Expression and clinical significance of TLR9 in pancreatic cancer
WU Hanqing ZHU Shi-Kai ZHANG Jian-Jun YANG Zhi-Yong WANG Chun-You WU He-Shui*
Abstract】Objective To detect the expression of TLR9 in pancreatic cancer tissues and explore its clinical significance. Methods Real-time quantitative PCR, immunoblotting and immunohistochemistry were used to detect the expression of TLR9 in 30 pancreatic cancer tissues and their adjacent paraneoplastic tissues and 10 normal pancreatic tissues, and to analyze the relationship between the expression of TLR9 in pancreatic cancer tissues and pathological grading, clinical stage and metastasis. Results The TLR9 mRNA amplification fold value was 2.32 (1. 41~3.22) in pancreatic cancer and 1.23 (1.18~1.28) in paraneoplastic tissues, and there was a significant difference in expression between the two groups (t=2.642, P=0.023). The positive expression rate of TLR9 protein in pancreatic cancer was 73.3%, 33.3% in paracancerous tissues and 20% in normal pancreatic tissues, with a decreasing trend (c2 =13.99, P=0.001). The high TLR9 expression in pancreatic cancer was positively correlated with tumor differentiation degree, TNM stage, and lymph node metastasis. western blot results were consistent with immunohistochemistry, and grayscale analysis showed significant differences. Conclusion TLR9 was highly expressed in pancreatic cancer tissues, and TLR9 expression was correlated with tumor grade, suggesting that it may be involved in the process of pancreatic cancer development through immune mechanism. Wu Hanqing, Department of Emergency Medicine, Wuhan Union Medical College Hospital
Keywords】pancreatic tumor, cancer, TLR9
TLR9 expression in pancreatic cancer and its clinic significance
Han-Qing Wu, Shi-Kai Zhu, Jian-Jun Zhang, Zhi-Yong Yang, Chun-You Wang, He-Shui Wu
Department of Pancreatic Surgery Center ,Union Hospital ,Tongji Medical College ,Huazhong University of Science and Technology ,Wuhan Hubei 430022,China
Correspondence to: Professor He-Shui Wu, Email:[email protected].
[Abstract] Objective: To detect the expression of Toll-like receptor 9 (TLR9) in pancreatic cancer, and to explore their clinical significance. Methods: The real-time RT-PCR technique, western blot method and immunohistochemical method were used to examine the expression of TLR9 in the pancreatic cancer, tissues near tumor, and normal pancreatic tissues which obtained during operation from 30 pancreatic carcinoma patients and 10 normal non-lump patient. The relationships with pathological grade, clinical stage, and metastasis of pancreatic cancer were statistically analyzed. Results The amplification value level of TLR9mRNA expression in human pancreatic tissues, paracancerous tissues were 2.32（1.41~3.22）and 1.23（1.18~1.28），respectively（t=2.642,p=0.023）.The highly expressed TLR9 correlated significantly with the degree of tumor differentiation, an [Key Words] Pancreatic neoplasms; Cancer; Toll-like receptor 9 Toll-like receptor (TLR) is a newly discovered ancient family of receptors that are not only important receptors involved in natural immunity, but also closely related to specific immunity, immune tolerance and the prevention and treatment of certain diseases. toll-like receptors have recently been found to be potentially involved in the development and progression of a variety of malignancies [1]. And TLR9 is an important member of this family that stimulates the expression of a large number of cytokines and chemokines such as interleukin (IL)-12, IL-6, interferon-γ, monocyte inhibitory protein and metalloplasmic proteases secreted by B cells and dendritic cells [2]. Many tumors show high TLR9 expression, but it is not clear whether TLR9 is associated with the development of pancreatic cancer. The aim of this study was to investigate the relationship between TLR9 expression in pancreatic cancer and the development of pancreatic cancer, and to explore its clinical significance in order to provide a new basis for the treatment of pancreatic cancer. Data and Methods I. Clinicopathological data Thirty human fresh pancreatic cancer tissues and corresponding paracancerous tissues (1-2 cm according to the tumor margin) were obtained from pancreatic cancer patients, 10 normal pancreatic tissue specimens were obtained from non-tumor patients who required partial pancreatic resection for other reasons, and all specimens were from pancreatic cancer patients who underwent surgical treatment from December 2008 to August 2009 at the Pancreatic Surgery Center of Union Medical College of Huazhong University of Science and Technology and were treated by Postoperative pathology confirmed that no preoperative radiotherapy or chemotherapy was administered. The specimens were collected immediately after surgical resection, in duplicate, and one copy was immediately placed in liquid nitrogen and then stored at -80°C. Another copy was fixed with 10% formaldehyde and embedded in paraffin for immunohistochemical analysis. 