| Background and objectiveHepatocellular carcinoma（HCC）is the most common malignant tumor of the liver,accounting for approximately 85%-90%of primary liver cancers.In China,the incidence of HCC is about 25.7/10000 and the number of new HCC cases ranks the first worldwide.Because HCC is not easy to be diagnosed at early stage,patients with HCC are often discovered at advanced stage that the clinical symptoms have been serious.The prognosis of HCC still remains poor.According to epidemiological data,the 5-year survival rate of HCC is less than 10%.To date,HCC has become the second most frequent cause of cancer-related death worldwide.In China,the mortality of HCC in urban cities ranks the second and that in rural areas ranks the first among all cancer mortality.Despite of the wide application of positive surgery combined chemotherapy and radiotherapy,the clinical efficacy of treatment for HCC still remains poor due to recurrence,metastasis and lack of efficient biomarkers for early diagnosis and treatment.Many risk factors have been identified for HCC,including virus infection（hepatitis B virus and hepatitis C virus）,aflatoxin exposure,smokingand excessive alcohol intake.Hepatocarcinogenesis is a complex multistep process in which many oncogenic genes（β-catenin,PI3KCA,RAS et al.）are activated,tumor suppressor genes（TRET,P53,CTNNB1,Axin1 et al.）are inactivated and various signaling pathways（WNT,JAK/STAT,PI3K/AKT,RAS/MAPK et al.）are altered.Although many altered pathways and aberrantly expressed genes involved in hepatocarcinogenesis were identified,the precise molecular mechanisms for HCC are not entirely clear.Thus,in order to improve the prognosis of patients with HCC,an urgent need for novel molecular markers that can help in early diagnosis,risk assessment and therapy appears to be imperative.Most of the eukaryotic genome is transcribed,yielding a complex network of transcripts.And those greater than 200nt in length with limited or no protein-coding capacity are generally defined as long non-coding RNAs（lncRNAs）.LncRNAs were used to be regarded as byproducts or noise of transcription.However,with the in-depth research,accumulating evidence has demonstrated that lncRNAs emerge as essential regulators in a diverse range of cellular functions,such as development,differentiation,and cell fate as well as tumorigenesis.Mounting evidence has linked mutation and dysregulation of lncRNAs to cancer initiation,growth and metastasis and they may act as oncogenic factors or tumor suppressors.To date,HCC-related lncRNAs are demonstrated to influence the life cycle of genes by various means including epigenetic silencing,splicing regulation,lncRNA-miRNA interaction,lncRNA-protein interaction and genetic variation.Moreover,they can participate in diverse biological processes involved in HCC progression through impacts upon cell proliferation,apoptosis,invasion and metastasis and angiogenesis.For example,lincRNA-UFC1,a target of miR-34a,interacts directly with the mRNA stabilizing protein HuR to increase the levels of beta-catenin in HCC cells,thereby promoting proliferation and cell-cycle progression and inhibited apoptosis.As for cell apoptosis,ANRIL was found to be up-regulated in HCC and regulate cell apoptosis by epigenetic silencing of KLF2.Moreover,Li,T.et al.found that ZEB1-AS1 is frequently upregulated in HCC samples,especially in metastatic tumor tissues and aberrant methylation of the promoter region of ZEB1-AS1 is tightly correlated with high expression levels of ZEB1-AS1 in HCC.ZEB1-AS1 positively regulates the ZEB1 expression,thus promoting tumor metastasis.In 2012,Yuan,S.X et al found MVIH promotes angiogenesis by reducing the secretion of PGK1 and serves as a predictor for hepatocellular carcinoma patients’ poor recurrence-free survival after hepatectomy.Additionally,lncTCF7 is shown to promote self-renewal of human liver cancer stem cells through activation of Wnt signaling.Other