The Role Of LncRNA H19 In Nasopharyngeal Carcinoma Cell Invasion And Underlying Mechanism

Objective:Nasopharyngeal carcinoma(NPC)is a common head and neck cancer in southern China.Although it is sensitive to radiotherapy and radiochemotherapy,a significant proportion of patients still develop neck and distant metastasis and the prognosis of these patients remains poor.Recurrence and metastasis of NPC is a complicated process and the underlying molecular mechanisms remain largely unclear.Tumor metastasis is a continuous dynamic process,including multiple stages with interaction of a variety of factors.A key step of that is tumor invasion to basement membrane,which involves changes in cell morphology,secretion of matrix-degrading enzymes,cell migration.An important way for tumor cell to obtain capability of invasion and metastasis is epithelial-mesenchymal transition(EMT).Long non-coding RNA(lncRNA)is an important class of regulatory RNA.Recent studies have shown that they are associated with a variety of human diseases,especially with cancer.Many lncRNAs have been found expressing disorderly,and acting as tumor promoters or tumor suppressors.H19,one of the lncRNAs,is overexpressed in a variety of tumors,regulating cancer proliferation,apoptosis,invasion and EMT.H19 is specifically upregulated in the undifferentiated NPC cells,not in the well differentiated NPC cells,implying H19 may be related to the differentiation and metastasis of NPC.However,the effects of H19 on NPC invasion and metastasis have not been reported.A recent study found that H19 could promote bladder cancer metastasis through interaction with EZH2.Interestingly,EZH2 is also overexpressed in NPC,especially in poorly differentiated NPC cells.Taking into account that EZH2 is an important tumor metastasis-related genes,we speculated that H19 may also interact with EZH2 in NPC.EZH2 gene is an important member of PcG family,and as an important component of PRC2,functions as histone methyltransferase,which is closely related with cancer development.The recent study has shown H19 could inhibit the expression of E-cadherin and promote EMT through direct interaction with EZH2 in bladder cancer.In addition to direct interaction,previous reports showed that H19 could regulate a variety of miRNA function through miRNA sponge,and EZH2 was regulated by miRNA.However,whether miRNAs are involved in regulation of H19 on EZH2 is unclear.Whether EZH2 also mediates the function of H19 in NPC to promote metastasis deserves further study.Therefore,this part first detected the expression of H19 and EZH2 in NPC tissues and cells,and analyzed the relationship between the expression of EZH2 and H19.To explore the role of H19 and EZH2 in NPC invasion and metastasis,we then observed the effects of H19 or EZH2 down-regulation on cell invasion using lentivirus-mediated H19 and EZH2 shRNA.Furthermore,we revealed the underlying mechanism of H19 in NPC invasion.Method:1.31 cases of NPC tissues and 30 cases of nasopharyngeal chronic inflammation tissues were collected and total RNA was extracted.After reverse transcription,Real-time PCR was performed to detect the expression levels of H19 and EZH2.The first case of nasopharyngeal chronic inflammation tissue was used as control and the relative RNA expression was calculated using the 2-△△Ct method.The Pearson correlation coefficient was used to analyze the correlation of H19 and EZH2 expression in NPC tissue.2.Normal nasopharyngeal epithelial cells NP69 and nasopharyngeal carcinoma cell lines CNE1,CNE2 and HONE1 were cultured.After total RNA extraction and everse transcription,Real-time PCR was performed.NP69 was used as control and the relative H19 and EZH2 expression was calculated.Protein of NP69,CNE1,CNE2 and HONE 1 cells was extracted and western blot was performed to detect EZH2 protein expression.3.Lentivirus with H19 shRNA(Lv-H19 shRNA)or Lv-EZH2 