MiR-124-3p Reduces Cell Invasiveness And Metastasis Of Hepatocellular Carcinoma Cell Via Targeting CRKL

Liver cancer is the cancer that begins from the cells of liver,there are several types of liver cancers,but most primary type of liver cancer is hepatocellular carcinoma(HCC).Liver cancer is a leading cancer occurs frequently worldwide and the fifth most prevalent human malignancy globally.There are around 630,000 new HCC cases yearly internationally,the morbidity and mortality are increasing with HCC predominantly in China.Even though surgery resection pooled with radiation,chemotherapy and biological-therapy.Reoccurrence of tumor and its progression is the leading cause in HCC patients with high mortality rate up to 20-30%.HCC is a complex multistep mechanism together with invasion of ECM(extracellular matrix),intravasation,invasion and migration,which ultimately form the metastatic nodules.HCC molecular pathogenesis and prognosis are complicated and merely understood,henceforth detection for therapeutic targets possess critical importance and management of clinical data for HCC patient have integral importance in clinical research.Early diagnosis for HCC is still in a veil because of the poor understandings of its biomarkers despite the fact that there are sufficient advancements in its cellular signaling events and pathways.To control the HCC mortality and progression,the early biomarkers for analysis and treatment are to be detected.Recent studies show microRNAs perform a vital role in the progression of HCC,thus providing new clues to HCC diagnosis and therapeutics.MicroRNAs are noncoding regulatory RNAs with the size of 18-25 nucleotides produced by RNA polymerase 2 and 3.These small noncoding RNAs bind directly with 3’-untranslated region(3’UTR)of target genes regulating their expressions by suppressing or degrading the target mRNA through post-transcriptional protein translation.Many studies have proved that microRNAs are playing a vital role in the beginning and development of tumor or cancer.These small noncoding RNAs perform its role in critical biological mechanisms like cell proliferation,differentiation,metastasis,apoptosis and metabolism.Abnormal regulations of microRNAs in variety of cancers revealed their role as tumor suppressor or promoter in carcinogenesis.In precedent,studies have promised that abnormal miR-124-3p expressions linked in diverse cancers including bladder cancer,breast cancer,glioblastoma,prostate cancer,lung cancer and esophageal cancer.MiR-124-3p is essential brain augmented microRNA,miR-124-3p is involved in neural development and gastrulation regulation.Recent studies reported that miR-124-3p served as a tumor suppressor via targeting ROCK2 and EZH2 genes.Moreover,miR-124-3p could reduce tumor progression by decreasing the malignant invasion of prostate cancer by blocking TGF-α-induced EMT(epithelial-mesenchyme transition).However,miR-124-3p were found downregulated in bladder cancer,breast cancer,esophageal cancer and glioblastoma cancer,while its restoration could prevent the malignant invasion capacities of neoplastic cells.Furthermore,the decrease of miR-124-3p expression level was correlated with weak prognosis of glioblastoma patients.CRK adapter protein family member V-CRK sarcoma virus CT10 oncogene homologue(Avian)-like(CRKL)is transversely expressed in a eukaryotic organism.It is composed by one conserved N-terminal Src homolgy2(SH2),one N-terminal SH3 and one C-terminal SH3 domains including the wide range of linkage for anchoring and proline-rich proteins.It can form localized complexes with BCR-ABL,ABL-1,SOS,BCAR1,C3G,GEF,GAB and Pax to play vital function in cell adhesion,proliferation,migration and survival.CRKL can assists in the regulation of tyrosine kinase activity or cellular signaling events via making complex along with downstream receptor protein or act as the upstream facilitator for signaling initiations.The aberrant expression of CRKL has been involved in a malignant invasion,EMT and apoptosis in various cancers.Our previous study reported that CRKL is linked with cell proliferation and malignant invasion in murine hepatocarcinoma Hca-P.Though the miR-124-3p-CRKL axis in HCC’s molecular mechanism and its unique role in HCC is need to be evaluated.Objectives:To investigate the clinical