Expression of cytokine and apoptosis-related genes in murine macrophages and dendritic cells stimulated with Brucella melitensis recombinant proteins

doi:10.21203/rs.3.rs-1151478/v1

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Expression of cytokine and apoptosis-related genes in murine macrophages and dendritic cells stimulated with Brucella melitensis recombinant proteins

https://doi.org/10.21203/rs.3.rs-1151478/v1

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Background

Brucellosis, a zoonosis of public health and economic importance globally. Vaccination is the most efficient way to prevent the infection. Many protein antigens have been selected as diagnostic antigens recently, whereas, the exact mechanisms at the early stage of immune response are remain unknown. In this study, four Brucella melitensis cellular proteins were cloned, and the expressed recombinant proteins were purified. The expression of several cytokine genes (IFN-β, IFN-γ, IL-4, IL-10, IL-12p40, and TNF-α) was analyzed in a murine macrophage cell line (RAW264.7) and murine dendritic cells line(DCs) after stimulation with the recombinant proteins. Two apoptosis-related genes, Bax and Bcl-2, were also included in the analysis to find out the adverse effects of the proteins on the cells.

Results

Each protein induced a different pattern of cytokine expression depending on the antigen dose and cell type. The expression of Th1-type cytokine genes (IFN-γ, TNF-α, and IL-12p40), Th2-type cytokine genes (IL-10 and IL-4), and IFN-β were induced with all of the proteins in RAW264.7 and DCs. The expression of apoptosis-related genes (Bax and Bcl-2) was also induced with most of the proteins in RAW264.7 and DCs. Specifically, the expression of two apoptosis-related genes was not altered with OMP25 protein treatment in DCs; prpA, wadC, and RomA can induce apoptosis-related gene expression both in RAW264.7 cell and DCs.

Conclusions

These results suggest that the four recombinant antigens of B. melitensis can induce pro- or anti- inflammatory cytokine genes at the mRNA level and these changes should be confirmed at the protein level. OMP25 might be a promising antigen for developing a vaccine, but the immunogenicity needs to be further assessed in vivo.

Brucella melitensis

recombinant protein

macrophage

cytokine

apoptosis

Brucellosis is a highly contagious zoonosis disease that is transmitted from livestock and wild animals to humans. This disease is mainly caused by Brucella abortus or Brucella melitensis, which are Gram-negative, facultative intracellular pathogens [1]. Ten bacterial species have been identified based on the differences in pathogenicity and host preference [1, 2]. Brucellosis in animals often causes spontaneous abortion, reduced milk production, and impaired fertility, leading to significant economic loss worldwide [3, 4]. Human brucellosis is not usually fatal, but it can be debilitating with undulating fever. During chronic infection, it leads to lifelong complications, including endocarditis, meningitis, sacroiliitis, and arthritis [5]. Annually, more than 500,000 human cases are reported globally, and it has been an important public health concern [6].

The innate immune response forms the first-line defense against brucellosis. However, Brucella develops multiple strategies to subvert or evade the immune responses for its adaption to the host. Protection against Brucella infection requires both innate and adaptive immune systems, involving an inflammatory response that promotes Th1-type immunity and a cell-mediated immune response that is considered mainly responsible for clearing intracellular bacteria [7, 8].

Cytokines play a pivotal role in the response to infection because they can amplify and coordinate the proinflammatory signals they induce the effector molecules to secrete, thus regulating the innate immunity against infection [9]. For this reason, the cytokine-mediated inflammatory responses to Brucella infection have been explored in recent years.

PrpA belongs to the family of proline racemases and it has been identified as a potent IL-10 inducer required for the establishment of chronicity after Brucella infection[10]. OMP25 is considered a controversial protein. Many researchers suggested that the OMP25 is a key virulence involved in the intracellular survival of Brucella [11–13], whereas it also has been reported that the protein is an immunogenic antigen that can effectively induce a cell-mediated and humoral immune response [14–16]. wadC and RomA proteins are involved in synthesis of Brucella lipopolysaccharide (LPS) and these two proteins acts as a shield against immune response via downregulation of the inflammatory response triggered by Brucella[17, 18].

