oncolytic adenovirus expressing survivin shRNA and TRAIL plus gemcitabine. Together, our findings provide a strong rationale for treating pancreatic cancer patients with both gemcitabine and oncolytic adenovirus armed with survivin shRNA and TRAIL.

Lentiviral vector-mediated survivin shRNA delivery in gastric cancer cell lines significantly inhibits cell proliferation and tumor growth.

It has been well documented that survivin has multiple functions including cytoprotection, inhibition of cell death, and cell cycle regulation, particularly at the mitotic stage of the cell cycle, all of which favor cancer survival. Its expression in normal tissue is developmentally regulated, and any type of deregulation in survivin expression favors cancer survival. Gastric cancer is one of the most common malignancies and the second most common cause of cancer-related mortality worldwide. The molecular mechanisms involved in the transformation and progression of gastric cancer remain unclear. In the present study, we investigated the effect of lentiviral vector-mediated survivin shRNA delivery in gastric cancer cell lines. Lentiviral-mediated survivin shRNA was used to knock down survivin expression in gastric cancer cell lines SGC-7901, MGC-803 and MKN-28. The Τranswell chemotaxis and the CCK-8 assays were used to assess the migration and proliferation of the tumor cells, respectively. TUNEL assay was used to detect apoptosis. Quantitative real-time PCR and western blot analysis were used to quantify mRNA and protein levels, respectively. Our results demonstrated that lentiviral-mediated RNAi markedly suppressed the survivin expression in all three gastric cancer cell lines. Significant decrease in survivin mRNA and protein expression were detected in the gastric cancer cell lines stably transfected with the lentiviral survivin shRNA vector, and knockdown of survivin also significantly inhibited the proliferation and migration in the gastric cancer cells and tumorigenicity in a xenograft animal model. Our results indicated that aberrant high cytoplasmic survivin expression in gastric cancer cells is associated with increased proliferation index and tumor growth. In conclusion, our results suggest that lentiviral-mediated gene therapy has the potential to be developed into a novel therapeutic strategy for the treatment of gastric cancer.

Effect of lentiviral-mediated delivery of Survivin shRNA with adenomatous polyposis coli fragment on Survivin expression and cell proliferation in HT-29 cells

目的：观察Survivin sh RNA、结肠腺瘤息肉易感基因（adenomatous polyposis coli,APC）片段联合对HT-29细胞Survivin表达及细胞增殖的影响.方法：构建Survivin sh RNA慢病毒载体、A P C有效片段慢病毒载体,对H T-29细胞分别采用单慢病毒载体侵染及联合侵染.实验分阴性对照组、空载组、sh RNA组、APC组、sh RNA＋APC联合组,对侵染48 h后的HT-29细胞进行real-time PCR、Western blot及CCK8细胞增殖检测,检测Survivin m RNA、蛋白表达水平及对细胞增殖的影响.结果：（1）sh RNA＋APC联合组与其余各组相比,Survivin m RNA表达量显著下降（P〈0.05）;（2）s h R N A＋A P C联合组与其余各组相比,其Survivin蛋白抑制率明显高于其余各组（P〈0.05）;（3）sh RNA＋APC联合组与其余各组相比,细胞增殖速度低于其余各组（P〈0.05）.结论：Survivin sh RNA与APC片段联合能抑制HT-29细胞内Survivin m RNA及蛋白的表达,同时能够抑制细胞增殖能力,并且优于单个基因侵染.

Simultaneous Inhibition of Tumor Growth and Angiogenesis for Resistant Hepatocellular Carcinoma by Co-delivery of Sorafenib and Survivin Small Hairpin RNA

The development of multidrug resistance (MDR) in human hepatocellular carcinoma (HCC) is one of the major obstacles for successful chemotherapy of HCC. Co-delivery of sorafenib (SF) and survivin shRNA (shSur) was postulated to achieve synergistic effects in reversing MDR, suppressing tumor growth and angiogenesis. For this purpose, in this work, SF and shSur co-loaded pluronic P85-polyethyleneimine/d-α-tocopheryl polyethylene glycol 1000 succinate nanocomplexes (SSNs) were first designed and developed for the treatment of drug resistant HCC. The experimental results showed that SSNs could achieve effective cellular internalization and shSur transfection efficiency, induce significant downregulation of the survivin protein, and cause remarkable cell arrest and cell apoptosis. The tube formulation assay demonstrated that SSNs completely disrupted the enclosed capillary networks formed by human microvascular endothelial cells. The in vivo antitumor efficacy showed that SSNs were superior to that of other treatments on drug resistant hepatocellular tumor models. Therefore, it could be an efficient strategy to co-deliver SF and shSur for therapy of drug resistant HCC.