examples include DREH,H19,HEIH,HOTAIR,HOTTIP,LALR1,MALAT-1 and RERT.However,investigations on the function and clinical significance of the majority of HCC-related lncRNAs still remain limited.Interestingly,some types of IncRNAs have been demonstrated to be present in body fluids,like urine and plasma,which can be easily obtained by the least invasive methods.As for HCC,some circulating IncRNAs have been identified and may be useful as novel potential biomarkers for diagnosis and therapy.Tang,J.et al.discovered three circulating IncRN A,RP11-160H22.5,XLOC014172 and LOC149086,which were up-regulated in HCC comparing with the cancer-free controls and might be the potential biomarker for the tumorigenesis prediction.XLOC 014172 and LOC149086 were confirmed highly increased in metastasis HCC patients,which may serve as potential biomarker for metastasis prediction in the future.Although the exact mechanism for the release of IncRNAs into body fluids is still not well-defined,lncRNAs may be potential biomarkers for HCC due to their specificity and good accessibility.Totally,lncRNAs play critical roles in HCC progression and are suggested to be useful as novel potential biomarkers for HCC diagnosis,prognosis and prediction of response to therapy.Therefore,it is of great importance to study the role and mechanism of lncRNAs in HCC.In this study,we identified a novel lncRNA PTTG3P（pituitary tumor-transforming 3,pseudogene）via microarray analysis.We show that PTTG3P was frequently up-regulated in HCC by RT-PCR and high levels of PTTG3P were positively correlated with tumor size and negatively with age.Further investigations on the role of PTTG3P and its molecular basis in HCC revealed that PTTG3P promoted cell growth,metastasis and tumorigenicity via activation of PI3K/AKT signaling pathway.These results indicated that PTTG3P harbors great potential significance as a prognostic biomarker and a therapy target for HCC.Contents and methods1.The screening,validation and clinical significance of lncRNA PTTG3PThe differentially expressed lncRNAs and mRNAs were screened by microarray analysis.Among them,we noticed a novel lncRNA PTTG3P.Then,the level of PTTG3P was validated by RT-PCR in 46 HCC tumor tissues and adjacent tissues.Subsequently,the associations between PTTG3P expression and clinicopathologiccharacteristics（gender,age,tumor size,AFP levels,HBsAg,liver cirrhosis,Edmonson grade）were analyzed.2.The role of PTTG3P on cell growth and metastasis in HCCHepG2 and Hep3B cells were transfected with lentiviral shRNA vectors.Then cells with stably silenced PTTG3P expression were selected and enriched by applying puromycin in culture medium.The efficiency of sh-PTTG3P was confirmed by RT-PCR.HepG2 and Hep3B cells stably over-expressing PTTG3P were constructed by the same methods.Cell proliferation was evaluated by CCK-8 assays,colony formation assays,EdU incorporation assays and cell cycle analysis.To investigate the effect of PTTG3P on cell apoptosis,flow cytometry was performed by APC/7AAD staining.Additionally,HepG2 cells stably transfected with sh-PTTG3P or sh-con,Lv-PTTG3P or Lv-con were injected subcutaneously into nude mice for xenoplantation to determine the effects of PTTG3P on tumor growth in vivo.,Transwell migration assays and Boyden chambers assay were performed to determine the function of PTTG3P in HCC cell migration and invasion,respectively.As for cell migration and invasion in vivo,HepG2 cells with stably decreased PTTG3P expression and control cells were independently injected into the spleens of nude mice to assess the migration and invison ablilty in vivo.3.The molecular mechanism of PTTG3P on cell growth and metastasis in HCCTo obtain further insight into the mechanisms of PTTG3P in cell growth,the levels of C-myc,CyclinDl,CDK4,CDK6,Rb and p-Rb were assessed by western blot analysis.The protein levels of caspase3 and cleaved caspase3 were evaluated by western