shRNA(Lv-EZH2 shRNA),lentivirus negative control(Lv-NC)were used to transfect HONE1 of CNE2 cells.After puromycin screening and detection of H19 or EZH2 expression by Realtime PCR,stable transfected HONE1 of CNE2 cells was constructed.EZH2 protein expressions were also detected by western blot.4.CNE2 and HONE1 cells(control group,Lv-NC group and H19 or EZH2 interference group)was added into Matrigel coated Transwell chambers,after culturing for 24 hours,invaded cells were counted to assess the cell invasion capability.5.EZH2 expressions in H19 shRNA stably transfected CNE2 and HONE1 cells were detected by Realtime PCR and western blot.6.RNA immunoprecipitation(RIP)assay was performed to determine the interaction of H19 and EZH2.CNE2 cells were lysed and then incubated with magnetic beads conjugated with anti-EZH2 antibody or IgG as a negative control.H19 in the immunoprecipitated RNA was detected by RT-PCR.An input RNA without immunoprecipitation was a positive control.7.Potential miRNA-binding sites of EZH2 3’ UTR were predicted using online database TargetScan(http://www.targetscan.org),microRNA.org(http://www.microrna.org)and miRDB(http://www.mirdb.org/miRDB).The complementary sequences of these miRNAs and H19 were analyzed by blast tool(http://blast.ncbi.nlm.nih.gov/Blast.cgi).8.The luciferase assay system was used to test whether EZH2 is a target gene of these miRNAs in NPC cells.Luciferase reporter vectors with complete EZH2 3’ UTR(pGL3-wt EZH2 3’ UTR)or mutated EZH2 3’ UTR(pGL3-mut EZH2 3’ UTR)were construct.CNE2 cells were co-transfected with pGL3-wt EZH2 3’ UTR or pGL3-mut EZH2 3’ UTR,corresponding miRNA mimics and pRL-TK as an internal control.48 h post-transfection,the cells were lysed and luciferase activity was measured.The relative luciferase activity was analyzed by normalization with a control group.9.The expressions of corresponding miRNAs in miRNAs mimics or mimics NC transfected cells were detected by Realtime PCR.10.Pulldown and northern blot assay were performed to determine the interaction of H19 and miRNAs.The cell lysate was incubated with H19 probe-coated beads and the RNA complexes bound to the beads were extracted.Northern blot assay was then performed to analyze the levels of miR-101 and miR-630 using their probes in the RNA complexes.11.MiRNA sensor and luciferase reporter assay were performed to determine the effect of H19 on miR-630 activity.To construct the miR-630 sensor,the complementary human genomic sequence flanking pre-miR-630 was synthesized and inserted into the psiCHECK vector.To overexpress H19,pcDNA-H19 and pcDNA-mut H19 were constructed.CNE2 cells or H19 interfering CNE2 cells were co-transfected with rmiR-630 sensor and miR-630 mimics,mimics NC,pcDNA-H19 or pcDNA-mut-H19.After 48 h,luciferase activity was measured and Renilla luciferase activities were normalized to firefly luciferase activities.12.CNE2 and HONE1 cells were transfected with mimic NC,miR-630 mimics or co-transfected with miR-630 mimics and pcDNA-H19,miR-630 mimics and pcDNA-mut H19.EZH2 expressions were detected by Realtime.PCR and western blot.13.Control NPC cells(CNE2 and HONE1)or H19-interfered NPC cells were transfected or co-transfected with miR-630 mimics,pcDNA-H19,pcDNA-mut-H19,miR-630 inhibitor or Lv-EZH2-shRNA.48h after transfection,cell invasion assay was performed to assess the cell invasion capability.14.After above transfection,E-cadherin protein expressions were detected by western blot.15.Western blot was performed to detect the expression levels of IKKa,p-ERK1/2,ERK1/2 and MMP-9 in control CNE2 cells,Lv-NC or EZH2 shRNA transfected CNE2 cells.Results:1.H19 and EZH2 expression in NPC tissues was higher than in nasopharyngeal chronic inflammation tissues,and the difference was statistically significant(P<0.05).2.Compared with in NP69 cell,H19 and EZH2 expression in well differentiated NPC cell CNE1 was slight increased