| Hepatocellular carcinoma(HCC)is a critical problem to human health,mortality of which ranks second in cancers.Patiens diagnosed with HCC in China accounts for 55%of the world and it is the third most cancer in our country.Among multiple factors causing HCC,chronic HBV infection is the very reason leading to 80%HCC.HBV(Hepatitis B Virus),as partially double-strand DNA virus,alters cellular gene expression and signaling pathways to promote development of HCC directly or indirectly.First,the integration of HBV DNA in cellular genome causes genomic instability and activates oncogenes or deactivates tumor suppressors,which regulate cell proliferation and apoptosis.Meanwhile,HBV-encoded protein HBx or preS/S regulate expression of genes controlling cell proliferation or metastasis through trans-activation or interaction with protein.While,HBV indirectly leads to liver immune cells dysfunction and mediates inflammation in liver microenvironment to promote HCC.Multiple studies have demonstrated the process and mechanisms of HBV-associated HCC,however,there still remains needs for cure of HCC.It is of great significance to study the mechanisms of HBV inducing HCC foundamentally.ZHX2 plays role of tumor suppressor in HCC and leukemia.The previous studies demonstrated that oncofetal genes such as AFP(alpha fetoprotein)and Gpc3(Glypican 3)are suppressed by ZHX2.Our lab proved that expression of nuclear ZHX2 in liver paratumor tissues was significantly higher compared to tumor tissues.Meanwhile,ZHX2 inhibits expression of Cyclin A and Cyclin E to suppress the proliferation of hepatoma cells.Data from our lab also showed that ZHX2 down-regulates expression of MDR1(Multi-Drug Resistance 1)via binding with NF-Y to enhance sensitivity of hepatoma cells to chemotherapeutics.The studies above strongly prompt that ZHX2 plays a vital role as a novel tumor suppressor in development of HCC.While,it still remains unclear whether ZHX2 participates in HBV-associated HCC.Recent studies reported that chromosomal rearrangement leads to decreased expression of ZHX2 in Hodgkin Lymphoma cells.In familial corticobasal degeneration,upstream miR-4277 is possibly responsible for the decreased expression of ZHX2.However,it still remains unknown whether and how ZHX2 is dysregulated in HBV-related HCC.In summary,clinical liver tumor tissues,mice experiments and hepatoma cell lines were used to foundamentally study expression and regulation of ZHX2 in HBV-associated HCC,which contributes to further disclose the mechanism of HBV mediating HCC development.Data shown as below:1.ZHX2 is downregulated in HBV positive liver tissuesTo study the possible regulation of HBV on ZHX2,the expression of ZHX2 was firstly tested in clinical liver tumor specimens and liver tissues of HBV transgenic mice to compare the difference caused by HBV infection.1.1.ZHX2 is significantly decreased in HBV-positive liver tumor samples.Thirty-six liver tumor and paratumor specimens were collected and ZHX2 was analyzed with qRT-PCR.Our data indicated that proportion of high-ZHX2-expression tumor tissues from HBV-positive patients was significantly lower compared to HBV seronegative patients(11/17,65%vs.1/7,14%,χ2=54.42,df=1,P<0.0001).Among HBV+ specimens,the expression of ZHX2 in tumor tissues was significantly decreased compared to paratumors(4/16,25%vs.9/12,75%,χ2=50,df=1,P<0.0001),which is consistent with the tumor suppressor role of ZHX2.1.2.ZHX2 is significantly decreased in liver cells of HBV transgenic mice.Six C57B/6 wild-type mice and six HBV transgenic(HBV-Tg)mice were euthanized.The liver tissues were retained and hepatocytes were purified with centrifuge.ZHX2 in liver tissues and hepatocytes were analyzed by qRT-PCR and Western blot.Our data showed that ZHX2 mRNA in liver tissues of HBV-Tg