HBV PreS2 Enhances The Expression Of TAZ By MiR-338-3p To Promote The HCC Cell Growth

Hepatocellular carcinoma is a kind of malignant tumor that affects human health severely. It is the fifth most prevalent cancer and the third most frequent cause of cancer-related death. Each year, HCC is diagnosed in more than half a million people worldwide. The incidence of this disease is different between male and female. Liver cancer is the fifth most common cancer in men and the seventh in women. Most of the new diagnosed disease is distributed in the developing countries, especially in regions where infection with HBV is endemic:East Asia, Southeast Asia and sub-Saharan Africa. The major risk factors of HCC include infection of HBV or HCV, alcoholic liver disease and nonalcoholic fatty liver disease. Other causes include hereditary hemochromatosia, alpha-antitrypsin deficiency, autoimmune hepatitis, and Wilson’s disease. However, chronic and persistent HBV infection is the most risk factor of the development of HCC.In the worldwide, HBV infection is the most prevalent chronic virus infection. Roughly 30% of the world’s population show serological evidence of current or past infection and more than 350 million people are chronically infected. Globally, chronic HBV infection accounts for approximately 50% of all cases of HCC. Especially in China with the most HBV infections, the HCC cases takes up 50% of the world. Thus, it is significant to investigate the molecular mechanism of HBV associated HCC development.The pathological mechanism of HBV related HCC is complicated and the integration of HBV DNA to the host genome is one of the most important mechanisms. The integrated HBV-DNA not only can trigger cis-activation directly, but also express the regulatory protein HBx and preS2 to transactivate the host oncogene expression. The regulation mechanism of HBx in the progression of HCC is relatively clearly understood. However, the role of preS2 in HCC development is still largely unknown. Previous study in our lab revealed that preS2 could transactivate the oncoprotein Foxp3 and hTERT to promote the progression of HCC. It is still interesting to investigate whether preS2 could promote HCC progression by regulate other oncogene or signal pathway.The Hippo pathway, first discovered in Drosophila in 2003, is critical in controlling organ size by regulating both cell proliferation and apoptosis. YAP and TAZ are transcriptional co-activators and the major downstream effectors of Hippo pathway. Accumulated data demonstrated that dysregulation of Hippo pathway is actively involved in tumorigenesis. As a key transducer of Hippo pathway, TAZ has been demonstrated as oncoprotein in many cancers, including breast cancer, non-small cell lung cancer. TAZ not only promotes the proliferation and epithelial-mesenchymal transition of cancer cells, but also confers the cancer stem cell. Previously, zhang reported that HBx enhances the expression of YAP by CREB to promote HCC progression. However, whether HBV could regulatedTAZ to promote the progression of HCC is still largely unknown.ObjectivesIn order to investigate the mechanism of HBV related HCC and the gene regulation during HCC development, we focus on the following aspects:(1) Whether HBV could regulate the Hippo pathway effective molecule TAZ and the associated molecular mechanism.(2) The effect of TAZ in the development of HCC.Methods and ResultsI. HBV preS2 promotes TAZ expression by repressing miR-338-3p1. The different endogenous expression of TAZ in the HCC cell lineTo investigate the role of TAZ in HCC tumorigenesis, the HCC cell line SMMC7721, BEL7402 and HepG2 was collected and the total RNA and protein was harvested. RT-PCR and Western Blot were applied to examine the endogenous expression of TAZ. The results indicated that the endogenous expression of TAZ is different among these four HCC cell lines. At the protein level, HepG2 has the lowest endogenous expression while HepG2.2.15 integrated with 4 copies of HBV DNA has the highest TAZ expression. At the mRNA level, however, TAZ expression in HepG2 and HepG2.2.15 was at the same level. This result indicated that TAZ expression may be upregulated by HBV at the posttranscriptional level.2. HBV encoded protein HBx and preS2 upregualte the expression of TAZ atthe posttranscriptional levelTo further investigate the regulation of TAZ by HBV, two important functional protein HBx and preS2 encoded by HBV was transfected into the HCC cell line SMMC7721, BEL7402 and HepG2. The total RNA and protein was harvested 48h after transfection, and RT-PCR, qRT-PCR and Western Blot was applied to detect TAZ expression. The PCR result indicated that TAZ mRNA level was not significantly upregulated by overexpression of HBx or preS2. However, the western blot result indicated that TAZ protein level was significantly upregulated by HBx and preS2.This result indicated that TAZ was upregulated by HBV encoded protein at the posttranscriptional level.preS2 is the minimal functional fragment of MHBs1, an important transactivator encoded by HBV. Clinical study indicated MHBs’/preS2 integrated into 1/3 of HBV associated HCC genome. Previous