without significant differences.Two poorly differentiated NPC cell lines,CNE2 and HONE1,were observed to have significant higher H19 and EZH21evels than NP69 cell(P<0.05).3.Compared with the control,H19 or EZH2 expressions in Lv-H19 shRNA or Lv-EZH2 shRNA-transfected CNE2 cells and HONE1 cell were significantly decreased.Lv-NC transfection has no effect on H19 or EZH2 expression in both cells.4.Cell invasion assay showed that compared with control,H19 or EZH2 interference significantly suppressed the invasive ability of CNE2 and HONE1 cells(P<0.05).5.Expression of H19 and EZH2 was positively associated in NPC tissues(R2=0.3037,P<0.01).6.Real-time PCR and western blot assay showed that compared with control,EZH2 expression in H19 interference group significantly reduced.7.RIP assay showed that H19 expression was only detected in total RNA sample,but not in sample immunoprecipitated with EZH2 antibody or IgG control.8.Five miRNAs were identified that contain complementary binding sites of both the EZH2 3’ UTR and H19.They were miR-101,miR-630,miR-340,miR-559 and miR-498.9.Real Time PCR analysis showed miRNAs mimics transfection significantly increased the level of the corresponding miRNAs.10.Luciferase reporter assay showed that the relative luciferase activities in cells transfected with pGL3-wt EZH2 3’ UTR were significantly reduced in the presence of miR-101 and miR-630(P<0.05),but not of other three miRNAs.Moreover,no significant response to miR-101 and miR-630 was observed in cells transfected with pGL3-mut EZH2 3’ UTR.11.Pull-down and northern blot assay showed that miR-101,miR-630 and U6 could be detected in cell lysates without pull-down by the H19 probe,but not in cell lysates with pull-down by random probe.miR-630,but not miR-101,could be pulled down by the H19 probe.12.Real Time PCR analysis showed,H19 expression was significantly increased in pcDNA-H19 or pcDNA-mut H19 transfected cells.13.MiRNA sensor and luciferase reporter assay showed that the miR-630 activity was increased in cells treated with miR-630 mimics(P<0.05).This was suppressed by pcDNA-H19 transfection(P<0.05),whereas the pcDNA-mut-H19 had no effect.The H19 knockdown also induced miR-630 activity(P<0.05).14.Realtime PCR and Western blot showed that miR-630 could inhibit EZH2 expression(P<0.05 in Realtime PCR),which was rescued by pcDNA-H19(P<0.05 in Realtime PCR),but not by pcDNA-mut-H19.15.Realtime PCR showed that miR-630 inhibitor could significantly reduce the expression level of miR-630.16.H19 or the miR-630 inhibitor attenuated the suppression of cell invasive ability by miR-630 or H19 interference,respectively(P<0.05).After EZH2-shRNA was used for transfection,NPC cells with silenced H19 and low levels of miR-630 showed significantly reduced cell invasive ability(P<0.05).17.H19 interference and the miR-630 mimic increased the E-cadherin level in NPC cells.This.increase of E-cadherin level was attenuated by the miR-630 inhibitor or WT H19,but not by mut-H19.EZH2 interference abolished the effect of the miR-630 inhibitor and increased the E-cadherin level.18.Western blot detection showed that compared with control,IKKa levels were markedly increased,total ERK1/2 protein level had no change,while p-ERK1/2 and MMP-9 protein levels were obviously decreased in EZH2-interfered cells.Conclusion:1.H19 and EZH2 is overexpressed in NPC tissues and poorly differentiated NPC cells.H19 or EZH2 down-regulation significantly inhibited invasion of NPC cells.2.Expression of H19 and EZH2 was positively associated in NPC tissues.H19 interference inhibited EZH2 expression.3.H19 regulated EZH2 expression by suppressing the activity of miR-630,which is a repressor of EZH2 and interacts with H19 in a sequence-specific manner.4.H19/miR-630/EZH2 pathway promoted cell invasion probably through inhibition of E-cadherin expression or IKKα/ERK pathway-mediated up-regulation of MMP-9 expression.