Understanding the immune mechanisms is crucial for developing a vaccine because many bacterial proteins have been selected as diagnostic antigens without considering the immune response in vivo [19–21]. The mechanisms underlying the apoptotic activities involved in B. melitensis infection have not been revealed in the expression of cytokines and apoptotic-related genes. Therefore, for understanding the mechanism of the immune responses to B. melitensis, a murine macrophage cell line (RAW264.7) and dendritic cell line (DCs) were stimulated with four recombinant B. melitensis proteins. Cytokine expression and the expression of apoptotic-related genes were analyzed for understanding the induction of immune responses.

Bacterial strains

BL21(DE3) (Invitrogen, USA) was used for the expression of the outer membrane protein 25 (OMP25), proline-racemase protein A (prpA), glycosyltransferase (wadC), and periplasmic protein (RomA). The bacterial strains were grown at 37°C in Luria-Bertani (LB) broth or agar (Difco Dickinson). B. melitensis M5-90 was cultured in Brucella broth or agar (BD, USA).

Production and purification of the recombinant proteins

Cloning, expression, and purification of recombinant proteins have been described previously [22]. Briefly, the target genes were amplified by PCR from genomic DNA of B. melitensis M5-90 (Table 1). The PCR products were subjected to sequencing for confirmation of the nucleotide sequences. The amplified DNA fragment was cloned into a pET32a + vector (Novagen, Madison, WI, USA). Thirty milliliters of the culture were inoculated into 1 L of LB broth and supplemented with 250 µg ampicillin. After culturing at 220 rpm, 37°C for 3 h, 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG; Amresco, USA) was added to induce expression of recombinant proteins, and the culture was further incubated for 7 h under the conditions described previously. Cells were harvested by centrifugation at 4,400 g for 15 min. The pellets were suspended in 25 mL of binding buffer (20 mM Tris–HCl, 8 M urea, 500 mM NaCl, 20 mM imidazole, 1 mM β-mercaptoethanol, pH 8.0), and sonicated at 10,000 Hz in ice water (50% pulse, 25 s pulse/50 s steps, 20 cycles) and centrifuged at 4,400 g for 10 min to collect supernatants. The recombinant proteins were purified using a His SpinTrap (GE Healthcare, UK) according to the manufacturer’s protocol. The concentration of purified recombinant proteins was measured through a BCA kit (Bio-Rad, USA). The purity and identity of the recombinant proteins were analyzed by SDS-PAGE and western blotting with an anti-His antibody (Abcam, UK). Endotoxin contamination in the purified proteins was eliminated by 0.1% Triton X-114 in washing buffer in the purification process and was further confirmed using an endotoxin assay kit (Toxin Sensor™ Chromogenic LAL endotoxin Assay kit, GenScript) [23–25].

Table 1 Primer sequences for cloning Brucella melitensis genes

RAW264.7 and DCs culturing and stimulation

The murine macrophage RAW264.7 cell line (ATCC, TIB-71) and DCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco Life Technologies, Rockville, MD, USA) supplemented with 10% fetal bovine serum (Gibco, USA), 100 U/mL penicillin, and 100 µg /mL streptomycin at 37°C with 5% CO₂. The RAW264.7 and DCs were respectively adjusted to 1 × 10⁶ cells/mL in a 6-well plate and then stimulated with 30 or 50 µg/mL of the four different recombinant proteins for 24 h. Concanavalin A (ConA, 1 µg/mL) was used as a positive control.

Total RNA extraction from RAW264.7 and DCs

Total RNA was extracted from RAW264.7 and DCs using an RNeasy mini kit (CWBIO, Beijing, China), according to the manufacturer’s protocols. Genomic DNA was eliminated by a TURBO DNA-free DNAse (Ambio). Total RNA concentration was measured via Nanodrop 2000 (Thermo, USA). cDNA was synthesized from 2 µg of total RNA with a HiFiScript cDNA kit (CWBIO, Beijing, China), as per the manufacturer’s instructions. The reaction products were used to analyze gene expression with real-time PCR.

