Effects on the STAT3-shRNA in Non-Small-Cell Lung Cancer Therapy:Design, Induction of Apoptosis, and Conjugation with Chitosan-Based Gene Vectors

ZHANG Wangwang, ZHANG Yan, JIANG Zhiwen, SUN Le, WANG Litong, GU Zhiyang, LI Wenya, GUO Lili, CHEN Xiaotong, ZHANG Haibin, HAN Baoqin, and CHANG Jing, *

Effects on the STAT3-shRNA in Non-Small-Cell Lung Cancer Therapy:Design, Induction of Apoptosis, and Conjugation with Chitosan-Based Gene Vectors

ZHANG Wangwang1), #, ZHANG Yan1), #, JIANG Zhiwen1), 2), SUN Le1), WANG Litong1), GU Zhiyang1), LI Wenya1), GUO Lili1), CHEN Xiaotong1), ZHANG Haibin1), HAN Baoqin1), 2), and CHANG Jing1), 2), *

1),,266003,2),,266235,

STAT3 plays a particularly important role in several cancer-related signal transduction pathways. Silencing STAT3RNA interference or small molecule inhibitors induces the apoptosis of tumor cells, thereby inhibiting the growth of the tumors. In this study, short-hairpin RNA sequences targeting the STAT3 genes were designed, synthesized, and then connected to pGPU6/GFP/Neo plasmids as the shRNA-expression vectors. The expression of STAT3-shRNA was analyzed by real-time PCR, western blotting, and cell apoptosis assay to study the growth and apoptosis of the cells. Then, the effect of STAT3 knockdown on the NCI-H1650 cells was studied in a tumor mouse model. The results revealed that, after antransfection, the proliferation of NCI-H1650 cells was inhibited, and the cells were induced to apoptosis. The mRNA and protein expression levels of STAT3 were downregulated in the STAT3-shRNA group., the tumor mass and volume in the STAT3-shRNA group were significantly lower than in the other two groups. Both theandresults demonstrated a long-period inhibiting effect on NSCLC, especially, when the tumor inhibition rate could reach 50% in the STAT3-shRNA group, which is an exciting outcome. Moreover, the study of the conjugation of STAT3-shRNA and chitosan-based vectors revealed that they could be combined steadily with good cytocompatibility and transfection efficiency. These results together provide convincing evidence for the application of STAT3-shRNA used in the treatment of non-small lung cancer, which could be a promoting prospect for the development of gene therapy.

signal transducer and activator of transcription 3; short-hairpin RNA; target downregulation; chitosan-based vectors; gene therapy

1 Introduction

The overall 5-year survival rate of all lung cancer patients is approximately 14%, and most of them are diagnosed with non-small-cell lung cancer (NSCLC) (Steelman., 2004; Howard., 2005; Siegel., 2017).However, the methods of treatment are limited to surgery, radiotherapy, and chemotherapy (Sundar, 2014; Kot- mak??., 2017; Russo., 2017). Although chemotherapy has been reported to increase a patient’s lifespan, it kills healthy cells in the patient’s body, subsequently creating additional complications for the patients. Unfor- tunately, >70% of these patients are diagnosed with unresectable advanced tumors, and their immune functions are greatly reduced due to radiotherapy (Li., 2018).Since the metastasis of cancer cells has not been specifical- ly studied, the prevention and treatment of NSCLC is becoming increasingly difficult and needs to be urgently re- solved (Lin., 2017; Xi., 2017; Kim., 2018).

In order to improve the treatment effect and avoid the resultant side-effects, gene therapy has been developed to target the transfers or restrictions in cancer cells (Uch., 2003), which can introduce hereditary materials into cells to activate a normal function or correct abnormal function by a virus or a non-virus vector. Thus, re- cognition molecules play a key role in cancer progression and promise to provide new therapeutic targets and reasons for lung cancer treatment (Grisch-Chan., 2017; Yu., 2018). To date, several NSCLC treatments using gene therapy have been studied. For example, the hsa- miR-124a-targeting USP14 has been shown to enhance NSCLC cells’ sensitivity to Gefitinib drug, and the miR- 9-5p has been reported to promote cell growth and metas- tasis in NSCLC by repressing TGFβR2 (Han., 2015).