blot analysis.As for the molecular mechanism of PTTG3P on cell metastasis,several EMT-associated regulators including Snail,Slug,E-cadherin and N-cadherin were detected by western blot analysis.Since PI3K/AKT plays important role in HCC progression,we also investigated the effect of PTTG3P on PI3K/AKT signaling pathway.The protein levels of PI3K,P-PI3K,AKT and P-AKT were analyzed by western blot.Results1.Through microarray analysis,we noticed an lncRNA PTTG3P.PTTG3P was up-regulated in HCC.The high level of PTTG3P in patients with HCC was negatively correlated with age and positively correlated with tumor size.1.1.Lots of differentially expressed IncRNAs were screened by microarray analysis.And data revealed that lncRNA PTTG3P was significantly upregulated in HCC.The differentially expressed IncRNA and mRNA profiles between HCC tumor tissues and adjacent tissues were analyzed by microarrays.As compared with adjacent tissues,822 IncRNAs exhibited more than 1.5-fold change（P<0.05）;513 were up-regulated,and 209 were down-regulated;979 mRNAs exhibited more than 1.5-fold change（P<0.05）;449 were up-regulated and 530 were down-regulated in HCC tumor tissues.Pathway analysis of the differentially expressed mRNAs revealed that all differentially expressed genes were mainly involved in cell cycle progression.Among all the differentially expressed lncRNAs,we noticed a novel lncRNA—PTTG3P with 11.87 fold elevation in HCC tumor tissues.1.2.LncRNA PTTG3P was frequently up-regulated in HCC.The levels of PTTG3P were assessed in 46 HCC tumor tissues and adjacent tissues by RT-PCR.The results showed that the levels of PTTG3P were higher in HCC tumor tissues than adjacent tissues.1.3.The high level of PTTG3P in patients with HCC was positively correlated with tumor size and negatively correlated with age.Chi-square test showed that the high levels of PTTG3P in patients with HCC positively correlated with tumor size and negatively correlated with age and there was no significant association between PTTG3P expression and other characteristics.2.LncRNA PTTG3P promotes HCC cell growth and metastasis in vivo and in vitro.2.1 HepG2 and Hep3B cells with stably silenced PTTG3P expression and HepG2 and Hep3B cells stably over-expressing PTTG3P were successfully constructed by lentivirus infection.The levels of PTTG3P in HepG2 and Hep3B cells transfected with Lv-PTTG3P and Lv-con vectors was detected by RT-PCR.The results revealed that HepG2 and Hep3B cells transfected with Lv-PTTG3P vectors had a higher level of PTTG3P than those transfected with Lv-con vectors,suggesting that HepG2 and Hep3B cells stably over-expressing PTTG3P were successfully constructed.Similarly,the efficiency of the sh-PTTG3P vector was confirmed by RT-PCR.The efficiency of sh-PTTG3P was 34.53%in HepG2 cells and 35.03%in Hep3B cells.These results indicated that HepG2 and Hep3B cells with stably silenced PTTG3P expression were successfully constructed.2.2 LncRNA PTTG3P promotes HCC cell growth in vivo and in vitro.CCK-8 assays and colony formation assays revealed that over-expression of PTTG3P promoted cell proliferation while knockdown of PTTG3P inhibited cell proliferation.In EdU incorporation assays,enhanced PTTG3P expression resulted in an increase of the number of S-phase cells.Cell cycle analysis by flow cytometry also revealed that over-expression of PTTG3P results in a significant decrease of G1-phase cells and an increase of S-phase and G2/M phase cells.However,suppressed PTTG3P expression had the opposite effects.These results indicated that PTTG3P accelerate cell proliferation by promoting G1/S transition.Additionally,we also observed that the percentage of apoptotic cells in HepG2 and Hep3B cells with stably blocked PTTG3P expression was higher than control cells while over-expression of PTTG3P can protect HepG2 and Hep3B cells from 5-fluorouracil treatments,suggesting PTTG3P inhibited cell apoptosis.To evaluate the