significance of miR-124-3p and CRKL expression in HCC.To detect the differential expressions of miR-124-3p and CRKL in cell lines HCCLM3 and Huh7.To establish the co-relationship between miR-124-3p and CRKL.To investigate the effects of level changes of miR-124-3p and CRKL on the proliferative,migratory,invasive,motility and apoptotic capacities of HCCLM3 and Huh7.To explore miR-124-3p-CRKL axial regulation mechanism in HCC.Methods:1.Western blotting and qRT-PCR assays were done to detect the relative expression levels of CRKL protein,miR-124-3p and CRKL mRNA in HCC clinical samples and HCC cells.2.Dual luciferase gene reporter assay was used to detect the targeting of miR-124-3p to CRKL.3.Transient transfections were done to overexpress miR-124-3p and knockdown CRKL in HCC cell lines.4.MTT assay detected the effect of miR-124-3p on the proliferation of HCC cells.5.The effect of miR-124-3p overexpression on the colony forming abilities of HCC cells.6.The effect of miR-124-3p on CRKL expression level was detected using immunofluorescence assay.7.The effect of miR-124-3p overexpression on the migration and invasion capabilities of HCC cells through regulating CRKL was detected by trans well chamber assay.8.Wound healing assay was done to check the overexpression of miR-124-3p on the motilities of HCC cells.9.Western blotting method were used to detect CRKL and signaling pathways proteins.Results:1.23 cases of liver cancer clinical samples showed that CRKL expression level in liver cancer tissue were significant highly expressed(69.5%,P=0.0129)compared with their adjacent normal liver tissue samples.And miR-124-3p was significantly downregulated(37.7%,P=0.0453)in liver cancer clinical samples compared with their adjacent normal liver tissue samples.2.The expression of CRKL protein and mRNA in HCCLM3 cells were found significantly increased by 83%(P=0.0314)and 17.4-fold(P=0.0164),and in Huh7 by 31%(P=0.0093)and 14.7-fold(P=0.0178)compared with a normal liver cell LO2,and expression levels of miR-124-3p in HCCLM3 cells were found significantly downregulated by 74%(P=0.0130)and 75%(P=0.0085)in Huh7 compared with the normal liver cells LO2.3.Bioinformatics analysis indicated that CRKL was a potential target gene of miR-124-3p with putative binding sites at 2283-2289 and 3785-3791 at CRKL-3’-UTR region.Luciferase reporter assay was performed to investigate the in vitro relationship between the miR-124-3p and CRKL-3’-UTR in 293T cells,the activity of luciferase were declined by 67.9±2.6%(P=0.0002)or 53.2±22.4%(P=0.0226)in 293T cell co-transfected with the PsiCHECK-2-CRKL-3’-UTR-WT or PsiCHECK-2-CRKL-3’-UTR-WT2 luciferase reporter assay and miR-124-3p mimic in compared with negative control cells.4.For investigating effect of miR-124-3p overexpression on CRKL by qRT-PCR,the HCC cells were transiently transfected with miR-NC and miR-124-3p mimics led the expression levels increased by 59347-,6699-and 4363-fold in HCCLM3 cells,and by 3292-,29551-and 2990-fold in Huh7 cells,at the transfection times of 24,48 and 72 h.correspondingly,MiR-124-3p overexpression affected the mRNA expression levels of CRKL decreased by 65.56%(P=0.00001),31.8%(P=0.0002)and ns in HCCLM3 cells,and by 39.3%(P=0.00001),72.3%(P=0.0004)and 47%(P=0.0058)in Huh7 cells consequently.MiR-124-3p upregulation resulted in decreases of CRKL protein expression levels in HCCLM3 by 67.7%(P=0.00001),32.7%(P=0.0002)and 11.7%(ns)and by 65.7%(P=0.0001),45.7%(P=0.0018)and 39.4%(P=0.0025)at 24,48,and 72 h,respectively.5.Immunofluorescence staining assay,for the identification of CRKL expression,exhibited dull and poor CRKL expression pattern in HCCLM3 and Huh7 cell transfected with miR-124-3p mimics compared with miR-NC.6.MTT assay was performed to determine the rate of proliferation of HCC cells with miR-124-3p overexpression.Proliferation arrest was observed in HCCLM3 decreased by 15.58%(P=0.0426),21.95%(P=0.0110)and 31.14%(P=0.0227)at time intervals of 48,72 and 96 h and Huh7 cells by 10.72%(P=0.162),11.45%(P=0.0002),17.02%(P=0.00016)and 26.08%(P=0.0004)at 24,48,72 and 96 h respectively,following miR-124-3p overexpression.7.To investigate the migration and invasion capacities of HCC cells,Boyden transwell chamber assays were performed.Due to overexpressed miR-124-3p,average quantity of cell migrated cells per field was