RNA interference (RNAi) has been demonstrated to be an influential tool for downregulating genes and re-sensitizing drug-resistant cancer cells. According to a past study on RNAi, more than 20 different drugs known as small- interfering RNAs (siRNAs) or short-hairpin RNAs (shRNAs)have been studied (Burnett., 2011; Ni., 2011). The mechanism of RNAi is the use of powerful polymerase III promoters to induce the expression of stem-loop shRNAs, and the dicer can make it into RNAi triggers (Brummelkamp., 2002; Paddison., 2002; Adams., 2017). shRNAs refers to RNA sequences with a hairpin loop structure that can overcome the shortcomings of the siRNA expression and can exert a relatively stronger effect of gene silencing than siRNA. This expression has excellent continuity and can last for several months. It is more suitable for gene function research and downregulation of the target genes underconditions (Aagaard and Rossi, 2007; Rao., 2009; Zhao., 2018).

STAT3, a member of the signal transducer and activator of the transcription family, plays a critical role in a variety of cancer cells. STAT3 activation is conducive to induce the proliferation of tumor cells, thereby deteriorating cancer (Spitzner., 2014; Xu and Lu, 2014). The activation of STAT3 can also affect several other ma- lignancies, such as lymphomas, ovarian cancer, prostate cancer, and others (Scott and Gandhi, 2015). Inhibiting the STAT3 expression by RNAi or small molecule inhibitors can influence the proliferation, invasion, and apoptosis of tumor cells (Lin., 2015; Cho., 2016). The study in NSCLC determined that the downregulation of the STAT3 expression can inhibit tumor growth, revealing that the STAT3 may be a potential target for the treatment of NSCLC. Based on the understanding of thegene, RNAi can target the gene and inhibit the expression of genes through interfering signaling pathways related to the tumor, which in turn inhibit the metastasis of the tumor and leading to satisfying treatment of the tumor disease (Klink., 2012; Sun., 2013).

The gene vectors are important vehicles to help shRNA enter the cells. It is well known that liposome and PEI 25k possess strong cytotoxicity (Feng., 2014), which can affect the normal physiological activities of cells in several ways, such as by participating in the regulation of the protein kinase C (PKC) pathway (Movassaghian., 2013), inhibiting the activity of ATPase (Jiang and Aliasger, 2012) and interacting with the mitochondrial membrane (Pinnapireddy., 2017). Therefore, it is necessary to select a safe and effective gene carrier. Chitosan (CTS) is a linear copolymer of β-1,4 linked N-acetyl-D- glucosamine (GlcNAc) and D-glucosamine (GlcN), and it is the only polysaccharide that carries positive charges (Hattori and Ishihara, 2015). Several CTS-based gene vec-tors have been reported to exhibit good biocompatibility, good biodegradability, and low toxicity (Oliveira and Silva, 2015). Herein, a new typeof CTS-based gene vector called vitamin E succinate-CTS-histidine (VCH) was fabricated to assists shRNAs when entering the cells.

As depicted in Scheme 1, specific methods for RNAi were studied to downregulate the STAT3 expressionandin this study. The targeting gene sequences were synthesized, and the shRNA could enter the cells and associate with the nuclear region to influence the mRNA. Various indicators on the evaluation effect were explored to study the influence of shRNA on the apoptosis of lung cancer cells. Both the results ofandexperiments demonstrated a long-period inhibiting effect on NSCLC, especially the tumor inhibition rate could reach 50% in the STAT3-shRNA groupundercondition. The conjugation of STAT3-shRNA and CTS- based vectors were studied, and the results revealed that they could be combined steadily to demonstrate good cy- tocompatibility and transfection efficiency. The results can provide some experimental data for use in the treatment of lung cancer by gene therapy as well as provide evident ideas for the future study of different signal transduction pathways in an organism and their impact on cell proliferation and migration.

2 Materials and Methods

2.1 Materials

CTS and the vitamin E succinate-CTS-histidine (VCH) were prepared and provided by our laboratory. Lipofecta- mine 2000 was purchased from Invitrogen. pGPU6/GFP/ Neo was purchased from Gene Pharma Co., Ltd. (Shanghai,China). PEI 25k, Hoechst 33258, and 3-(4,5-dime-thyl-thia- zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were pur- chased from Sigma-Aldrich (USA). NCI-H1650 cells and A549 cells were cultured in RPMI 1640 medium supplemented with 10% FBS (Gibco?) in a humidified incubator at 37℃with 5% CO2. Goat anti-rabbit 1gG-HRP was purchased from Abcam (Cambridge, UK). Goat anti-mouse 1gG-HRP was purchased from Key GEN (Key GEN BioTECH, China). All other chemicals and reagents used in this study were of the highest commercial grade available.

