| BackgroundLong non-coding RNA Actin filament-associated protein 1 antisense RNA 1(LncRNA-AFAP1-AS1)has attracted attention in cancer research.LncRNA AFAP1-AS1 is transcribed from antisense strand of AFAP1 protein.The biological functions and regulation mechanism of IncRNA AFAP1-AS1 in hepatocarcinoma still unclear.CRK was initially originated in avian sarcoma virus CT10 as an oncogene protein and comprised of SH2 and SH3 domains.CRKI,CRKII and CRKL,the three members of CRK family,are broadly expressed in many tissues.Recent studies show that abnormal expression of CRKL is closely associated with the variety of cancers and their progression via the regulation of cytoskeleton reorganization and signal transduction process.ObjectiveWe investigated the positive bidirectional correlation of CRKL and lncRNA AFAP1-AS1.The present research inquired the role of IncRNA AFAP1-AS1 in the regulation of HCC proliferation,migration,invasion and metastasis through c-Src/CRKL regulation mechanism.The present study investigated the role of IncRNA AFAP1-AS1 in HCC metastasis via Ras,MEK,c-Jun-EMT signaling pathway.MethodsThe IncRNA AFAP1-AS1 expression in HCC tissues and cell lines were investigated by qRT-PCR assay.CRKL mRNA and protein expressions were investigated by qRT-PCR and western blotting.LncRNA AFAP1-AS1 knockdown effects on HCC proliferation,colony formation,migration-invasion and metastasis were investigated by MTT assay,colony formation,transwell chamber and wound healing assays respectively.And c-Src,Ras,MEK,c-Jun and EMT markers were measured by western blot analysis.ResultsSignificant upregulation of lncRNA AFAP1-AS1 was found in hepatocarcinoma tissues comparing with paired paracancerous non-tumor liver tissues,as well as in hepatocellular carcinoma cell lines Huh7,HCCLM3 and HepG2 as compared to normal non-malignant liver cell LO2.Conversely,downregulation of lncRNA AFAP1-AS1 by siRNA interference led to significant decreases in proliferative,colony forming,migratory,invasive and metastatic capabilities of HepG2 and HCCLM3.It was also found a positive correlation of lncRNA AFAP1-AS1 with oncogenic CRKL expression in hepatocarcinoma patient's specimens.Bidirectional regulation of CRKL and lncRNA AFAP1-AS1 in hepatocellular carcinoma was evidenced experimentally.Knockdown of CRKL by siRNA significantly suppressed expression of AFAP1-AS1 in HepG2 and HCCLM3,and in turn,upregulation of CRKL significantly elevated AFAP1-AS1 expression in PCDH-CRKL HepG2 and PCDH-CRKL HCCLM3.Contrarily,loss of lncRNA AFAP1-AS1 downregulated expression of CRKL in HepG2 and HCCLM3,and knockdown of lncRNA AFAP1-AS1 diminished the c-Src kinase expression in both cell lines.Silencing of lncRNA AFAP1-AS1 positively altered the expressions of Ras,MEK and c-Jun,and also effected epithelial-messenchymal transition(EMT)process through increasing expression of E-cadherin and decreasing expression of N-cadherin and vimentin in HepG2 and HCCLM3.ConclusionTogether,these results provide an understanding that lncRNA AFAP1-AS1 is a critical oncogenic biomarker.LncRNA AFAP1-AS1 promotes proliferation,migration,invasion and metastasis via c-Src-regulated CRKL mechanism in hepatocarcinoma.LncRNA AFAP1-AS1 also regulates Ras/MEK/c-Jun and EMT pathway through c-Src/CRKL axis.As a consequence,lncRNA AFAP1-AS1 and CRKL can be potential candidates for therapeutic targets in hepatocarcinoma. |
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