although novel treatment protocols have been developed , the prognosis for colon cancer remains poor , with a 5-year survival rate lower than 40% .
patients with colon cancer mainly die of metastasis and recurrence ( 2 ) . despite numerous experimental and clinical studies on the pathogenesis of colon cancer ,
its mechanisms have not yet been elucidated , though they are of great clinical significance for the diagnosis and treatment of this cancer .
tumor necrosis factor receptor - associated factor 6 ( traf6 ) has been reported to be highly expressed in many tumor tissues ( 3 ) , but it is still of value to detect its expression in patients with colon cancer and to clarify its clinical significance ( 4 , 5 ) .
the aim of this study was to detect the expression levels of traf6 protein in tumor and paracancerous tissues using immunohistochemical methods , to identify correlations between the clinicopathological characteristics and prognosis of colon cancer , and to provide evidence for future prognostic determinations and target therapies .
a total of 135 patients with primary colon cancer , who underwent surgery at our hospital from february 2008 to march 2013 , were selected .
informed consent was obtained from each patient , and the study protocol conformed to the ethical guidelines of the 1975 declaration of helsinki .
patients who had received radiotherapy , chemotherapy , or biological immunotherapy before surgery , or who were lost to follow - up , were excluded .
the sample size equation is : where n is the sample size , z is the confidence interval ( for a level of confidence of 95% in this study , z = 1.96 ) , p is the probability value , and e is the error value .
tumor and paracancerous tissues resected during surgical procedures were immersed in neutral formalin at 4c for 2 days , and embedded with paraffin into 6-m - thick sections ( leica biosystems , usa ) , which were then deparaffinized with dimethyl benzene and hydrated with ethanol solutions at gradient concentrations .
after antigen retrieval ( beijing zhongshan golden bridge biotechnology co. , ltd . , china ) and blocking with rabbit immune serum ( beijing zhongshan golden bridge biotechnology co. , ltd . , china )
, the sections were incubated with traf6 antibody ( santa cruz , 1 : 50 ) in a 37c incubator for 2 hours , washed with pbs , incubated with secondary antibody for 1 hour , washed again with pbs , and incubated with horseradish peroxidase - labeled streptavidin ( beijing zhongshan golden bridge biotechnology co. , ltd . ,
afterwards , the sections were washed with pbs , colored with dab ( beijing zhongshan golden bridge biotechnology co. , ltd . ,
pbs was used to replace primary antibody as the negative control . with regard to standards for the determination of immunohistochemical staining ,
traf6 was determined to be positively expressed in the case of brownish - yellow precipitates in the cytoplasm .
the procedure was completed by two experienced pathologists . by using the double - blind method ,
15 fields of vision were selected for each section with a high - powered lens (  400 ) , and the ratio of the number of traf6-positive cells to 100 average cells was calculated as the positive rate .
positive results of immunohistochemical staining were determined according to the following two aspects : 1 ) percentage of traf6-positive cells : < 1% , 0 ; 1% - 10% , 1 point ; 10% - 30% , 2 points ; 30% - 60% , 3 points ; > 60% , 4 points , and 2 ) staining results of traf6-positive cells : negative , 0 ; yellowish , 1 point ; yellow , 2 points ; brownish yellow , 3 points . for the determination of results ,
tumor and paracancerous tissues resected during surgical procedures were immediately stored in liquid nitrogen , from which total rna was extracted by using trizol reagent according to the kit s instructions .
upstream primer for traf6 : 5-gaatcacttggcacgacactt-3 , downstream primer for traf6 : 5-gagtttccattttggcagtca-3 and length of amplified fragment : 227 bp .
upstream primer for gapdh : 5-accacagtccatgccatcac-3 , downstream primer for gapdh : 5-tgctgtagccaaattcgttg-3 and length of amplified fragment : 450 bp .
rt - pcr was performed under the following conditions : reverse transcription at 50c for 30 minutes , reaction at 95c for 15 minutes ( cycling was started ) , denaturation at 94c for 30 d , annealing at 57c for 30 seconds , and extension at 72c for 30 seconds , for a total of 30 cycles .