2.2 shRNA-Mediated Downregulation of STAT3

STAT3-targeting and negative-control shRNAs were sourced from Gene Pharm Technologies (Shanghai, China). The sequences of STAT3-targeting and negative-control shRNAs are shown in Table 1. Stable culture ofDH5α was transformed with the plasmids (pGPU6/GFP/ Neo STAT3-shRNA) that encoded shRNA and targeted the mRNA of STAT3, and the plasmids (pGPU6/GFP/Neo scramble-shRNA) were obtained using the same method. H650 cells were transfected with STAT3 shRNA or scramble-shRNA at 85% confluence using Lipofectamine 2000. After 24h of incubation, the cells were checked for gene expression. The H1650 cells were transfected with STAT3 shRNA or scramble-shRNA and then selected with puro- mycin to form stable cell lines.

Table 1 STAT3-targeting and negative control shRNAs

2.3 Cell Transfection in vitro and the Effect on the Induction of Apoptosis

2.3.1 Cell transfection

The H1650 cells were plated in 6-well plates (1×105cellsperwell) and transfected with STAT3-shRNA or scramble-shRNA using Lipofectamine 2000 when the cultured H1650 cells were up to 85% confluence. The H1650 cells of the control group were incubated without transfection. After 24, 48, and 72h of incubation, the fluorescence of the three groups was assessed by confocal laser scanning microscopy (Nikon, Japan).

2.3.2 Cell proliferation assay

The cell proliferation was evaluated by the MTT assay, and the NCI-H1650 cells were seeded in 96-well culture plates (4×103cellsperwell) and allowed to attach for 24h. Next, the cells were transfected with STAT3-shRNA or scramble-shRNA. The cells were then cultured for an ad- ditional 24h or 48h after the transfection. These cells were incubated with MTT for 4h and then treated with dime- thyl sulfoxide. The absorbance of each well was analyzed at 492nm on the Multiskan Go 151 Microplate Scanning Spectrophotometer (Thermo Fisher Scientific, INC, USA). The cell relative viability (%) was finally calculated.

2.3.3 Real-time PCR

The H1650 cells were plated in 6-well plates and transfected with STAT3-shRNA or scramble-shRNA as descri- bed earlier. After incubation of 24 and 48h, the total RNA of the cells was extracted with Trizol (Takara, Dalian,China) following the manufacturer’s protocol. The reverse transcription was achieved using the Prime Script RT reagent kit (Takara). The primers used for STAT3 and the β-actin gene were as follows: STAT3 forward, 5’-GCTA CAGCAGCTTGACACACG-3’ and STAT3 reverse 5’-G TGGCATGTGATTCTTTGCTGGC-3’; β-actin forward, 5’- AGAAAATCTGGCACCACACC-3’ and β-actin reverse, 5’-TAGCACAGCCTGGATAGCAA-3’. β-actin was used as an endogenous control. The PCR setcycle conditions were 98℃ for 5min, 98℃ for 10s, 50℃ for 15s, 72℃ for30s, after which the relative STAT3 expression was esti- mated by qRT-PCR (Roche,Germany) and obtained based on the 2?ΔΔCtmethod (Schmittgen and Livak, 2008).

2.3.4 Western blotting

The cells transfected with STAT3-shRNA or scramble- shRNAs were cultured for an additional 48h. After the transfection, the proteins in the three groups were extracted with RIPA buffer. The different samples were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then, the membranes were incubated with different antibodies. The antibodies used were as fol- lows: rabbit anti-human STAT3 (1:600), mouse anti-human β-actin (1:200), goat anti-rabbit 1gG-HRP (1:1000), and goat anti-mouse 1gG-HRP (1:1000). At last, the protein bands of the three groups were visualized using DBA (Lunavat., 2016), and the bands were analyzed by the Image J software.

