Relationships between interleukin-12B and interleukin-10 gene polymorphisms and hepatitis C in Chinese Han hemodialysis patients

Abstract

Background and Aims: Hepatitis C virus (HCV) commonly causes a chronic infection but portion of hemodialysis patients are able to resistant to HCV infection, even clear the virus naturally. Interleukin-10 (IL-10) and interleukin-12 (IL-12) are crucial regulators of the immune response to T-helper 1 (Th1) or T-helper 2 (Th2) categories and play a role in autoimmune and infectious diseases. This study was undertaken to investigate the possible association of genetic polymorphisms of 3′ untranslated region (3′UTR) of the IL-12B (−1188) and IL-10 (−592 and −819) and hepatitis C in Chinese Han hemodialysis (HD) patients. Methods: The genotyping of IL-12B 3′UTR and IL-10 −592 and −819 were performed by polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) method. Results: Compared with the IL-12B-AA genotype, CC and combined CC/AC genotypes were associated with a significant decreased risk of HCV infection in Chinese hemodialysis patients (p < 0.001). However, the IL-10−592 and −819 genotypes were not found significant difference both between the anti-HCV (+) and anti-HCV (−) patients (p > 0.05) and the persistent infection and viral clearance HD patients (p > 0.05). Conclusions: The present study indicated that the polymorphisms of IL-12B 3′UTR might contribute to the susceptibility of HCV infection in Chinese HD population.

• Hepatitis C
• hemodialysis
• Interleukin-12B gene
• IL-10 gene
• polymorphisms

Introduction

Hepatitis C virus (HCV) has been recognized as the major cause of acute and chronic hepatitis among patients with end-stage renal disease (ESRD) undergoing hemodialysis (HD), although the prevalence of the infection differs among countries and regions, ranging from 7% to 40%.1^,2 And the mortality rate increased in the HD patients with Hepatitis C.3^,4 The natural outcome of HCV infection varies dramatically among individuals, including spontaneous clearance in a minority and persistent infection for the most patients which may progress to cirrhosis, eventually hepatocellular carcinoma.5 Although the determinants of susceptibility to infection, self-limiting or development of chronicity have not been well defined, many investigators have drawn attention to the significance of immune-related genes.

The uremic patients have an impaired cell-mediated immunity and phagocytic activity, which account for their susceptibility to infection and malignancies.3 This condition may disrupt the cytokine network by depressing the Th1 response, enabling Th2 type mediators to prevail. The balance between Th1 pro-inflammatory cytokines (such as IL-2, TNF-α, IFN-γ, IL-12) and Th2 anti-inflammatory cytokines (such as IL4, IL-5 IL-10) have been proposed to play vital role in modulation of host’s response to HCV infection.6

Being involved in the proinflammatory cytokine network, IL-12 is a key cytokine which exert a synergic effect and is thought to be regulator of cytokine expression, inducing interferon gamma (IFN-γ), and enhancing the cytotoxicity of NK cells. It is a heterodimer composed of a light chain (p35) and a heavy chain (p40), and is an immunomodulatory cytokine that is the primary inducer of the development of Th1 cells, with down-regulation of Th2 cytokine. On the other hand, IL-10, a momentous immunoregulatory cytokine, shifts the Th1/Th2 balance by down regulating the Th1 responses and by suppression of pro-inflammatory cytokines, such as TNF-α and IFN-γ secretion.6^,7

Studies have demonstrated that cytokine genetic polymorphism plays an important role in the natural clearance of HCV. Most of these polymorphisms are in the cytokine gene regulatory regions and are consequently involved directly in controlling the transcription rates of these genes. IL-10 possesses a highly polymorphic promoter with variations at −1082(G/A), −819(C/T), −592(C/A), that have been extensively studied and implicated in altering the rate of IL-10 gene transcription.8^,9 Some reporters have indicated that in patients infected with HCV, the production of inappropriate amounts of IL-10 could facilitate viral evasion and result in chronic infection.10 In the general patients infected with HCV, the associations between IL-10 promoter genetic polymorphisms and self-limiting infection, response to therapy have been sufficiently reported.11^,12

