Abstract Objective To investigate the transketolase-like gene TKTL1 role in the development of human colon cancer. Method using real-time quantitative PCR detection of colorectal cancer and paraneoplastic organizations transketolase-like gene TKTL1 mRNA expression levels, continuous monitoring of total transketolase activity detected by the organizations. Results TKTL1 mRNA expression in colon cancer tissue than adjacent tissues significantly enhanced and enhanced invasive colon cancer than non-invasive colon cancer expression. Similarly, colon cancer tissue total transketolase enzyme activity in the high (P <0.01) than the adjacent tissues, invasive colon cancer compared with non-invasive colon transit ketone activity increased (P <0.01). Conclusion TKTL1 mRNA expression in human colon cancer development are closely related.
Key words colon cancer; transketolase-like gene 1; quantitative real-time polymerase chain reaction
Expression of TKTL1 mRNA in Colon Cancer and Paracancer Tissue
YANG Shu xiong1, ZHENG Li duan2, YANG Ju hong2
1.Department of Pathology, The People’s Hospital of Songzi City in Hubei Province, Songzi 434200, China; 2.Department of Pathology, Union Hospital of Tongji Medical College, Huazhong University of Science & TechnologyAbstract: Objective To explore the effect of TKTL1 on occurrence and development of colon cancer. Methods Real time PCR was used to determine the mRNA expression of TKTL1 gene in human colon cancer and para cancer tissue. Continuous monitoring assay was used to determine total transketolase activity in the human colon cancer and para cancer tissue. Results The expression of TKTL1 gene was significantly higher in the colon cancer tissues than para cancer tissue. Further more, the expression of TKTL1 gene was significantly up regulated in the invasive colon cancer compare with the noninvasive colon cancer. The total transketolase activity significant increase in the colon cancer tissues compare with para cancer tissue. The total transketolase activity significant increase in the invasive cancer tissue compare with the noninvasive colon cancer. Conclusion Expression of TKTL1 mRNA is related to the occurrence and development of human colon cancer.
Key words: Colon cancer; Transketolase like gene 1 (TKTL1); Real time PCR
Colon cancer is a common human malignancies, its causes are not fully understood, it may be due to a combination of factors. Recent studies have found that some malignant cells in the presence of metabolic anomalies, especially abnormal enhancement of glucose metabolism . The recent development of positron emission tomography technique (PET), the principle of the application is to monitor the changes in the metabolism of tumor cells by tracer – Major changes in glucose metabolism to the early diagnosis of tumor. In this study, using real-time quantitative PCR detected 65 cases of colon cancer and adjacent tissue transketolase-like gene the 1 (transketolase like gene 1, TKTL1) expression, continuous detection and observation of the organizations of the transit the ketone activity change, explore the TKTL1 role in the development of colon cancer.
1 Materials and Methods
1.1 Clinical data
In this study, 65 patients with colon cancer tissue and corresponding paraneoplastic organization from January 2005 to January 2006 in Wuhan Xiehe Hospital surgical resection specimens, including 11 cases of non-invasive colon cancer (carcinoma in situ), invasive colon cancer 54 cases of cancer tissue taken from the edge of the tumor 5cm outside the area for the naked eye under “normal" tissue after the tissue removal immediately placed in -70 ℃ refrigerator spare; according to the TNM classification of the International Union Against Cancer (1997) 54 cases of invasive colon cancer is divided into: Ⅰ 12 cases, Ⅱ 13 cases, Ⅲ of 13 cases, Ⅳ of 16 cases. All patients, 34 males and 31 females; youngest patient was 36 years old, maximum 79 years, the median age was 56 years, with an average of 60.3 years of age, and all patients without preoperative chemotherapy or radiotherapy.
1.2 Reagents and Instruments
D 5 ribose disodium salt, 5 xylulose phosphate, glyceraldehyde phosphate iso purchased enzyme and NADH (Sigma, USA); Trizol (Invitrogen Corporation, USA); ReverTraAce α TM reverse transcription kit (Japan Toyobo Company); exam brilliant blue G 250 (United States Amresco Company); Taq enzyme (American MBI); ELX-800 microplate reader (the U.S. BIO TEK); UV 1601 UV spectrophotometer (Shimadzu Corporation, Japan) ; nucleic acid electrophoresis instrument (Beijing, China sixty-one Instrument); Lightcycler the fluorescence PCR instrument (the Swiss company Roche).
