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Abstract

Although gene therapy has brought new insights into the treatment of malignant melanoma, targeting delivery of nucleic acid which targets critical oncogene/anti-oncogene in vivo is still a bottleneck in the therapeutic application. Our previous in vitro studies have found that the oncogene Livin could serve as a potential molecular target by small interfering RNA for gene therapy of malignant melanoma. However, how to transport Livin small interfering RNA into malignant melanoma cells specifically and efficiently in vivo needs further investigation. Cumulative evidence has suggested that single-chain antibody–mediated small interfering RNA targeted delivery is an effective way to silence specific genes in human cancer cells. Indeed, this study designed a protamine–single-chain antibody fusion protein, anti-MM scFv-tP, to deliver Livin small interfering RNA into LiBr cells. Further experiments confirmed the induction of cell apoptosis and suppression of cell proliferation by anti-MM scFv-tP in LiBr cells, along with efficient silence of Livin gene both in vitro and in vivo. Altogether, our findings provide a feasible approach to transport Livin small interfering RNA to malignant melanoma cells which would be a new therapeutic strategy for combating malignant melanoma.

Keywords

Small interfering RNA Livin single-chain antibody targeting delivery malignant melanoma

Introduction

Human melanoma of the skin is the eighth most frequently diagnosed cancer among both male and female in developed countries per year.¹ In the United States, 73,870 new cases of melanoma skin cancer and 9,940 deaths were estimated to occur in 2015.² The switch of human melanoma phenotype from non-metastatic to highly metastatic, developing to distant metastasis which is the leading cause of death, is related to different molecular mechanisms that have not yet been elucidated.^3–5 Therefore, the molecular mechanisms of malignant melanoma (MM) development and metastasis need further exploration which will be helpful for developing effective therapies for this disease.

RNA interference (RNAi) is found to be a considerable therapeutic approach to silence disease-causing genes and is a promising strategy in cancer therapy.^6,7 This gene therapy has opened a new window on MM treatment. However, the main obstacle in therapeutic application to achieve gene silencing by RNAi technologies in vivo is targeted delivery of nucleic acid drug to appropriate cell populations in vivo, improving its therapeutic index, and minimizing potential side effects and cellular toxicity.^8–10 Until now, a significant amount of evidence has suggested that single-chain antibody–mediated small interfering RNA (siRNA) targeted delivery is an effective way to silence specific genes in particular human cancer cells.^11–14 This single-chain antibody–protamine fusion protein has a Fab fragment that can recognize and target cell membrane receptors, and a fusing nucleic acid binding domain—protamine, which has a strong nucleic acid binding ability, being able to deliver siRNA to specific cells and silence specific genes efficiently.

Livin/melanoma inhibitor of apoptosis (ML-IAP), an IAP family member, is selectively overexpressed in most human cancers, including melanoma.^15–17 High level of Livin expression appears to be involved in tumor progression, resistance to apoptosis induced by a variety of stimuli, and resistance to chemotherapy both in vitro and in melanoma patients receiving chemotherapy.^16,18,19 Published studies also show that Livin/ML-IAP is a promising therapeutic target for human cancer treatment.¹⁶ In our previous study, we found that Livin could serve as a potential molecular target by siRNA for gene therapy of MM in vitro, and deserved further investigation in vivo.²⁰ Therefore, in this study, we intend to design a single-chain antibody–protamine fusion protein to deliver siRNA specifically to knockdown Livin expression in MM cells and investigate the effect of this strategy for gene therapy of MM.

In this study, we designed a single-chain antibody–protamine fusion protein combining with nucleic acid by protamine and directly targeting appropriate cells by single-chain antibody against ganglioside in MM. We demonstrated that siRNA could be specifically delivered into LiBr cells by the fusion protein and knockdown Livin expression efficiently. Further experiments confirmed the induction of cell apoptosis and suppression of cell proliferation by anti-MM scFv-tP in LiBr cells, along with efficient silencing of Livin gene both in vitro and in vivo. Thus, our findings describe a feasible approach to transport targeting siRNA into MM cells, suggesting single-chain antibody–mediated targeted silencing Livin gene as a new therapeutic strategy for gene therapy for MM.

