Effects Of RNAi On PIDD In HT-29 Cell And The Changes Of Drug Sensitivity

Background & AimsThe activation of nuclear factor kappa B (NF-κB) is important in the development of tumorigenesis. Activated NF-κB plays a pivotal role in tumour formation,angiogenesis,metastasis and anti-apoptosis. The transcription of survival gene of tumour cell can be up-regulated with the activation of NF-κB , the activated NF-κB accounts for the phenomenon of acquired chemo-resistance that impedes effective cancer therapy.During the past years, numerous groups have contributed to our understanding of cell surface-receptor-induced NF-κB activation. In un-stimulated cells, NF-κB is sequestered in the cytoplasm through interaction with an inhibitory protein.Activated by some activators,free NF-κB translocated into nucleus and activated transcription of target genes. However, some investigation revealed recently the so-called'nuclear-to-plasmic'pathways might be more important in the activation of NF-κB for most of the chemotherapeutics can damage DNA directly,and the damaged DNA is just the activator of NF-κB. PIDD (p53-induced protein with a death domain) is one of the mediators of the'nuclear-to- plasmic'pathways. PIDD mainly located in cytoplasm,the full-length protein has a molecular mass of approximately 100 kDa. PIDD is constitutively processed giving rise to a N-terminal fragment containing the leucine-rich repeats (LRRs, PIDD-N) and a C-terminal fragment containing the death domain (DD,PIDD-C). Somebody found that PIDD-C enters into nuclear forms PIDDosome containing RIP1 (receptor-interacting protein 1) and NEMO (NF-κB essential modulator, also known as IKKγ) to promote SUMO-1 (small ubiquitin-like modifier)attachment on NEMO in response to genotoxic and perhaps other stresses.These modifications cause NEMO translocation from the nucleus to the cytoplasm, where it causes the displacement of IκB (which gets degraded). This leads to the release of free NF-κB in the cytoplasm, which then enters the nucleus to induce prosurvival gene transcription and makes cell resist to apoptosis. Even if it is important of PIDD in the process inducing appoptosis,there is no investigation about PIDD in drug sensitivity of colon carcinoma. We try to explain the effect of PIDD on colon carcinoma drug sensitivity and consummate the mechanism of colon carcinoma drug resistance .Methods1. Construction, transfection and detection of specific PIDD siRNABased on the sequence of human PIDD mRNA, we chose four gene loci and designed four PIDD siRNA by using the software of RNAi Designer for genome wide scanning and sequence homology analysis. A negative control with identical sequence but not targeting at PIDD was also included. Transfect efficacy was detected by fluorescence microscope after HT-29 cells were transfected 24,48,72 hours later respectively. Cell protein was extracted and Western blotting was employed to detect PIDD protein. The group with best transfect efficacy will be used in the following experimentation.2. Cultured HT-29 cells were divided into following 3 groups:A. blank group,B. negative control group,C. transfect group .All of these groups were incubated by 5-Fu.The cytoplasm and nuclei proteins of every group were extracted respectively. Western blotting was employed to detect PIDD protein. MTT was applied to detect the sensibility of HT-29 cell to chemotherapeutics. The activation of NF-κB was tested by EMSA (electrophoretic mobility shift assay). The apoptosis of cells was detected by FCM.Results1. The transfect efficacy of PIDD-360 showed a tendency to be higher than the other three siRNA, but no significant difference was observed. Fluorescence expression was observed at different time points after transfection, showing the efficiency of transfection all reached 90%. Western blot showed that PIDD in HT-29 cells decreased significantly after RNA interfering. The suppressive effect manifested as a time-effect relationship, namely: inhibitory effect could be detected 48h after transfection and reached peak at about 72-96h.2. Western blot showed PIDD distributed mainly in cytoplasm and with only a small quantity in nucleus while most PIDD translocated from cytoplasm to nucleus after being stimulated by 5-Fu in blank group and negative control group. However, in transfect group, the PIDD were found fewer both in cytoplasm or nucleus than the other two groups and had no changes after being treated with 5-Fu. 3. MTT assay showed that IC50 of transfect group was lower than that of other two groups with statistically significant difference(P<0.05), indicating the drug sensitivity was significantly increased in transfect group.4.EMSA showed the NF-κB activity of all these groups was negative before 5-Fu treatment,whereas the activity of NF-κB in blank group and negative control group became positive after being stimulated by 5-Fu.There was no change in transfect group.5. FCM showed the apoptosis rate of the three groups were similar without statistically difference (P>0.05). However, the apoptosis rate in transfect group increased significantly after being treated with 5-Fu(P<0.05).Conclusions1. The expression of PIDD-C in HT-29 could be effectively blocked by siRNA. The suppressing effect is significantly and manifested as a time-effect relationship.2. The nuclear PIDDosme causes the activation of NF-κB in HT-29 cell,which then induces prosurvival gene transcription and the process of anti-appoptosis.The drug sensitivity of HT-29 cell to 5-Fu decreased obviously.3. Interfering the express of PIDD-C in HT-29 cell can attenuate the PIDDosome in nuclear,leading to reduced activity of NF-κB,depressed pathway of anti-apoptosis and increased drug sensitivity to of HT-29 cell to 5-Fu.