IRGD Conjugated TPGS Mediates Codelivery of Paclitaxel and Survivin shRNA for the Reversal of Lung Cancer Resistance

Multidrug resistance (MDR) is one of the major obstacles in tumor treatment. Herein, we reported an active targeting strategy with peptide-mediated nanoparticles deep into tumor parenchyma, which iRGD conjugated d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) mediated codelivery of paclitaxel (PTX) and survivin shRNA (shSur) for the reversal of lung cancer resistance. Pluronic P85-polyethyleneimine/TPGS complex nanoparticles incorporated with iRGD-TPGS conjugate codelivering PTX and shSur systems (iPTPNs) could induce effective cellular uptake, RNAi effects, and cytotoxicity on A549 and A549/T cells. In particular, iPTPNs showed superiority in biodistribution, survivin expression, tumor apoptosis, and antitumor efficacy by simultaneously exerting an enhanced permeability and retention (EPR) effect and iRGD mediated active targeting effects. iPTPNs significantly enhanced the accumulation of PTX and shSur, down-regulated survivin expression, and induced cell apoptosis in tumor tissue. The in vivo antitumor efficacy showed the tumor volume of iPTPNs group (10 mg/kg) was only 12.7% of the Taxol group. Therefore, the iRGD mediated PTX and shSur codelivery system could be a very powerful approach for the reversal and therapy of lung cancer resistance.

Impact of Co-transfection with Livin and Survivin shRNA Expression Vectors on Biological Behavior of HepG2 Cells

To construct short hairpin RNA (shRNA) eukaryotic expression vectors targeting Livin and Survivin genes, and to explore the impact of co-transfection of Livin and Survivin shRNA expression vectors on the biological behavior of HepG2 cells. shRNA eukaryotic expression vectors pSD11-Livin and pSD11- Survivin were designed and constructed then transfected into HepG2 cells separately or in combination. mRNA and protein expression in transfected cells was assessed by quantitative fluorescence PCR and Western blotting, respectively. Cell proliferation was measured by MTT assay and cell apoptosis by TUNEL assay. The Livin and Survivin shRNA eukaryotic expression vectors were successfully constructed and transfected into HepG2 cells. The relative mRNA expression levels of Livin and Survivin in HepG2 cells co-transfected with pSD11-Livin and pSD11-Survivin were 0.12 ± 0.02 and 0.33 ± 0.13, respectively, which was significantly lower than levels in cells transfected with either pSD11-Livin or pSD11-Survivin (P<0.05). The relative protein expression levels of Livin and Survivin in the co-transfected cells were also significantly decreased compared to single- transfection (P<0.05). The inhibition rate of cell growth in the co-transfection group was higher than that in the single-transfection groups at 48 h, 60 h, or 72 h after transfection (P<0.01). The apoptotic rate increased to the greatest extent in the co-transfection group relative to any other group (P<0.05). Co-transfection with pSD11-Livin and pSD11-Survivin was more efficient than transfection with either vector alone in reducing the mRNA and protein expression of Livin and Survivin genes in HepG2 cells. Co-transfection also inhibited the proliferation of transfected cells more than the other groups, and induced cellular apoptosis more effectively.

MiR-203 regulates the proliferation, apoptosis and cell cycle progression of pancreatic cancer cells by targeting Survivin

MicroRNAs have emerged as crucial regulators of tumorigenesis. However, the mechanism by which miR‑203 is involved in the pathogenesis of pancreatic cancer (PC) remains elusive. In the present study, PC cell lines were used as an experimental model to investigate the expression and functional role of miR‑203 in PC. miR‑203 mimic virus, miRNA negative control virus and Survivin shRNA virus were transfected into the PC cell line, CFPAC‑1. mRNA and protein levels of Survivin were detected using qPCR and western blot analysis. Proliferation, apoptosis and cell cycle profiles were detected by an MTT assay and flow cytometry. Female BALB/cA‑nu nude mice were used to validate the role of miR‑203 in vivo. The protein levels of Survivin were found to negatively correlate with miR‑203 levels in four PC cell lines. A luciferase assay revealed that Survivin was a direct target of miR‑203. Transfection with miR‑203 mimic inhibited CFPAC‑1 cell proliferation and induced apoptosis and G1 phase cell cycle arrest, similar to knockdown of Survivin. In the in vivo nude mouse model, the downregulation of Survivin by knockdown of Survivin or transfection with miR‑203 mimic inhibited tumor growth. Results of the current study indicate that miR‑203 regulates the proliferation, apoptosis and cell cycle progression of PC cells by targeting Survivin.

Cotransfection of Survivin and CD44v3 Short Hairpin RNAs Affects Proliferation, Apoptosis, and Invasiveness of Colorectal Cancer

Colorectal cancer is one of the common malignant tumors in humans, and the incidence rate is gradually increasing year by year. Survivin and CD44v3 are ideal targets for gene therapy due to their overexpression in colorectal cells. Studies show that downregulation of survivin could promote apoptosis and depress proliferation, and reduction of CD44v3 expression could inhibit tumor invasive capacity. It is difficult to achieve satisfactory curative effect. In this study, we use survivin and CD44v3 short hairpin RNAs (shRNA) combined transfection into colorectal cancer cell line SW480 to investigate its effects on the cell apoptosis, proliferation and invasiveness. ShRNA plasmids targeting survivin and CD44v3 were singly or co-transfected into SW480 cells. The co-transfection group exhibited the most significant inhibitory effect on cell growth (P < 0.05) and the highest apoptosis rate (P < 0.05). In addition, the invasive capacity in the co-transfected group was the least. The tumor inhibition rate of the cotransfected group in xenograft tumor mice was significantly higher than other groups (P < 0.05). Moreover, the microvessel density of the co-transfected group was significantly decreased compared with other groups (P < 0.05). These results suggest combined transfection of survivin shRNA and CD44v3 shRNA may produce a synergistic effect on gene therapy in colorectal cancer.