Long Noncoding RNA TFAP2A-AS1 Suppressed Hepatitis B Virus Replication by Modulating miR-933/HDAC11

Objective. Studies have shown that long noncoding RNAs (lncRNAs) play crucial roles in multiple tumor types and regulate various biological processes. The present study tried to study lncRNA TFAP2A-AS1 in HBV infection hepatocellular carcinoma. Methods. The level of TFAP2A-AS1 and miR-933 in HCC cell and samples were detected by qRT-PCR assay. Luciferase reporter gene assay was carried out to study the mechanism of TFAP2A-AS1 and miR-933. Cell proliferation was measured by CCK-8 assay. HBV DNA replication was detected by RT-qPCR. Results. We ﬁ rstly demonstrated that TFAP2A-AS1 was downregulated in HCC cell lines and HBV-infected HCC samples compared with nontumor tissues. However, miR-933 was upregulated in HCC cell lines and HBV-infected HCC samples compared with nontumor tissues, and miR-933 was negatively associated with the expression of TFAP2A-AS1 in HBV-correlated HCC samples. TFAP2A-AS1 and HDAC11 expression was decreased and miR-933 was upregulated in the HBV-infected cell HepG2.2.15. TFAP2A-AS1 acted as a sponge for miR-933 and HDAC11 was one direct target gene for miR-933. Overexpression of TFAP2A-AS1 suppressed cell growth, HBV DNA replication, HbeAg, and HbsAg expression, while knockdown of TFAP2A-AS1 enhanced cell proliferation, HBV DNA replication, HbeAg, and HbsAg expression in HepG2.2.15 cell. In addition, ectopic expression of miR-933 promoted cell growth, HBV DNA replication, HbeAg, and HbsAg expression in HepG2.2.15 cell. TFAP2A-AS1 suppressed HBV replication and infection through regulating HDAC11. Conclusion. These data demonstrated that TFAP2A-AS1 acted crucial roles in the modulation of HbeAg and HbsAg expression and HBV replication and may be one potential target for HBV infection treatment.


Introduction
HBV (hepatitis B virus) could cause acute or chronic infection of hepatitis B, which was supposed to be the most risk factor for hepatocellular carcinoma and chronic liver cirrhosis [1][2][3][4].Epidemiological reports show that there are 400 million people with HBV infection worldwide and the number is still increasing.Importantly, more than 30% HBV infection cases were from China [5][6][7].HBV can relax genome of rcDNA (circular DNA) in the nucleocapsid and then converted into cccDNA (covalently closed circular DNA), which acted important roles in viral persistence [8][9][10].Thus, it is of great significance to investigate the mechanism of HCC development induced by HBV.
We manifested that TFAP2A-AS1 was downregulated in HCC cell lines and HBV-infected HCC samples and ectopic expression of TFAP2A-AS1 suppressed HBV replication and infection.

Materials and Methods
2.1.Clinical Samples.HBV-induced HCC specimens and nontumor tissues were collected from our hospital and were stored in liquid nitrogen until used.This research was agreed by the Clinical Ethics Committee of The Fourth Hospital of Harbin Medical University and informed consents were obtained.

HBV Replication and Gene Expression Analysis.
The DNA of HBV from cell supernatants was extracted by the Viral Column DNA out reagent (TIANDZ, China) following to the product's information and measured with RT-qPCR method.The HBsAg (hepatitis B surface antigen) and HBeAg (hepatitis B e antigen) expression in supernatants of HCC cell was determined with the ELISA reagent (Biotech, Shanghai).
2.6.Statistical Analysis.Data were analyzed with SPSS-19.0 (Chicago, USA) and showed as the mean ± SD (standard deviation).Student's t-test was applied to measure comparison difference between two groups.p data of <0.05 was represented as significant.

miR-933
Level in HBV-Associated HCC Samples.RT-qPCR analysis was carried out to detect miR-933 expression in HCC cell, HBV-infected HCC samples, and nontumor tissues.As indicated in Figure 2(a), miR-933 was upregulated in HCC cell lines compared to HL-7702.MiR-933 level was higher in HBV-infected HCC samples compared to nontumor tissues (Figure 2   3.6.TFAP2A-AS1 Inhibited HBV Replication and Infection.

Discussion
To improve the treatment of HBV infection and associated HCC, new therapies must be explored.We firstly showed that TFAP2A-AS1 was downregulated in HCC cell lines and HBVinfected HCC samples compared to nontumor tissues.However, miR-933 was upregulated in HCC cell lines and HBV-infected HCC samples compared to nontumor tissues, and miR-933 was negatively associated with the expression of TFAP2A-AS1 in HBV-correlated HCC samples.The expression of TFAP2A-AS1 and HDAC11 was decreased while miR-933 was overexpressed in HBV-infected cell HepG2.2.15.TFAP2A-AS1 acted as one sponge for miR-933 and HDAC11 was one direct target gene for miR-933.Ectopic expression of TFAP2A-AS1 suppressed cell growth, HBV DNA replication, HbeAg, and HbsAg expression, while knockdown of TFAP2A-AS1 enhanced cell proliferation, HBV DNA replication, HbeAg, and HbsAg expression in HepG2.2.15 cell.In addition, ectopic expression of miR-933 promoted cell growth, HBV DNA replication, HbeAg, and HbsAg expression in HepG2.2.15 cell.TFAP2A-AS1 suppressed HBV replication and infection through modulating HDAC11.These data illustrated that TFAP2A-AS1 acted cru-cial roles in the modulation of HBV replication and may be one potential therapeutic target for HBV infection.

Disease Markers
Multiple studies have shown that lncRNA participated in tumor progression and occurrence by acting as one sponge for miRNAs and their correlated genes [35][36][37].Zhao et al. [38] found that lncRNA TINCR inhibited HCC invasion and growth through sponging miR-218-5p/DDX5.Wu et al. [39] showed that lncRNA SUMO1P3 induced HCC development by promoting Wnt/β-catenin pathway via sponging miR-320a.Dai et al. [40] demonstrated that lncRNA TUG1 enhanced HCC progression through modulating DLX2/miR-216b-5p axis.Zheng et al. [41] demon-strated that linc00467 promoted HCC development through sponging NEDD9/miR-18a-5p.A recent study showed that TFAP2A-AS1 suppressed breast cancer progression through sponging miR-933 [28].Based on the bioinformatics TargetScan system, HDAC11 can potentially bind to miR-933.Overexpression of miR-933 suppressed luciferase activity of HDAC11-Wt but not HDAC11-Mut.Overexpression of TFAP2A-AS1 repressed miR-933 in HepG2.2.15 cell.MiR-933 expression level was higher in HBV-infected HCC samples compared to nontumor tissues.Disease Markers miR-933 expression was negatively associated with the expression of TFAP2A-AS1 in HBV-correlated HCC samples.Moreover, HDAC11 suppressed HBV replication via epigenetic inhibition of cDNA transcription [42].Furthermore, we showed that TFAP2A-AS1 suppressed HBV replication and infection through modulating HDAC11.Disease Markers Our manuscript has some limitations.The sample size is small and more samples are needed to study the expression of TFAP2A-AS1.Secondly, the function of TFAP2A-AS1 needs to be verified in vivo.

Conclusion
Collectively, our data showed that TFAP2A-AS1 was downregulated in HCC cell lines and HBV-infected HCC samples, and ectopic expression of TFAP2A-AS1 suppressed HBV replication and infection through modulating miR-933/ HDAC11.These data suggested that TFAP2A-AS1 may be a potential therapeutic target for HBV infection.