mice were significantly lower than that in wild-type mice(P<0.05).Consistent with this,mRNA and protein expression of ZHX2 in hepatocytes were also decreased compared to wild-type mice(P<0.01).2.HBV,specially HBx,significantly downregulates ZHX2 expression in cultured HCC cell lines.To further investigate modulation of HBV on ZHX2,expression levels of ZHX2 were tested in HBV and HBV-encoded protein overexpressed hepatoma cell lines in vitro.2.1.ZHX2 is suppressed in HBV-infected hepatoma cell line.Purified HBV virions were added into medium of HepG2-NTCP,in which NTCP stably expresses.The cells and supernatant were harvested 3 dpi and 5 dpi(day post infection).HBsAg(HBV surface antigen)in supernatant was tested by ELISA and pgRNA,HBx and ZHX2 in cells were respectively determined by RT-PCR、qRT-PCR and Western blot.Our data showed that infection of HBV in HepG2-NTCP was successful.The mRNA and protein expression of ZHX2 in cells 3 dpi and 5 dpi were significantly decreased(P<0.01).2.2.ZHX2 is downregulated in full length HBV and HBV-encoded proteins overexpressed hepatoma cell lines.Liposome transfection was applied to overexpress 1.1 full length of HBV genome pcDNA3-HBV1.1 and HBV-encoded protein containing plasmids pcDNA3-preS2-HA,pcDNA3-HBx-HA,pcDNA3-HBc-HA and empty vector pcDNA3.48 hours after transfection,cellular ZHX2 was examined by RT-PCR and Western blot.Our data showed that ZHX2 mRNA and protein levels were inhibited by the fulllength HBV1.1 and all HBV-encoded proteins,of which,reduction of ZHX2 was most significant in HBx-overexpressed cells.2.3.ZHX2 is increased in HBV-encoded proteins knock-down hepatoma cell line.The antisense oligos against preS2,HBx and HBc were composed and transfected in HepG2.2.15 in which HBV genome is integrated.Our data demonstrated that addition with as-S2,as-HBx and as-HBc led to decreased levels of their target transcripts.Compared to control cells transfected with as-Ran,ZHX2 mRNA and protein levels were significantly increased in all three HBV-encoded transcripts knock-down cells.Collectively,these data demonstrate that HBV and HBV-encoded proteins inhibit expression of ZHX2,with HBx being the most potent repressor.3.HBV restricts inhibition of ZHX2 on hepatoma cell proliferation via HBxTo further investigate whether HBV modulates function of ZHX2,mice models and hepatoma cell lines were used for experiments.Data shown as below:3.1.HBx abrogates ZHX2-mediated inhibition of hepatoma cell growth in vivo.Activated mouse hepatoma cell line H22 was injected subcutaneously at groin of fifteen BALB/c male mice.The mice were randomly devided into 3 groups when tumor diameter reached 3 mm.The same amount of plasmids,pcDNA3(20ug),pcDNA3+pcZHX2(10ug+10ug),pcHBx+pcZHX2(10ug+10ug)were respectively injected into tumors.The plasmids were injected every other day and tumors were measured every two days until twelfth day.Increased mRNA and protein levels of ZHX2 and HBx in injected tumors were confirmed by RT-PCR and Western blot.The data of tumor growth curve and tumor weight showed that compared to pcDNA-treated control tumors,ZHX2 significantly inhibit growth of tumors,whereas tumors treated with addition of pcDNA-HBx grew faster at the same rate as control tumors(P<0.05).3.2.HBx negates ZHX2-mediated inhibition of hepatoma cell growth in vitro.The same plasmids as intratumorous injection were transfected with liposome into HepG2.EdU staining and Western blot were used after 48h.With Fluorescence Inverse Microscope,percentage of EdU positive cells in ZHX2 overexpressed cells was significantly decreased compared with negative control cells,prompting downregulation of cell proliferation.While proportion of EdU positive cells in pcHBx +pcZHX2 cells rebound(P<0.05),it demonstrates that HBx deactivates