1.3 Total RNA extraction and cDNA synthesis
Colon cancer tissue and cancer tissue taken at -70 ℃ and storing each specimen was cut about 50mg into the homogenizer, add 1ml TRIzol homogenate extraction of total RNA from cells, then the mRNA by reverse transcription kit transcribed into cDNA, synthetic system, reaction conditions, with reference to the instructions in the kit, and the system are as follows: RNase Free H2O 6μl, 5 × by RT of Buffer 4μl, dNTPs, (10mmol / L) 2 μl, 1 μl of RNase Inhibitor (107U / L), a random primer (25μmol / L) 1μl, RNA 5μl, Rever TraAce (2 × 108u / L) 1μl, the total volume of 20μl; reaction conditions: 30 ℃ 30min, 42 ° C 20min, 99 ° C 5min, 4 ° C 5min.
1.4 Real-time quantitative RT PCR
Real-time fluorescence quantitative of TKTL1 gene amplification in the the Lightcycler fluorescent PCR instrument amplification conditions: 94 ° C denaturation 5min; followed by 40 cycles of 94 ℃ for 5s, 57 ℃ annealing 5s and 72 ° C extension 10s fluorescence detection. And then the melting curve analysis, and rose to 95 ° C to 0.2 ° C / s from 65 ℃; finally cooled to 40 ° C, and to record the melting curve (Figure 1) and the Ct value of each gene, and the ultimate expression of each gene amplification results with 2 – △ △ ct relative quantification method . The formula is as follows: △ Ct = Ct (target)-Ct (β actin)] △ △ Ct = △ Ct (target) – △ Ct (control) mRNA relative amount = 2 – △ △ Ct. Transketolase (TKTL1) gene amplification primer required Reference Document : 5 ‘ TAA CAC CAT the GAC GCC TAC TGC 3’; 5 ‘ CAT CCT AAC AAG CTT the TCG CTG 3’, amplification product fragment of 150bp. β actin gene amplification primers required based on the gene sequences in the Genbank application software primer premier 5.0 design, the sequence is as follows: 5 ‘ GTG CGT GAC ATT AAG GAG 3’ (upstream); 5 ‘ CTA AGT CAT AGT CCG CCT 3 ‘(downstream), the amplification product fragment was 520bp.
It showed primer dimmer that the melting out temperature (80.6 ℃) of spinnacle is low. It show objective production that the melting out temperature (84.8 ℃) of spinnacle is high
Figure 1 melting curve
Fig 1 Solubility curve
1.5 Determination of transketolase activity
Preparation of tissue extracts: each group with a homogenizer was homogenized, and the cells of cold lysis buffer was added after washing with PBS solution (50 mM Tris / HCl, 150 mM NaCl, 1% Triton X 100,100 μg / ml PMSF in 5μg/ml Leupeptin, 5μg/ml Aprotinin), 0 ° C cracking 30min, 15min, 10 000r/min centrifuged supernatant as transketolase activity was measured of samples. Determination of transketolase activity: The enzymatic activity measurement reagent containing 50 mM Tris / HCl, 2mM 5 of 5 ribose, 1mM 5 xylulose phosphate, 5 mM MgCl2, 0.2u/ml the TPI, 0.2 mm NADH and 0.1mM the TPP. Transketolase determination method according to the literature . The protein concentration was determined by the method of Bradford tissue extract samples. The above experiment was repeated three times, The results are the average of three experiments.
1.6 statistical methods
Statistical analysis using statistical software SPSS10.0, each group should analyze independent samples t test, P <0.05 for the difference was statistically significant.