Materials and methods

Construction, expression, and purification of the anti-MM scFv-tP fusion protein

The anti-MM scFv fragment sequence was provided by Dr Ziling Wang (Academy of Military Medical Sciences, Beijing, China). The anti-MM scFv-tP fusion protein sequence was obtained by polymerase chain reaction (PCR), with the human protamine encoding sequence designed in the primers of anti-MM scFv. Then, it was cloned into the pGEX-4T-1 vector (provided by Dr Yuling Wang, Academy of Military Medical Sciences, Beijing, China). Next, the fusion protein was expressed in Escherichia coli BL21 system and purified by Ni²⁺-NTA (nitrilotriacetic acid) agarose (Qiagen, Valencia, CA, USA), according to the manufacturer’s protocol.

Electrophoretic mobility shift assay

The anti-MM scFv-tP fusion protein was mixed with increasing amount of biotin-labeled siRNA in the mixing ratio of 1:3, 1:6, and 1:9 at room temperature for 30 min and was run on a 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). For auto-radiography, the polyacrylamide gel was dried and exposed on radiographic film at −70°C for 24 h.

Cell lines and cell culture

MM cell line LiBr was a kind gift from Dr Tianwen Gao (The Fourth Military Medical University, Xi’an, China). The LiBr cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) medium (Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine serum (Invitrogen) in a humidified chamber at 37°C with 5% CO₂.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay

LiBr cells (5 × 10³/well) were seeded on 96-well culture plates and incubated overnight. Then, the cells were incubated with 200 nM anti-MM scFv-tP-Livin-siRNA mixture for 24, 48, 72, and 96 h. Once the plates were washed, 0.5 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added and incubated at 37°C. After 4 h, the culture medium was removed carefully and dimethyl sulfoxide (DMSO) was added to solubilize the formazan crystals. Finally, the absorbance was measured at a wavelength of 490 nm using a microplate autoreader (BioTek Instruments Inc., Winooski, VT, USA). Independent experiments were repeated in triplicate.

Flow cytometry

The anti-MM scFv-tP fusion protein was labeled by fluorescein isothiocyanate (FITC) and incubated with LiBr cells. After incubating for 1 h, the cells were collected and fixed with ice-cold 70% ethanol in phosphate-buffered saline (PBS) before flow cytometry (FACSCalibur, BD Biosciences, Franklin Lakes, NJ, USA). For apoptosis analysis, LiBr cells, incubated with 200 nM anti-MM scFv-tP-Livin-siRNA mixture for 72 h, were centrifuged, washed with PBS, and incubated with Annexin V-conjugated FITC (Jingmei Biotech, Shenzhen, China) before analysis by flow cytometer according to the manufacturer’s protocol.

Immunofluorescence and immunohistochemistry

For Immunofluorescence, LiBr cells were incubated on coverslips with tetramethylrhodamine (TRITC)-labeled anti-MM scFv-tP fusion protein for 24 h at 4°C, fixed, and stained using 4′,6-diamidino-2-phenylindole (DAPI). For immunohistochemistry, formalin-fixed mouse tumor tissues were embedded in paraffin and serial 5-µm-thick sections were obtained and stained by hematoxylin & eosin staining (H&E) staining.

siRNA design and preparation

The siRNA targeting Livin was designed according to the study of siRNA by Reynolds et al.²¹ Three siRNAs were designed and identified using an online design tool, the siRNA Selection Web Server (http://jura.wi.mit.edu/bioc/siRNA), as described in a previous study.²⁰ They are siRNA-1 (sense: 5′-GGC CUG GAC ACC UGC AGA GdTdT-3′ and antisense: 5′-CUC UGC AGG UGU CCA GGC CdTdT-3′), siRNA-2 (sense: 5′-GGU GCU UCU UCU GCU AUG GdTdT-3′ and antisense: 5’-CCA UAG CAG AAG AAG CAC CdTdT-3′), and siRNA-3 (sense: 5′-GAG AGG UCC AGU CUG AAA GdTdT-3′ and antisense: 5′-CUU UCA GAC UGG ACC UCU CdTdT-3′), targeting to sites 294–312, 541–559, and 790–808 of human Livin messenger RNA (mRNA) sequences, respectively. The negative control of siRNA (siRNA-NC), with random sequences, did not target any known mammalian gene.

