Influence Of HCT-116 Colon Cancer Cells Chemo-sensitivity Mediated By Ubiquitination Of P21^WAF1/CIP1

Objective: To knock out p21waf/cip1 gene expression in HCT-116 cell line, and establish a HCT-116-p21-/- cell line. Compare the cancrous chemotherapeutic sensitivity difference in the HCT-116 and HCT-116-p21-/- cell lines. Compare the ubiquitination expression of p21waf/cip1 in the HCT-116 and HCT-116-p21-/- cell lines. To investigate the effection of p21waf/cip1 ubiquitination on the chemotherapeutic sensitivity in HCT-116 cell lines.Methods: 1. The si RNA technology was used to silence p21 gene expression in the HCT-116 cell line, and total RNA was extracted from HCT-116-p21-/- cells, the si RNA fragment of target region was amplified by RT-PCR, and the target gene sequences was analysed for single nucleotide polymorphism(SNP) that may existed, to avoid the inactivation of si RNA caused by SNP.2.The chemotherapeutic effections were indecued by 5-fluorouracil(5-Fu), oxaliplatin(L-OHP),irinotecan(CPT-11) on the HCT-116 and HCT-116p21-/- cells proliferation, were evaluated by the MTT method.3. Flow cytometry(Annexin V/PI staining) was used to detect apoptosis on human colon cancer cell line HCT-116 and HCT-116-p21-/- after treated with 5-fluorouracil(5-Fu), oxaliplatin(L-OHP) and irinotecan(CPT-11).4. Western blot method was used to detect the protein expression of MDM2, P53 and ubiquitin(Ub) in the HCT-116 and HCT-116-p21-/- cells, after the treatment of 5-fluorouracil(5-Fu), oxaliplatin(L-OHP) and irinotecan(CPT-11).Results: 1. The si RNA targeted gene fragments were sequenced, compared with BLAST sequence and no common SNP were detected. Western blot results showed that the expression of p21 was disappeared,it proved that si RNA had been silenced the gene p21 expression on HCT-116 cells2.MTT results showed cell proliferation changed between HCT-116 and HCT-116-p21-/- cells with significant difference,p<0.05. The OD of HCT-116 cell proliferation inhibitions were : 0.73±0.27( nc)、 0.34±0.19( 5-Fu)、 0.12±0.15(L-OHP)、0.23±0.16(CPT-11);The OD of HCT-116-p21-/- cell inhibition were: 0. 92±0.25(nc)、0.56±0.26(5-Fu)、0.35±0.11(L-OHP)、0.45±0.03(CPT-11); p<0.001, paired t-test,The Chemotherapeutic inhibition on the HCT-116 cell line was higher than the HCT-116-p21-/-.3. Annexin V / PI double stainingfor the HCT-116 cell apoptosis rate: 0.086±0.027(nc)、0.562±0.082(5-Fu)、0.372±0.075( L-OHP)、0.457±0.023(CPT-11); For HCT-116-p21- /- cells, apoptotic rate: 0.069±0.024(nc)、0.305±0.031(5-Fu)、0.377±0.083(L-OHP)、0.266±0.073(CPT-11); by paired t test, two groups apoptosis rate had significantly different, p<0.001, apoptosis on HCT-116 cells is more pronounced.4. Western Blot assay showed that: HCT-116-p21 cells expressed higher level p53 protein, compared with HCT-116 cells there was a statistically significant difference, p <0.05; HCT-116-p21- /- cells expressed a lower level of MDM2 protein, had a statistically significant difference, p<0.05; Ub ubiquitin protein expression was no significant difference between the two. After the using of chemotherapy drugs, HCT-116-p21- /- and HCT-116 cells compared with the control group: P53 protein expression was up-regulated, compared with the control group(not chemotherapy drugs were used), there are significant differences; and after treated 5-Fu the MDM2 protein expression levels were rising in the two groups, while the MDM2 protein level decreased after using CPT-11, the difference was statistically significant; the express of MDM2 increased after treated with L-OHP in HCT-116,while it went down in HCT-116-p21- /- cells.Ubiquitin protein levels had no statistically significant between HCT-116 and HCT-116-p21- /- cells.Conclusion: 1. p21 deficiency in human colon cancer cells showed less sensitive to chemotherapy, while the expression of p21 could enhance the sensitivity of human colon cancer cells to chemotherapeutic drugs.2. The activation of p53 signaling pathway is the main mechanism for chemotherapy in human colon cancer cells, and p21 expression could enhance the activation of p53 signaling pathway.3. Ubiquitination of p21 was mainly dependent on MDM2 protein, p21 ubiquitination is also one of the important mechanism regulating cancer cell sensitivity to chemotherapy, while the role of ub protein in regulating p21 still remains to be further defined.