inhibition of ZHX2 on hepatoma cell proliferation.The expression of PCNA(proliferating cell nuclear antigen)in cells further confirms that cotransfection of HBx and ZHX2 in cells restored PCNA expression compared with ZHX2-treated cells.In vivo and in vitro data above demonstrated that HBx inhibits expression of ZHX2 and abrogates ZHX2-mediated suppression of liver tumor cell growth,however,the mechanisms of HBx regulating ZHX2 remains unclear.Previous studies showed that multiple microRNAs possibly regulate expression of ZHX2,therefore,we hypothesize that some microRNA mediates the process of HBV inhibiting ZHX2.4.HBV,specially HBx,induces upregulation of miR-155 to promote cell proliferation.4.1.MiR-155 might be the key miRNA mediating HBx suppressing ZHX2 through microRNA microarray analysis combining with bioinformatics prediction.To seek out the miRNA involving in HBV inhibiting ZHX2,pcDNA3-HBx was overexpressed in BEL-7402 and differential expressing microRNAs were analyzed through microRNA microarray.The microRNAs,such as miR-501-5p,miR-320,miR-155,etc.,were two-fold upregulated in HBx-overexpressed cells.The microRNAs targeting ZHX2 were analyzed in the microRNA prediction website(www.microrna.org),and miR-106b,miR-17-5p,miR-155,etc.,were included.Combining microarray data and predicted microRNAs,miR-155 is the only microRNA which is 2-fold upregulated in HBx-overexpressed cells and binds with ZHX2 3’UTR via seed sites.This prompts HBx suppresses ZHX2 expression through miR-155.To confirm this hypothesis,in vitro assays and mice and clinical liver tissues were tested.4.2.HBV,specially HBx,promotes miR-155 in vitro.To confirm HBV regulating miR-155,HepG2-NTCP were harvested at 3 dpi and 5 dpi,and miR-155 was tested with qRT-PCR.The miR-155 levels were higher in HBV-infected HepG2-NTCP cells compared to cells without infection.Higher levels of miR-155 were determined in HepG2,SMMC7721 and QSG7701 cells 48 hours after transfection with pcDNA-HBx,compared to control cells,prompting that HBx upregulates miR-155.To further confirm this,as-HBx was transfected into HepG2.2.15 cells and miR-155 RNA levels were dramatically diminished in HBx-knockdown cells,consistent with previous data.4.3.MiR-155 is elevated in liver tissue of HBV transgenic mice.To confirm HBV regulating miR-155 in vivo,liver tissues were collected from C57B/6 wild-type and HBV transgenic mice and hepatocytes were purified.Total RNA was isolated and miR-155 levels were tested with qRT-PCR.Our data showed the miR-155 levels in liver tissue and purified hepatocytes of HBV transgenic mice were significantly higher compared to wild-type mice(P<0.05,P<0.01),demonstrating HBV regulates miR-155 in vivo.4.4.MiR-155 promotes proliferation of hepatoma cells in vitro.Multiple studies show that miR-155 is involved in inflammation-mediated oncogenesis.To investigate modulation of miR-155 on proliferation of liver cancer cells,miR-155 was firstly overexpressed or inhibited in SMMC7721 and percentage of EdU positive cells was determined via EdU staining.Then,miR-155 mimic/inhibitors and respective negative controls were respectively transfected in Huh7,HepG2,SMMC7721 as well,and expression of PCNA in cells were tested by Western blot.The data showed that the percentage of EdU positive cells and PCNA levels significantly increased in in miR-155-overexpressed cells,while reduced in miR-155-inhibited cells,indicating the role of miR-155 in promoting cell proliferation.4.5.The expression of PCNA and miR-155 in HBV+ liver tumor are higher,compared to HBV-tumors.To further confirm HBV-miR155 promoting proliferation of hepatoma cells in clinical specimens,PCNA and miR-155 in liver tumor tissues were analyzed with qRT-PCR.Both expression levels of PCNA and miR-155 were higher in HBV+tumor tissues