| 【Background and Objective】Hepatocelluar carcinoma (HCC) is one of the most common cancers in the world,especially in Asia. Because of its insidious pathological process, most patients have beendeprived of the opportunity to receive radical resection or liver transplantation. Therefore,despite great advances have been made in the dianosis and treatment of the disease, theprognosis of HCC is still unsatisfied. Hence, novel therapeutic strategies or targets forHCC are urgently needed.microRNAs (miRNAs) are a class of small non-coding RNA molecules which regulateposttranscriptional events. Many studies reveals that miRNAs have emerged as tumorsuppressive molecules or oncomiRs by targeting tumor oncogenes or suppressor genes.Therefore miRNAs have become a hotspot in the researches concerning many cancersincluding HCC.miR-370is located within the DLK1/DIO3imprinting region on human chromosome14or cognate mouse chromosome12. This region contains one of the largest miRNA clustersin the genome. Many miRNAs in this cluster including miR-370have been reported to beinvolved in cancer pathogenesis. Previous studies demonstrated that miR-370playsimportant roles in the development of hematological malignancies, gastrointestinal cancers,head and neck cancers, nervous system neoplasms, the urinary and genital system tumors.Nevertheless, there is no consensus about wether miR-370is a oncomiR or a tumorsuppressor, and even the role of miR-370in the same disease, such as leukemia or gasticcancer, remains controversial. In addition, the main physiology function of miR-370is theregulation of lipid metabolism. miR-370has been verified to affect the lipid metabolism inthe liver by directly targeting Cpt1α and up-regulating liver-enriched miRNA miR-122,indicating that miR-370may be important for the hepatic function. Moreover, someresearchers observed that expression of miR-370was significantly upregulated in the mice liver upon ischaemia-reperfusion injury and inhibition of miR-370efficiently attenuatedthe damage to the liver. These sudies indicated that as a important cancer-associatedmiRNA, miR-370also functions as key effector in the physiologica and pathologicalprocess in the liver. However, to date, the functional role of miR-370in HCC has not beenrepoted.In this study, we will investigate the role of miR-370in HCC by in vitro and in vivoassays and elucidate the in-depth mechanism through searhing its downstream target andthe related signal transduction pathways in the progression of HCC, which will shed newlight on the treatment of HCC.【Methods】1. Effect of miR-370on the malignant behaviour of HCC cells1) Effects of miR-370on the apotosis of HCC cells in vitroHCC cells were transfected with miR-370mimic and then cultured in DMEMsupplemented with10%FBS or transfected with miR-370inhibitor and then cultured inDMEM without FBS. Ninety-six hours after transfection, flow cytometry assay was usedto assess the apotosis rate of HCC cells.2) Effects of miR-370on the migration and invasion of HCC cells in vitroTranswell assays were performed in MHCC-LM3cells or YY-8103cells transfectedwith miR-370mimic or miR-370inhibitor to acess the migration and invasion of both ofthe two cells.3) Effect of miR-370inhibiton on the migration and invasion of IMHTranswell assays were performed in human nontransformed immortalized hepatocytes(IMH) transfected with miR-370inhibitor to acess the migration and invasion of IMH.4) Effect of miR-370on tumorigenesis of HCC cells in vivoMHCC-97H (4×106) or YY-8103(2×106) cells infected with Ad-miR-370orcontrol adenovirus Ad-GFP in100μl of serum-free medium were injected subcutaneouslyinto both flanks of Balb/c nude mice (n=8). Tumor formation and size were evaluatedperiodically and all mice were sacrificed40days after innoculation. Comparison of thexenograft size and weight was performed in the both groups. Real-time PCR was used todetect the level of miR-370in the