The 2.1 TKTL1 expression in colon cancer tissue
The groups organizations TKTL1 gene mRNA expression using real-time quantitative PCR using 2 – △ △ ct calculated relative quantification method in the relative amount of TKTL1 mRNA expression in colon cancer tissue relative to adjacent tissues. Adjacent tissues, 1.24 ± 0.11, non-invasive colon cancer was 3.26 ± 0.18, invasive colon cancer was 5.82 ± 0.39. Statistically the results show TKTL1 in colon cancer tissue than cancer tissue expression was significantly increased (t = 5.38, P <0.01), and invasive colon cancer than non-invasive colon cancer increased expression (t = 4.27, P <0.01 ). The amplification products from each organization TKTL1 agarose gel electrophoresis analysis chart, you can see the same results, as shown in Figure 2.
The 2.2 organization transit ketone activity of
The transketolase activity generated per minute amount of the product (ng) and the ratio of total protein (mg) of the cell. The above experiment was repeated 3 times, the average results of three independent experiments, each the tissue transit ketones the Average of enzyme activity are shown in Table 1. Table data suggest that human colon cancer (invasive and noninvasive) were converted to ketones enzyme activity compared to adjacent tissues markedly enhanced and the invasive colon tissue transit ketone activity than noninvasive. These results indicate that transketolase activity play an important role in colon cancer development. Table 1 colon cancer and adjacent tissues transit ketone enzyme activity
Non-invasive colon cancer tissue transit ketone activity of cancer tissue compared, P <0.01; invasive colon cancer tissue transit ketone next to the activity of cancer tissue compared, P <0.01; invasive colon cancer tissue transit one enzyme activity Compared with non-invasive colon cancer tissue, P <0.01
The present study, the malignant proliferation regulatory genes as a whole or by the damage caused by a complex phenomenon of gene function . The rapid growth and proliferation of the malignant cells require large amounts of energy and nucleic acid synthesis and nucleic acid synthesis, mainly from the non-oxidative portion of the pentose phosphate pathway in glucose metabolism, transketolase is a rate limiting enzyme in the non-oxidative portion of the pentose phosphate pathway. Boros et al  nucleotide synthesis necessary ingredients – more than 85% of the ribose directly or indirectly from the PPP pathway. Cascante et al  on the characteristics of tumor cell metabolism analysis showed: the transketolase activation of agents (thiamine) by adjusting the transketolase activity increased nucleotide ingredients – ribose synthesis, and nucleotide synthetic tumor cell survival and resistance to chemotherapeutic drugs and their growth and proliferation necessary transketolase inhibitors contrast.
The human genome currently known transketolase gene family the TKT, TKTL1 and TKTL2,. Langbein et al , a number of malignancies TKTL1 average at the mRNA and protein is upregulated TKT and TKTL2 not raised, and TKTL1 upregulation of bladder cancer prognosis. , Staiger  found, TKTL1 the upregulation is a common phenomenon in gastric, he to think TKTL1 as a new target for gastric cancer treatment research. V lker  that TKTL1 overexpression laryngeal prognosis. 65 cases of colon cancer and its corresponding paraneoplastic organize TKTL1 the expression we used quantitative real-time PCR assay results showed significantly higher expression in TKTL1 in colon cancer tissue than adjacent tissues, and the invasion of invasive colon cancer than non- colon cancer increased expression. The organizations interim ketone activity detection, total colon tissue transketolase activity than paracancerous organization the high invasive colon cancer compared to non-invasive the colon transit ketone activity increased. These results suggest that TKTL1 high expression may be related to the development of malignant tumors.
Since TKTL1 high expression of malignancy related to the occurrence and development, whether through the inhibition of the expression of TKTL1 of transketolase activity decreased, thereby inhibiting tumor cell nucleic acid synthesis, ultimately inhibit tumor cell growth and proliferation? Hu et al  found that the human hepatoma cell line (HepG2) TKTL1 the high expression, TKT and TKTL2, expression is not raised, the use of small interfering RNA technology to inhibit the expression after the TKTL1 total cell transketolase activity was significantly reduced, and cancer significantly inhibited the growth and proliferation. Zhang et al  in the human colon cancer cells (LoVo cells) also give similar results. These results illustrate TKTl1 total transketolase activity play a major role to play an important role in the growth and proliferation of the malignant cells.
Short, TKTL1 high expression may play an important role in the development of colon cancer, we will be more tumors study TKTL1 expression to believe TKTL1 become a new target for cancer treatment research.