Real-time quantitative PCR assay

Total RNA was extracted using TRIzol reagent (Life Technologies, Rockville, MD, USA) according to the manufacturer’s protocol. Then, the total RNA was reverse-transcribed to complementary DNA (cDNA) using PrimeScript™ RT Reagent Kit (TaKaRa, Dalian, China) according to the manufacturer’s protocol. The cDNA was studied using CFX96 real-time PCR system (Bio-Rad Laboratories, Hercules, CA, USA) with SYBR Green PCR Master Mix (TaKaRa). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used for normalization. The specific primers for Livin (forward: 5′-GTC AGT TCC TGC TCC GGT CAA-3′ and reverse: 5′-GGG CAC TTT CAG ACT GGA CCTC-3′) and for GAPDH (forward: 5′-GCA CCG TCA AGG CTG AGAAC-3′ and reverse: 5′-ATG GTG GTG AAG ACG CCA GT-3′) were designed and synthesized by TaKaRa. Relative gene expression was calculated using the 2^−ΔΔCt method.

Western blotting assay

Cells or tissues were washed three times with cold PBS and lysed using radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris pH 8.0, 150 mM NaCl, 0.1% SDS, 1% NP-40 (nonyl phenoxypolyethoxylethanol), and 0.5% sodium deoxycholate) containing protease inhibitors. Approximately, 30 µg of protein was separated with 10% SDS-PAGE and blotted onto nitrocellulose membranes. The membranes were blocked with 5% skim milk at room temperature for 1 h and then incubated with primary antibodies against β-actin (Zhongshan, Beijing, China), Livin (Alexis Biochemicals, Lausen, Switzerland), and caspase-3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight, followed by Tris buffered saline with Tween 20 (TBST) wash and 1 h incubation with horseradish peroxidase–labeled secondary antibodies at room temperature. Protein bands were visualized by a Molecular Imager ChemiDoc XRS System (Bio-Rad Laboratories).

Small animals living image detection and in vivo treatment with antibody–siRNA

For small animals living image detection assay, PBS and Cy5–siRNA alone or pre-incubated with the scFv-tP fusion protein in 6:1, were injected into LiBr subcutaneous xenograft nude mice via tail vein. After 24 h, the results were detected through the small animals living image detector To investigate the in vivo treatment efficiency of antibody–siRNA in nude mice, 1 × 10⁷ LiBr cells were injected subcutaneously into right side of the flank region. Three mice were used for each cell clone. From the second day onwards LiBr cells were injected into the flanks of nude mice to form ectopic tumors, 200 µL of PBS (Black group), anti-MM scFv-tP-siRNA-NC (Mock group), or anti-MM scFv-tP-siRNA (20 µg of siRNA) was injected into LiBr cell subcutaneous xenograft nude mice through the tail vein once in every 2 days. The tumor growth was monitored by measuring tumor volume every 2 days after 10 days. Xenograft tumors were harvested and fixed with 4% paraformaldehyde after 5 weeks. Animal care and protocols were in accordance with the guidelines of the Institutional Animal Care and Use Committee of Xi’an Jiaotong University.

Statistical analysis

GraphPad Prism version 6.0 software (GraphPad, San Diego, CA, USA) was used to analyze differences between two groups (student’s t test), calculate Pearson’s correlation, and perform linear regression analysis (p < 0.05 was considered to be significant).