xenografts.5) Effect of miR-370on the meatastasis of HCC cells in vivo MHCC-LM3cells (1.5×106) stably expressing luciferase and infected with Ad-GFPor Ad-miR-370were injected via the tail vein of6-week-old male NOD/SCID mice. Micewere monitored using the IVIS200(Caliper LS) imaging systems once a week. Specifically,mice were injected with D-luciferin, anesthetized with isoflurane, and imaged15min afterluciferin injection. Quantification was performed using Living Image version2.6software.Mice were sacrificed8weeks after cell transplantation and tumors nodules on the lungswere counted and histopathologically analyzed with hematoxylin-eosin staining (H&E).6) Effect of miR-370on the subcutaneous tumor through intratumoral injectionTo investigate the proliferation-suppression effect of miR-370in vivo, asubcutaneously implanted HCC model was established by injecting MHCC-97H cells (4×106) into Balb/c nude mice. When tumors became visible, mice were treated byintratumoral injection of2×109plaque-forming units of Ad-miR-370or Ad-GFP twice aweek for up to3weeks, and sacrificed4days after the last treatment. Tumors wereweighed and histopathologically analyzed with H&E staining. Immunohistochemical assaywas used to assess the expressio of Ki-67and TUNEL assay was performed to evaluate theapotosis of HCC cells in vivo. Real-time PCR was used to detect the level of miR-370.2. The interaction between miR-370and LIN28A(1) The regulation of LIN28A by miR-3701) In silico complementarity search using TargetScan (http://www.targetscan.org/)and PicTar (http://picta.mdc-berlin.de/) was performed to screened for putative targets ofmiR-370. Real-time PCR and Western Blot were performed to detect the expression ofputative target LIN28A in the HCC cells with miR-370overexpression or inhibition.Immunohistochemical assay was used to assess the expression of LIN28A in theAd-miR370-treated MHCC-97H xenografts.2) Psicheck2-LIN28A3′UTR luciferase reporter plasmid was constructed bysub-cloning the LIN28A3′UTR to the downstream of psicheck2vector. Reporter assay wasperformed to detect the effect of miR-370on the wild type psicheck2-LIN28A3′UTRluciferase reporter plasmid. Deletion or point mutation of miR-370binding site on LIN28A3′UTR reporter vector were performed using overlap extension by PCR. Reporter assaywas also performed to detect the effect of miR-370on the mutated psicheck2-LIN28A3′UTR luciferase reporter vector to prove the direct binding of miR-370to the LIN28A3′UTR. 3) Transwell assays were performed in HCC cells transfected with siRNA againstLIN28A or HCC cells infected with lentivirus expressing LIN28A without3′UTR to acessthe migration and invasion of HCC cells. Using this lentivirus to up-regulate the expressionof LIN28A in HCC cells which were then transfected with miR-370mimic. Transwellassays were then performed to acess the migration and invasion of cells.(2) The effect of LIN28A on the maturation of miR-3701) Real-time PCR was carried out to determine the expression of mature miR-370,let-7(positive control) and miR-21(negative control) in HCC cells transfected with siRNAagainst LIN28A. Flag-tagged LIN28A was constructed by cloning the coding sequence ofLIN28A gene to pFlag-CMV-2vector. Plasmid for the expression of LIN28A proteincontaining a single amino acid substitution (C161A) which is requied for the RNA bindingaffinity of LIN28A was constructed using an approach of overlap extension by PCR.Real-time PCR was carried out to detect the level of mature miR-370, let-7and miR-21in HCC cells transfected with empty vector, Flag-LIN28A vector and Flag-LIN28A vectorcontaining C161mutation.2) RNA binding protein immunoprecipitation (RIP) assay and the followed Real-timePCR was performed to detect the binding between endogenous LIN28A and the precusorof miR-370in PLC/PRF/5cells.3) MHCC-97H cells was transfected with empty vector, Flag-LIN28A vector andFlag-LIN28A vector containing C161mutation. Forty