Establishment And Reversion Of An Oxaliplatin-induced Human Colon Carcinoma Drug-resistant Cell Line With Its Transcriptome Analysis

Human colon cancer is the world’s third largest common tumors, most patients can be cured with surgery, colon cancer patients with recurrence always follow with poor prognosis, low 5-year survival rate,and poor life. Oxaliplatin is recommended into the first-line chemotherapy drugs, which can improve the patient’s 5-year survival rate and quality of life, but still many patients is not sensitive to the chemotherapy drug, it make its curative effect is severely affected, so the study of drug resistance in L- OHP has become an urgent problem in the clinic.For study the resistance mechanism, the stepwise increasing dose of L – OHP and intermittent administration methods was used to establish the HCT-8 / L –OHP cell line. Using Illumine technology to sequence the HCT- 8 colon cancer cells and HCT-8 / L –OHP, screening the differentially expressed genes and analysis by GO and KEGG. Finally using RNA interference technology to build KLK11 small interference RNA(KLK11- si RNA) slow virus expression vector and silencing KLK11 type cells validation with drug resistance gene reversal, thus validation and explore the oxaliplatin resistance mechanisms in cell and molecular level. Based on the above purposes, we will describe the study with the following three parts:PART ONE ESTABLELISHMENT AND REVERSION OF AN OXALIPLATIN-INDUCED HUMAN COLON CARCINOMA DRUG-RESISTANT CELL LINEProjective Establishment the human colon cancer cells of resisting oxaliplatin, to lay the foundation for the research of oxaliplatin resistant and find the target molecules associated with the resistance of oxaliplatin. Methods the stepwise increasing dose of L – OHP and intermittent administration method was used to establish the HCT-8/L–OHP cell line. Parent strains HCT-8/L–OHP cell line’s cell growth curve, cross resistance, and resistant index were determined by MTT method. Using light microscopy and electron microscopy to observe cell morphology of parental cells and drug resistant cells. Flow cytometry(FCM) method to observe the distribution of cell cycle and apoptosis. By adopting the method of RT-PCR to detect the expression of ABCC1(MRP or MRP1), ABCB1(P- gp), GSTP1, ERCC1, ANPEP, HOXB8 genes. Results Built the stable HCT-8/L-OHP cell model. Its IC50 was 58.21μmol/L, RI was 10.07. Morphological changes metabolic rate reduce Biological activity weakening were happened in HCT-8 cell.HCT-8/ L-OHP cells not only for L-OHP resistance, but also for 5-fluorouracil and cisplatin, vincristine, irinotecan. HCT- 8 cells acquired drug resistance induced by L-OHP in vitro induction. The logarithmic phase distribution of cell cycle analysis by FCM showed that HCT-8 / L-OHP’s G2/M phase cells increased S phase, GO/G1 phase cells decreased. Statistically significant is existing when compared with HCT- 8 cell and HCT-8 / L-OHP(P < 0.01). The total apoptosis rate of HCT-8/L-OHP cells was lower than that of HCT-8 cells at early and late stage. ABCC1(or MRP1 MRP), ABCB1(P-gp) and other genes related drug resistance show high expression in the resistant cell lines. Conclusion This study successful build HCT-8/L–OHP cell line model. In addition, ABCC1(MRP or MRP1), ABCB1(P- gp), ERCC1, ANPEP, HOXB8 genes expression elevated may have asoccisation with oxaliplatin resistance in the molecular mechanisms.PART TWO THE STUDY OF DIFFERENEC GENE OF AN OXALIPLATIN-INDUCED HUMAN COLON CARCINOMA DRUG-RESISTANT CELL LINEObjective To screen the differentially expressed genes. Analysis the genes which have significant differences on the expression of oxaliplatin into resistance to provide clues of colon cancer drug-resistant mechanism and theory of oxaliplatin into resistance and to further predict rectal cancer oxaliplatin resistance related molecular target to provide more evidence. Method Firstly, using Trizol Kit technology to extract the RNA, and treat c DNA fragments by PCR amplification. Secondly, Using Illumina technology to sequence the HCT- 8 colon cancer cells and HCT-8 / L –OHP, screening the differentially expressed genes and analysis by GO and KEGG. Trinity program was use to integrate the transcriptome and DE novo assembly. Finally using Blast2 GO WEGO and software to execute GO analysis and gene function annotation. Results Compared with HCT- 8 cell lines and HCT- 8 / L- OHP cell lines, depending FDR≤0.05 and |log2 Ratio|≥1,totally screened 1829 differentially expressed genes. There are 922 and 907 high and low expression genes respectively. Differentially expressed genes were enriching, which included 39 Biological processes metabolism and so on. Enzyme catalytic activity, regulation 35 molecular functions and so on, stress response(Molecular Function) and the Function of Cellular connection, membrane 16 cells(Cellular Component). Map the 35.9%(21) differentially expressed genes into the KEGG database found that the largest concentration of pathway is associated