Results

The anti-MM scFv-tP fusion protein possessed siRNA binding ability and could directly target MM LiBr cells

The anti-MM scFv-tP encoding sequence was constructed and was inserted into the pGEX-4T-1 vector. Then, the recombinant fusion protein was expressed in E. coli and purified by Ni2⁺-NTA agarose (Figure 1(a) and (b)). The ability of purified s-tP fusion protein to carry siRNA was confirmed by the electrophoretic mobility shift assay (EMSA) experiment. As shown in Figure 1(c), when s-tP fusion protein was mixed with increasing amount of biotin-labeled siRNA, migration of the complexes was slower, indicating the binding of s-tP protein with siRNA. Our results also suggested that s-tP protein reached the maximum siRNA binding activity when the dilution ratio of siRNA was 1:6. To further test the interaction between the complexes (anti-MM scFv-tP-siRNA) and cells, we labeled the fusion protein with FITC or TRITC, and the results of flow cytometry and confocal microscopy assays indicated that the fusion protein could bind to the LiBr cell surface tightly and specifically (Figure 1(d) and (e)).

Figure 1.

The construction of anti-MM scFv-tP fusion protein which possessed siRNA binding ability and could directly target MM cells. (a) A schematic map of pGEX-scFv-tP vector. (b) Western blot (WB) assay confirmed the expression and purification of anti-MM scFv-tP fusion protein. (c) Detection of siRNA binding ability of the anti-MM scFv-tP fusion protein by EMSA experiment. When the dilution ratio of siRNA is 1:6, s-tP protein reached the maximum siRNA binding activity. (d) The anti-MM scFv-tP fusion protein was first labeled by fluorescein isothiocyanate (FITC) and incubated with MM LiBr cells for 1 h. Then, FITC protein was detected by flow cytometry analysis. (e) The fusion protein was labeled by tetramethylrhodamine (TRITC) and incubated with LiBr cells. The results of confocal microscopy showed tight and specific binding of the fusion protein with the cell membrane. These data were representative of three independent experiments.

The scFv-tP fusion protein specifically delivered siRNA into MM LiBr cells in vitro and in vivo

To determine whether the anti-MM scFv-tP fusion protein could specifically deliver siRNA into MM LiBr cells, the MM LiBr cells and prostate cancer DU145 cells were treated with the mixture of s-tP protein and different dose of FITC–siRNA. The results showed an efficient uptake of FITC–siRNA in the MM LiBr cells but not in DU145 cells, indicating that FITC–siRNA could be specifically delivered into MM LiBr cells by the anti-MM scFv-tP fusion protein (Figure 2(a)). To further determine the delivery of siRNA by scFv-tP fusion protein in vivo, LiBr subcutaneous tumors in nude mice were established, then PBS, Cy5–siRNA alone, or Cy5–siRNA pre-incubated with the scFv-tP fusion protein (anti-MM scFv-tP-siRNA-Cy5) was intravenously injected into mice bearing LiBr tumors via tail vein. After 24 h of the injections, the distribution of anti-MM scFv-tP-siRNA-Cy5 was detected by the small animals living image system, and the results showed that anti-MM scFv-tP-siRNA-Cy5 was specifically taken up by those LiBr subcutaneous tumors (Figure 2(b)), indicating an effective way to specifically deliver siRNA to MM cells by single-chain antibody fusion protein both in vitro and in vivo.

Figure 2.

The specific delivery of siRNA by scFv-tP fusion protein into MM LiBr cells. (a) The MM LiBr cells and prostate cancer DU145 cells were treated with the mixture of s-tP protein and different dilutions of FITC–siRNA. Cells under the light (upper panel) and fluorescence (lower panel) were observed. (b) PBS, Cy5–siRNA alone or pre-incubated with the scFv-tP fusion protein (6:1) were injected via tail vein into mice bearing LiBr subcutaneous tumors. After 24 h of injections, fluorescence was detected by small animals living image system. These data were representative of three independent experiments.

