Antitumor Effect of Membrane-type PBLs on Non-Small Cell Lung Cancer Cell of ICR Mice

This study aims at probing the inhibitory effect of transmembrane PBLs on non-small cell lung cancer (NSCLC) H446 cells and the potential application of PBLs on immune system of the experimental mice loaded with H446 cells. The changes of the gene expression of microRNA-25 and 223 in ICR mice with NSCLC were also investigated. Sixty ICR mice were randomly divided into experimental and control groups. The animal model was established via inoculation of NSCLC H446 cells at the hind thigh of mice. The plasmid PBLs was dissolved in saline solution and injected into the muscle of left thigh of the mice in experimental groups with different doses (0.1 mg, 0.2 mg and 0.3 mg per ICR mouse) using in situ injection method. After injection of PBLs solution, each three mice were killed at 12 h, 24 h, 36 h, and 48 h, respectively. The expression of microRNA-25 and 223 were detected by semi-quantitative reverse transcription-polymerase chain reaction. Tumor Necrosis Factor-γ (TNF-γ), Interleukelin-2 (IL-2) and Heat Shock Protein 70 (HSP70) in Bronchoalveolar Lavage Fluid (BALF) were detected by enzyme-linked immunosorbent assay. The expression of TNF-γ and IL-2 protein in lung tissue were detected by western blotting. The expression of microRNA-25 was up-regulated in the tissues and BALF with a dose- and time-dependent manner while microRNA-223 was down-regulated. The difference were statistically signicant comparing the control group (P<0.05). The TNF-γ and IL-2 levels in BALF of ICR mice in experimental group were increased comparing the control group with a dose-dependent manner (P<0.05). Synergistic effect between PBLs and HSP70 was also studied. It was found that the growth of tumor was signicantly suppressed after the transfection of PBLs. In the presence of PBLs, the proliferation of splenocytes and cytolysis in early phase of tumor development was signicantly enhanced. Thus, such anti-tumor effect was


Introduction
Lung cancer, as one of the common malignancies, can be divided into two categories: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) [1,2]. The latter is the most general pathological type of lung cancer. Basically, lung cancer is a common malignant tumor that in icts serious damage. In China, the morbidity rates of this disease are quite high. Lung cancer cells have been reported to have very strong invasion and migration abilities. The patients easily suffer relapse, as well as lung cancer cell migration and in ltration, which are life-threatening. The ve-year survival rate of lung-cancer patients is only 16.8%, and 50%-60% patients cannot be treated by surgery [3][4][5][6]. Moreover, most of the NSCLC patients are insensitive to radiotherapy and chemotherapy, it is urgent to develop anti-cancer drugs or methods with high e ciency and low toxic side effects based on sub-biological research.
MicroRNAs (miRNAs) are small, stable RNAs that critically modulate post-transcriptional gene regulation.
The miRNAs can be ubiquitously or variably expressed in different tissues and cell types and they are known to have cell type speci city [6]. At present, miRNAs have been reported as potential biomarkers for various types of disease. It was reported that miRNAs are present in human plasma in a remarkably stable form that is not prone to RNase degradation and established the measurement of tumor-derived miRNAs in serum or plasma [7]. These discoveries generated enormous enthusiasm for the potential use of miRNAs as biomarkers of neoplastic and non-neoplastic disease. Many studies have reported that plasma miR-25 may be a candidate biomarker for early detection and diagnosis of cancer [8][9][10]. The miR-223 promotes endotoxin-mediated in ammation in endothelial cells and it can be released from dendritic cells and are subsequently taken up by recipient dendritic cells [11]. In summary, miR-25 and 223 are involved in in ammation and considered as potential markers. However, the expression of miR-25 and 223 of NSCLC is still unknown. The aim of this study was to understand the changes of the gene expression of miR-155 and 127 in ICR mice with NSCLC.
PBLs were found to act as a ligand for reverse transmission of signals to affect tumor immunity correspondingly [12][13][14][15][16][17][18][19][20][21]. In order to investigate this new mechanism of PBLs molecules, NSCLC H446 cell strains of ICR mice were used to build a solid tumor model in thigh muscle to study the inhibitory effect of PBLs molecule on NSCLC solid tumor and the in uence on immune system of tumor bearing ICR mice.