decreased from 293.6±23.2 to 141.6±58.3 for HCCLM3 cells(P=0.0138),and from 197.6±11 to 83±9 for Huh7 cells(P=0.0002).Consistently,the mean number of invaded cells per field was decreased from 305.6±40 to 175±25 for HCCLM3 cells(P=0.0087),and from 252.3±13.6 to 125±28.6 for Huh7 cells(P=0.0022).The significant reductions were observed in HCCLM3 migration capacity(48.23%P=0.0138)and in Huh7(41.99%P=0.0002).The invasive capacity of HCCLM3 were decreased by(57.25%P=0.0087)and in Huh7(49.53%P=0.0022).8.Wound healing assay were performed to investigate the cell motility abilities of HCCLM3 and Huh7 cells,the percentages of wound closures were 30%,52%and 65%for miR-NC group HCCLM3 cells at 24,48 and 72 h,while,decreased to 18%(P=0.0223),26%(P=0.0009)and 38%(P=0.0008)for miR-124-3p mimic-transfected HCCLM3 cells.Consistently,compared with the miR-NC-transfected Huh7,the measurements of wound closures of miR-124-3p mimic-transfected Huh7 cells reduced from 47%to 25%(P=0.002),from 56%to 48%(P=0.0265)and from 82%to 58%(P=0.00001)at the time intervals of 24,48 and 72 h,correspondingly.9.Colony formation assay showed that higher expression of miR-124-3p reduced the colony forming efficiency of HCCLM3 and Huh7 cells.The total number of colonies formed in HCCLM3 is 157.66±55.51 per view that was the only about 46.44%of that miR-NC(339.33±53.72,P=0.0152).And the total number of colonies formed in HUH7 is 210.33±20.03 per that was the only about 49.02%of that miR-NC(429±14.93,P=0.0001).10.The indications mentioned above verified that CRKL expression levels were inversely regulated by miR-124-3p through attaching with its 3’-UTR region.Axial regulation mechanism was investigated through using WB assay,we found compared with the miR-NC-transfected HCCLM3 cells,the protein levels of RAF,MEK,ERK1/2,p-ERKl/25 C-JUN,BCL-2,Vimentin and N-cadherin were decreased by 64%(P=0.0001),78%(P=0.0001),44%(P=0.0128),52%(P=0.0027),59%(P=0.0003),57%(P=0.0007),53%(P=0.0047)and 37.33%(P=0.0008),meanwhile,the levels of BAX and E-cadherin were amplified by 29%(P=0.034)and 33%(P=0.0284)into miR-124-3p overexpressing HCCLM3 cells via miR-124-3p mimic transfection.Concordantly,overexpressing miR-124-3p led to decreased expressions of RAF,MEK,ERK1/2,p-ERK1/2,C-JUN,BCL-2,Vimentin and N-cadherin with 51%(P=0.0054),40%(P=0.0013),49%(P=0.0001),33%(P=0.0031),47%(P=0.0003),30%(P=0.0001),37.3%(P=0.0099)and 55%(P=0.0006),whereas,led to the expression increases of BAX and E-cadherin with 86%(P=0.0054)and 50%(P=0.0133)in Huh7 cells.MiR-124-3p-CRKL axis mediates the malignant properties of HCCLM3 and Huh7 via C-JUN in regulating cell proliferation,via RAF/MEK/ERK1/2 and Vimentin/N-cadherin/E-cadherin(EMT)in regulating cell invasion and metastasis,and via BAX/BCL-2 in regulating cell apoptosis.In current study,we found miR-124-3p deficiency antagonistically interrelated with CRKL overexpression within tumorous tissues contributed in the clinical development and progression of HCC patients.Paralleled with LO2,a normal liver cell line,we found downregulated miR-124-3p and the upregulated CRKL in both HCC cell lines HCCLM3 and Huh7.MiR-124-3p directly targets CRKL.By binding with CRKL’s 3’-UTR,overexpressed miR-124-3p led to reduced CRKL’s expressions at messenger RNA(mRNA)and protein levels.Our results concluded that overexpressed miR-124-3p affects HCC cells’ growth,migration,invasion as well as motility abilities.Mechanistic investigations indicated CRKL downregulation through miR-124-3p resulted in suppressed ERK pathway as well as EMT process,and decreased invasiveness and metastasis of HCC cells.The expressions of RAF,MEK,ERK1/2 and pERK1/2,key molecules in ERK pathway,of N-cadherin and vimentin,key promoters of EMT,were all downregulated.While E-cadherin,a key suppression indictor of EMT was upregulated.MiR-124-3p-mediated CRKL downregulation led to BAX/BCL-2 increase and C-JUN downregulation,which might synergically result in suppressed growths of HCCLM3 and Huh7 cells through promoting apoptosis and inhibiting proliferation.Collectively,our information illustrates miR-124-3p acted as an important tumor-suppressive microRNA suppressing HCC carcinogenesis and progression through targeting CRKL.The miR-124-3p-CRKL axial regulation pathway offers certain valuable clues to cancer research,diagnosis and treatment.