The scFv-tP fusion protein could successfully deliver siRNAs targeting Livin and efficiently cause gene silencing in MM LiBr cells

Livin is reported to be selectively overexpressed in MM and plays critical roles in the development and progression of this disease. Thus, we tested the efficacy of delivery of Livin-specific targeting siRNAs by scFv-tP fusion protein. Three designed siRNA oligos targeting human Livin mRNA (Figure 3(a)) were pre-incubated with the scFv-tP fusion protein and incubated with LiBr cells. After 48 h, Livin expression was detected using real-time quantitative PCR (RT-qPCR) and Western blot assays. The results showed that the anti-MM scFv-tP binding with Livin siRNAs (siRNA-1, siRNA-2, and siRNA-3) could significantly reduce the mRNA and protein levels of Livin compared to the control siRNA (siRNA-NC; Figure 3(b) and (c)). In particular, we found that siRNA-3 which targets to the 790–808 region of Livin mRNA, exhibited the highest efficacy of silencing Livin expression (Figure 3(b) and (c)). Then, we applied this result to our further experiments, and the results demonstrated that anti-MM scFv-tP-Livin-siRNA-3 could inhibit both Livin mRNA and protein expression in dose-dependent and time-dependent manners (Figure 3(d) and (e)). All these data suggested that the scFv-tP fusion protein could successfully deliver Livin siRNAs and efficiently cause gene silencing in MM cells.

Figure 3.

The scFv-tP fusion protein could successfully deliver Livin siRNAs and efficiently cause gene silencing in MM cells. (a) Three designed siRNA oligos targeting human Livin mRNA were shown. (b) RT-qPCR and (c) Western blot detecting Livin expression after the delivery of Livin siRNAs by the scFv-tP fusion protein. (d) RT-qPCR (upper panel) and Western blot (lower panel) showing dose-dependent inhibitory effect of the most efficient siRNA, siRNA-3, on Livin silencing. (e) RT-qPCR (upper panel) and Western blot (lower panel) showing time-dependent inhibitory effect of siRNA-3 on Livin silencing. The results were representative of three independent experiments. The values were shown as the mean ± SD (*p < 0.05).

The scFv-tP fusion protein delivered Livin siRNA efficiently induced cell apoptosis and inhibited the growth of LiBr cells in vitro

Based on the understanding of the oncogenic roles of Livin gene, we studied the biological functions of anti-MM scFv-tP-Livin-siRNA-3 in LiBr cells. Cell apoptosis analysis using Annexin V-FITC/propidium iodide (PI) assay showed that 200 nM of anti-MM scFv-tP-Livin-siRNA was able to significantly induce apoptosis in LiBr cells (Figure 4(a) and (b)). In addition, MTT assay showed that the proliferation of cells treated with anti-MM scFv-tP-Livin-siRNA was remarkably suppressed compared to the control cells (Blank or Mock group; Figure 4(c)).

Figure 4.

The scFv-tP fusion protein delivered Livin siRNA efficiently induced apoptosis and inhibited the growth of LiBr cells in vitro. (a and b) LiBr cells incubated with 200 nM anti-MM scFv-tP-Livin-siRNA for 72 h were centrifuged, washed with PBS, and incubated with Annexin V-conjugated FITC. Annexin V-FITC/PI apoptosis analysis indicated that the apoptosis index, early and late apoptotic rate was significantly increased in cells of scFv-tP-Livin-siRNA group (UL: upper left area, UR: upper right area, LL: lower left area, LR: lower right area). (c) MTT assay showed that the proliferation of cells of scFv-tP-Livin-siRNA group was remarkably inhibited. The results were representative of three independent experiments. The values were shown as the mean ± SD (*p < 0.05).