2.3.5 Cell apoptosis assay

The apoptosis of the H1650 cells was checked at 48h after treatment. First, the cells were added with Hoechst 33258 and left at room temperature for 10min in the dark and then photographed by laser confocal scanning microscope (Nikon). The images were randomly selected in each sample and reported as an average.

2.3.6 Flow cytometry

The cells were obtained using trypsin without EDTA to perform a single cell suspension. Then, the harvested cellswere treated by Annexin V-FITC/propidium iodide apoptosis detection kit (Key GEN Biotech, China) by flow cytometry (BD Biosciences). The cell apoptosis ratio was analyzed using the Cell Quest software (BD Biosciences). For the three groups of samples, the mean fluorescence intensity of 10000 cells was determined using the software FlowJo.

2.4 Anti-Tumor Experiment in vivo and Histopathological Examination

2.4.1 Anti-tumor experiment

We purchased 20 BALB/c nude female mice (weight 18–22g) from the Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The experimental mice were fed in a sterile laminar flow at the Qingdao University Animal Experimental Center. A total of 18 nude mice were randomly divided into three groups. Stable STAT3 knockdown or control H1650 cells were collected and su- spended with RPMI-1640 medium (without FBS). Then, the cells were injected into the flanks of BALB/c nude mice (1×107cellspermouse). The size of the tumor was measured every 3d by using a caliper, and the volume of tumors was calculated according to the following formula:=(length×width2)/2. After 28d, the tumor tissues were removed and weighed.

2.4.2 Histopathological examination of the tumor samples

The mice tumors were removed and weighed. The tumor tissues of the three groups were maintained in 4% paraformaldehyde solution. We excised the tumor tissues as a 5-μm section and performed HE staining to assess the tumor histology. The sections were then subjected to pho- tomicroscope observations. All photomicrographs for the three groups were taken with digital camera software.

2.5 The Conjugation of STAT3-shRNA and CTS-Based Vectors

2.5.1 Morphologies and agarose gel retardation assay

The morphology of VCH was tested using TEM (JEOL JEM-100CX, Japan) and the zeta potential was immediately obtained by using the zeta sizer Nano ZS (Malvern, UK). The VCH and STAT3-shRNA plasmids were mixed according to different N/P ratios, and the gel retardation test was performed to determine the DNA binding ability of VCH. The complexes were mixed softly and then placedat 37℃ for 30min. Then, 10μL of different complexes pre- pared were electrophoresed on 1% (w/v) agarose gel at 80V for 70min.

2.5.2 The cytotoxicity of the VCH/pDNA complexes

The cytotoxicity of VCH/pDNA complexes was studied using the MTT assay. The L929 cells were seeded in 96-well plates at a density of 2×104cellscm?2and cultured in a 5% CO2incubator for 24h. The culture medium was discarded and added in DMEM (without FBS) at different concentrations of CTS-based materials and materials/pDNAcomplexes (N/P=55). After culturing for 24h and 48h, MTT assay was performed to quantify the viability of the cells.

2.5.3transfection of VCH/pDNA

The gene transfection efficiency of the VCH was eva- luatedwith A549 cells. The VCH/pDNA complexes and CTS/pDNA complexes were obtained at the N/P ratios of 45, 55, and 65, respectively. The A549 cells were added to 24-well plates (8×104cellsperwell) and then cultured under an atmosphere of 5% CO2for 24h. The VCH/ pDNA complexes containing 0.8μg pDNA were added to 24-well plates in DMEM without FBS. After 4h of cultivation, the complexes were removed and replaced with fresh DMEM, after which the well plates were incubated at 37℃ for another 48h. The results were obtained using a fluorescence microscope (Leica, Germany) and by using the flow cytometry method (FACE Vantage, BD, USA). Next, the relative STAT3 expression was assayed by qRT- PCR (Roche) using the method described in Section 2.3.3. The transfected HEK-293 cells with CTS and the transfected HEK-293 cells with PEI 25k served as negative and positive controls, respectively.

2.6 Statistical Analyses

The student’s t-test was used to determine whether the difference observed between the two groups was significant. All data were presented as the mean±standard deviation (SD) from at least 3 independent experiments.P <0.05 was considered to be statistically significant.

