Effects Of Circular RNA Circ-0070934 On The Biological Behavior Of Hepatocellular Carcinoma And Its Molecular Mechanism

1.Integrated bioinformatics analysis reveals role of the miR-1247-5p acts as a tumor suppressor in hepatocellular carcinomaObjective: This study aims to identify miRNA that plays a tumor suppressor role in hepatocellular carcinoma(HCC)and explore whether it have inhibitory effects on HCC cell proliferation,invasion and migration in vitro,and find miRNA-targeted genes.Methods:(1)Using bioinformatics analysis to mine possible miRNA which acts as a tumor suppressor in HCC and find miRNA-targeted genes.(2)The expression levels of KIF18 B in HCC tissue microarrays and clinical HCC tissue samples were detected using immunohistochemistry(IHC)and Western Blot,respectively.(3)Both Hep G2 and Huh7 cells were transfected with mimics and inhibitor of miR-1247-5p.After transfection,miR-1247-5p expression was quantitated by quantitative real-time PCR(q RT-PCR).(4)Both Hep G2 and Huh7 cells were transfected with overexpressed plasmid and small interfering RNA(si RNA)of KIF18 B.After transfection,KIF18 B expression was quantitated by q RT-PCR and Western Blot.(5)Colony formation assay and Cell Counting Kit-8(CCK8)assay were performed to verify the influence of miR-1247-5p and KIF18 B overexpression and knockdown on the proliferation of Hep G2 and Huh7 cells.(6)Wound-healing assay and Transwell chambers assay were performed to verify the influence of miR-1247-5p and KIF18 B overexpression and knockdown on the invasion and migration of Hep G2 and Huh7 cells.(7)This study constructed wild-type and mutant KIF18 B 3′UTR luciferase reporter vectors.q RT-PCR,Western Blot and dual-luciferase reporter assay were conducted to verify whether miR-1247-5p binds to and regulates the expression of KIF18 B.(8)This study performed gain-of-function experiments to verify whether KIF18 B can rescue the influence of miR-1247-5p on the biological behavior of HCC cell lines.Results:(1)A low level of miR-1247-5p was expressed in the HCC tissues compared with normal tissues based on TCGA database and lower miR-1247-5p expression correlated with progressive clinicopathological characteristics and shorter overall survival of HCC patients.(2)A high level of KIF18 B was expressed in the HCC tissues compared with normal tissues,which predicted poor prognosis in HCC patients.Higher KIF18 B expression correlated with progressive stage and poor differentiation grade.(3)Compared with the control group,the expression of miR-1247-5p in HCC cells was increased and decreased at different levels after transfecting with miR-1247-5p mimics/inhibitor.(4)Compared with the control group,the m RNA and protein expression of KIF18 B in HCC cells was increased and decreased at different levels after transfecting with KIF18 B overexpressed plasmid and si RNA.(5)miR-1247-5p overexpression and KIF18 B knockdown exerted significantly suppressive effects on the proliferation,invasion and migration of Hep G2 and Huh7 HCC cells.Conversely,the ability of above biological behavior in HCC cells was strengthened when the expression miR-1247-5p was silenced and KIF18 B was enhanced.(6)KIF18B was a direct target of miR‐1247‐5p in HCC cells.miR-1247-5p regulated KIF18 B m RNA and protein expression levels.(7)KIF18B partially reversed the effects of miR-1247-5p on the biological behavior of HCC cell lines.Conclusions: miR-1247-5p suppressed the HCC cell proliferation,invasion and migration through inhibiting the expression of KIF18 B.2.Circular RNA circ-0070934 accelerates proliferation and metastasis of hepatocellular carcinoma by regulating miR-1247-5p/KIF18 B axisObjective: This study aims to clarify the role of circRNA in the regulation of miR-1247-5p expression in HCC cells and investigate whether this circRNA could as a “sponge” of miR-1247-5p to decrease the binding of miR-1247-5p and KIF18 B 3′UTR,and finally promotes HCC cell proliferation,invasion and migration.Methods:(1)The circRNA was screened by bioinformatics analysis,Ago2-RIP qPCR,and dual-luciferase reporter assay.