Quantitative RT-PCR (qRT-PCR) analysis

Real-time RT-PCR was performed using ThermoFisher QuantStudio 3 RT PCR-well Q3 (Thermo Fisher, USA) with SYBR (CWBIO, Beijing, China). The reaction mixture contained 5 µL SYBR Green PCR Master Mix, 1 µL of the primers, 1 µL of the template cDNA, and 3 µL of RNase-free water. The mixture reaction conditions were as follows: initial denaturation at 95°C for 5 min followed by 50 cycles of 95°C for 30 s and annealing at 57°C or 60°C for 30 s. The expression levels of target genes were normalized against the expression of the GAPDH gene. The sequence primers for target genes are shown in Table 2.

Table 2 Murine primer sets for real-time PCR

Statistical analysis

R version 3.6.1 was used to analyze the data. The student’s t-test was used to compare the data between the two groups when the data conform to the normal distribution and homogeneity of the test of variance. If the data failed to obey the normal distribution and homogeneity of the variance test, the rank sum of two independent samples was tested using the Mann–Whitney U test. The Kruskal–Wallis rank-sum test was used to analyze the significance of the data between two groups. Statistical significance was defined as p < 0.05.

The recombinant four different proteins of B. melitensis (OMP25, RomA, prpA, and wadC) were purified and analyzed by SDS-PAGE. The OMP25, RomA, prpA, and wadC proteins were approximately 25 kDa, 31 kDa, 30 kDa, and 27 kDa in size (Fig. 1A). Western blotting was used to confirm the specificity of the expressed proteins (Fig. 1B).

The total RNA of RAW264.7 cells and DCs was extracted after stimulation with four recombinants proteins, followed by cDNA synthesis using the total RNA template. The mRNA expression of cytokines and apoptosis-related genes was confirmed using real-time PCR. Generally, the different patterns of cytokine expression were induced after stimulation with different doses of recombinant proteins. In RAW264.7 cells, the expression of apoptotic-related genes (Bax and Bcl-2) was not significantly affected by treatment with the OMP25 protein, except for the upregulation of the Bax gene after stimulation with 30 µg OMP25 (p < 0.05) (Fig. 2A and 2I). IL-4 gene was significantly downregulated in a dose-dependent way after stimulation with 30 µg and 50 µg of OMP25 protein (p < 0.05, p < 0.01) (Fig. 2E and 2I). The IFN-beta (IFN-β) and IL-12p40 genes were also downregulated after stimulation with 30 µg or 50 µg of OMP25, although not significantly (Fig. 2C, 2G, and 2I). The IFN-gamma (IFN-γ) gene was dose-dependently upregulated after stimulation with 30 µg or 50 µg of OMP25 protein (p < 0.01) (Fig. 2D and 2I). The IL-10 and TNF-alpha (TNF-α) genes were efficiently induced in the cells treated with 30 µg and 50 µg of the OMP25 protein, but not in a dose-dependent way (Fig. 2F, 2H, and 2I). However, most cytokines and apoptotic-related genes were not greatly affected by stimulation with the OMP25 protein in DCs. Only the IL-12p40 gene was induced significantly by treatment with 30 µg of OMP25 protein, and the IL-10 gene was induced efficiently in a dose-dependent way. Additionally, the TNF-α gene expression was inhibited by stimulation with 50 µg of OMP25 protein in DCs (p < 0.05, p < 0.01) (Additional file 1F, 1G, 1H, and 1I).

Interestingly, the Bax gene was downregulated significantly in the RAW264.7 cells after treatment with 30 µg of prpA protein (p < 0.01), whereas it was greatly induced by stimulation of 50 µg of prpA (p < 0.01) (Fig. 3A and 3I). Although 50 µg of prpA protein was also able to induce higher Bcl-2 gene expression, there was no statistical difference in contrast to the PBS group. IL-10 gene expression was induced efficiently when the cells were stimulated with 30 µg and 50 µg of prpA protein (p < 0.01, p < 0.001) (Fig. 3F and 3I), and the TNF-α gene was also upregulated significantly by stimulation with 50 µg of prpA (p < 0.05) (Fig. 3H and 3I). However, the expression of IFN-β, IL-4, and IL-12p40 genes were significantly inhibited dose-dependently after stimulation with prpA protein (Fig. 3C, 3E, 3G, and 3I). Intriguingly, the expression of the IFN-β, IL-4, and IL-12p40 genes was significantly upregulated in the DCs treated with 50 µg of prpA protein (p < 0.05) (Additional file 2C, 2E, 2G, and 2I). In the DCs, only 50 µg of prpA protein-induced Bax and Bcl-2 gene expression (p < 0.05) (Additional file 2A, 2B, and 2I). The expression of IL-10, TNF-α, and IFN-γ was not affected in the DCs by stimulation with the prpA protein.