study indicated that preS2 could transactivate oncogene hTERT and Foxp3 to promote HCC tumorigenesis. We are also interested in preS2 induced HCC and associated molecular mechanism, so preS2 was selected for further investigation.3. preS2 represses miR-338-3p to promote the expression of TAZmicroRNA is a kind of small non-coding RNA about 21-25 nucleotides, which functions in RNA silencing and post-transcriptional regulation of gene expression. Accumulating data indicated that HBV regulate the host cell function via interaction with miRNA. To investigate the role of miR in the regulation of TAZ by preS2, Target Scan was used to predict the potential miR targeting TAZ. The prediction data indicated that TAZ may be targeted by miR-338-3p. To examine the effect of miR-338-3p, the following experiment was conduced:3.1.Transfection and luciferase assay indicated thatTAZ is a potential target gene of miR-338-3p(1) miR-338-3p represses TAZ expressionmiR-338-3p mimics and inhibitor was transfected into the HCC cell line and the NC mimics or inhibitor was used as control. Western blot result indicated that miR-338-3p mimics significantly repressed the expression of TAZ at the protein level, while the miR-338-3p inhibitor restored TAZ expression.(2) miR-38-3p interacts with the 3’UTR of TAZ to repress its expressionTo further investigate the regulation of TAZ by miR-338-3p, the wild or mutant TAZ 3’UTR was cloned and infused into the plasmid pGL3-promoter. The miR-338-3p mimics or inhibitor was contransfected with the wild or mutant TAZ 3’UTR plasmid. The luciferase assay result indicated that miR-338-3p mimics inhibited the luciferase activity while the miR-338-3p inhibitor restored the activity. Meanwhile, the activity of mutant TAZ 3’UTR was not significantly affected by mimics or inhibitor.3.2.preS2 repressed the expression of miR-338-3pPlasmid pcDNA3 and pcDNA3-preS2 was transfected into the HCC cells. The total RNA was harvested 48h after transfection. The qRT-PCR results indicated that miR-338-3p expression was repressed by preS2.II.TAZ promoted the proliferation of HCC cells1. The knockdown efficiency of TAZ siRNA in different HCC cell line.The TAZ siRNA was transfected into the HCC cell line and the total RNA and protein were harvested 48h after transfection. The RT-PCR and qRT-PCR result indicated that TAZ expression was significantly knocked down by TAZ siR-2 and siR-3. The western blot result also indicated that TAZ protein expression was repressed by TAZ siR-2 and siR-3. Therefore, TAZ siR-2/3 was used in the follow experiment.2. Growth curve experimentThe TAZ siRNA was transfected into BEL7402 and SMMC7721 cells. The proliferation rate was determined by CCK-8 assay. The result indicated that: compared with NC siRNA, the proliferation rate of BEL7402 and SMMC7721 was significantly repressed by TAZ siRNA.3. Colony formation assayTAZ siRNA was transfected into the HCC cell line, and the colony formation assay result indicated that:compared with NC siRNA control, the colony formation ability was significantly repressed by TAZ siRNA.4. preS2 promoted the HCC cell proliferation, migration and invasion by TAZ4.1 TAZ is involved in the HCC cell proliferation mediated by preS2Plasmid pcDNA3+NC siRNA, preS2+NC siRNA and preS2+TAZ was transfected into the HCC cell line, the proliferation rate assay and the colony formation assay indicated that:compared with the pcDNA3+NC siRNA transfected cells, preS2 significantly promoted the proliferation rate and the colony formation ability, and meanwhile, TAZ siRNA repressed the preS2 induced proliferation and colony formation.4.2 TAZ is involved preS2 mediated HCC cell wound healing, migration and invasionThe wound healing assay indicated that the wound healing ability of BEL7402 and SMMC7721 was promoted by overexpression of preS2. Co-transfection of TAZ siRNA significantly repressed the wound healing ability promoted by preS2. Meanwhile, the transwell assay also indicated that TAZ siRNA reduced the migration and invasion ability induced by preS2 overexpression.Conclusions1. The cell experiment indicated that HBV functional protein preS2 up regulated the expression of TAZ at the posttranscriptional level. Western Blot and luciferase assay indicated that TAZ is a direct gene of miR-338-3p. preS2 suppressed the expression of miR-338-3p2. Hippo pathway effective molecule TAZ promoted the proliferation of HCC cells.3. preS2 promoted the HCC cell proliferation, migration and invasion by TAZ.Innovations and significances1. Our study first reported that preS2 promote the expression of TAZ by repressing miR-338-3p, which provided new insight of the pathogenesis of HBV associated HCC2. Accumulated data indicated that Hippo pathway is involved in the progression of tumor. However, the effect of TAZ in the development of HCC is still unclear. Our study first reported that TAZ promoted the progression of HCC3. Hippo pathway is precisely regulated in the physical condition. Previous study reported that YAP was regulated by miR-375. However, whether TAZ could also be regulated by a miR is not clear. In this study, we reported that TAZ could be targeted by miR-338-3p.