PBLs expression and detection
Eighty ICR mice were taken to conduct the measurements. The plasmid PBLs was dissolved in saline solution and injected into the muscle of left thigh of the mice using in situ injection method. The mice were randomly divided into PBLs 0.1 mg, 0.2 mg, 0.3 mg groups and the control group with 20 mice in each group. After injection of PBLs solution, each three mice were killed after 12 h, 24 h, 36 h, and 48 h, respectively. RT-PCR method was applied to detect the expression of PBLs in the muscle tissue of ICR mice.
Tumor cell inoculation NSCLC H446 cells (1 × 10 6 /mL) were inoculated into the muscle of left thigh of mice (0.1 mL per mouse).
The mice were randomly divided into normal saline group (the blank control group), pcDNA group (the plasmid control group) and PBLs group (the treatment group) with 20 mice in each group. Plasmid was injected on alternate day after the second day of inoculation with each injection of 0.1 mL (1 mg/mL), the injection site was the position of inoculated tumor cells. These inoculated mice were dissected after two weeks, the tumor was taken and weighed to observe the growth of tumor.
Based on above experimental groups, HSP70 group (the treatment group) and PBLs/HSP70 group (the combined treatment group, injection was half for each plasmid) were added with 20 mice in each group, NSCLC H446 cells were inoculated with the same method and the same treatment was conduct for examination of synergistic effect of PBLs with HSP70.

Sample collection
The ICR mice were killed at 12 h, 24 h, 36 h, and 48 h after anesthesia (2% iso urane inhalation) employed. The fasting tissue and Bronchoalveolar Lavage Fluid (BALF) samples from the subjects were collected in containing vacationers, and transferred into a micro tube. The samples were centrifuged at

Polymerase chain reaction
The PCR conditions were used as follows: initial denaturation at 95 °C for 5 min followed by 40 cycles, annealing at 95 °C for 15 s, and extension at 60 °C for 20 s and nal extension at 72 °C for 40 s.

Gel electrophoresis
The PCR products of each interested gene were loaded into the same ethidium bromide-stained agarose gels (1%). A 1-kb DNA ladder molecular weight marker was run on each gel to con rm expected molecular weight of the ampli cation product. Stained gels were recorded and the band intensity was evaluated using the Tanon 1600 software. Band intensity was expressed as relative absorbance units. The sample RNA was determined and calculated to normalize for initial variations in sample concentration and as a control for reaction e ciency. Mean and standard deviation of all experiments performed were calculated after normalization.
The ELISA assay IL-2 and TNF-γ levels in BALF were measured by enzymelinked immunosorbent assay kit manufactured by Tiantan Biological Products co., Ltd (Beijing, China) according to manufacturer's instructions.

Western blot analysis
Lung tissues were harvested and homogenized using a homogenizer and tissue lysis/extraction reagent containing a protease inhibitor cocktail (Sigma-Aldrich). Protein concentrations were determined using a Bradford reagent (BioRad Laboratories, Inc.).

In vitro proliferation activity
Single cell was prepared under sterile conditions for determination of proliferation of the cells in vitro. The concentration of mice cell was adjusted to 1 × 10 6 /mL, suspended in culture medium containing 15% fetal calf serum followed by stained with CFSE. The solution was divided into two samples, one added NSCLC H446 antigen peptide and HSP70 protein complex. Both samples were transferred to a hole plate (100 µL per hole) at 37 o C in an incubator for 7 days. Flow cytometry was used to detect the cell proliferation index.

Statistical analysis
Statistical analyses were performed using the SPSS software version 23.0, and all variables were expressed as mean ± Standard Deviation (SD). Statistical signi cance between groups was analysed by the ANOVA when the variables between groups were normally distributed. A p value less than 0.05 was considered statistically signi cant.

