Study On ERCC1 Expression And Single Nucleotide Polymorphism Related To Colorectal Carcinoma Resistance To Oxaliplatin

Study on ERCC1 Expression and Single Nucleotide Polymorphism Related to the Colorectal Carcinoma Resistance to OxaliplatinIntroductionColorectal cancer is one of the most common malignant tumor with high incidence and mortality rate all over the world. In China, new cases of colorectal cancer increased in recent years. Incidence and development of colorectal cancer is related to family genetic background, diet habit and balance of intestinal microbiota. Usually some patients in earlier stage of colorectal carcinoma were cured by surgery, but most of patients developed metastasis and recurrent disease need a comprehensive treatment including chemotherapeutic agents. Oxaliplatin is new antitumor platinum as a chemotherapeutic agent to treat colorectal cancer. However whether Oxaliplatin is used alone or in combination, its objective response rate is closed to 20-55%. Unfortunately, no predictive index of response to them is yet available in clinical treatment.Cellular toxicity of Oxaliplatin is mainly due to specific platinum DNA adducts which induced cross-links in DNA single or double strands, inhibit DNA replication and end in cell death.. Therefore cell resistance to Oxaliplatin is normally related to it inherent DNA damage repair activity. Oxaliplatin carries a 1,2-diamino-cyclohexane ring (DACH) leading to DNA damage by forming DNA-platinum adducts with guanines In contrast to cisplatin, Oxaliplatin-induced adducts are recognized or processed by being predominantly repaired by the nucleotide excision repair pathway. About 30 identified polypeptides that participate in the nucleotide excision repair process, Excision Repair Cross Complementation group 1 (ERCC1) protein has a crucial role, in the incision process by stabilizing the XPF endonuclease. Deficiency in ERCC1protein will affect DNA repair activity in cell and induce high sensitivity to Oxaliplatin.Two common single nucleotide polymorphisms(SNPs) of ERCC1 have been reported. A synonymous polymorphism at codon 118 converting a common codon usage(AAC) to an infrequent one(AAT), both coding for asparagine, The codon 118 C/T polymorphism is associated with differential mRNA levels of ERCC1 and has been found to be a predictive factor for tumor response to platinum chemotherapy in patients with advanced colorectal cancer.In our study, we compare the relationship between ERCC1 expression level in colorectal tumor or in eolorectal carcinoma cell line and the sensitivity to Oxaliplatin in the first place. And then as a cell model contrast, we use CHO cell line AA8(wild type) and UV20(mutant type and ERCC1 defective cell line) to study the toxicity mechanism to Oxaliplatin through the established method to test cell toxicity and DNA damage repair. In addition, we create several plasmids containing different genotype of ERCC1 codon 118 SNPs and transfect to UV20 cell line. The transfected cells were selected for further testing ERCC1 expression and DNA damage repair cability and sensitivity to Oxaliplatin. Therefore, our purpose is finding a biomarker of Oxaliplatin treatment in advanced colorectal cancer and selecting optimization individual treatment in order to improve security and efficienty of Oxaliplatin. In addition, we attempt to elucidate the toxicity mechanism of Oxaliplatin, also we establish a experimental technology to study on the polymorphism of DNA damage repair gene. It will be benefit to overcome the problem about resistance of Chemotherapeutic agents and important to enrich related research field.Methods1. Materials(1) Colorectal carcinoma tumor preparation: Collect tumor sample in the process of operation. Fresh tissue was divided into two parts, one was for RNA isolation and the other part was for primary carcinoma cell culture. Patients were record natural condition such sex, age, clinical stage and pathology type of colorectal tumor.(2) Primary colorectal carcinoma cell culture: Regularly prepare the cells suspend solution, adjust cell density with RPMI-1640 culture medium containing 10% FCS, and seed in 96-well culture plate. No contamination cells for further test.(3) Cell line and cell culture. Five colorectal carcinoma cell lines including LOVO, Colo320, HCT81, DLD-1, SW48 and CHO cell line including AAS, UV20, grew in DMEM culture medium containing 5% FCS, Penicillin and Streptomycin.