3 Results

3.1 Cell Transfection

STAT3-targeting and negative-control shRNAs are successfully obtained. Subsequently, the H1650 cells were transfected with STAT3-shRNA and scramble-shRNA plasmids. The expression of plasmids in the cells was de- tected by confocal laser scanning microscopy (CLSM) at 24 and 48h, respectively. pGPU6/GFP/Neo possessed the gene of green fluorescent protein, such that the transfectedcells could be observed with green fluorescence. As shown in Fig.1, intense green fluorescence was detected in both the scramble-shRNA group and the STAT3-shRNA group. No significant difference was noted concerning morphological change or reduction in fluorescence between 24h and 48h of incubation in both the groups. However, the cells in the STAT3-shRNA group showed much less green fluorescence than the negative-control cells, indicating that the expression of STAT3-shRNA could affect the state of cells.

Fig.1 Laser confocal scanning microscopy of H1650cells (scale bar: 100μm). (a), scramble-shRNA group; (b), STAT3- shRNA group. A, bright field; B, fluorescence field; C, merged field; 1, 24h; 2, 48h.

3.2 Silencing of STAT3 Suppresses Cell Proliferation in H1650 Cells

As shown in Fig.2(a), the cells in the control group exhibited a proper morphology without any apoptosis appearance. No significant change was noticed in the scram- ble-shRNA group and the control group. However, after 24h of incubation, evident apoptosis could be noted in the STAT3-shRNA group, such as cell shrinkage and the dis- appearance of ligation between the cells. After 72h of in- cubation, the apoptosis in the STAT3-shRNA group became more evident. When compared with that in the other two groups, the number of cell apoptosis in the STAT3- shRNA group was increased, and the basic cell morphology had disappeared.

Fig.2 (a), microscope photographs of H1650 (100×). A, control group; B, scramble-shRNA group; C, STAT3-sh- RNA group; 1, 24h; 2, 48h; 3, 72h. (b), the relative growth rate of H1650. n=6, *P<0.05, **P<0.01, when compared with the control and the scramble-shRNA groups, respectively.

As shown in Fig.2(b), the relative growth rate of the control group was >100%. The relative growth rate showed a slight decrease for the scramble-shRNA group than for the control group, possibly due to the toxicity of Lipofectamine 2000. However, the relative growth rate of the STAT3-shRNA group was reduced at 24h to reach appro- ximately 30% at 72h. These results showed that STAT3- shRNA could significantly inhibit the proliferation of NCI- H1650 cells.

3.3 The mRNA and Protein Expression Levels of STAT3

The qRT-PCR resultsin Fig.3(a) demonstrate that the gene expression of the STAT3 in the STAT3-shRNA group was significantly less than that of the control group and the scramble-shRNA group. The control group and the scramble-shRNA group showed no significant difference in their results. As shown in Fig.3(b), the results of western blotting demonstrated that the levels of STAT3 in the STAT3-shRNA group weresignificantly lower than inthe other two groups. These results suggested that STAT3- shRNA could cause STAT3 downregulation.

Fig.3 (a), the detection of STAT3 mRNA levels via quantitative real-time PCR (**P<0.01 when compared with the control group and the scramble-shRNA group); (b), detecting the cell STAT3 levels by western blotting.

3.4 Downregulation of STAT3 Induces the Apoptosis of H1650 Cells

After 48h of incubation, the cells in the three groups were stained with a Hoechst 33258 dye solution. The fluorescence distribution of the cells in the control group and thescramble-shRNA group were relatively uniform (Fig.4(a)).The cells were found to be intact, with no apparent cell apop- tosis and a stable normal form. However, the STAT3- shRNA group exhibited a large number of strong nuclear fluorescence, nuclear fragmentation, marginalization, and other typical morphological changes indicative of apoptosis.

Fig.4 (a), targeting STAT3 promotes cellular apoptosis in H1650 cells. A, control group; B, scramble-shRNA group; C,STAT3-shRNA group; 1, bright field; 2, fluorescence field. (b), the apoptosis rate of cells in every group; (c), flow cytometry pattern of H1650 cells.

The cells of the three groups were then assessed by flowcytometry at 24 and 48h, respectively (results in Figs.4(b),(c)). The apoptosis rates of the STAT3-shRNA group cells (33% at 24h and 39% at 48h) were higher than those of the control group and scramble-shRNA group. The results cumulatively suggest that STAT3-shRNA can promote the apoptosis of H1650 cells.