Results
The expression of miR-25 in mice of induced lung cancer To gure out the change of miR-25 expression of NSCLC H446 lung cancer in vivo, the membrane-type PBLs were administered into ICR mice through injection using established methods. Littermate neonatal rats were used as controls. At 12 h, 24 h, 36 h, and 48 h after injection, lung tissues and BALF were evaluated to determine the expression of miR-25. As shown in Figs. 1 and 2, for BALF the miR-25 levels were signi cantly increased after PBLs stimulation in PBLs groups. These results suggested that the expression of miR-25 increased with extension of time after injection of PBLs.
The expression of miR-223 in mice of induced lung cancer As displayed in Fig. 3, as well as Fig. 4 into BALF-the miR-223 level of NSCLC H446 lung cancer were signi cantly decreased after PBLs stimulation in PBLs groups. Compared with the control group, miR-223 expression of lung tissue and BALF in the PBLs groups was signi cantly decreased with extension of time after injection of PBLs. There is statistical signi cance between the PBLs groups and the control group or among PBLs groups comparison (P < 0.05).
Pro-in ammatory cytokine production in mice of induced lung cancer In ammatory cytokines TNF-γ and IL-2 (Fig. 5) levels in the BALF increased with PBLs stimulation.
Induction of PBLs resulted in a signi cant increase in TNF-γ production compared with the control group, moreover, IL-2 signi cantly increase by PBLs induction in a dose-dependent manner, the difference was statistically signi cant (P < 0.05). Consistently, in ammatory cytokines TNF-γ and IL-2 ( Fig. 6) protein levels production in lung tissues were all increased in ICR mice. The more doses of PBLs, the higher TNFγ and IL-2 level, the difference was statistically signi cant (P < 0.05).

Histological examinations of lung tissue
We also performed the Hematoxylin and eosin stain to examine lung pathological changes. As shown in Figs. 7-10, PBLs induced lung injury was promoted in experiment groups, as manifested by increase in ltration of neutrophils, increased thickening of interstitial alveolar regions, and minimal structural damage compared with that seen in the control group.
Effects of PBLs on enhancement of splenocytes proliferation and cytotoxicity After inoculation for one week, the spleen cells were taken for proliferation experiment. Under the conditions of adding speci c antigenic peptide stimulation and without speci c antigenic peptide stimulation, ow cytometry was used to detect the mice splenocyte proliferation index of each experimental group. The results (see Fig. 11) showed that the PBLs group had signi cant difference (P < 0.05) comparing the saline control group and the pcDNA group with speci c antigenic peptide stimulation. Also, the mice splenocyte proliferation index of the PBLs group without speci c antigenic peptide stimulation did not have signi cant difference comparing the other two groups. The ratios of proliferation index obtained by adding speci c antigenic peptide stimulation and without speci c antigenic peptide stimulation for the three groups were 0.97, 0.99, and 1.62, respectively. This result indicates that the larger the ratio is, the higher the activity of spleen cell proliferation is under antigen stimulation. The killing activity experiment of spleen cells further con rmed the speci c killing activity of T cells. The speci c killing function of PBLs group was stronger than pcDNA group (P < 0.05) and saline control group (P < 0.01). The killing rate was 32%, 23%, and 14%, respectively. Two-color analysis of the expression of PBLs on mouse spleen B cells was shown in Fig. 11. ICR mice splenocytes were simultaneously stained with anti-mouse IgM[a] mAb DS-1 and puri ed anti-mouse PBLs mAb 1D3. The staining of primary antibody was detected with FITC-conjugated goat anti-mouse lg.
In vivo expression of HSP70 and PBLs in mice muscle tissues after 12 h, 24 h, 36 h, and 48 h were shown in Fig. 12. Western Blot used denaturing polyacrylamide gel electrophoresis (SDS-PAGE) for separation of protein mixture, the proteins were transferred to solid supports after separation and solid phase carrier absorb protein with non covalent interaction, which maintains the invariant properties of antigen protein or polypeptide onto membrane. Solid carrier protein or polypeptide behave as antigen and react with its corresponding antibody and enzyme labeled antibody to detect the expression of speci c proteins through chromogenic substrate.