19 of 40 patients were male and 11 were female, aged 39-75 years, with a mean age of 57 years. Histopathological grading of tumors: 12 cases with high differentiation and 18 cases with low to moderate differentiation. 10 cases of lymph node metastasis and 20 cases of non-metastasis. II. real-time quantitative PCR to detect TLR9 gene expression Total tissue RNA was extracted according to the instructions of Trizol kit (sigma), and the purity and concentration of RNA were determined by UV spectrophotometer. cDNA was then synthesized by reverse transcription with ReverTra Ace (Toyobo). primers were designed according to the GenBank human TLR9 mRNA sequence, and the upstream of TLR9 primer sequence is: 5´- GCCCAAATCCCTCATATCCC-3´, downstream: 5´-AACAGTTGCCGTCCATGAATAG-3´, amplification product was 113 bp. primers for internal reference β-actin, upstream: 5´-GTCCACCGCAAATGCTTCTA-3´, downstream: 5´- TGCTGTCACCTTCACCGTTC-3´, amplification product of 190bp (Shanghai Biotechnology Service Co.) Real-time quantitative PCR (SYBR GREEN) was used to detect TLR9mRNA. amplification curve reaction procedure: 50°C for 2 min, 95°C for 2 min, then denaturation at 95°C for 15 s, annealing for 15 s, extension at 72°C for 45 s, total 40 cycles. The △CT=CT(target gene)-Ct(β-actin) for pancreatic cancer or corresponding paracancerous tissues and 10 normal pancreatic tissues, △△CT=△CT experiment-△CT control, we used the average value of △CT of 10 normal pancreatic tissues as control. The amplification ploidy = 2-△△CT was obtained by calculation. III. Immunoblotting to detect TLR9 protein expression Tissue protein extraction was performed by conventional method, and protein concentration was measured by Komas Brilliant Blue method. 15 μl of protein was taken for 10% polyacrylamide gel electrophoresis, electrotransferred to nitrocellulose membrane, 5% skim milk powder was used to close the non-specific antigen, rabbit anti-human TLR9 monoclonal antibody (cell signaling company) diluted 1:750 was added, incubated overnight at 4°C, the membrane was washed with TBST containing 0.1% Tween 20, horseradish peroxidase (HRP) was added The labeled goat anti-rabbit secondary antibody was incubated at room temperature for 2 h. After washing the membrane, the enhanced chemiluminescence substrate EcL Plus reagent was added and exposed to x-film for 30 s. The film was routinely developed and fixed. Image scanning and analysis were performed using automated electrophoresis gel imaging analysis software (Band scan v5.0). IV. Immunohistochemical detection of TLR9 protein expression by immunohistochemistry SP (streptavidin-perosidase) method, referring to the instructions of the immunohistochemistry kit (Wuhan PhD Co., Ltd.). Sections of known TLR9-positive lung cancer tissues [3] were used as positive controls, and PBS instead of primary antibody was used as a negative control. Microscopic observation of cells stained brownish yellow was positive, and the staining material was granular or flaky, mainly present in the cytoplasm of tumor cells. The staining results were evaluated by double-blind, randomly observing 10 high magnification fields, avoiding tumor margins and necrotic areas, and semi-quantitative analysis was performed according to the intensity of color development and the percentage of staining, which were classified according to the degree of positive cell staining: negative as 0, weakly positive as 1, moderately positive as 2, and strongly positive as 3. The percentage of positive staining was divided into: 0 for no staining, 1 for total positive cells <25%, 2 for positive cells between 25% and 49%, and 3 for total positive cells above 50%. The two scores were summed and 0-1 was classified as negative (C), 2-3 as weakly positive (), 4-5 as positive (), and 5 or more as strongly positive (). () with () for high expression and (C) with () for low expression. V. Statistical methods SPSS 13.0 statistical analysis software was used to apply t-test, χ2 test, Fisher's exact probability method and rank sum test for the analysis of differences and correlations between values. with α=0. 