effect of PTTG3P on HCC cell growth in vivo,we inoculated Lv-PTTG3P and Lv-con,sh-PTTG3P and sh-con HepG2 cells subcutaneously into nude mice for xenoplantation.Our data showed that depletion of PTTG3P reduced the mass and volume of tumors and over-expression of PTTG3P promotes tumor growth in vivo.Taken together,lncRNA PTTG3P promotes cell growth in vitro and in vivo.2.3 LncRNA PTTG3P promotes HCC cell metastasis in vivo and in vitro.Transwell migration assays revealed that the cell migration ability was markedly reduced in cells with stably silenced PTTG3P and increased in cells over-expressing PTTG3P.Futhermore,Boyden chamber assays confirmed that stably suppressed PTTG3P expression decreased the number of invaded cells in both HepG2 and Hep3B cells.On the contrary,enhanced PTTG3P expression increased the number of invaded cells.These data indicated that PTTG3P promoted cell metastasis in vitro.To evaluate the effect of PTTG3P on metastasis in vivo,sh-PTTG3P and sh-con HepG2 cells were independently injected into the spleens of nude mice.After a month,much less metastatic nodules in livers of nude mice were observed in sh-PLOD2 group than sh-con group.3.LncRNA PTTG3P modulates multiple cell-cycle-related genes,key regulators involved in cell apoptosis and EMT-associated factors:activation of PI3K/Akt signaling in HCC cells was involved.To obtain further insight into the mechanisms of PTTG3P in cell growth,the levels of several cell-cycle associated regulators,including C-myc,CyclinDl,CDK4,CDK6,Rb and p-Rb,were detected by western blot analysis.Enhanced PTTG3P expression up-regulated the protein levels of C-myc and CyclinDl while depletion of PTTG3P down-regulated the protein levels of C-myc and CyclinDl.Meanwhile PTTG3P did not have a measurable effect on the protein levels of CDK4 and CDK6.Additionally,the level of p-Rb was significantly higher in cells over-expressing PTTG3P than control cells while sh-PTTG3P cells had a lower level of PTTG3P than sh-con cells.These data suggested that PTTG3P promoted cell proliferation by up-regulating C-myc and CyclinD1.We also detected the protein levels of Caspase3 and Cleaved Caspase3 by western blot analysis.The proportion of cleaved caspase3/caspase3 was higher in sh-PTTG3P cells than sh-con cells and lower in Lv-PTTG3P cells than Lv-con cells treated with 5-fluorouracil.To further investigate the molecular mechanisms of PTTG3P in cell migration and invasion,the levels of several EMT-related regulators were detected by western blot analysis.Significant increases in the protein levels of E-cadherin and decreases of slug and snail were observed in cells with stably suppressed PTTG3P expression.Oppositely,over-expression of PTTG3P resulted in up-regulation of Snail and Slug and down-regulation of E-cadherin.Transgenic expression of PTTG3P or knockdown of PTTG3P did not affect the levels of N-cadherin.These results revealed that PTTG3P prometed cell metastasis by upregulating Snail and Slug and down-regulating E-cadherin.Knockdown of PTTG3P resulted in decreased levels of phosphorylated PI3K and AKT whereas total levels remained unchanged.Increased levels of p-PI3K and p-AKT were observed in cells with enhanced PTTG3P expression.These data indicated that PTTG3P can activate PI3K/AKT signaling pathway.Conclusion1.LncRNA PTTG3P is frequently up-regulated in HCC.And high levels of PTTG3P positively correlate with tumor size and negatively correlate with age in patients with HCC.2.LncRNA PTTG3P promotes HCC cell growth and metastasis in vivo and in vitro.3.LncRNA PTTG3P activates PI3K/AKT signaling pathway,regulating lots of cell-cycle associated genes（up-regulating C-myc and CyclinDl）,key factors involved in cell apoptosis（reducing the levels of cleaved caspase3）,EMT-related regulators（up-regulating Snail and Slug,down-regulating E-cadherin）,thus promoting cell growth and metastasis. |
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