Annals of Hepatology (Jul 2021)
LncRNA CASC9 promotes proliferation, migration and inhibits apoptosis of hepatocellular carcinoma cells by down-regulating miR-424-5p
Abstract
Introduction and objectives: CASC9 and miR-424-5p are closely related with hepatocellular carcinoma (HCC) progression. This study aimed to evaluate the effect of CASC9 involved with miR-424-5p on the development of HCC. Materials and methods: qRT-PCR was performed to determine the mRNA expressions of CASC9 and miR-424-5p in HCC tissues/cells and adjacent normal tissues/human hepatic epithelial cells, and to analyze the relationship of CASC9 with the clinico-pathological characteristics and prognosis of HCC patients. Then, cell proliferation was measured by CCK-8 and11 LncRNAs, long non-coding RNAs; HCC, hepatocellular carcinoma; AFP, alpha-fetoprotein; CASC9, cancer sensitivity 9; ESCC, esophageal squamous cell carcinoma; qRT-PCR, quantitative reverse transcription polymerase chain reaction; ATCC, American Type Culture Collection; DMEM, Dulbecco's Modified Eagle Medium; CCK-8, cell counting kit-8; RIPA, radio-immunoprecipitation assay; SDS-PAGE, sodium dodecylsulphate polyacrylamide gel electrophoresis; PVDF, polyvinylidene fluoride; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; siNC, small interfering RNA of negative control; M: miR-424-5p mimic; IC, inhibitor control; I, miR-424-5p inhibitor. clone formation assays. Apoptosis of HCC cells was measured by flow cytometry. Besides, cell migration and invasion were determined by scratch wound-healing and Transwell assays, respectively. DIANA-LncBase V2 and dual luciferase reporter gene assay were used to verify the targeted relationship between CASC9 and miR-424-5p. Bcl-2, Bax and cleaved caspase-3 expressions were detected by Western blot. Results: Higher expression of CASC9 was observed in HCC tissues/ cells than in adjacent normal tissues/ human hepatic epithelial cells, and was closely linked to poor prognosis of HCC, tumor size, TNM stage, differentiation degree, lymph node metastasis and alpha-fetoprotein (AFP). Down-regulation of CASC9 decreased the proliferation, invasion and migration of HCC cells while enhancing apoptosis. Besides, CASC9 was negatively correlated with miR-424-5p. MiR-424-5p inhibitor enhanced cell proliferation, invasion and migration while decreasing apoptosis. Interestingly, siRNA-CASC9 partially offset the effects of miR-424-5p inhibitor on HCC cells. Conclusion: CASC9 promoted proliferation, invasion and migration and inhibited apoptosis in HCC cells by inhibiting miR-424-5p.