After stimulation of the RAW264.7 cells with the wadC protein, the Bax gene was significantly induced by the treatment with the 30 ug and 50 µg of protein (p < 0.05) (Fig. 4A and 4I), whereas the Bcl-2 and IFN-γ gene expressions were not influenced by the wadC protein. The IL-10 gene expression was markedly inhibited when the cells were stimulated with 30 µg and 50 µg of wadC protein (p < 0.001, p < 0.0001) (Fig. 4F and 4I). IL-12p40 and IL-4 gene expressions were also downregulated, but there was no statistical difference between the PBS and treatment groups. However, the IFN-β and TNF-α expressions were greatly upregulated after stimulation with the wadC protein (p < 0.05, p < 0.05) (Fig. 4C, 4H, and 4I). When the DCs were stimulated by the wadC protein, on the contrary, Bax and Bcl-2 were both significantly downregulated (p < 0.01, p < 0.01) (Additional file 3A, 3B, and 3I). IFN-β and TNF-α were greatly inhibited in the DCs in contrast to the RAW264.7 cells (Additional file 3C, 3H, and 3I). However, the expression of IFN-γ and IL-4 was induced by the treatment with 50 µg of wadC protein (p < 0.05) (Additional file 3D, 3E, and 3I). Interestingly, the expression of IL-10 and IL-12p40 was significantly downregulated in both the DCs and RAW264.7 cells. (p < 0.05, p < 0.01) (Additional file 3F, 3G, and 3I).

In the case of RomA protein stimulation, Bax gene expression was significantly downregulated in the RAW264.7 cells stimulated with 30 µg of protein (p < 0.01); however, Bcl-2 gene expression was upregulated by the treatment with 50 µg of protein (p < 0.001) (Fig. 5A, 5B, and 5I). There were no meaningful changes in the gene expression of IFN-β and IL-12p40 in RAW264.7 cells (Fig. 5C, 5G, and 5I). The IFN-γ gene expression was markedly induced in RAW264.7 cells stimulated with 30 µg and 50 µg of RomA (p < 0.01, p < 0.01) (Fig. 5D and 5I). The IL-4 gene expression was not affected by the RomA protein stimulation, whereas the IL-10 and TNF-α gene expressions in RAW264.7 cells were significantly downregulated by the treatment of the RomA protein (p < 0.0001, p < 0.001) (Fig. 5F, 5H, and 5I). In the DCs, both of the two apoptotic-related genes were upregulated by the stimulation of RomA protein (p < 0.01, p < 0.01) (Additional file 4A, 4B, and 4I). However, the expression of IFN-β and IL-4 was efficiently induced after the treatment with RomA in the DCs, but not in the RAW264.7 cells (Additional 4C, 4E, and 4I). TNF-α gene expression was also significantly inhibited in the DCs treated with 50 µg of RomA protein (p < 0.01) (Additional file 4G and 4I). IFN-γ, IL-10, and IL-12p40 gene expressions were not affected in the DCs after stimulation with RomA protein (Additional 4D, 4F, 4G, and 4I).

Brucellosis is a re-emerging zoonosis that causes huge world economic losses to animal production and human health. However, the real burden of brucellosis remains underestimated. Vaccination is the most efficient way to eradicate animal disease and prevent human brucellosis [26]. Recently, Brucella antigens have been screened and assessed for vaccine development, given their important role during infection [27]. In addition, it can also help to disclose the mechanism underlying the pathogenesis of Brucellosis.