eight hours after transfection, RIPassay and the followed Real-time PCR was performed to detect the binding betweenLIN28A and the precusor of miR-370.3. The effect of miR-370-LIN28A-NF-κB regulatory circuit on HCC cells(1) LIN28A activates NF-κB pathway by post-transcriptionally regulating RelA/p651) HCC cells were transfected with siRNA against LIN28A or infected with lentivirusexpressing LIN28A. The cells were collected for reporter assay of luciferase activity ofNF-κB luciferase reporter, Real-time PCR analysis of mRNA level of RelA/p65and thedownstream genes of NF-κB including IL6, TNFα and MMP9as well as Western Blotanalysis of RelA/p65protein.2) RIP assay and the followed Real-time PCR was performed to detect the bindingbetween endogenous LIN28A and RelA/p65mRNA in PLC/PRF/5cells.3) MHCC-97H cells was transfected with empty vector, Flag-LIN28A vector and Flag-LIN28A vector containing C161mutation. Forty eight hours after transfection, RIPassay and the followed Real-time PCR was performed to detect the binding betweenLIN28A and RelA/p65mRNA.4) psicheck2-RelA/p653′UTR luciferase reporter plasmid was constructed bysub-cloning the RelA/p653′UTR to the downstream of psicheck2vector. Reporter assaywas performed to detect the effect of LIN28A overexpression or depletion onpsicheck2-RelA/p653′UTR luciferase reporter plasmid.5) HCC cells were infected with lentivirus expressing LIN28A and then treated withinhibitors of RelA/p65NF-κB transcriptional activity, Oridonin and JSH-23. Transwellassays were then performed to acess the migration and invasion of these cells.(2) Effect of miR-370-LIN28A-NF-κB regulatory circuit in the development of HCC1) HCC cells were transfected with miR-370mimic or inhibitor. The cells were thencollected for reporter assay of luciferase activity of NF-κB luciferase reporter, Real-timePCR analysis of mRNA level of RelA/p65and the downstream genes of NF-κB includingIL6, TNFα and MMP9as well as Western Blot analysis of RelA/p65protein.Immunohistochemical assay was used to assess the expression of RelA/p65in theAd-miR370-treated MHCC-97H xenografts. Real-time PCR was performed to detect thelevel of IL6, TNFα and MMP9mRNA in these xenografts.2) HCC cells were infected with lentivirus expressing LIN28A without3′UTR andthen transfected with miR-370mimic. The cells were collected for analysis of NF-κBluciferase reporter activity, Real-time PCR analysis of mRNA level of RelA/p65and thedownstream genes of NF-κB as well as Western Blot analysis of RelA/p65protein.3) HCC cells were transfected with miR-370inhibitor and then treated with NF-κBtranscriptional activity inhibitors, Oridonin and JSH-23. Transwell assays were thenperformed to acess the migration and invasion of these cells.4) HCC cells were treated with human recombinant IL6. The cells were then collectedfor Real-time PCR analysis of miR-370level and Western Blot analysis of LIN28A protein.Real-time PCR analysis was performed to detect the expression of primary miR-370indifferent HCC cells treated with methylation inhibitor5-aza-2′-deoxycytidine (5-aza-CdR).5) Real-time PCR was used to detect the level of miR-370, LIN28A mRNA and IL6mRNA in86paired primary HCC and adjacent non-tumorous liver tissues from primaryHCC patients. The following correlation analysis was performed to evaluate the relation between miR-370and LIN28A, miR-370and IL6as well as between LIN28A and IL6.4. Statistical analysesStatistical analyses were performed with SPSS software (18.0version), with a P value<0.05considered significant. For experiments involving only two groups, data wereanalyzed with two tail Student’s t test or Mann-Whitney U test.