3.5 Downregulation of STAT3 Impairs Tumorigenesis of H1650 Cells in Nude Mice

Finally, the effects of STAT3 downregulation underconditions were determined. The H1650 cells transfected with STAT3-shRNA or scramble-shRNA were injected into nude mice. Fig.5(a) demonstrates that the body- weight of each mouse was the same, indicating that the expression of STAT3-shRNA had no adverse effect on the bodyweight of nude mice. As shown in Fig.5(b), the tumor formation time was approximately 7d in the scramble- shRNA group and control group, but 12d in the STAT3- shRNA group. The daytime in the treatment group was significantly later than in the other two groups. Meanwhile, the trend of tumor growth in this group was relatively slow. The tumor volume (approximately 600mm3) before the final sacrifice was smaller than that of the control and scramble-shRNA groups. After sacrifice, the tumors in the nude mice were detached and weighed. As shown in Figs.5(c–d), the tumor weight of the STAT3- shRNA group was lower than that of the other two groups, and that the graft tumors from the STAT3-shRNA group grew significantly slowly when compared with those in the scramble-shRNA group and control group. The results cumulatively revealed that the expression of STAT3-sh- RNA could significantly inhibit the growth of lung cancer H1650 cells in nude mice and that the STAT3-shRNA group was statistically significant when compared with the other two groups (<0.05).

Fig.5 (a), body weight changes in the three groups of nude mice; (b), the growth of xenograft tumor in the three groups; (c), the comparison of average tumor weight in the three groups (**P<0.01 when compared with the control and scramble-shRNA groups); (d), macroscopic photographs of the tumors in the three groups; (e), HE staining optical micrographs of tumor tissues (scale bar: 10mm). A, control group, B, scramble-shRNA group; C, STAT3-shRNA group.

The tumors’ pathological changes in the three groups were further analyzed. Fig.5(e) displays the HE images in the three groups. The tumor cells of the control and scram- ble-shRNA groups were deeply stained and tightly arranged. However, the tumor cells of the STAT3-shRNA group were arranged sparsely, with local necrosis areas, and the nucleus was broken and dissolved.

3.6 The Morphology, Zeta Potential, and pDNA Combined Ability of VCH

Fig.6(a) presents the morphology of VCH, indicating that the shape of the particles was a regular sphere and the size was approximately 150nm. It is well known that the particles with the sizes 50 to a few 100nm are most likely to enter into the cells. The zeta potential of VCH is displayed in Fig.6(b), with the value +38.6mV. The positive charge makes it easier for VCH to bind to pDNA. The ability of VCH and CTS binding with the plasmids are displayed in Figs.6(c) and (d), respectively. These results together demonstrate that the VCH material could be fully bounded with the plasmids at the N/P ratio of 2 and that there was no significant difference between VCH and CTS.

3.7 The Cytotoxicity of VCH/pDNA Complexes

The cytotoxicity of VCH and VCH/pDNA complexes in the L929 cells was studied by MTT assay. As shown in Fig.7, when the concentration of the material was in the range of 10–100μgmL?1, the relative growth rate of L929 cells in each group was >85% at 24h, almost reaching > 100% in 48h. This result indicated that CTS-based gene vectors had good cell compatibility and showed better cell compatibility with the commercial Lipofectamine 2000 and PEI 25k. These results provide a basis for the deve- lopment of new and safe CTS-based non-viral vectors that can be used to develop different types of drug-delivery carriers.

Fig.6 (a), the morphology and size distribution of VCH; (b), the zeta potential of VCH; (c), agarose gel electrophoresis of VCH/pDNA with different N/P values; (d), agarose gel electrophoresis of CTS/pDNA with different N/P values.

Fig.7 (a), microscopic photographs of L929 cells after 24h of transfection; (b), microscopic photographs of L929 cells after 48h of transfection; (c), the relative growth rate of VCH and CTS groups at various concentrations in the L929 cells. The data are presented as mean±standard deviation (n=6).

