MicroRNA-340 Inhibits The Proliferation And Invasion Of Hepatocellular Carcinoma Cells By Targeting JAK1

Background and aimsHepatocellular carcinoma(HCC)is the fifth most common malignancy and third most frequent cause of cancer related death worldwide.Various strategies are currently used to treat patients with HCC,including surgical resection,chemotherapy,and liver transplantation;however,the 5-year overall survival rate of HCC patients remains low,mostly because of intrahepatic and extrahepatic metastases or postsurgical recurrence.The molecular mechanisms underlying HCC development and progression are not clear.Janus kinase(JAK)belongs to a family of non-receptor tyrosine kinases consisting of JAK1,JAK2,JAK3,and TYK2 that activates signal transducer and activator of transcription(STAT)proteins in response to various cytokines and growth factors.The JAK/STAT signaling pathway is tightly regulated in normal cells,and it is persistently activated as a result of the aberrant activation of JAK family kinases or other tyrosine kinases in cancer cells.Among the JAK/STAT family members,JAK1/STAT3 plays a crucial role in many biological processes such as cell growth,apoptosis,migration,and invasion.STAT3 is aberrantly activated in different types of human cancer including HCC,head and neck cancer,breast cancer,and lung cancer.Recent studies showed that persistently activated JAK1/STAT3 signaling is associated with tumorigenesis and cancer progression.Thus,the JAK1/STAT3 signaling pathway may be a promising molecular target for the treatment of human cancer.MicroRNAs(miRNAs),a class of endogenous noncoding small RNAs of approximately 21–25 nucleotides in length,participate in the regulation of many biological processes by binding to the 3′ untranslated region(3′-UTR)of target mRNAs,causing mRNA degradation or post-transcriptional inhibition.Emerging evidence shows that miRNAs affect cancer initiation,promotion,and progression and represent potential diagnostic markers and therapeutic targets.Given that one miRNA can regulate one or more target genes,it is of particular interest to identify miRNAs that might interfere with the JAK1/STAT3 signaling pathway,thereby contributing to the regulation of tumor cell proliferation,migration,and invasion.Increasing evidence indicates that dysregulation of microRNAs(miRNAs)contributes to tumorigenesis.MicroRNA-340(miR-340)is downregulated in several types of cancer.However,the functional mechanism of miR-340 in hepatocellular carcinoma(HCC)remains unclear.To explore the regulatory role and the underlying mechanisms miR-340 in HCC invasion and metastasis,with the aim of laying a foundation for formulating novel therapeutic target for treatment of HCC.Materials and methods1.Cell lines and cell cultureHuman HCC cell lines(SK-Hep-1,Hep G2,HCCLM3,and MHCC97-H),the human immortalized liver cell line L02,and HEK-293 T cells were purchased from the Cell Resource Center,Chinese Academy of Science Committee(Shanghai,China).All cells were maintained in DMEM-high glucose media(Invitrogen,Carlsbad,CA)supplemented with 10% FBS at 37°C in a humidified incubator containing 5% CO2.2.Patients and tissue specimensMatched fresh HCC specimens and adjacent normal tissues were obtained from 32 clinically confirmed HCC patients who underwent hepatic resection at Eastern Hepatobiliary Surgery Hospital(Shanghai,China).None of the HCC patients had received radiotherapy or chemotherapy before surgery.This study was approved by the Ethics Committee of Eastern Hepatobiliary Surgery Hospital and all patients gave informed consent.3.Oligonucleotides,plasmid construction,lentivirus and cell transfectionmiR-340 mimics,antagomir-340 and their negative control were purchased from RiboBio(Guangzhou,China).Lentiviruses overexpressing miR-340 and negative control were purchased from GenePharma(Shanghai,China).The wild-type 3′-UTR of JAK1,which contains a miR-340 binding site,was amplified by PCR.Mutations were generated in the miR-340 binding site sequence.PCR products were inserted into the pGL3-control luciferase vector(Promega,Madison,WI).For JAK1 over-expression,the open reading frame of JAK1 was inserted into the pcDNA3.1 vector.Transfection was performed using the Lipofectamine 2000 Reagent(Invitrogen)according to the manufacturer’s protocol.4.RNA extraction and quantitative real-time PCR(qRT-PCR)Total RNA was extracted from cell lines and frozen tumor specimens using the mirVANA RNA Isolation Kit according to the manufacturer’s protocol(Ambion,Austin,TX).Complementary DNA synthesis was performed using the Prime Script RT Reagent Kit(TaKaRa,Dalian,China).qRT-PCR was performed with the SYBR green Premix Ex Taq II(TaKaRa)in an ABI 7900 system(Applied Biosystems,Carlsbad,CA).