SNPs at position 1188A/C in the 3′UTR of the IL-12 gene mapped to chromosome 5q31–33 was found to correlate with many diseases. Cytokine gene sequences are polymorphic at specific sites, and some mutations located within the coding and regulatory regions could powerfully control the production of specific cytokines.13 In the 3′UTR of the IL-12B gene, the gene encoding IL-12B is polymorphic and a functional single nucleotide polymorphism (A/C) of the 3′UTR at position 1188 has been identified recently.7^,13–15

Although there are many investigations on certain aspects of HCV infection in restricted areas, there are no data available on the association of the IL12B 3′UTR and IL-10 genetic polymorphisms with HCV infection among HD patients. Our previous study have demonstrated that TNF-α−238, −308 and IL12B 3′UTR-1188 genetic polymorphisms might be associated with HBV susceptibility and IL-10 genetic polymorphisms might be related to the HBV persistence infection in Chinese Han HD patients.16 And it also has found that TNF-α −238, −308 gene polymorphisms were not associated with HCV infection in Chinese hemodialysis patients. The essay has been published in Renal Failure.17 In this article, we want to investigate further the possible associations between HCV infection and IL-10, IL12B 3′UTR genetic polymorphisms in Chinese HD patients.

Subjects and methods

Patients

The study patients were recruited from 14 different HD units in Jiangsu, China. Clinical and epidemiological data such as age, sex, primary causes of ESRD, blood transfusion, kidney transplantation, HD duration and dialyzer reuse were investigated. The Institutional Ethical Committee approved the protocol for sample collection as well as biochemical, viral, and genetic analyses. Informed consent was obtained before enrolling the patients for conducting the various blood tests including studies on genetic polymorphism.

A total of 884 HD patients were enrolled in this study, which included 556 males and 328 females. The mean age was 52.62 ± 13.44 years and the mean duration of HD was 3.56 ± 3.81 years. They were classified into two groups, anti-HCV (+) and anti-HCV (−). And the anti-HCV (+) individuals were further divided into the persistent infection group and the viral clearance group. The persistent infection group was defined as positive for anti-HCV antibodies and HCV-RNA at the first detection and after 6 months HCV-RNA was still positive, while the criteria for the viral clearance group was positive for anti-HCV and HCV-RNA, but the HCV-RNA became negative after 6 months.

Serology

Five milliliters of blood was collected, and the serum and peripheral blood mononuclear cells were separated and stored in aliquots at −70 °C until assay. Liver biochemistry tests including alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyltransferase (GGT) were measured with a HITACHI 7170 automatic biochemistry analyzer. Serum anti-HCV antibodies were detected by the ELISA kit (Shanghai Kehua biotech Co., Ltd, Sanghai, China). The detection of HCV-RNA was by the diagnostic kit for quantification of HCV-RNA with polymerase chain reaction (PCR)-Fluorescence probing (Shanghai Kehua biotech Co.) at the department of clinical biochemistry laboratory of the First Affiliated Hospital of Nanjing Medical University. The detection limit of HCV RNA was 10³IU/mL.

Genomic DNA extraction and PCR amplification

Genomic DNA of each subject were extracted from 5 mL of peripheral blood leukocytes using sodium dodecyl sulphate (SDS) lysis and proteinase K digestion followed by standard phenol-chloroform methods according to standard protocols as previously described. After genomic DNA were extracted from the blood samples, the region containing the polymorphic site was amplified by polymerase chain reaction (PCR), as reported by Van et al. Briefly, PCR was carried out with primers in a 20-mL volume with buffer (10 equals 500 mM KCl, 100 mM Tris-HCl (pH 8.8), 25 mM MgCl2), 1 pM of specific oligonucleotide primers, 100 mM dNTPs, 50 ng of genomic DNA, and 1U Taq DNA polymerase (Boehringer Mannheim, GmbH, Mannheim, Germany). PCR was carried out using a MJ-PTC-200 Thermal Cycler under the specified PCR conditions. The sequences of the primers used in the assays are provided in Table 1. The parameters for thermocycling of the IL-12B is as follows: 94 °C for 6 min, 33 cycles of 94 °C for 30 s, 52 °C for 30 s and 72 °C for 1 min, final 72 °C incubation for 10 min, and the parameters for thermocycling of the IL-10–592 and −819 were as follows: 94 °C for 6 min, 33 cycles of 94 °C for 30 s, 57 °C for 30 s and 72 °C for 1 min, a final 72 °C incubation for 10 min. The amplified PCR products were digested at 37 °C with the specified restriction endonuclease RsaI (IL-10–592), SspI (IL-10–819) and 70 °C for IL-12B digested by TaqI to detect the polymorphism of the gene nucleotide and the above specified restriction endonucleases were all bought from New England Biolabs. After digestion, under different conditions recommended by the manufacturer’s instructions, the fragments electrophoresed on a 3% agarose gel (Biowest Agarose Gene Tech Company, Ltd., Shanghai, China) and stained with ethidium bromide.16 The sizes of fragments estimated by comparison with previously known size markers (Tiangen Biotech, Beijing, China).