3.8 In vitro Transfection of STAT3-shRNA Plasmids Conjugated with VCH

The fluorescence images of A549 cells transfected with STAT3-shRNA plasmids for 48h are exhibited in Fig.8(a). The VCH group showed obvious green fluorescence. The transfection efficiency of VCH and CTS increased with the N/P ratios, indicating the most obvious fluorescence with 55 N/P ratios. The cells of the four groups were detected by flow cytometry at 55 N/P ratios, and the results are shown in Fig.8(b). The fluorescence intensity of the VCH group was significantly higher than that of the CTS group, and it achieved a similar outcome to that of the PEI 25k group, indicating the satisfactory gene transfection efficiency of the VCH vector. Then, the relative STAT3 expression of each group with the 55 N/P ratios was stu- died by the qRT-PCR method. The results in Fig.8(c) con- firmed that the gene expression of STAT3 in the VCH group was significantly lower than that of the control group and the CTS group, reaching similar results to those of the PEI 25k group. All the results of thetransfection experiments revealed the high efficiency of STAT3-shRNA pDNA conjugated with VCH vector in gene transfection, thus providing a broad prospect for the application of STAT3-shRNA and VCH in gene therapy.

4 Discussion

It is already known that STAT3 plays an essential role in tumor progression and development and that STAT3 is overexpressed in NSCLC tissues. It has also been accepted that the inhibition of STAT3 signaling accounts for the suppression of tumor cell migration, invasion, and proliferation (Waldmann, 2017). In addition, as RNA in- terference can degrade specific mRNAs, it is used as a cancer molecular diagnostic and therapeutic method. The methods of RNA interference included chemically synthesized siRNAs and shRNA expressed by vectors. Although both siRNA and shRNA can silence specific m- RNAs, the mechanism is not precisely the same. Regardless of persistence or effectiveness, shRNA was superior to siRNA. It was also found that when using RNA interference treatment of viral hepatitis C, the inhibition of lu- ciferase expression could be up to 92.8% by shRNA (Mc- Caffrey., 2002). Past studies have shown that <1% ofthe siRNA double-stranded molecules were retained within the cells for 48h after the transfection. However, shRNA could be synthesized in the cells continuously (McAnuff., 2007). Furthermore, it has been shown that the knockdown of STAT3 with shRNA could potentially re- duce tumor propagation and migration, which is consistent with the results of the present study (Spitzner., 2012).

Fig.8 (a), fluorescence images of the VCH/pDNA complexes at different N/P ratios of 45, 55, and 65; (b), flow cytometry results of fluorescence intensity of VCH/pDNA at N/P ratios of 55; (c), flow cytometry results of fluorescence intensity of VCH/pDNA at N/P ratios of 55; (d), the detection of STAT3 mRNA levels via quantitative real-time PCR (*P<0.05, **P<0.01). CTS, chitosan; VCH, vitamin E succinate-chitosan-histidine; PEI, polyethyleneimine; linear, MW 25000.

Supporting this result, the knockdown of STAT3 in this study reduced the proliferation of H1650 cells by RNAi. Moreover, RNA interference was noted to degrade speci- fic mRNAs, making it a cancer molecular diagnostic and therapeutic method. First, the recombinant pGPU6/GFP/ Neo STAT3-shRNA plasmids were successfully constru- cted, which were dissected with BamH I and Pst I enzymes and further screened for positive clones. Then, the recom- binant plasmid was preliminarily identified by agarose electrophoresis, and, finally, further confirmed by DNA sequencing. The results showed that the designed inserted vector fragment was consistent with the sequencing results. LipofectamineTM2000 was used to transfect recombinant plasmids into H1650 cells. Laser confocal scanning microscope images indicated that the H1650 cells had a large amount of green fluorescence expression after trans- fection of the experimental group and the negative plasmid group, signifying that the transfection effect of the two groups was satisfied.