β-actin or U6 was used as an endogenous control.Relative expression levels of target gene were calculated according to the 2-△ △ CT method.5.MTT assay and colony formation assayCell viability was assessed by the MTT assay.Cells were seeded in 96-well plates at a density of 4 × 103 cells per well and cultured for 1,2,3,and 4 days after transfection.A volume of 100 μl MTT(0.5 mg/ml,Invitrogen)was added to each well of the 96-well plates,and cells were incubated for another 4 h at 37°C,followed by removal of the supernatants and addition of 150 μl of dimethyl sulfoxide(Sigma,St.Louis,MO).The absorbance at 490 nm was measured with a microplate reader(Thermo Fisher Scientific,Rockford,IL).For colony formation assay,transfected cells were cultured in six-well plates at a density of 1,000 cells per well and grown for 12 days.After fixation with methanol,colonies were stained with 0.1% crystal violet for 15 min and washed.The number of colonies was counted under a light microscope.6.Invasion assayCell invasion was detected using 24-well Transwell chambers coated with Matrigel(BD Biosciences,San Jose,CA).A total of 1 × 104 transfected cells in serum-free medium were seeded in the upper chamber.The lower chamber was filled with DMEM containing 10% FBS as a chemoattractant.After incubation for 48 h,cells that adhered to the underside of the membrane were fixed,stained and counted using a microscope.7.Wound healing assayTransfected cells were cultured in 24-well plates.The cell layer was scratched with a 200 μL pipette tip.The healing process was observed for 0 and 48 h.The wound width was measured at 0 and 48 h after scratching to assess the migratory ability of tested cells.8.Western blottingTotal protein was extracted,separated by SDS-PAGE,and transferred onto PVDF membranes(Millipore,Billerica,MA).Then,the membrane was incubated with antibodies and detected by chemiluminescence.9.Luciferase reporter assayCells were transfected with 100 ng wild-type or mutant luciferase reporters and 50 nM miR-340 mimic,along with 20 ng Renilla luciferase vector with the Lipofectamine 2000 reagent.After 48 h,luciferase activity was detected by the Dual-luciferase Reporter Assay System(Promega)and relative luciferase activity was normalized to Renilla luciferase activity.10.Tumorigenicity assay in nude miceInfected HCCLM3 cells(2×106)were injected subcutaneously into 5-week-old BALB/C nude mice.The tumor volume was measured with a caliper once every 5 days using the following formula: V(mm3)= 0.5 × length × width2.After 30 days,all tumor grafts were collected,photographed,fixed and embedded.11.Statistical analysisData are expressed as the mean ± SD.Significance between two groups was assessed with the Student’s t-test.The Spearman correlation test was used to examine the correlations between miR-340 and JAK1 expression.All statistical analyses were performed using SPSS13.0.The statistical significance was set to P <0.05.Results1.mi R-340 is downregulated in HCC tissues and cell linesThe expression levels of miR-340 were evaluated in 32 pairs of HCC specimens and matched non-tumor liver tissues by qRT-PCR.The results showed that miR-340 expression was significantly lower in HCC specimens than in the noncancerous counterparts.Next,the expression level of miR-340 was examined in four human HCC cell lines(HepG2,SK-HEP-1,HCCLM3,and MHCC97-H)and one human non-transformed hepatic cell line,L02.Consistent with the expression in tumor tissues,miR-340 was downregulated in all four HCC cell lines compared with its expression in L02 cells.These findings indicated that downregulation of mi R-340 may play a role in the pathogenesis and development of HCC.2.Effects of miR-340 on HCC cell proliferation,migration,and invasionTo examine the biological function of miR-340 in HCC progression,miR-340 mimic or control non-targeting miRNA(Ctrl)was transfected into the HCC cell lines HCCLM3 and MHCC97-H,and cell viability and colony-formation ability were measured.The MTT assay showed that the viability of cells transfected with miR-340 mimics was significantly decreased compared with that in control cells.The colony formation assay consistently revealed that enforced expression of mi R-340 dramatically reduced the number of colonies in the two HCC cell lines.Next,we investigated the effect of miR-340 on HCC cell migratory and invasive capabilities.Data from the wound