【Results】1. miR-370inhibits the malignant phenotype of HCC cells in vitro and in vivo1) Flow cytometry assay showed that overexpression of miR-370promoted theapoptosis of HCC cells, while inhibition of miR-370attenuated serum starvation-inducedapoptosis of HCC cells.2) miR-370overexpression markedly reduced while miR-370inhibition increased themigration and invasion of MHCC-LM3cells and YY-8103cells in vitro.3) miR-370inhibitor markedly enhanced the migration and invasion of IMH in vitro.4) MHCC-97H or YY-8103cells infected with Ad-miR-370or control adenovirusAd-GFP were subcutaneously transplanted into the flanks of Balb/c nude mice. Xenograftswere detected in37.5%(3/8) of mice as early as day14, and in all subjects by day33afterinoculation in mice receiving MHCC-97H cells infected with Ad-GFP. No xenografts wereobserved until day33in mice receiving MHCC-97H cells infected with Ad-miR-370, andonly small nodules were identified in50%(4/8) of mice by day38. The size and weight ofxenografts were significantly smaller in the Ad-miR-370group compared with the controlgroup. Real-time PCR analysis showed a significant increase in miR-370levels in theAd-miR-370group, relative to the Ad-GFP control. Similar results were obtained withYY-8103cells.5) To investigate the effect of miR-370on HCC metastasis in vivo, luciferase-labeledMHCC-LM3cells infected with Ad-miR-370or Ad-GFP were transplanted intoNOD/SCID mice through the tail vein. Eight weeks after transplantation, luciferase signalsin lung were observed in all of the mice in Ad-GFP group but in only2of5of the mice inAd-miR-370group via ex vivo imaging. Histologic analysis confirmed the reduced tumorfoci in Ad-miR-370group.6) We then explored the anti-tumor effect of miR-370on an established HCC cellstransplanted subcutaneous tumor model in Balb/c nude mice. Intratumoral injection of Ad-miR-370significantly reduced the growth and weight of MHCC-97H xenografts.Real-time PCR confirmed the increased expression of miR-370in Ad-miR-370-treatedtumor nodules. Histological analysis revealed that the tumor nodules were composed ofHCC cells arranged in trabecular pattern as proved by H&E staining. Additionally, theAd-miR-370-treated tumor nodules displayed decreased Ki-67expression and containedmore apoptotic cells.2. The double negative feedback loop between miR-370and LIN28A plays animportant role in the progression of HCC(1) LIN28A is a direct target and functional mediator of miR-3701) In silico complementarity search using Target Scan (http://www.targetscan.org/)and PicTar (http://picta.mdc-berlin.de/) identified LIN28A, an evolutionarily conservedmolecule across many species, as a potential downstream target for miR-370. LIN28AmRNA and protein levels in HCC cells were decreased by ectopic expression of miR-370and increased by miR-370inhibitor. Immunohistochemical analysis also revealeddecreased LIN28A in the Ad-miR370-treated MHCC-97H xenografts.2) Reporter assay revealed that overexpression of miR-370decreased the luciferaseactivity of wild-type LIN28A3′UTR by59.4%(P <0.0001). Deletion or point mutation ofthe target sequence on LIN28A3′UTR diminished the impact of miR-370on LIN28A,indicating that LIN28A is a direct downstream target for miR-370.3) Overexpression of LIN28A significantly augmented, whereas down-regulation ofLIN28A suppressed the migration and invasion of HCC cells. Suppressive effects ofmiR-370on the migration and invasion of HCC cells were substantially reduced upon theinfection of a lentiviral expression vector of LIN28A without3′UTR. These findingsdemonstrate that down-regulation of LIN28A contributes to the functional role of miR-370in HCC cells.(2) LIN28A blocks the maturation of miR-370by binding to its precusor1) Overexpression of LIN28A significantly decreased miR-370level whilesubstitution of a single amino acid (C161A) required for RNA binding affinity of LIN28Aefficiently reversed the effect of LIN28A on miR-370. As a positive control, let-7levelwas also reduced upon ectopic expression of LIN28A but not C161A mutation. However,as a negative control, miR-21level was not influenced by LIN28A. In contrast, knockdownof LIN28A by siRNA substantially raised the level of miR-370and let-7but not miR-21. 