after final extension at 72c for another 10 minutes , the pcr products were subjected to agarose gel electrophoresis .
the numerical data were compared by  test , and the survival rates were estimated by the kaplan - meier method .
a total of 135 patients with primary colon cancer , who underwent surgery at our hospital from february 2008 to march 2013 , were selected .
informed consent was obtained from each patient , and the study protocol conformed to the ethical guidelines of the 1975 declaration of helsinki .
patients who had received radiotherapy , chemotherapy , or biological immunotherapy before surgery , or who were lost to follow - up , were excluded .
the sample size equation is : where n is the sample size , z is the confidence interval ( for a level of confidence of 95% in this study , z = 1.96 ) , p is the probability value , and e is the error value .
tumor and paracancerous tissues resected during surgical procedures were immersed in neutral formalin at 4c for 2 days , and embedded with paraffin into 6-m - thick sections ( leica biosystems , usa ) , which were then deparaffinized with dimethyl benzene and hydrated with ethanol solutions at gradient concentrations .
after antigen retrieval ( beijing zhongshan golden bridge biotechnology co. , ltd . , china ) and blocking with rabbit immune serum ( beijing zhongshan golden bridge biotechnology co. , ltd . , china )
, the sections were incubated with traf6 antibody ( santa cruz , 1 : 50 ) in a 37c incubator for 2 hours , washed with pbs , incubated with secondary antibody for 1 hour , washed again with pbs , and incubated with horseradish peroxidase - labeled streptavidin ( beijing zhongshan golden bridge biotechnology co. , ltd . ,
afterwards , the sections were washed with pbs , colored with dab ( beijing zhongshan golden bridge biotechnology co. , ltd . ,
pbs was used to replace primary antibody as the negative control . with regard to standards for the determination of immunohistochemical staining
, traf6 was determined to be positively expressed in the case of brownish - yellow precipitates in the cytoplasm .
the procedure was completed by two experienced pathologists . by using the double - blind method ,
15 fields of vision were selected for each section with a high - powered lens (  400 ) , and the ratio of the number of traf6-positive cells to 100 average cells was calculated as the positive rate .
positive results of immunohistochemical staining were determined according to the following two aspects : 1 ) percentage of traf6-positive cells : < 1% , 0 ; 1% - 10% , 1 point ; 10% - 30% , 2 points ; 30% - 60% , 3 points ; > 60% , 4 points , and 2 ) staining results of traf6-positive cells : negative , 0 ; yellowish , 1 point ; yellow , 2 points ; brownish yellow , 3 points . for the determination of results ,
tumor and paracancerous tissues resected during surgical procedures were immediately stored in liquid nitrogen , from which total rna was extracted by using trizol reagent according to the kit s instructions .
upstream primer for traf6 : 5-gaatcacttggcacgacactt-3 , downstream primer for traf6 : 5-gagtttccattttggcagtca-3 and length of amplified fragment : 227 bp .
upstream primer for gapdh : 5-accacagtccatgccatcac-3 , downstream primer for gapdh : 5-tgctgtagccaaattcgttg-3 and length of amplified fragment : 450 bp .
rt - pcr was performed under the following conditions : reverse transcription at 50c for 30 minutes , reaction at 95c for 15 minutes ( cycling was started ) , denaturation at 94c for 30 d , annealing at 57c for 30 seconds , and extension at 72c for 30 seconds , for a total of 30 cycles .
after final extension at 72c for another 10 minutes , the pcr products were subjected to agarose gel electrophoresis .
the numerical data were compared by  test , and the survival rates were estimated by the kaplan - meier method .
the he staining results for the tumor and paracancerous tissues are shown in figure 1 .