(4) Gateway_TM cloning Technology to create ERCC1 expression vector. According to Bacteriophage lambda Recombination BP reaction, an Entry clone: pDONR-ERCC1(C/C or T/T) containing ERCC1 target gene was established. Pstâ… and PSQ confirmed ERCC1 sequence. Express clone: pIRES-GFP-ERCC1(C/C or T/T) was also established through LR recombination.(5) pIRES-GFP-ERCC1(C/C or T/T) transfect UV20 cell and get stable transfect cell line. pIRES-GFP plasmid as a empty control. All transfected cells can grow in DMEM containing 10% G418 and express Green Fluorescent Protein(GFP). Confirm the genotypes.2. Experimental Methods(1) RNA isolation in tumor and cell line and eDNA synthesis. TRIZOL orβ-Mercaptoethanol lyse cells and Isopropanol extract total RNA. cDNA synthesis reaction system 20μl include 1μg RNA, random primer, dNTP, RNA inhibator and 5×reverse buffer.(2) ERCC1 mRNA level measurement in colorectal tumor and cell lines. Reverse transcriptase PCR(RT-PCR) expand about 894bp ERCC1 target segment in tumor tissue, agarose electrophoresis distinguish the target gene segment. ERCC1 mRNA level was compared according to light density. ERCC1 mRNA level in colorectal cancer cell line were measured by probe method through 7500 Taqman Real-time PCR. Endo-reference gene such as GAPDH was measured by SYBR green. (3) ERCC1 protein level in transfected cell lines were measured by Western blot. The total protein extracted by frozen and melting method, SDS-PAGE electrophoresis test ERCC1 protein expression. 1st Antibody is dilution 1:1000. 2nd Antibody is diluted as 1:10000. Regularly make gel, transfer membrance, blotting, ECL reaction and exposing films.(4) Pyrocequencying for ERCC1 SNIPs genotype. A 80bp PCR product containing codon 118 SNP were checked by Pyrocequencying and analysed by SQA software.(5) Measurement of cell inhibition rate to Oxaliplatin (MTT and SRB method) According to the experimental requirement, we seed cells in 96-well plate at a certain cell density for 16-24h attachment. Oxaliplatin treatment for another 24h, change medium and continue cell culture for another 72h. Cell inhibition rate of colorectal tumor to Oxaliplatin was measured by MTT, and sensitivity to Oxaliplatin of all cell lines was measured by SRB Assay.(6) DNA damage test: the Modified Comet Assay. According to the experimental requirement, we seed cells in 6-well plate at a certain cell density for 16-24h attachment, 195, 3125nM Oxaliplatin treatment for 1h, Change Medium. Cell culture for another 6h and 24h. After 30μM H₂O₂ induce DNA single strand break, Cells are collected, then Making slides, Lysis, Denaturing, Electrophoresis, Neutralization and EB staining, counting and evaluation under Fluorescensis Microscope.(7) Rad51 Immunity Fluorescence test for DNA damage repair ability. According to the experimental requirement, we seed cells in 24-well plate at a certain cell density for 16-24h attachment. 195, 3125nM Oxaliplatin treatment for 1h. After fixation, 1st Antibody of Rad51 diluted 1:1000 and incubated for 90 min, washing with PBS and 2nd antibody incubate for another 60min, DAPI staining. Counting FOCI and evaluation under immunity Fluoresensis microscope.3. Data analysisSPSS 11.5 software used for data analysis. Statistics descriptive as Mean±SD. Statistics inference including Pearson Correlation Analysis, Spearman Rank Correlation Analysis, ANOVA, Kruskal-Wallis according to data Normality and Variance equality.Results1. The mRNA level in colorectal tumor checked by Reverse transcriptase PCR(RT-PCR)has a positive relationship with primary culture cells IC₅₀ to Oxaliplatin through Spearman Rank Correlation analysis(r_s=0.711, P=0.032). ERCC1 mRNA level in colorectal cancer cell line were measured by probe method through 7500 Taqman Real-time PCR. Similar to the results in tumor tissue(before Oxaliplatin treatment r=0.910, P=0.032; after Oxaliplatin treatment r=0.907, P=0.037) Therefore, whether colorectal tumor or colorectal cancer cell line, Cell sensitivity to Oxaliplatin decreased following the increasement of ERCC1 level.2. Comparation between before and after 195nM Oxaliplatin treatment ERCC1 mRNA level have no significant difference through the Quantitive software analysis of 7500 Real-time