Table 1. Sequences of PCR amplifying primers.

Gene	PCR amplifying primers	Annealing temperature(°C)
IL10–592	forward 5′-CCTAGGTCACAGTGACGTGG-3′	57
C/A	reverse 5′- GGTGAGCACTACCTGACTAGC-3′
IL10–819	forward 5′-TGCTGGAGATGGTGTACA-3′	57
C/T	reverse5′-TAGTGAGCAAACTGAGGCACAGAAAT-3′
IL12B-1188	forward 5′-GATATCTTTGCTGTATTTGTATAGTT-3′	52
3′ UTR C/A	reverse 5′- AATATTTAAATAGCATGAAGGC-3′

Notes: IL, interleukin; PCR, polymerase chain reaction; TNF, tumor necrosis factor.

Genotyping analysis was done without knowing the subjects cases. When genotypes were analyzed, two research assistants independently read the gel images. If they did not reach a consensus on the tested genotypes (<2%), they repeated the genotyping independently to reach a consensus.

Statistical analysis

Results were expressed as mean ± standard deviation (SD). The difference in the distribution of gender between the two cohorts was tested by chi-squared test, the age and duration of hemodialysis were tested by independent t-test. Multivariate analysis of variance was used to analyze the difference of biochemistry index among patients. A chi-squared test was used to compare the distribution of IL-12B and IL-10 genetic polymorphisms. Odds ratio (OR) and 95% confidence interval (95% CI) were calculated using unconditional logistic regression analysis that was adjusted by gender and age. The p-value was two-sided and values of p < 0.05 were considered statistically significant. All statistical analyses were performed with SPSS 16.0 statistical package (SPSS version 16.0, SPSS, Chicago, IL).

Results

The primary diseases

The primary diseases of these patients were chronic glomerulonephritis 527 (59.6%), diabetic nephropathy 119 (13.5%), hypertensive renal disease 111 (12.6%), polycystic renal disease 48 (5.4%), obstructive nephropathy 22 (2.5%), gouty nephropathy 16 (1.8%) and the others 41 (4.6%).

Demographic and clinical features

Among the 884 patients, 179 (20.2%) showed anti-HCV (+), while 705 (79.8%) were anti-HCV (−). The anti-HCV (+) and anti-HCV (−) patients displayed similar demographic characteristics in terms of age (53.26 ± 11.64 vs. 52.46 ± 13.86 years) and gender (p > 0.05). However, the anti-HCV (+) group showed longer duration of hemodialysis (6.98 ± 4.79 year vs. 2.68 ± 2.94 year, p < 0.001), higher rate of blood transfusion (63.7% vs. 44.1%, p < 0.001), kidney transplantation (5.0% vs. 1.6%, p < 0.01) and dialyzer reuse (39.1% vs. 7.8%, p < 0.001) than the anti-HCV (−) group (Table 2). Persistent infection was found in 143 of 179 (79.9%) of the anti-HCV (+) samples. Age and gender did not differ between the persistent infection group and the viral clearance groups either (p > 0.05) (Table 3).

Table 2. Comparison of clinical features between anti-HCV(+) and anti-HCV(−) groups.

	Anti-HCV (+) (179)	Anti-HCV (−) (705)	p^a Value
Gender (%)
Male	122 (68.2%)	434 (61.6%)	>0.05^b
Female	57 (31.8%)	271 (38.4%)
Blood transfusion
Yes	114 (63.7%)	311 (44.1%)	<0.001^b
No	65 (36.3%)	394 (55.9%)
Kidney transplantation
Yes	9 (5.0%)	11 (1.6%)	<0.01^b
No	170 (95.0%)	694 (98.4%)
Dialyzer reuse
Yes	70 (39.1%)	55 (7.8%)	<0.001^b
No	109 (60.9%)	650 (92.2)

Note: ^at-test; ^bX²-test.