significantly more traf6 protein was expressed in tumor tissues than in paracancerous tissues . in tumor tissues ( figure 1 a - c ) ,
traf6 protein was mainly expressed in the cytoplasm , showing dark brown particles . meanwhile , tumor stromal cells were also positively stained . in paracancerous tissues
( figure 1 d - f ) , however , traf6 protein was mainly scattered in the cytoplasm , exhibiting light brown particles . in the meantime ,
traf6 was weakly positively expressed in the cytoplasm of normal tissues ( positive rate : 33.3% ) , which was significantly less than in tumor tissues ( positive rate : 66.7% ; p < 0.05 ) ( table 1 ) .
a - c , traf6 expression in tumor ; and d - f , paracancerous tissues .
a and d (  100 ) : he staining ; b and e (  200 ) , c and f (  400immunohistochemical staining .
tumor and paracancerous tissues all expressed traf6 mrna . the traf6 mrna expression level in tumor tissues
( 15.21  2.43 ) was significantly higher than in paracancerous tissues ( 4.23  1.13 ; p < 0.01 ) ( figure 2 ) .
the positive expression rate of traf6 was not correlated with the age or gender of the patients ( p > 0.05 ) , whereas it was correlated with dukes staging , degree of differentiation , and lymphatic metastasis ( p < 0.05 ) .
traf6 was highly expressed in patients with later stages , lower degrees of differentiation , and lymphatic metastases ( table 2 ) .
the expression of traf6 was negatively correlated with the 5-year survival rate ( figure 3 ) ; the patients in whom more traf6 was expressed survived longer .
the he staining results for the tumor and paracancerous tissues are shown in figure 1 .
significantly more traf6 protein was expressed in tumor tissues than in paracancerous tissues . in tumor tissues ( figure 1 a - c ) ,
traf6 protein was mainly expressed in the cytoplasm , showing dark brown particles . meanwhile , tumor stromal cells were also positively stained . in paracancerous tissues
( figure 1 d - f ) , however , traf6 protein was mainly scattered in the cytoplasm , exhibiting light brown particles . in the meantime ,
traf6 was weakly positively expressed in the cytoplasm of normal tissues ( positive rate : 33.3% ) , which was significantly less than in tumor tissues ( positive rate : 66.7% ; p < 0.05 ) ( table 1 ) .
a - c , traf6 expression in tumor ; and d - f , paracancerous tissues .
a and d (  100 ) : he staining ; b and e (  200 ) , c and f (  400immunohistochemical staining .
tumor and paracancerous tissues all expressed traf6 mrna . the traf6 mrna expression level in tumor tissues ( 15.21  2.43 ) was significantly higher than in paracancerous tissues ( 4.23  1.13 ; p < 0.01 ) ( figure 2 ) .
the positive expression rate of traf6 was not correlated with the age or gender of the patients ( p > 0.05 ) , whereas it was correlated with dukes staging , degree of differentiation , and lymphatic metastasis ( p < 0.05 ) .
traf6 was highly expressed in patients with later stages , lower degrees of differentiation , and lymphatic metastases ( table 2 ) .
the expression of traf6 was negatively correlated with the 5-year survival rate ( figure 3 ) ; the patients in whom more traf6 was expressed survived longer .
as a member of the traf family , traf6 consists of 530 amino acids , including a highly conservative traf6 domain at the c terminus and an activated coiled - coil domain at the n terminus .
in particular , the activated domain comprises a ring finger and five zinc fingers ( 6 ) . out of all of the trafs
, traf6 is the only member that directly binds to interleukin receptor - associated kinases , nf-b receptor activator , and cd40 . through the signaling pathways mediated by membrane surface receptors , such as lps , il-1 , tlr4 , and cd40 ,
traf6 is able to finally activate nf-b , activator protein 1 , and pkb / akt .
since traf6 can activate several signaling pathways simultaneously , it plays a crucial role in the growth , proliferation , differentiation , and death of cells ( 7 ) .
it was previously reported that traf6 dominantly controlled the onset , progression , invasion , and metastasis of tumors ( 3 , 8 , 9 ) .
