The Role And Mechanism Of MiRNA-146b-3p And DBN1 In Regulating Hepatocellular Carcinoma Metastasis

Background and objectivePrimary liver cancer refers to the liver cells or intrahepatic cholangiocarcinoma cancer,which is one of the most common malignant tumors in China.Recently,the incidence of primary liver cancer in the world is on the rise.Primary liver cancer is divided into hepatocyte type,bile duct cell type and mixed type.Hepatocellular carcinoma(HCC)is the most common type of primary liver cancer and it's the world's fifth-ranked cancer.In the world,approximately 1 million to 2.5 million people die of HCC each year.HCC is a highly aggressive malignancy with rapid progress and poor prognosis.In recent years,although HCC patients survival rate has been improved due to the progressed radiotherapy/chemotherapy and surgery,metastasis and recurrence are still the main threat for HCC patients' life.MicroRNAs(miRNAs)are a class of 19-22 nt evolutionarily conserved small non-coding RNAs.Mature miRNAs are obtained by continuous digestion of longer primary transcripts,which play a negative role in transcription and translation of genes.Numerous studies have shown that miRNAs play an important role in cancer-related processes such as cell proliferation,cell cycle arrest,apoptosis,invasion and metastasis of cancer.Aberrant expression of miRNAs can target the relevant signaling molecules and play a regulatory role in the occurrence and development of HCC.miRNAs are highly specific in different diseases and tissues,suggesting that it might be a new possible target for cancer diagnosis and treatment.Developmental regulated brain protein(DBN1),also known as Drebrin,is a highly conserved protein,especially in the amino terminus containing the actin depolymerization homology domain and the actin binding domain(residues 1-315).DBN1 was reported in neuronal cells at first time,but emerging evidence has shown that DBN1 was also expressed in a variety of non-neuronal cells,mainly locating in the actin rich pseudo-foot region..The connection between cells and cells and the formation of pseudopods play important roles in the metastasis and invasion of cancer.DBN1 is mainly associated with cell processes and cell junctions,and it is combined with fibroblast actin(F-actin).What's more,F-actin can affect the ability of tumor cells movement.Therefore,we speculate that DBN1 may play an important role in the metastasis and invasion of cancer.This study aimed to investigate the expression of miR-146b-3p and DBN1 in HCC and their related functions and explore the underlying mechanism of miR-146b-3p/DBN1/F-actin in HCC metastasis and invasion.Research contents and methodsIn this study,we use GEO HCC datasets,HCC clinical samples,HCC cell lines and nude mice as the objects.Firstly,the low expression of miR-146b-3p in HCC was screened from the GEO HCC datasets.Survival analysis was used to compare the effect of the expression level on the prognosis of HCC.The expression of miR-146b-3p in HCC was detected by real-time PCR.The influence of miR-146b-3p overexpression on the migration,invasion,proliferation,apoptosis and cycle of HCC cells was detected by Transwell,Matrigel-Transwell,CCK8 and flow cytometry.This part mainly discusses the role of miR-146b-3p in HCC.Secondly,the miR-146b-3p potential target gene(DBN1)was predicted based on the site of miRNAs.The expression level of DBN1 was detected by real-time PCR and Western blot after miR-146b-3p overexpressed.Double luciferase reporter assay was used to detect whether miR-146b-3p directly target DBN1.We used GEO HCC datasets to analyze the expression of DBN1 in HCC and its impact on tumor volume,metastasis,staging and prognosis.Transwell,Matrigel-Transwell,CCK8 and flow cytometry were used to detect the effects of DBN1 on the migration,invasion,proliferation,apoptosis and cell cycle of HCC cells.Laser confocal and CO-IP were used to investigate the effects of miR-146b-3p on DBN1 and F-actin complex formation,the regulation of cell polarization and pseudopodia formation,thus affecting the migration and invasion of HCC.This part mainly discusses the role and mechanism of miR-146b-3p/DBN1 in HCC invasion and metastasis.Finally,we established the stablely transfected LV-NC shRNA-HepG2 and LV-DBN1 shRNA-HepG2 cells by lentivirus packaging.The effect of DBN1 on the metastasis and invasion of HepG2 cells in nude mice was detected by flow cytometry.This part mainly elucidates the role of DBN1 in HCC cells metastasis and invasion in vivo.ResultsFirst,in this study,bioinformatics analysis and clinical specimen detection showed that miR-146b-3p was lowly expressed in HCC and its low expression was correlated with poor prognosis of HCC.CCK8 and flow cytometry results showed that miR-146b-3p had no significant relationship with the proliferation,cell cycle and apoptosis of HCC cells.Transwell,Matrigel-Transwell and wound healing assays showed that miR-146b-3p inhibited the invasion and metastasis of HCC.These results demonstrated that miR-146b-3p played an important role in the prognosis,invasion and metastasis of HCC.Furthermore,miRNAs related webs predicted that DBN1 might be the miR-146b-3p potential target gene.Real-time PCR and Western blot showed that miR-146b-3p could inhibit the expression of DBN1,while the double luciferase reporter assay showed that miR-146b-3p did not directly target DBN1.Bioinformatics analysis showed that DBN1 was highly expressed in HCC,and its high expression was correlated with poor prognosis,staging and metastasis of HCC.Gene set enrichment analysis revealed that DBN1 was associated with cytoskeletal protein-related gene enrichment,indicating that DBN1 may regulate HCC through cytoskeletal related protein pathway.The expression of miR-146b-3p was negatively correlated with DBN1 in HCC clinical samples.CCK8 and flow cytometry results showed that DBN1 had no significant relationship with the proliferation,cell cycle and apoptosis of HCC cells.Transwell,Matrigel-Transwell and wound healing results showed that the high expression of DBN1 promoted the invasion and metastasis of HCC.CO-IP experiment confirmed that DBN1 directly combined with F-actin,laser confocal experiment demonstrated that overexpression of miR-146b-3p or inhibition of DBN1 weakened HepG2 cell polarization and filopodia pseudo-contraction.These results suggested that miR-146b-3p blocked the binding of DBN1 to F-actin by inhibiting the expression of DBN1,decreasing the polarization of cells and the formation of filopodia,and ultimately reducing the migration and invasion of HCC cells.Finally,lentiviral packaging experiments successfully constructed LV-NC shRNA-HepG2 and LV-DBN1 shRNA-HepG2 stable transfected cell lines.Nude mice in vivo experiments showed that the invasion ability of DBN1 stablely silenced HepG2 cells to mice lung was decreased versus wild type HepG2 cells,further confirming the important role of DBN1 in HCC invasion and metastasis.Conclusion1.miR-146b-3p is lowly expressed in HCC tissues and cell lines,and is closely related to the prognosis of HCC.Low expression of miR-146b-3p promotes the invasion and metastasis of HCC.2.DBN1 is highly expressed in HCC tissues and cell lines,and its high expression is correlated with staging,metastasis and prognosis of HCC.High expression of DBN1 promotes the invasion and metastasis of HCC.3.In HCC,miR-146b-3p blocks the binding of DBN1 to F-actin through inhibiting DBN1,which regulates cell polarization and pseudopodium formation,and finally contribute to migration and invasion of HCC cells.4.The invasive ability of LV-DBN1 shRNA-HepG2 cells to mice lung tissue was lower than LV-NC shRNA-HepG2 cells.It demonstrates that DBN1 plays a crucial role in HCC invasion and metastasis in vivo.