Table 3. Demographic and clinical features of the persistent, viral clearance and anti-HCV (−) groups.

	Anti-HCV (+) (n = 179)
Characteristics	Persistent infection (n = 143)	Viral clearance (n = 36)	P^1	Anti-HCV (−) (n = 705)	P^2	P
Age (mean ± SD)	52.71 ± 11.75	55.44 ± 11.08		52.46 ± 13.86		>0.05
Gender, n (%)
Male	99 (69.2%)	23 (63.9%)		434 (61.6%)		>0.05
Female	44 (30.8%)	13 (36.1%)		271 (38.4%)
ALT (U/L)	27.58 ± 32.23	11.14 ± 7.14	<0.001	11.74 ± 12.30	<0.001	<0.001
≥40	30 (21.0%)	1 (2.7%)		15 (2.1%)		<0.001
<40	113 (79.0%)	35 (97.3%)		690 (97.9%)
AST (U/L)	25.42 ± 21.32	14.97 ± 7.86	=0.001	16.36 ± 14.93	<0.001	<0.001
≥40	18 (12.6%)	2 (5.5%)		37 (5.2%)		<0.01
<40	125 (87.4%)	34 (94.5%)		668 (94.8%)
GGT (U/L)	50.10 ± 44.32	26.66 ± 18.75	=0.001	29.64 ± 35.99	<0.001	<0.001
≥50	48 (33.6%)	5 (13.9%)		98 (13.9%)		<0.001
<50	95 (66.4%)	31 (86.1%)		607 (86.1%)

Notes: P¹: Persistent infection group versus viral clearance group, P²: Persistent infection group versus Anti-HCV (−) group.

P: Persistent infection group, viral clearance group and anti-HCV (−) group.

ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, γ-glutamyltransferase.

Several previous studies covered that the average levels of serum aminotransferase were lower in HD patients.17 Interestingly, our data showed that the mean levels of serum ALT, AST and GGT were still within the normal range in the persistent infection group (27.58 ± 32.23, 25.42 ± 21.32 and 50.10 ± 44.32 U/L, respectively), and only 30 (21.0%), 18 (12.6%) and 48 (33.6%) patients had an increase in serum ALT, AST and GGT levels, respectively (Table 3).

Associations between the IL12B 3′UTR and IL-10 gene polymorphisms and HCV susceptibility

As shown in Table 4, compared with the IL12B-AA genotype, the CC genotype had a significantly reduced risk of susceptibility to HCV infection. When we combined CC and CA genotypes, assuming a dominant allele effect, the C allele was associated with a 61.6% (adjusted OR = 0.40, 95% CI = 0.31–0.52) marginally decreased risk of susceptibility to HCV infection.

Table 4. Comparison of frequencies of genotypes and alleles between the hepatitis C virus (HCV) infection and HCV non-infection groups.

	Anti-HCV (+)	Anti-HCV (−)
Genes	179 (20.2%)	705 (79.8%)	p^a Value	OR^a (95% CI)
IL-12B
AA	92 (51.4%)	113 (16.1%)	–	1.00 (reference)
CC	45 (25.1%)	276 (39.1%)	<0.001	0.23 (0.15–0.37)
CA	42 (23.5%)	316 (44.8%)	<0.001	0.19 (0.12–0.30)
CC + CA	87 (48.6%)	592 (83.9%)	<0.001	0.21 (0.14–0.31)
A	226 (63.1%)	542 (38.4%)	–	1.00 (reference)
C	132 (36.9%)	868 (61.6%)	<0.001	0.40 (0.31–0.52)
IL-10-592
CC	25 (14.0%)	89 (12.6%)	–	1.00 (reference)
CA	80 (44.7%)	348 (49.4%)	0.520	0.69 (0.22–2.13)
AA	74 (41.3%)	268 (38.0%)	0.549	1.41 (0.46–4.38)
IL-10-819
CC	25 (14.0%)	89 (12.6%)	–	1.00 (reference)
CT	79 (44.1%)	350 (49.6%)	0.177	0.47 (0.16–1.41)
TT	75 (41.9%)	266 (37.7%)	0.993	0.99 (0.33–2.99)

Note: ^aLogistic regression model, adjusted by age, gender, duration of hemodialysis, blood transfusion history, kidney transplantation and dialyzer reuse.