compared to HBV-tumors(10/17,59%vs.2/7,29%,X2=18.26,df=1,P<0.0001),indicating higher level of miR-155 and increased cell proliferation in HBV+ liver tumor.5.MiR-155 suppresses ZHX2 expression via binding with its 3’UTRThe bioinformatics analysis indicated miR-155 seed site in ZHX2 3’UTR,prompting ZHX2 is one target gene of miR-155.In vitro assays,dual luciferase system and clinical specimens were used to confirm the hypothesis.5.1.MiR-155 inhibits ZHX2 expression in vitro.MiR-155 mimic/inhibitor and respective negative controls were transfected with liposome in Huh7、HepG2、SMMC7721.Cells were harvested after 48h and cellular ZHX2 was tested with Western blot.The data showed addition of miR-155 mimic resulted in lower expression levels of ZHX2 in all three cell lines.5.2.MiR-155 aligns with ZHX2 3’UTR.Multiple studies demonstrate that miRNAs promote mRNA degradation or suppress translation to regulate gene expression through binding with seed sites in target mRNA 3’UTR.To investigate the mechanism of miR-155 regulating ZHX2,a 603 bp fragment of the ZHX2 3’ UTR containing the miR-155 seed site was inserted at Xba I of the pGL3-promoter vector(pGL4-promoter-ZHX2 3’UTR).Meanwhile,the ZHX2 3’ UTR with mutated miR-155 site and the ZHX2 3’ UTR with deleted miR-155 site were also cloned into pGL3-promoter(pGL4-promoter-ZHX2 3’UTR site mut and pGL4-promoter-ZHX2 3’UTR del mut).These three plasmids were respectively co-transfected with the miR-155 mimic or microRNA mimics control into HeLa cells.Dual luciferase assay was used for measuring cell extracts after 48 hours.Our data indicated that luciferase activity was significantly reduced in cells with the wild-type ZHX2 3’ UTR and miR-155 mimic compared to cells co-transfected with microRNA mimics control(P<0.01),whereas,no significant decrease were shown in cells transfected with ZHX23’UTR site mutation or deletion mutation plasmid,demonstrating that miR-155 aligns with seed site in ZHX2 3’UTR.In vitro assays indicate that miR-155 inhibits expression of ZHX2 via binding with seed site in ZHX2 3’UTR.5.3.ZHX2 is negatively correlated with miR-155.To further confirm that miR-155 regulating ZHX2,miR-155 and ZHX2 mRNA were analyzed in a series of liver cancer cell lines(HCCLM3,MHCC97H,BEL-7402,Huh7,SMMC7721 and HepG2)and twelve HBV+ paratumor tissues with qRT-PCR.Our data indicated an inverse correlation between miR-155 and ZHX2 mRNA levels in both hepatoma cell lines and liver tissues.6.HBx inhibits ZHX2 expression via upregulating miR-155.The data above demonstrate that HBx promotes miR-155 levels and miR-155 suppresses expression of ZHX2.To confirm the role of miR-155 as a key factor in HBx regulating ZHX2,rescue assays were performed.pcDNA3+ microRNA inhibitor control,pcDNA3-HBx+ microRNA inhibitor control,pcDNA3-HBx+miR-155 inhibitor were respectively co-transfected in HepG2 and SMMC7721.Cells were harvested after 48h and ZHX2 expression level was analyzed with qRT-PCR and Western blot.The data showed that compared to control cells,expression of ZHX2 was lower in HBx-treated cells,which was reversed in addition of miR-155 inhibitor,indicating HBx represses ZHX2 expression via activating miR-155.Conclusions and significances:In summary,our study firstly demonstrates HBV,specially HBx,inhibits expression of tumor suppressor ZHX2 to promote development of HCC via oncomiR miR-155.Data here show that miR-155 is activated to high expression level by HBx,promoting proliferation of hepatoma cells and ZHX2 is identified as its new target gene.The study here indicates high level of miR-155 and decreased expression of ZHX2 are possible key factors in HBV-associated HCC and provides novel theory basis for exploring efficient therapeutic strategies. |
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