The scFv-tP fusion protein delivered Livin siRNAs efficiently inhibited MM tumor growth in vivo

To confirm whether the anti-MM scFv-tP-Livin-siRNA can efficiently inhibit tumor growth in vivo, LiBr cell subcutaneous xenografts were first generated in nude mice. Then, 200 µL of PBS (Black group), anti-MM scFv-tP-siRNA-NC (Mock group), or anti-MM scFv-tP-Livin-siRNA (20 µg siRNA) was injected into LiBr subcutaneous tumor bearing mice through the tail vein for every 2 days. The results showed that anti-MM scFv-tP-Livin-siRNA significantly reduced tumor growth (Figure 5(a) and (b)) compared to the Blank and Mock groups. In addition, pathological analysis of the tumor tissues showed that tumor cell mass was significantly reduced in anti-MM scFv-tP-Livin-siRNA treated mice compared to Black and Mock groups (Figure 5(c)). Furthermore, silenced Livin expression in anti-MM scFv-tP-Livin-siRNA treated tumor tissue was confirmed by RT-qPCR and Western blot assays (Figure 5(d) and (e)). Elevated apoptosis was also observed in anti-MM scFv-tP-Livin-siRNA treated tumor tissues than Black and Mock groups, which was confirmed by Western blot analysis of apoptotic markers procaspase-3 and activated caspase-3 (Figure 5(f)). Taken together, these results indicated that the anti-MM scFv-tP fusion protein delivered Livin-siRNA could efficiently inhibit tumor growth in vivo.

Figure 5.

The scFv-tP fusion protein delivered Livin siRNAs efficiently inhibited MM tumor growth in vivo. (a) A volume of 200 µL of PBS (Black group), anti-MM scFv-tP-siRNA-NC (Mock group), or anti-MM scFv-tP-Livin-siRNA (20 µg siRNA) was injected into LiBr cell subcutaneous xenograft nude mice through the tail vein for every 2 days. A reduced tumor size in the scFv-tP-siRNA group was observed compared to the Black and Mock groups after 5 weeks of treatment. (b) The tumor growth was monitored by measuring tumor volume for every 2 days. (c) H&E stain of the pathological sections showed that tumor cell mass was significantly reduced in LiBr xenograft tissues of the scFv-tP-siRNA group compared to the Black and Mock groups. (d) RT-qPCR and (e) Western blot showed the Livin expression in tumor tissue of the indicated groups. (f) Western blot showed the expression of procaspase-3 and activated caspase-3 in tumor tissue of the indicated groups. The values were shown as the mean ± SD (*p < 0.05).

Discussion

Despite increasing advances in the management of human melanoma of the skin, there remain many challenges to be overcome in the treatment of this disease, such as preventing the switch of human melanoma phenotype from non-metastatic to highly metastatic, and the problem of resistance to current treatments. Therefore, it needs further exploration to develop more effective therapies for this disease.

RNAi is found to be a powerful tool to silence disease-causing genes and is a promising strategy for cancer therapy.^22–24 This gene therapy has opened a new window for MM treatment. However, the key barrier in therapeutic application is how to specifically and efficiently transport siRNA to MM cell silencing specific gene in vivo. To date, researchers have designed several strategies for targeted delivery of siRNA to specific cell populations, such as using of specialized liposomes,^25–27 viral vectors,²⁸ and single-chain antibody–protamine fusion protein,^29–33 among which, single-chain antibody–protamine fusion protein delivery has the greatest potential for its observed efficiency of delivering siRNA to specific target cells and inducing the desired biological effect on those cells.³⁴ The single-chain antibody–protamine fusion protein can recognize and target cell membrane receptors by a Fab fragment, and has a strong nucleic acid binding ability by a fusing nucleic acid binding domain—protamine, being able to deliver siRNA to specific cells and silence specific genes efficiently.

Many researchers have reported that Livin/ML-IAP is selectively overexpressed in most human cancers, including melanoma.^15–17,35 It has become clear that the aberrant overexpression of Livin contributes to the pathogenesis of MM, and Livin is a promising therapeutic target for human cancer treatment. In our previous study, we found that silencing Livin by siRNA could significantly knockdown the expression of Livin, induce apoptosis, and inhibit proliferation of MM LiBr cells, revealing that Livin could serve as a potential molecular target by siRNA for gene therapy of MM in vitro, and deserving further investigation in vivo.²⁰ Therefore, in this study, we intend to design a protamine–single-chain antibody fusion protein to deliver siRNA that specifically knockdown Livin gene expression and evaluate the effectiveness of this strategy for gene therapy of MM both in vitro and in vivo.