PCR.3. DNA damage caused by Oxaliplatin and DNA damage repair ability in cell related to ERCC1 were measured by the Comet Assay. Whether AA8 or UV20, DNA damage tend to be serious following the increase of Oxaliplatin Concentration. Compared with AA8, UV20 expressed more serious DNA damage whether for 3125nM or 195nM Oxaliplatin. AA8 showed a perfect repair DNA system at 24h checkpoint compared with ERCC1 defective cell line.4. Rad51 immunity fluoresense test show a significant difference between AA8 and UV20 at 6h and 24h check point. DNA damage break caused by 3125nM Oxaliplatin in UV20 had much more FOCI than AA8 at 24h checkpoint. But at 6h, the difference in those cell lines had no significance.5. ERCC1 entry vector pDONR-ERCCI(3150bp) selected by 1% kanamycin, got 533bp and 2617bp expected fragment after restricted enzyme Pstâ… incubation. On the other hand, ERCC1 expressed vector pIRES-GFP-ERCC1(6626bp) selected by 1% Ampicillin, got 1227 and 5039bp expected fragment by the incubation of Hindâ…¢, or got 891 and 5735bp expected fragmant by the incubation of Bglâ…¡. Examination the genotype of the constructed plasmid, and we got the right DNA neucleotide genotypes.6. pIRES-GFP-ERCC1(C/C) and pIRES-GFP-ERCC1(T/T) transfected to UV20 cell line meanwhile pIRES-GFP as a negative control. Transfected cell line can grow up in 10% G418 DMEM medium, next day we change 5%G418 DMEM. After 5 days, all transfected cell line can show green fluorencesis protein(GFP) under immunity Fluorecensis Microscope.7. ERCC1 protein expression in all cell lines checked by Western-blot analysis. 38KD ERCC1 protein can be seen in transfected cell line UV20-ERCC1(C/C) and UV20-ERCC1(T/T), confirmed the successful transfection and ERCC1 expression protein.8. Check the genotype of different cell line is C or T by PSQ 96 MA analysis.9. ERCC1 mRNA level in all cell line including transfected cell line, ERCC1 transfected cell line show a high ERCC1 expression similar to AA8. But ERCC1 defective cell line UV20 and UV20-GFP showed a low level and had no significant difference between each other. ERCC1 level in UV20-ERCC1(C/C) and UV20-ERCC1(T/T) had no difference compared with each other.(One way ANOVA, LSD, P=0.479.)10. Experimental results of SRB cell inhibition test and clonogenetic assay relected cell toxicity to Oxaliplatin. Higher sensitivity to Oxaliplatin was been seen in ERCC1 defective cell line UV20 and UV20-GFP, and had a 16 times IC₅₀ drop comparing with AA8. ERCC1 Over-expression cell line UV20-ERCC1(C/C) and UV20-ERCC1(T/T) had a 32 times higher than UV20. In addition, there was no significant difference of Oxaliplatin incubation rates in two ERCC1 transfected cell lines. Clonogenitic Assay confirmed the above result. For 3125nM Oxaliplatin, there was about 50% cell inhibation rate in AA8 and two ERCC1 transfected cell line. For 781nM, there was about 80% cell alive rate in AA8 and two ERCC1 transfected cell line, and until 195nM, UV20 and UV20-GFP had a about 45% cell alive rate. As the two ERCC1 transfected cell lines were concerned, there was no significant difference in all different concentration Oxaliplatin treatment.11. Comet assay showed a high DNA damage repair ability in all transfected cell line because of ERCC1 over-expression. It seems no significant difference in two different genotype transfected cell lines UV20-ERCC1(C/C) and UV20-ERCC1(T/T). Rad51 test also confirmed the above results that DNA strand break focus dot(FOCI) standing for the DNA damage center showed no difference in two transfected cell lines comparing each other at 24h check point after 1h Oxaliplatin treatment.Conclusions1. There is a positive relationship between antitumor resistance of Oxaliplatin and inherent ERCC1 expression level in colorectal tumor or cell lines.2. Oxaliplatin acts its cell toxicity as a DNA cross-links agent and relates to the DNA damage repair ability in cells. Protein ERCC1 is a key element in DNA damage repair process in cells.3. Construction of Plasmid containing ERCC1 different SNPs by the Gateway Technology and transfection to UV20 cell line successfully provide a technique platform to study on the polymorphisms of different DNA damage repair genes.4. ERCC1 codon 118 SNPs didn't show any difference in expression of ERCC1, DNA damage repair ability and sensitivity to Oxaliplatin.