Macrophages are considered to be a key element of the innate immune response against intracellular pathogens such as Brucella. Most pathogens are killed by macrophages at the early stage of infection; however, some of them still can reach replicative niches for survival [28]. Although several protein antigens have been identified and can stimulate a protective immune response in a murine model [29–32], the roles of some immunogenic proteins still remain unclear. Hence, three different antigens (prpA, wadC, and RomA) with unknown potential in the induction of immune responses and OMP25 protein were selected for analysis of the effect on RAW264.7 and DCs in this study.

Inflammation is a well-known host response to microbial challenges during which various cytokines and chemokines are produced. Cytokines are crucial in innate immunity against infection because of their role as a soluble mediator of cell–cell interaction and amplify or coordinate proinflammatory signals to induce the secretion of effector molecules [33].

Apoptosis is an excellent host defense response against intracellular pathogens. However, Brucella has evolved multiple strategies that allow it to regulate the function of immune cells, such as the promotion or inhibition of apoptosis. BAX and BCL-2 proteins belong to the BCL-2 protein family that are respectively pro-apoptotic and anti-apoptotic [34]. In this study, prpA, wadC, and RomA proteins had a mild effect on Bax or Bcl-2 gene expression in RAW264.7 cells, whereas these three proteins can obviously affect the Bax or Bcl-2 gene expression in DCs. Hence, prpA, wadC, and RomA might affect apoptosis in DCs.

IFN-γ and TNF-α are two crucial cytokines for the host control of Brucella infection [35, 36]. IFN-γ can activate macrophages and promote antigen presentation to CD4 + T cells by enhancing MHC-II expression on their surfaces [37, 38]. It has been demonstrated that IFN-γ neutralized with monoclonal or IFN-γ gene knocked out mice was more susceptible and died in shorter days compared with the control group after infection with B. abortus 2308 [36, 39]. TNF is an important proinflammatory cytokine for the host to combat microbial infection [40]. It has been shown that TNF can recruit neutrophils to the infection site through induction of chemokines and adhesion molecules expression [33]. The macrophages from TNF receptor knock-out mice could not produce NO and IL-12, which resulted in a higher susceptibility to virulent Brucella [41]. In this study, most of the proteins efficiently induced IFN-γ and TNF-α gene expression in RAW264.7 cells, except RomA protein, which significantly inhibited TNF-α gene expression (Fig. 5H and 5I). However, most proteins remarkably downregulated TNF-α gene expression but did not affect IFN-γ gene expression in DCs. This result concurs with findings from a previous study showing that TNF-α production was negatively regulated in human DCs by stimulation with the Brucella suis and B. suis omp25 mutants triggered TNF-α production upon DCs invasion[11]. IL-12 is a cytokine that promotes the differentiation of Th1 cells and inhibits the cytokines required for Th2 cell development and proliferation [42]. Many studies reported that IL-12 is the predominant cytokine against Brucella infection [43–45]. Our results suggest that all of these recombinant proteins downregulate IL-12p40 gene expression in RAW264.7 cells. Furthermore, the wadC protein could inhibit IL-12p40 gene expression both in RAW264.7 and DCs, whereas OMP25 and prpA could induce IL-12p40 gene expression in DCs.

IL-10 and IL-4 are Th2-type cytokines with a strong ability to suppress the Th1 immune response. Many studies revealed that IL-10 is crucial for intracellular pathogens’ survival and chronic infection [46–49]. A major mechanism used by Brucella is to affect TLR signaling and then damp antigen-presenting cells (APCs) activation via induction of IL-10 [50]. IL-4 is an important cytokine that promotes M0 macrophage differentiation into M2 macrophages [51]. It has been reported that chronic brucellosis is associated with IL-10–mediated M2 polarization and the levels of IL-4 in the peripheral blood of patients with chronic brucellosis are higher than in patients with acute brucellosis [52, 53]. Hence, IL-4 is also used by Brucella to regulate the function of immune cells for its survival. In our study, the OMP25 and prpA proteins are effective inducers of IL-10 but significantly inhibit IL-4 gene expression in RAW264.7 cells (Fig. 2E, 2F, 3E, and 3F). In addition, the prpA, wadC, and RomA proteins could upregulate IL-4 gene expression in DCs (Additional file 2E, 3E, and 4E). Thus, the OMP25 protein may be associated with chronic infection in mice; the wadC and RomA proteins participate in Brucella chronic infection in humans, and the prpA protein is a key virulence factor in both mice and humans. However, the underlying mechanism needs to be further explored.