Apoptosis, a phenomenon of programmed cell death, plays a key role in tumor control and therapy. Past studies have shown that STAT3 does not directly cause carcinogenesis and that STAT3 can be activated to enter the nucleus and combine with the DNA phase, thereby affecting the transcription and expression of downstream genes (Dorritie., 2014). The study of the relative growth rate of H1650 cells revealed that the cell proliferation observed in control and scramble-shRNA groups had a significant difference when compared with that in the STAT3-shRNA group.The apoptosis ratio increased in the STAT3-shRNA group with an increase of the incubation time, indicating the long-time inhibition effect of STAT3-shRNA. Hoechst33258 fluorescence staining and Annexin V-FITC/PI fluorescent double-staining kit were used to detect the apoptosis of cells, showing the evident apoptosis phenomenon in the STAT3-shRNA group. The results of cell observation by laser confocal microscopy showed that the expression of STAT3 was silenced by the transfection of STAT3-shRNA plasmid, such that a large number of cells with strong nuclear fluorescence and other obvious apoptosis phenomena could be observed. After 48h, the apoptosis rate of H1650 cells in the STAT3- shRNA group was 39%. The protein and RNA samples extracted from H1650 cells were treated for 48h with STAT3-shRNA and then subjected to western blotting and qRT-PCR. The results of gene silencingfurther suggested that the constructed STAT3-shRNA plasmids could significantly downregulate the expression of STAT3. In addition, STAT3-shRNA significantly downregulated the mRNA and protein expression of STAT3qRT-PCR and western blotting., all results of the STAT3-shRNA group indicated a significant downregulation of cell viability and proliferation ability, while cell apoptosis was significantly upregulated. These results are also related to the JAK/STAT3 signal transduction pathway to target gene transcription.

Subsequently, the establishment of a tumor model of verified gene silencingwas launched. Makowiecki(2014) suggested the siRNA approach to persona- lized treatment of NSCLC. When compared to the report on the use of siRNA, only a select few studies on NSCLC have used the shRNA methods. In this study, the tumor weight in the STAT3-shRNA group was lower than in the other two groups, indicating that tumors in the STAT3- shRNA group had a less moderate proliferation activity. The growth of lung cancer H1650 cells in nude mice was further suppressed and the tumor inhibition rate could reach 50% in the STAT3-shRNA group,which was an exciting result. These results also demonstrated that STAT3-sh- RNA could achieve a good silencing effectand demonstrated a more satisfying anti-tumor outcome.

Finally, the STAT3-shRNA plasmids were conjugated with VCH materials, and the gene transfection efficiency of the composites was studied in further detail. The sizes of the VCH particles were approximately 150nm and the particles carried obvious positive charges. These 2 main properties of VCH could facilitate it to bind well with plasmids and assist the plasmids in entering into cells. The ability to combine with naked DNA was essential to use VCH as a gene vector, and the agarose gel retardation demonstrated that the VCH material could be bounded fully with the plasmids at the N/P ratio of 2. The VCH/ pDNA composites revealed good cytocompatibility; this feature could overcome the shortcomings of other gene vectors, such as Lipofectamine 2000 and PEI 25k, indicating severe cytotoxicity during the process of gene transfection. The transfection efficiency of VCH/pDNAwas studied by fluorescent observation, flow cytometry, and qRT-PCR methods. The cumulative results validated the consistent conclusions that the STAT3-shRNA pDNA conjugated with VCH had a high efficiency in gene transfection, which implies a promising future for the STAT3- shRNA application in gene therapy.

5 Conclusions

In conclusion, our experimental results demonstrated that pGPU6/GFP/Neo STAT3-shRNA could effectively downregulate STAT3 in H1650 cells and promote it to apoptosis, thereby inhibiting the proliferation of H1650 cells bothand. The CTS-based gene vector could facilitate theSTAT3-shRNA to enter into cells with successful transfection. These results suggest a therapeutic potential of STAT3 in the treatment of poor prognosis cases of NSCLC and lay a good foundation for further gene therapy for lung cancer treatment.

Acknowledgements

The authors wish to thank the National Natural Science Foundation of China (No. 51773188), the Natural Science Foundation of Shandong Province (No. ZR2017MC072), the National Key Research and Development Program (No. 2018YFC1105602), the Key Research and Development Program of Shandong Province (No. 2016YYSP018), the Second Maker Program of Marine Biomedical Research Institute of Qingdao (No. MGTD20170002M), the Scientific and Technological Innovation Project Financially Sup- ported by Qingdao National Laboratory for Marine Science and Technology (No. 2015ASKJ02).

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#The two authors contributed equally to the work.

. E-mail: jingchouc@163.com

July 30, 2020;

September 23, 2020;

November 4, 2020

? Ocean University of China, Science Press and Springer-Verlag GmbH Germany 2021

(Edited by Chen Wenwen)