Synergistic effect of PBLs with HSP70
So far, we have demonstrated that transfection of PBLs into tumor-bearing mice signi cantly inhibited the growth of tumor. Furthermore, the growth was further inhibited when PBLs and HSP70 were simultaneously transfected. After inoculation for two weeks, of the average weight of tumor was only 0.42 g, signi cantly lower than that of PBLs transfected mice (0.84 g) or HSP70 transfected mice (1.57 g) and normal saline group (1.61 g). The experimental group treated with combined PBLs and HSP70 has signi cant difference (P < 0.05), suggesting that PBLs and HSP70 have stronger synergistic antitumor effect.

Discussion
Previous studies demonstrate that PBLs has been used as the receptor molecule on the surface of T3 cells in the studies of membrane-type PBLs [5][6][7]. Generally, HSP70 is considered as the ligand of PBLs [8,9]. The signal produced by synergistic effect of HSP70 and membrane-type PBLs can prevent the activation of the cell and negatively regulate the cell immunity. However, if membrane-type PBLs inhibits receptor only, soluble PBLs should enhance the immune response by blocking effect. Previous studies have found that soluble PBLs does not produce an immune enhancing effect, but generates an immunosuppressive effect [10][11][12].In addition, some studies have found that membrane-type PBLs is not only expressed on the surface of cells, but also on the surface of other tissue cells, including tumor cells [13,14]. The function of expression of membrane-type PBLs in these cells is unclear. However, all these studies suggest that PBLs might have multiple biological effects.
In this study, it was found that T cells can enhance the immune function by transfecting the recombinant membrane-type PBLs eukaryotic expression plasmid into NSCLC H446 cells of vaccinated mice, which had signi cant antitumor effect. This result is not consistent with a previous publication which reported that membrane-type PBLs can induce T cell immune tolerance. The membrane-type PBLs is a receptor of the T cell surface, after binding with ligand for signal transduction, PBLs produced a series of changes within the cell. In current work, intramuscular injection of recombinant carrier of membrane-type PBLs was directly conducted, this carrier effectively expressed in muscle cells but was di cult to transfect T cells. The possibility that membrane-type PBLs as cell surface receptor expression in mature muscle cell surface to induce the produce of positive immune effect by muscle cells is very small. Another possibility is that the membrane-type PBLs has ligand properties, which produced positive immune regulatory effect through the interaction with receptor of immune cells surface. Because only HSP70 molecules are known to bind with membrane-type PBLs, the existence of other receptor molecules remains unclear. In this research, the method of local transfection was taken to allow the expression of membrane-type PBLs only in speci c position -the thigh muscle cells of ICR mice. The free cells that can contact membranetyp PBLs or even generate biological effects include macrophages, T cells, B cells and granulocytes [18,19]. We speculate that in this case the membrane-type PBLs may not have a direct effect on T cells, but indirectly activate T cells via activation of macrophages or antigen cells. Because HSP70 that combined with PBLs can express in a variety of tissue cells including macrophages, the PBLs molecule will react with these cells. The RT-PCR results obtained with pure NSCLC H446 cell samples showed very weak expression of HSP70 only, the possiblity that PBLs molecules reacted with the NSCLC H446 cells locally inoculated can be excluded.

Conclusions
In this study, we have found that the membrane-type PBLs and HSP70 could have synergistic effect. HSP70 as a molecule having clear physiological functions can interact with its receptor (expressed on the surface of activated T cells) to maintain the activity of T cells or further enhance the activity and turn it into memory T cells. The transformation is independent and not restricted by other molecules, suggesting that the mechanisms of membrane-type PBLs signal is activation of T cells eventually, and produce synergistic effect with HSP70 based on the activation. After 2 weeks of tumor cells inoculation, HSP70 molecule started to function. Although there are T cells activation at this moment, the effect of PBLs molecules is not further strengthened, suggesting that PBLs molecules do not in uence the functions of T cells after activation.

Declarations
Ethics approval and consent to participate All the experimental procedures mentioned in the present study were approved by the Science Research Department (in charge of animal welfare issue) of The Fourth Hospital of Hebei Medical University (Shijiazhuang, Hebei, China) and First Hospital of Qinhuangdao (Qinhuangdao, Hebei, China). Ethical approval for animal surgery was given by the animal welfare committee of both hospitals.

Consent for publication
All authors have consented the manuscript been published.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.