Regulation Of The Microrna Processor DGCR8by Hepatitis B Virus Protein Via The Transcription Factor YY1

ObjectiveTo study the effect of hepatitis B virus (HBV) on the expression ofDGCR8, to reveal the mechanism of HBV modulating the expression ofDGCR8.Methods1. The expressions of DGCR8in HepG2and HepG2.2.15cells weredetected using RT-PCR, Real-Time PCR and Western blot. Then, HBVexpression plasmids (pCH9/3091) was transfected into HepG2cells atdifferent dose and DGCR8expressions were detected with RT-PCR andWestern blot analysis.2.We constructed DGCR8expression plasmid (pGL3-DGCR8-P).Then, pGL3-DGCR8-P and pCH9/3091were transiently cotransfected intoHepG2cells to detect the effect of HBV on DGCR8promoter activities.3. HBV four protein expression plasmids(pCMV-sport6-HBc, HBp,HBs, HBx) were constructed. Then, these plasmids were respectivelycotransfected with pGL3-DGCR8-P into HepG2cells to detect DGCR8promoter activities. To further determine the result, shRNA plasmids wereconstructed to disturb HBV proteins expressions. And the effects of HBV protein shRNA plasmids on the expression of DGCR8were confirmed byDual-luciferase Report Assay and Western blot.4. RT-PCR, Western blot were carried out to check the expressions oftranscription factor YY1in HepG2and HepG2.2.15cells. HepG2cellswere cotransfected with pCMV-Sport6-YY1and pGL3-DGCR8-P, thenluciferase activities were detected. HepG2cells were transfected withpCMV-Sport6-YY1, then Western blot were performed. Finally, wetransfected YY1-shRNAs into HepG2.2.15cells and used Dual-luciferaseReport Assay and Western blotting to detect the DGCR8expressions.Results1. HBV could downregulate the miRNAs processor DGCR8mRNAand protein expressions in stable or transient HBV-expressing cells. AndHBV down-regulated the DGCR8expressions in a dose-dependent manneras pCH9/3091was transfected into HepG2cells at different dose.2. pGL3-DGCR8-P was successfully constructed and confirmed itsactivities by transfecting into HepG2cells. Then pGL3-DGCR8-P andpCH9/3091were transiently cotransfected into HepG2cells, HBV inhibitedDGCR8promoter activities.3. HBs and HBx could significantly inhibit the DGCR8promoteractivities. Meanwhile, shRNA plasmids of HBs or HBx were transfectedinto HepG2.2.15cells, the expressions of DGCR8were increased. 4. HBV could increase the expressions of transcription factor YY1inboth mRNA and protein level, and YY1could inhibit expressions ofDGCR8in HepG2cells. The activities of DGCR8promoter were enhancedby repressing the expression of YY1under the HBV expression conditions.ConclusionHBV repress the DGCR8promoter activities via upregulating theexpression of transcription factor YY1, and in this process, HBx and HBsproteins may play important roles.