traf6 has also controlled the proliferation and invasion of tumor cells in lung cancer and esophagus cancer tissues by regulating the activity of nf-b ( 3 , 10 ) , and it has been shown that inhibiting the activity of traf6 in multiple myeloma cells could suppress their proliferation and promote apoptosis ( 11 ) .
the expression of akt was shown to be significantly elevated in colon cancer tissues ( 12 ) , and inhibiting the activity of the akt enzyme could promote the apoptosis of tumor cells and suppress their proliferation ( 13 ) .
moreover , nf-b is highly expressed in 40% of colon cancer tissues , which is closely associated with the infiltration and metastasis of these tissues . however , inhibiting the activity of nf-b could suppress the proliferation of these cells .
therefore , we postulated that traf6 , as the key enzyme for the activation of akt and nf-b , may be highly expressed in colon cancer tissues .
compared with paracancerous tissues , the expression of traf6 in tumor tissues was significantly increased , suggesting that traf6 may play a critical role in the onset , development , invasion , and metastasis of colon cancer .
patients with dukes stage c or d , and those with lymphatic metastases , had significantly higher traf6 expression levels than those with stage a or b disease , and those without colon cancer .
in addition , the 5-year survival rate of the patients with positive traf6 expression was significantly lower than in those with negative expression .
hence , it is of value to detect the expression level of traf6 in colon cancer tissues in clinical practice . with regard to the mechanism behind the regulatory effect of traf6 on the onset and progression of colon cancer cells , sun et al .
found that after binding to hif-1 , traf prevented it from degradation by polyubiquitination linked with lys63 ( 14 ) . as a result ,
hif-1 protein was destabilized and induced to accumulate in cells , which controlled the proliferation of tumor cells and angiogenesis by regulating the expression of many genes .
although primarily the effects of traf6 on colon cancer have been studied , this disease s pathogenesis , and the possibility of treating it by down - regulating the expression of traf6 , should be further evaluated in animal models in order to provide valuable theoretical evidence for colon cancer therapy using traf6 interventions . in summary
, traf6 was abnormally expressed in colon cancer tissues , which was closely associated with the prognosis .
targeted traf6 therapy , as a novel protocol , may be effective for mitigating colon cancer .
 background : tumor necrosis factor receptor - associated factor 6 ( traf6 ) has been reported to be highly expressed in many tumor tissues , but it is still of value to detect its expression in patients with colon cancer and to clarify its clinical significance.objectives:to study the expression and clinical significance of traf6 in patients with colon cancer.patients and methods : a total of 135 patients with colon cancer who underwent surgery at our hospital from february 2008 to march 2013 were selected . 
 the expression levels of traf6 protein in tumor and paracancerous tissues were detected with the immunohistochemical method to evaluate their clinical significance.results:the positive expression rate of traf6 in tumor tissues was 66.7% , which was significantly higher than in normal paracancerous tissues ( 18.5% ) . 
 the positive expression rate of traf6 was significantly correlated with dukes staging , degree of differentiation , and lymphatic metastasis ( p < 0.05 ) , but not gender or age ( p > 0.05 ) . the expression level of traf6 was negatively correlated with the 5-year survival rate ; patients with high traf6 expression levels had significantly decreased survival compared to those with low levels ( p < 0.05).conclusions : traf6 plays an important role in the onset of colon cancer ; therefore , drugs targeting it may have great clinical significance in the treatment of this disease . 
1. Background
2. Objectives
3. Patients and Methods
3.1. General Information
3.2. Immunohistochemical and HE Staining
3.3. Detection of TRAF6 mRNA Expression in Tumor and Normal Tissues by RT-PCR
3.4. Statistical Analysis
4. Results
4.1. TRAF6 Expression in Tumor and Paracancerous Tissues
4.2. RT-PCR Results
4.3. Correlation Between TRAF6 Expression and Clinicopathological Characteristics, Staging of Colon Cancer
4.4. Correlation Between TRAF6 Expression and 5-Year Survival Rate
5. Discussion