Traditionally, type I IFNs have been assigned a minor role in antibacterial host defenses [54]. However, it has been shown that the induction of type I IFN is detrimental to the host during Listeria monocytogenes infection and Mycobacterium tuberculosis [55, 56]. Leonardo A et al. [57] found that Brucella DNA can induce splenic apoptosis through induction of Ifn-β and Ifn-αβR KO mice are more resistant to infection. Hence, IFN-β can be utilized by Brucella to interfere with the immune response for intracellular survival. It has been shown that the wadC and RomA proteins are involved in the synthesis of the Brucella lipopolysaccharide (LPS) [17, 18]. In our study, the wadC and RomA proteins could markedly induce IFN-β gene expression in RAW264.7 cells (Fig. 4C and 5C), whereas the prpA and RomA proteins could upregulate IFN-β expression in DCs (Additional file 2C and 4C). This finding agrees with a report showing that Brucella LPS can induce IFN-β gene expression, and wadC and RomA may play a key role in LPS-mediated IFN-β production during Brucella infection [57].

Intriguingly, in our results, OMP25 can significantly downregulate IL-12 gene expression in RAW264.7 cells, but upregulate IL-12p40 gene expression in DCs. The explanation for this result may be attributed to the different cellular properties [58, 59]. In addition, it has been reported that Escherichia coli O55:B5 LPS or p38MAPK inhibitor also has different effects on different cell types [60, 61].

These results suggest that the recombinant proteins can induce proper immune responses in the RAW264.7 and DCs. OMP25 might be a promising candidate antigen for developing a vaccine due to its effectiveness in the induction of IFN-γ, TNF-α, and IL-10, and inhibition of IL-12p40, IFN-β, and IL-4 in RAW264.7 cells. Furthermore, the expression of IL-12p40 was induced by the stimulation of OMP25 in DCs without adverse effects (such as apoptosis). However, further research is needed to validate whether OMP25 is involved in intracellular survival via the induction of IL-10 and the these cytokine changes should be confirmed at the protein level. The remaining three recombinant proteins (prpA, wadC, and RomA) can also induce an immune response in these two different cells. Most of the three proteins can significantly inhibit or not affect IL-12, TNF-α, and IFN-γ gene expression, while inducing IFN-β, IL-10, and IL-4 in RAW264.7 or DCs. In addition, these three proteins can induce Bax or Bcl-2 in both RAW264.7 and DCs. Hence, these three proteins might be involved in macrophages and DCs apoptosis. In conclusion, the four recombinant B. melitensis proteins examined in this study appear to induce effective pro- or anti-inflammatory cytokine genes at the mRNA level and these changes should be confirmed at the protein level. Specifically, the immunogenicity of OMP25 protein needs to be further assessed in vivo.

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Availability of data and materials

All of the data generated or analyzed in this study are available in the additional supporting files.

Competing interests

The author declare that they have no competing interest.

Funding

This work was supported by the National Natural Science Foundation of China (Granted No.U1803236, 32002245), the Key Scientific Plan in Bingtuan (Granted No. 2017AA003)

Authors’ contribution

H Z, Y-L W and Y-F W carried out the laboratory works, J-F Y, X-Y D, Z C-M and H-H L made contributions to the interpretation of data, N-N Y, Y-M W, Z-Q L and J-L S made contributions to the analysis of data, Z W, Y W, Y-Z W and C-F C made contributions to the design of the work, H Z,Y-Z W and C-F,C drafted and revised the manuscript. All authors have read and approved the final manuscript.

Acknowledgments

We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.

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No competing interests reported.

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Expression of cytokine and apoptosis-related genes in murine macrophages and dendritic cells stimulated with Brucella melitensis recombinant proteins

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Abstract

Background

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Introduction

Materials And Methods

Bacterial strains

Production and purification of the recombinant proteins

RAW264.7 and DCs culturing and stimulation

Total RNA extraction from RAW264.7 and DCs

Quantitative RT-PCR (qRT-PCR) analysis

Statistical analysis

Results

Discussion

Abbreviations

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References

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