In univariate and multivariate analysis of IL-10 in the distributions of IL-10 genotypes at the −592 and −819 positions, no significant differences were detected between the anti-HCV (−) group and anti-HCV (+) group (Table 4).

Associations between the IL12B 3′UTR and IL-10 gene polymorphisms and HCV persistent infection

Then, the distribution of genotypes in persistent infection group and viral clearance group were analyzed. As shown in Table 5, the distribution of the IL12B 3′UTR-1188 and IL-10 −592, −819 genotypes all have no differences in persistent infection group and viral clearance group (p > 0.05).

Table 5. Comparison of frequencies of genotypes between the hepatitis C virus (HCV) persistent infection and HCV clearance groups.

Genes	Persistent infection 143 (79.9%)	Viral clearance 36 (20.1%)	p^a Value	OR^a (95% CI)
IL-12B
AA	77 (53.8%)	15 (41.7%)	–	1.00 (reference)
CC	35 (24.5%)	10 (27.8%)	0.44	0.70 (0.28–1.75)
CA	31 (21.7%)	11 (30.5%)	0.14	0.50 (0.20–1.27)
CC + CA	66 (46.2%)	21 (58.3%)	0.19	0.60 (0.28–1.29)
A	185 (64.7%)	41 (56.9%)	–	1.00 (reference)
C	101 (35.3%)	31 (43.1%)	0.26	0.73 (0.43–1.26)
IL-10-592
CC	21 (14.7%)	4 (11.1%)	–	1.00 (reference)
CA	66 (46.2%)	14 (38.9%)	0.808	0.86 (0.25–2.92)
AA	56 (39.2%)	18 (50.0%)	0.366	0.57 (0.17–1.92)
IL-10-819
CC	20 (14.0%)	5 (13.9%)	–	1.00 (reference)
CT	65 (45.5%)	14 (38.9%)	0.847	1.12 (0.36–3.52)
TT	58 (40.6%)	17 (47.2%)	0.084	0.82 (0.26–2.54)

Note: ^aLogistic regression model, adjusted by age, gender.

Discussion

Despite strict infection control policies, high prevalence of HCV infection was still covered in HD centers. As it is well known that the determinants of HCV infection may not only depend on external factors but also the host’s condition. The susceptibility and elimination of HCV depends on the integrated activities in immune systems and the cytokine network of the patients.

Whereas, the factors lead to viral clearance or persistence are poorly understood. Some research disclosed that the outcome might already be determined at an early time point after infection [18]. In early HCV infection, a vigorous CD4+ T cells response is associated with viral clearance. Th1/Th2 balance plays a pivotal role in modulation of virus–host immune system interaction.6 Th1-mediated immune response are critical for host anti-viral effects, favor virus elimination and exacerbate inflammation, while Th2 responses suppress the Th1 response.19^,20 These findings manifest that the ability to mount an efficient cellular immune response is the main mechanism responsible of HCV elimination, while a defect in this response leads to chronicity.18^,21 As the indispensability of cytokines and the cellular immune response in the eradication or pathogenesis of HCV infection has been clearer, the significance of the immune response has also been proposed. The production of cytokine in individuals has a major genetic component. This has been ascribed to be polymorphic within the regulatory regions of cytokine gene. We chose to examine polymorphisms in IL12 and IL10 genes, which have demonstrated correlations with cytokine production and have been associated with disparate clinical outcomes in HCV infection in general people.

IL12 is a pivotal cytokine for promoting and maintaining anti-viral immune responses. It might participate in the pathogenesis of HCV infection by affecting Th1/Th2 balance. Many chronic infections, including HCV and HIV, are associated with impaired IL-12 production. Whether this lower IL-12 level is a cause or consequence of chronic infection remains unclear, but an individual’s genetically influenced ability to produce IL-12 in reaction to antigen exposure is likely to be important in influencing the outcomes of infection. The SNP at position 1188 in the 3′UTR of IL12 is associated with immune-mediated diseases, such as asthma, type 1 diabetes, psoriasis, and so on. Hegazy et al.22 found that the course of HCV infection was related with single nucleotide polymorphisms in the 3′UTR of IL-12B. A study on the functional characteristics of this SNP in HCV-infected Chinese patients demonstrates an increased frequency of the CA genotype and a decreased frequency of the AA genotype in the self-limited infection group.22 Mueller et al.23 revealed that subjects who are chronically infected and carriers of the C allele seem to have a better outcome of clearing HCV infection with anti-viral treatment. In accordance with these findings, our study concluded that the CC/AC genotype might have a decreased risk of HCV infection.