In this study, we designed an anti-MM scFv-tP fusion protein, which could combine with nucleic acid by protamine and directly target appropriate cells by single-chain antibody against ganglioside in MM. The purified s-tP fusion protein appeared to exhibit ability to carry the siRNA confirmed by the EMSA experiment. The flow cytometry and confocal microscopy results indicated that this protein could directly target and bind to the LiBr cell surface tightly and specifically. Further experiments revealed that the anti-MM scFv-tP fusion protein could specifically deliver siRNA into MM LiBr cells. In addition, results from small animals living image detector showed that the anti-MM scFv-tP-siRNA-Cy5 could be specifically enriched in tumor tissues in MM-burdened nude mouse. These results indicated that it was an effective way to specifically deliver siRNA to MM LiBr cells by single-chain antibody fusion protein both in vitro and in vivo.

Moreover, we demonstrated that the anti-MM scFv-tP-Livin-siRNA targeting to the site 790–808 of Livin mRNA could effectively silence Livin expression at both mRNA and protein levels in MM LiBr cells. Then, Annexin V-FITC/PI analysis indicated that the apoptosis index, early and late apoptotic rate was significantly increased in cells with silencing Livin. MTT assay showed that the proliferation of cells with silencing Livin was remarkably inhibited. Next, inhibited tumor growth, downregulated Livin expression, and increased apoptosis were observed in vivo study.

Our data showed that single-chain antibody–mediated targeted delivery of Livin siRNA could inhibit human MM LiBr cell proliferation and tumor growth in vitro and in vivo. However, there are some problems yet needed to be resolved before its final clinical application. Although siRNA can be successfully delivered and remain functioning, the mechanism of how siRNA leaves the endosomes and enter the cytoplasm remains unclear, and further research is required to define the exact transport pathway.³¹ And it also needs to be further investigated whether this strategy affects other cancer biological processes besides apoptosis and proliferation.

In conclusion, we demonstrated that siRNA could be specifically delivered into LiBr cells by the fusion protein, and efficient knockdown of Livin was observed. Further experiments investigated and confirmed the effect of targeted delivery of Livin siRNA on apoptosis and proliferation of LiBr cells both in vitro and in vivo. Altogether, our findings describe a feasible approach to transport targeting siRNA to MM cells in vivo, revealing single-chain antibody–mediated targeted silencing Livin as a new therapeutic strategy for gene therapy of MM.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by National Natural Science Foundation of China (Grants: 30960349 to H.W.).

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Single-chain antibody–delivered Livin siRNA inhibits human malignant melanoma growth in vitro and in vivo

Abstract

Keywords

Introduction

Materials and methods

Construction, expression, and purification of the anti-MM scFv-tP fusion protein

Electrophoretic mobility shift assay

Cell lines and cell culture

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay

Flow cytometry

Immunofluorescence and immunohistochemistry

siRNA design and preparation

Real-time quantitative PCR assay

Western blotting assay

Small animals living image detection and in vivo treatment with antibody–siRNA

Statistical analysis

Results

The anti-MM scFv-tP fusion protein possessed siRNA binding ability and could directly target MM LiBr cells

The scFv-tP fusion protein specifically delivered siRNA into MM LiBr cells in vitro and in vivo

The scFv-tP fusion protein could successfully deliver siRNAs targeting Livin and efficiently cause gene silencing in MM LiBr cells

The scFv-tP fusion protein delivered Livin siRNA efficiently induced cell apoptosis and inhibited the growth of LiBr cells in vitro

The scFv-tP fusion protein delivered Livin siRNAs efficiently inhibited MM tumor growth in vivo

Discussion

Footnotes

Declaration of conflicting interests

Funding

References