Screening And Identification Of Effective MicroRNAs Of Hedgehog-Glil Signaling Pathway In Pancreatic Cancer

PartⅠ Screening effective mi RNAs of nuclear transcription factor Gli1 of Hedgehog signaling pathway in pancreatic cancerObjective: Affymetrix mi RNA3.0 and Affymetrix HTA2.0 microarray techniques are used to detect total RNA extracted from pancreatic cancer cells for candidate effective mi RNAs of nuclear transcription factor Gli1 of Hedgehog signaling pathway by differential screening. And on this basis,we attempt to acquire the target gene sets of the candidate effective mi RNAs.Methods: The RNA samples are respectively from pancreatic cancer SW1990 and Panc-1 cells. The experimental groups are SW1990 cells transfected with the gene Gil1 and Panc-1 cells transfected with the gene Gil1,and the control groups are SW1990 cells and Panc-1 cells. A total of 12 RNA samples of both experimental and control groups have a test. And to obtain the candidate effective mi RNAs and the target gene sets of and the corresponding effective mi RNAs based on the correlation coefficients and P value of the test results.Results: 1.The quality of RNA samples are all qualified. 2. Affymetrix mi RNA2.0 chip quality results are qualified. 3. Chip quality of Affymetrix HTA3.0 chip is qualified and hybridization conditions of that are reliable. 4.We acquire effective mi RNAs with Gli1 binding site,they are has-mir-301a-3p,has-mir-29b-1-5p and has-mir-1228-3p. 5. We obtain positive and negative effects associated mi RNAs target gene sets.Conclusion: with the use of Affymetrix mi RNA3.0 and Affymetrix HTA2.0 microarray techniques, the effective mi RNAs of HH- Gil1 and their corresponding target gene sets are obtained accurately,efficiently and completely,which is laying a solid foundation for our follow-up study.PartⅡ Identification of effective mi RNAs of nuclear transcription factor Gli1 of HH signaling pathway in pancreatic cancerObjective: Combine with Chromosome immunoprecipitation reaction and fluorescence quantitative polymerase chain reaction(Ch IP-QPCR) methods to verify that the candidate effective mi RNAs can bind with HH-Gli1. Gli1 overexpression vector was constructed to verify the candidate effective mi RNAs can also be a corresponding high expression. To verify the candidate mi RNAs are the direct effective mi RNAs of Gli1.Methods: ChIP-QPCR experiments are carried out with SW1990 pancreatic cancer cells transfected with Gli1 overexpression vector as the experimental group and SW1990 cells as the control group. In the experiments the experimental and control groups are individually divided into experimental group(with anti-Gli1 antibody), negative control group(with rabbit Ig G), positive control group(with anti-RNA polymerase Ⅱ antibody) and input group. Samples are purified into DNA fragments by cross-linked, sonication, antibody incubation and elution,that are finally tested by PCR. Finally, the results are displayed by the percentage of input. Differences are made by comparing the Gli1 group with negative control group. And to obtain the fold change in occupancy between experimental and the control groups by comparing Gli1 groups of the two groups. The expression of the corresponding effective mi RNAs are detected with SW1990 pancreatic cancer cells transfected with Gli1 overexpression vector as the experimental group and SW1990 cells as the control group.Results: 1. Gli1 over-expression vector is constructed successfully 2. ChIP-QPCR is carried out in SW1990 pancreatic cancer cells transfected with Gli1 gene(experimental group) and SW1990 cells(control group), respectively. There are no statistically significant differences(P> 0.05) of three effective mi RNAs(ie, mi R-301 a, mi R-29b-1, mi R-1228) between the Gli1 group and the Ig G group of control groups. However, the differences of three effective mi RNAs between Gli1 group and the Ig G group of experimental groups are statistically significant(P <0.01). 3. Comparison is made between the Gli1 groups of three effective mi RNAs(ie, mi R-301 a, mi R-29b-1, mi R-1228) in experimental and control groups. The mi RNA levels of experimental groups are three times, 2.7 times and 2 times than the control groups respectively. 4. Total RNA extracted from SW1990 cells transfected with Gli1 over-expression vector is detected highly expression levels of mi R-301 a, mi R-29b-1 and mi R-1228.Conclusion: From the above results, we can initially come to the conclusion that mi R-301 a, mi R-29b-1, mi R-1228 is direct effective mi RNAs of HH-Gli1, which laid the foundation for us to find the target genes of effective mi RNAs.