On the other hand, IL-10, a Th2 cytokines may interfere with the generation of an effective anti-viral response by inhibiting T-cell proliferation, major histocompatibility complex expression and the production of Th1 cytokines.24 This was followed by further proof that inhibiting the IL-10 and its receptor may enhance the immune responses against HCV and other viruses.25^,26 Some studies imply identical genetic background of patients and controls and suggest that IL-10 SNPs do not influence susceptibility to HCV infection. In this article, we also did not observe the associations between the IL-10 promoter polymorphisms of the −592C/A, −819C/T and the susceptibility to HCV and HCV self-limiting infection in the Chinese HD patients. But there were still conflicting results, and this speculation should be experimentally tested in the future study. A study in Pakistan general people suggested different IL-10 gene polymorphisms, 819(C/T), −592(C/A) and 1082(G/A) may lead to an imbalance between the pro-inflammatory and anti-inflammatory cytokine response which in turn influence the susceptibility to HCV infection. A study from UK showed that AA genotype at the IL-10−592 locus was associated with the self-limiting HCV infection in the general populations. And Mangia et al.27 reported that inheritance of −1082A, −819T and −592A alleles as an extended haplotype appeared to be associated with HCV eradication in Italian populations.

The discrepancy above could be attributed to the special HD population, who were different from the general population, since HD patients infected with HCV are under the influence of uremia, the HD procedure, chronic renal failure complications, therapeutic interventions for their treatment and the HCV infection itself. The chronic kidney disease could induce increased spontaneous TNF-α and IL-12 overproductions that imply an up-regulation of the inflammatory activity, which were most likely due to uremia. However, HD patients showed diminished cytokines production and in HD patients the immunity cells are characterized by decreased chemotactic, phagocytotic and bactericidal activities. The different clinical course between HD patients and the general population with HCV infection was probably a result of deficient CD8 cytotoxic T-lymphocyte function in the HD patients. Moreover, it also indicated deficient CD4 helper T-lymphocyte function, which provides help to CD8 cytotoxic T-lymphocytes and is also essential for antibody production by the B-lymphocyte. As we all know, Th1 lymphocytes cytokine promote the cellular part of acquired immunity by providing help to CD8 cytotoxic lymphocytes and/or by activating macrophages. Th2 lymphocytes cytokine promote the humoral part of acquired immunity providing help to B-lymphocyte for antibody production. So the above inconsistent results may result from the special immunity state of HD patients or the different ethnic population, therefore, studies with large samples of HD patients and different ethnic backgrounds are still needed. Furthermore, the genotypes studied here represent only one factor among complex immune systems. The serum levels of cytokines were associated not only with the genetic factors but also with the host’s condition such as the basic disease and with the exposure to environmental pathogens. So the exact mechanism of HCV susceptibility and HCV chronicity still needs to be further investigated.

In summary, our study suggested that the polymorphisms of IL-12B 3′UTR might contribute to the susceptibility to HCV infection in Chinese HD population. The roles of cytokine gene polymorphisms in the HD patients with HCV may have some differences with the general population. We have only observed the characteristics of gene polymorphisms of cytokine genes (IL-10 and IL-12B) in HD patients with or without HCV infection. It is very interesting to explore the influence of genetic and immunological factors on the HCV infection in patients with chronic uremia status and/or undergoing hemodialysis. We have just initiated our work in this area. In future, we will continue to get more data of pre-hemodialysis and post-hemodialysis patients, and do more subgroup analyses among those patients with HCV infection.

Declaration of interest

This work was supported by Project supported by the key lab of Jiangsu province MMB09KF04, Project supported by the Scientific Research Program of the Health Department of Jiangsu Province H200902 and XK16200903, Project supported by the National Natural Science Foundation of China H0511-81070588, Project supported by the international cooperation projects of Jiangsu province BZ2010058 and Project supported by the funding of blue project 2010 and Peak Talent Funding in six fields of Jiangsu province 2010-175. The authors declare no conflicts of interests.