healing assay showed that miR-340 mimics-treated cells had decreased wound closure ability compared with that in the control cells.The Transwell assay with Matrigel demonstrated that the number of invasive clones was significantly lower in the miR-340 over-expression group than in the control group.On the other hand,HepG2 cells that expressed high level of miR-340 were transfected with antagomir-340 or its control antagomir-NC.As expected,the results showed that suppression of mi R-340 significantly increased the proliferation,migration and invasion of HepG2 cells.These results suggest that miR-340 could impair the proliferation,migration,and invasion of HCC cells significantly.3.Effects of miR-340 overexpression on HCC xenograft tumor growth in nude miceTo validate the in vitro observations that overexpression of mi R-340 may have tumor-inhibitory effects,an in vivo xenograft tumor model of HCC was established.Stably expressing HCCLM3 cells were prepared by infection with lentivirus carrying the miR-340 gene or control,and their tumorigenic effects were examined in nude mice.Consistent with the in vitro results,miR-340 overexpression inhibited tumor growth in vivo,and the tumor size and volume derived from miR-340 overexpressing cells were dramatically smaller than those of the control group.The average tumor weight in the control group was significantly higher than that in the miR-340 group.qRT-PCR analysis of tumor tissues confirmed that mi R-340 expression levels were increased in mi R-340 overexpressing tumors.Analysis of HCC proliferation activity showed that mi R-340 over-expression decreased the levels of the cell proliferation marker Ki67 in tumor cells compared with those in the control group.These data support an important role for miR-340 in the suppression of HCC growth in vivo.4.miR-340 directly targets JAK1Next,we used online publicly available databases(TargetScan and mi Randa)to predict the candidate target genes of miR-340.JAK1,which plays an important role in the growth and metastasis of malignant cells,was identified as a potential target of miR-340.To assess whether JAK1 was a direct target of miR-340,the wild-type and mutant JAK1 3′-UTR binding sites were inserted into luciferase reporter vectors,and these vectors were subsequently co-transfected with mi R-340 mimics or negative control and pRL-TK Renilla plasmid into HCCLM3 and MHCC97-H cells.As shown in Fig.4B,miR-340 overexpression decreased the luciferase activity of the JAK1 3′-UTR,whereas the luciferase activity of the mutant JAK1 3′-UTR was not affected.The results showed that miR-340 mimics downregulated JAK1 expression in HCCLM3 and MHCC97-H cells,and that antagomir-340 increased JAK1 expression in HepG2 cells.Western blot analysis of the expression of JAK1 in subcutaneous tumor tissues showed that miR-340 over-expression downregulated JAK1 in tumor cells compared with the control group.These data suggested that miR-340 modulates JAK1 expression by directly targeting its 3′-UTR.5.Alteration of JAK1 expression influences the effects of mi R-340 on HCC cellsThe direct targeting of JAK1 by miR-340 led us to hypothesize that JAK1 mediates the effects of miR-340 on HCC cell growth and metastatic suppression.This possible mechanism was examined by gain-of-function analyses.HCCLM3 cells were transfected with negative control,miR-340,or miR-340 plus JAK1 plasmid.The expression of JAK1 was confirmed by western blotting.The results showed that restoration of JAK1 without the 3′-UTR partially abrogated miR-340-induced inhibition of HCC cell proliferation,migration,and invasion.These results supported that JAK1 is a downstream functional mediator of miR-340.6.miR-340 regulates the JAK1/STAT3 signaling pathwayTo explore whether mi R-340 regulates the JAK1/STAT3 signaling pathway,which is linked to tumor growth and metastasis,the expression of the main JAK1/STAT3 target genes,including Bcl-2,cyclin D1,and MMP2,was examined.As shown,p-STAT3,Bcl-2,cyclin D1,and MMP2 expression levels were markedly decreased in HCCLM3 and MHCC97-H cells transfected with mi R-340 mimics.Conversely,the expression levels of these molecules were significantly increased in cells co-transfected with miR-340 mimics and JAK1 plasmid.These results indicated that the JAK1/STAT3 signaling pathway is functionally involved in the effects of miR-340 on HCC cell growth and invasion.ConclusionsOur results showed that miR-340 suppressed HCC cell proliferation and invasion by regulating the JAK1/STAT3 pathway,suggesting its potential as a novel therapeutic target for HCC.