05 as the test level, and P<0.05 as the difference is statistically different. Results 1 Real-time quantitative PCR results TLR9 mRNA and β-actin mRNA were expressed in normal pancreatic tissue, pancreatic cancer tissue and paracancerous pancreatic tissue. The TLR9 mRNA in normal pancreatic tissues was used as a control, and the amplification fold value of TLR9 mRNA in pancreatic cancer was 2.32 (1.41~3.22) and 1.23 (1.18~1.28) in paraneoplastic tissues, and the difference in expression between the two groups was significant (t=2.642, p=0.023). 2 Immunoblotting results As seen in Figure 1, the expression of the internal reference GAPDH protein was observed in cancer and paraneoplastic tissues as well as in normal pancreatic tissues, with protein bands at 36 kDa and TLR-9 at 130 kDa. the expression of TLR9 in cancer tissues was significantly higher than that in the corresponding paraneoplastic and normal pancreatic tissues. Figure 1 TLR-9 protein immunoblotting results (1: normal pancreas, 2: paracancerous pancreatic tissue, 3 pancreatic cancer tissue) 3 Immunohistochemical results TLR9 protein was mainly expressed in the cytoplasm of cancer cells, which was tan or brown with uniform staining; TLR9 protein expression could also be seen in the cytoplasm of paracancerous and normal pancreatic tissues, but it was light yellow and less frequent than in pancreatic cancer tissues (Figure 2).The positive expression rates of TLR9 in normal pancreatic tissues, paracancerous tissues and pancreatic cancer tissues were 20% (2/10), 33.3% ( 10/30) and 73.3% (22/30), with statistically significant differences between the groups (χ2=13.99, P=0.001) (Table 1). No high expression of TLR9 was observed in normal pancreatic tissues, while 73.3% (22/30) of pancreatic cancer tissues showed high expression of TLR9 protein. The positive expression rate of TLR9 was increasing from normal tissue, paraneoplastic tissue to pancreatic cancer tissue. the high expression of TLR9 protein in 30 pancreatic cancer tissues was not related to the site of tumor and tumor size (P>0.05), but to the degree of tumor differentiation, TNM stage, and lymph node metastasis (P<0.05, Table 2). Figure 2 Expression of TLR9 protein in pancreatic cancer tissues, paracancerous pancreatic tissues and normal pancreatic tissues (×200); pancreatic cancer tissues (A), paracancerous pancreatic tissues (B), normal pancreatic tissues (C) Table 1 Expression of TLR9 protein in normal, paraneoplastic and pancreatic cancer tissues group Example TLR9 protein positive Positivity rate (%) positive negative normal tissues paraneoplastic tissues pancreatic cancer tissues 10 30 30 2 20 22 8
10 8 20.0 33.3 73.3 Table 2 Relationship between high TLR9 expression in pancreatic cancer tissues and pathological parameters Clinical characteristics Number of cases Positive TLR9 high expression TLR9 high expression negative Positivity rate (%) P value tumor site head of the pancreas in the tail of the pancreatic body 20 10 15 7 5 3 75.0% 70.0% 1.00 tumor size ≤3cm >3cm 17 13 13 9 4 4 76.5% 69.2% 0.68 degree of differentiation highly differentiated medium to low differentiation 12 18 6 16 6 2 50.0% 88.9% 0.034 TNM stage І CП Ш- 13 16 7 15 6 1 53.8% 93.8% 0.026 Lymph node metastasis no There was 20 10 13 9 7 1 65.0% 90.0% 0.024 Note: Statistical analysis was performed by applying the χ2 test, Fisher’s exact probability method Discussion TLRs are newly discovered pathogen pattern recognition receptors that recognize conserved molecular components of specific types of microorganisms, i.e. pathogen-associated molecular patterns (PAMPs). in 1997 Medzhitov [4] et al. first identified the first human Toll homologous variants (i.e. TLR4), and later 10 human TLR family proteins were identified and confirmed, named TLR1-TLR11. Among them, TLR9 is an important member of the TLR family, whose specifically recognized PAMPS are CpG motifs in bacterial and viral DNA or synthetic oligodeoxynucleotides (CpG- oligodeoxynucleotides, CpG-ODN) [5], TLR9 has an important biological significance in mediating the process of innate immunity activation by exogenous CpG DNA. in addition to its anti-infective effects, TLR9 is also associated with autoimmune disorders and malignant tumorigenesis [6]. It has been suggested that signaling pathways induced by TLRs, including TLR9, may play an important role in tumor formation, and upregulation of TLRs expression may be closely related to the occurrence and development of gastric, intestinal, and lung cancers [7].
It was also found that TLR9 expression on tumors could provide new approaches for chemotherapy and immunotherapy of tumors.