with cancer. A total of 56 differentially expressed genes. Additionally the enrichment of pathways involved different mechanism including P53 signal pathway, cell cycle, base mismatch repair, etc. Conclusion Successfully screened the HCT-8 cells and HC-8/L-OHP differentially expressed genes. Secondly L-OHP resistance of colon cancer cells may be associated with cancer of the pathway, P53 signaling pathways, cell cycle and base mismatch repair pathways involved in cancer treatment such as resistance mechanisms and related pathways of reverse. At the same time, the DNA damage repair pathways of MGMT, RFC5, PRIM1, MCM7, POLE, MCM3, MCM4, FEN1 and MCM6 associated gene expression, on the other hand, this study found the high expression of KLK11 may be associated with the drug resistance of oxaliplatin.PART THREE THE INFLUENCE OF CONSTRUCTION OF SLOW VIRUS KLK11-si RNA EXPRESSION VECTOR FOR REVERSING COLON CANCER CELL LIINES RESISTANTProjective Built slow virus carrier interference which the target genes is KLK11, and establish KLK11 defect type cell lines, in order to further specifically validate the correlation with the expression of KLK11 and the drug resistance of L-OHP in cellular and molecular level. Methods According to the design principle of sh RNA, designed three segments of interference against target genes KLK11, builds the corresponding slow virus expression vector, through microbial PCR and sequencing identification of recombinant plasmid, fluorescence and drug screening method to determine the best virus drops. According to the best drop degree of virus,using the three slow virus infect HCT- 8 / L- OHP cells to establish KLK11 defect type cell lines. With RT-PCR detect the gene expression of KLK11 after Interference the HCT-8/L OHP cells. Filter out the most efficient interference and suppression of KLK1-i RAN.Using Western blotting to detect the effect of KLK11 gene protein expression by the KLK11-si RNA which has highest inhibition efficiency. MTT method was used to detect the proliferation inhibition influence of HCT-8/L- OHP by KLK11-si RNA combination oxaliplatin. Adopting Western blotting detect the effect of KLK11-si RNA with the highest inhibition efficiency on the expression of KLK11 protein. The MTT method used for the determination of KLK11-si RNA combined with oxaliplatin on proliferation inhibition of HCT-8/L-OHP Using FCM analysis the cell cycle distribution before and after KLK11-si RNA infection HCT-8/L-OHP cells.Results The synthesis of 3 interference sequence groups successfully insert GV248 carrier. The construction of recombinant plasmid is successful. three Viral vector group were successfully infected 293 T cells, and infection rates were above 80%.The best virus droplet degrees of 3 slow virus expression vector groups were 3 x 108 TU/m L(LV- KLK11- RNAi(41637-1)), 5 x 108 TU/m L(LV- KLK11- RNAi(41638-1)), 1 x 109 TU/m L(LV- KLK11- RNAi(41640-1)) respectively. Compared with CON cell group, all the KLK11 cell interference group were shown as GFP positive, infection efficiency was almost 100%.Comparing HCT-8/L-OHP with the control cells and the negative control group, KLK11- RNAi(41637-1), the KLK11-RNAi(41638-1), the KLK11- RNAi(41640-1) three targets of KLK11 gene expression have significantly inhibitory effect(P < 0.01) in HT-29 cells.Among them, KLK11 m RNA expression quantity in the KLK11-RNAi(41638-1) set of cells was minimum.which has highest knock reduction efficiency(92.3%).KLK11-RNAi(41638-1) set of cells’ KLK11 protein expression level was minimum. Comparing the cells with the negative control group,the differences were statistically significant(P < 0.01).HCT-8/L-OHP cells of oxaliplatin IC50 = 54.72μmol/L.KLK11 silence HCT-8/L-OHP cells of oxaliplatin IC50 = 23.37μmol/L. inhibition rate of the two groups were compared under the action of the different concentration of L-OHP cell, the differences were statistically significant(P < 0.01).With the gradual increase of drug concentration, the proliferation inhibition effect of oxaliplatin was enhanced in HCT-8/L-OHP resistant cells and KLK11 silence HCT-8/L-OHP cell. There has a significant dose-effect relationship, and the inhibiting effect of oxaliplatin in cells of KLK11 silence is stronger than the HCT-8/L-OHP cells. When the concentration of L-OHP is 160 umol/L, almost completely inhibit the growth of the KLK11 silence cell.Cell cycle distribution have differences statistically significant between interference and noninterference cell(P < 0.01).Among them, the KLK11 silence HCT-8 /L-OHP cells’ G2 / M phase decreased, S phase and the GO/G1 phase cells increased. Conclusion The slow virus expression vector of KLK11-RNAi(41638-1) can effectively inhibit HCT-8/L-OHP cells express the KLK11 m RNA and KLK11 protein, improve the L-OHP inhibition rate in HCT-8/L-OHP. The resistance of L-OHP in colon cancer cell lines was reversed successfully. At the molecular level to further validate the high expression of KLK11 may be related to drug resistance of oxaliplatin...