2) RNA binding protein immunoprecipitation (RIP) assay and the followed Real-timePCR revealed that both miR-370and let-7precursors but not miR-21precursor werehighly enriched in LIN28immunoprecipitates from PLC/PRF/5cells.3) To confirm the specificity of the binding, MHCC-97H cells were transfected withFlag-LIN28A or the empty vector. The following RIP assay displayed significantenrichments of pre-miR-370and pre-let-7in Flag-LIN28A immunoprecipitates whichabolished by C161A mutation.3. The novel miR-370-LIN28A-NF-κB regulatory circuit is involoved in theprogression of HCC(1) LIN28A activates NF-κB pathway by post-transcriptionally regulating RelA/p651) LIN28A overexpression enhanced but LIN28A knockdown suppressed the activityof NF-κB luciferase reporter in HCC cells. LIN28A inhibition resulted in down-regulationof NF-κB target genes, including IL6, TNFα and MMP9, indicating that LIN28A isimplicated in the activation of NF-κB pathway in HCC. Real-time PCR failed to detect asignificant change of RelA/p65mRNA level by LIN28A. The protein level of RelA/p65,however, was drastically increased with LIN28A overexpression and decreased uponLIN28A knockdown.2) RIP assay and the followed Real-time PCR revealed that RelA/p65mRNA werehighly enriched in LIN28immunoprecipitates from PLC/PRF/5cells.3) MHCC-97H cells were transfected with Flag-LIN28A or the empty vector. Thefollowing RIP assay displayed a significant enrichments of RelA/p65mRNA inFlag-LIN28A immunoprecipitates which abolished by C161A mutation.4) Overexpression of LIN28A increased but repression of LIN28A decreased theactivity of the luciferase reporter gene carrying RelA/p653′UTR.5) The effects of LIN28A on the migration and invasion of HCC cells were reversedupon inhibition of RelA/p65NF-κB transcriptional activity with Oridonin and JSH-23.(2) Perturbation of miR-370-LIN28A-NF-κB regulatory circuit contributes to thedevelopment of HCC1) miR-370overexpression decreased but miR-370inhibition increased RelA/p65protein and the activity of NF-κB luciferase reporter in HCC cells, but RelA/p65mRNAwas not affected. Consistently, ectopic expression of miR-370led to a down-regulation ofNF-κB target genes (i.e., IL6, TNFα and MMP9) while inhibition of miR-370yielded an opposite effect. Reduced expression of RelA/p65protein levels repressed these NF-κBtarget genes were also observed in MHCC-97H xenografts treated with Ad-miR-370.2) The impacts of miR-370on RelA/p65protein level, the activity of NF-κBluciferase reporter and NF-κB downstream genes in HCC cells could be abrogated bynon-targetable LIN28A.3) The effects of miR-370on migration and invasion in HCC cells were abrogated byOridonin or JSH-23.4) Treatment of HCC cells with IL6significantly decreased miR-370level followedby an increase of LIN28A protein. The methylation inhibitor5-aza-2′-deoxycytidine(5-aza-CdR) markedly increased the expression of primary miR-370, suggesting thatmiR-370may be down-regulated in HCC through an epigenetic manner.5) Correlation studies displayed that miR-370levels were inversely correlated withLIN28A and IL6mRNA levels in human HCC specimens and LIN28A expression waspositively correlated with IL6expression. Specifically, low expression of miR-370wasmore likely in human HCC specimens with high levels of LIN28A and IL6mRNAs, whilehigh expression of LIN28A was more likely in human tissues with high IL6levels.【Conclusion】1. miR-370inhibits the malignant phenotype of HCC cells in vitro and in vivo.2. RNA binding protein LIN28A serves as a direct and functional mediator ofmiR-370.3. LIN28A blocks the maturation of miR-370by binding to its precusor and thusforming a double negative feedback loop with miR-370.4. LIN28A activates NF-κB pathway by post-transcriptionally regulating RelA/p65.5. Perturbation of miR-370-LIN28A-NF-κB regulatory circuit is involoved in theprogression of HCC. |
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