However, little is known about the expression and role of TLR9 in pancreatic cancer. In this study, TLR9 was detected in pancreatic cancer tumor tissues at the gene level and protein level as well as TLR9 protein size, and compared with paracancerous pancreatic tissues and normal pancreatic tissues, and the results at the gene level revealed that TLR9 mRNA was expressed in pancreatic cancer tissues, paracancerous pancreatic tissues and normal tissues, but the highest expression was found in cancerous tissues, which was significantly higher than non-cancerous tissues (P<0.05 ). On immunohistochemical staining pictures, TLR9 was found to be expressed mainly in cytoplasmic cells of pancreatic adenocarcinoma, and the TLR9 positive expression rate was significantly higher in pancreatic adenocarcinoma tissues than in paracancerous and normal pancreatic tissues, which is consistent with the positive correlation results of Droemann [3] in lung cancer. The high expression of TLR9 in pancreatic cancer tissues was not related to the patient's age, gender, tumor site as well as size, but to the degree of tumor differentiation, TMN stage, lymph node metastasis or distant metastasis, suggesting that TLR9 receptors are not only involved in physiological functions such as growth and development of normal pancreatic tissues, but also in the malignant transformation process of pancreatic tissues.A study by Schmausser [8] et al. confirmed the TLR9 is involved in gastric carcinogenesis together with other TLRs. The results of the present study are consistent with this. In recent years, numerous studies have confirmed that CpG ODN has a strong immune apoptotic effect in the Th1 direction, mainly by binding to TLR9 and inducing the secretion of Th1 polarity cytokines such as IFN-7 and IL-12 to promote the differentiation of Th0 cells to the Th1 direction. For example, CpG DNA enters dendritic cells through sequence non-dependent receptor-mediated endocytosis, interacts specifically with TLR9 in the lysosomal region, and then activates downstream signaling pathways through IRAK1, IRF7, TRAF6, MAPK, nuclear factor-KB (NF-KB) pathways and the articulator MyD88 [9]. Through downstream signaling pathways eventually lead to the activation of plasmacytoid dendritic cells and their maturation into specialized antigen-presenting cells. These mature antigen-presenting cells express co-stimulatory molecules (CD80, CD86), chemokine receptor CCR7 on their surface, secrete Th1-type cytokines, activate NK cells [10], and due to the increased expression of Fc receptors on the surface of neutrophils, cytotoxic activity is enhanced, which further causes more plasma cell-like and dendritic cells to activate and mature and antigen-present. Through the action of CpG and TLR9, antigen-presenting cells such as Dc cells can cross-present exogenous antigens, thus cross-activating the activation of CD8+ T cells, NK cells, and macrophages, generating a Th1-type immune response and activating the natural immune response [11]. And currently CpG ODN is used as an immune adjuvant to induce strong Thl-type immune response via TLR-9 on human lung adenocarcinoma A549 cells, which may be applied as monotherapy or adjuvant therapy to immunotherapy for tumor treatment [12]. In conclusion, the high expression of TLR9 in pancreatic cancer tissues suggests its potential as a new intervention target for tumor therapy. the expression of TLR9 protein is associated with the malignancy of pancreatic cancer cells and also with the presence of lymph node metastasis in pancreatic cancer, which provides a reference for the treatment and prognosis of pancreatic cancer. Immunotherapy of pancreatic cancer using CpG ODN, a TLR9-related ligand, may offer new therapeutic possibilities for pancreatic cancer treatment. Ref. 1. Jurk M, Vollmer J. Therapeutic Applications of Synthetic CpG Oligodeoxynucleotides as TLR9 Agonists for Immune Modulation[J]. BioDrugs,2007;21(6):387-401. 2. Theiner G, Rossner S, Dalpke A, et al. TLR9 cooperates with TLR4 to increase IL-12 release by murine dendritic cells [J]. Molecular Immunology,2008;45(1):244-252. 3. Droemann D, Albrecht D, Gerdes J ,et al. Human lung cancer cells express functionally active Toll-like receptor 9[J]. Respir Res,2005,6(1):1-10. 4. Medzhitov R, Preston - Hurlburt P, Janeway CA. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity[J].
Nature, 1997, 388: 394C397. 5. Hemmi H, Takeuchi O, Kawai T, et al. A Toll-like receptor recognizes bacterial DNA[J]. Nature,2000, 408(6813) :740. 6. Ishii KJ, Akira S. Innate immune recognition of, and regulation by, DNA[J]. Trends Immunol, 2006 Nov;27:525-532. 7. Coussens LM , Werb Z. Inflammation and cancer[J] . Nature , 2002; 420 (6917): 860-867. 8. Schmauber B, Andrulis M, Endrich Med Microbio, 2005,295(3):179-185. 9. Underhill DM. Toll – like receptors : networking for success[J]. Eur J Immunol , 2003 ,33 (7) :1767. 10. Ballas Z. Modulation of NK cell activity by CpG oligodeoxynucleotides[J]. Immunol Res, 2007;39(1-3):15-21. Krieg AM. Therapeutic potential of Toll-like receptor 9 activation[J]. Nat rev drug disc, 2006,5:471-484. 12. Krieg AM. Toll-like receptor 9 (TLR9) agonists in the treatment of cancer[J]. Oncogene, 2008; 27:161-167. This article was published in the Chinese Journal of Pancreatic Diseases, 2011, Vol. 3, No. 3, please see the original article This work was supported by the National Natural Science Foundation of China (Grant No. 30200272) Author Biography:H.Q. Wu, M., Ph.D., is a researcher focusing on pancreatic tumors. E-mail:[email protected] Correspondence should be addressed to Wu Heshui, Professor, PhD Supervisor, Center for Pancreatic Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China; Email:[email protected].