Screening Of ADAR1 Interactors And Detection The Complex Affects MiRNA Processing

ADAR1(adenosine to inosine acting on RNA enzyme 1) is one of the family members of A-to-I RNA editing enzyme. ADAR1 converts adenine to inosine on double stranded RNA. ADAR1 interacts with double stranded RNA binding proteins, which involved in the corresponding of signaling pathways. ADAR1 interactors have two classifications. One is dependent on RNA editing such as PKR. Another is independent on RNA editing to interact with ADAR1, such as NF45, NF90 and Dicer. The interaction between Dicer and ADAR1 promotes mi RNA processing. Various evidences indicate that ADAR1 and its interactors play an important role in biological processes.Hepatocellular carcinoma is one of high incidence tumors in China. At present, the effect of ADAR1 and its interactors on mi RNA processing in hepatocellular carcinoma(HCC) has not been reported. The let-7 family is overexpressed in HCC, which may be a biomarker for HCC. Some members of let-7 family are edited by ADAR1.The other is not dependent on RNA editing, it is the interaction between Dicer and ADAR1 to promote mi RNA processing. On the basis of this, we used proteomics technology to investigate the interactors of ADAR1 at first. Then we discussed the influence of the complex influenced on mature mi RNA processing. Finaly, we detected cells migration of HCC.Objective We had screened and detected the interactors of ADAR1. Then, we studied the correlation of ADAR1 interactors and clinico-pathological features of HCC. Finally, we preliminarily explore the effect of ADAR1 and interacting protein in hepatocellular carcinoma(HCC) of miRNA processing and cell migration.Methods1. Construction the ADAR1 overexpression vector: p GC-LV-ADAR1 vector was constructed and the correctness of the vector was identified by PCR and sequencing.2. Establishment of ADAR1 overexpression lentivirus: after identifing correctness of the vector, we packaged lentivirus, transient transfected 293 T cells, and then used q PCR to verify the lentivirus titer and m RNA expression.3. Construction of ADAR1 stable expression cell lines: ADAR1 overexpression lentivirus was stably transfected to normal human embryonic liver L02 cell line. The L02-ADAR1 stable transfection cell line was obtained after being screened, and detected ADAR1 m RNA and protein expression levels by q PCR and Western blot.4. Proteomics technology and bioinformatics analysis With co-immunoprecipitation(Co-IP) technology and mass spectrometry analysis,we mainly made use of Label free method to detect ADAR1 interactors. And biological information system analysis was used to obtain a novel ADAR1 interactor, which is DDX17. ADAR1 and DDX17 proteins seemingly colocalized both in the cytoplasm and nucleus by immunofluorescence.5. Western blot Expression of ADAR1/DDX17 proteins were detected the in nine hepatocellular carcinoma(HCC) cell lines by Western blot.6. Immunohistochemical staining.Anti-ADAR1 and anti-DDX17 stained consecutive cuts of hepatocellular cancer tissue in eight cases of patients with HCC and adjacent tissues by immunohistochemical method.7. Nano String The mi RNAs regulated by ADAR1 were screened, and target mi RNAs were detected by Nano String.8. si RNA ADAR1 and DDX17 interference sequence had been designed, and si RNA was used to transient transfection in MHCC97-H, L02 and ADAR1 stable expression cell lines to investigate the effects of ADAR1/DDX17 on mi RNA processing in cell lines.9. Transwell assay.Endogenous ADAR1/DDX17 expressions were knockdown in MHCC97-H by transfecting si RNA, expression of ADAR1/DDX17 decreased at protein level firstly.Preliminary exploration showed that down-regulating ADAR1/DDX17 inhibited migration of MHCC97-H cells by transwell assay.Results1. Successful construction of ADAR1 stably transfection in L02 cell lines: GV-166 vector was linearized, ADAR1 primer was designed, synthesized, and amplified by PCR. The GV-166 vector was right by sequencing and PCR. Then this plasmid vector was transfected into 293 T cells. Virus titer was determinated at 10. The lentivirus was stably transfected in L02 cells.24 h after infection, puromycin was added to the media at2mg/ml, and cell populations were selected for 2 weeks. The expression of ADAR1 was detected in stable cell lines by Western blot and q PCR. We found that the expression of ADAR1 in L02-ADAR1 cell line was about three times higher than in L02-control cell line. Finally, L02-ADAR1 stable cell lines were obtained.2. Identification the interaction between DDX17 and ADAR1: ADAR1 interactors were screened to find the target protein helicase DDX17 by using proteomics approach.The complex of ADAR1/ DDX17 was validated by co-immunoprecipitation. ADAR1 and DDX17 were colocalized in cells by immunofluorescence.3. ADAR1 and DDX17 were overexpressed in hepatocellular carcinoma(HCC):The expression of ADAR1 and DDX17 proteins overexpressed in HCC cell lines by Western blot. ADAR1 and DDX17 proteins also overexpressed in HCC tissues by immunohistochemistry. Because the sample was not enough, we will expand the HCC sample to analyze the clinic pathological features and statistical analysis.4. Screening the differential expression mi RNAs: 236 mi RNAs were detect by Nanostring.The screen principle was that fold change less than 1.5 was discarded.According to the selection principle, 37 mi RNAs were up-regulated; and 45 mi RNAs were down-regulated. Combining with reviews and studies before, 4 mi RNAs were screened: let-7c-5p, let-7e-5p, mi R222-3p and mi R27a-3p.5. The complex of ADAR1/DDX17 had no effect on mature mi RNA processing by si RNA and q PCR. Therefore, we will explore the influence of this complex on pri-mi RNA and pre- mi RNA processing in the next step.6. Knockdown ADAR1/DDX17 decreased MHCC97-H cells migration by transwell assay. In the next, this part of results will be supplemented.Conclusion1. The construction of ADAR1 overexpression vector was successful.2. L02-ADAR1 stable overexpression cell line was successfully established.3. A novel ADAR1 interactor was screened, which is DDX17.4. The proteins ADAR1 and DDX17 colocalized in the cytoplasm and nucleus.5. DDX17 and ADAR1 formed a complex by co-immunoprecipitation.6. ADAR1/DDX17 proteins were overexpression in HCC tissues and cell lines.7. ADAR1/DDX17 had no significant effect on mature mi RNAs in cells.8. Knockdown ADAR1/DDX17 inhibited migration of hepatocellular carcinoma cells.