(2)Sanger sequencing,RNase R digestion assay,and actinomycin D(Act D)chase experiment were used for circRNA identification in Hep G2 and Huh7 cells.(3)Fluorescence in situ hybridization(FISH)probes for circ-0070934 and U6 were synthesized.RNA isolation and FISH assay were employed to determine the localization of circ-0070934 in HCC cells.(4)This study constructed wild-type and mutant circ-0070934 luciferase reporter vectors.Dual-luciferase reporter assay,q RT-PCR,and co-localization FISH were conducted to verify whether circ-0070934 binds to and regulates the expression of miR-1247-5p.(5)Both Hep G2 and Huh7 cells were transfected with overexpressed plasmid and si RNA of circ-0070934.After transfection,circ-0070934 expression was quantitated by q RT-PCR.(6)Colony formation assay,CCK8 assay,5-ethynyl-2?-deoxyuridine cell proliferation assay,and cell cycle and apoptosis analysis kit were performed to verify the influence of circ-0070934 overexpression and knockdown on the proliferation of Hep G2 and Huh7 cells.Expressions of PCNA,cyclin D1,p21,Bcl-2,and c-caspase3 were detected by Western Blot analysis.(7)Transwell chambers assay was performed to verify the influence of circ-0070934 overexpression and knockdown on the invasion and migration of Hep G2 and Huh7 cells.Expressions of E-cadherin,N-cadherin,Vimentin,MMP2,MMP3 and MMP9 were detected by Western Blot analysis.(8)This study performed gain-of-function experiments to verify whether miR-1247-5p and KIF18 B can rescue the influence of circ-0070934 on the biological behavior of HCC cell lines.Results:(1)Circ-0082730,circ-0074817,circ-0070934,circ-0054715 and circ-0062517 enriched in Ago2 pellet compared to the Ig G immunoprecipitates.miR-1247-5p overexpression significantly reduced the luciferase activity of wide-type vector of circ-0082730 and circ-0070934.(2)The spliced mature sequence length of circ-0070934 derived from the LARP1 B gene is 745 bp.Circ-0070934 was resistant to RNase R digestion and actinomycin D degradation.(3)Circ-0070934 was mainly distributed in cytoplasm of HCC cells,except for a small amount in the nucleus.(4)miR-1247-5p was a direct target of circ-0070934 in HCC cells.(5)Compared with the control group,the expression of circ-0070934 in HCC cells was increased and decreased at different levels after transfecting with circ-0070934 overexpressed plasmid and si RNA.(6)Circ-0070934 knockdown markedly inhibited proliferation of Hep G2 and Huh7 HCC cells,induced an increase of the apoptosis rate and blocked G1-S entry,and reduced the protein expression of PCNA,cyclin D1,and Bcl-2.(7)Circ-0070934 overexpression exerted significantly promoted migration and invasion of Hep G2 and Huh7 HCC cells,induced the epithelial to mesenchymal transition(EMT),and increased the protein expression of matrix metalloproteinases(MMPs).(8)miR-1247-5p and KIF18 B partially reversed the effects of circ-0070934 on the biological behavior of HCC cell lines.Conclusions: Circ-0070934 was localized predominantly in the cytosol.Circ-0070934 inhibited apoptosis and regulated cell cycle progression,and promoted EMT in HCC cells by competitively bind miR-1247-5p and upregulating oncogene KIF18 B.3.Circular RNA circ-0070934 promotes the malignant progression of hepatocellular carcinoma by inhibiting p53-dependent growth arrestObjective: This study aims to deeply explore biological effects and the potential molecular mechanism of circ-0070934 in HCC in vitro and in vivo.Circ-0070934,miR-1247-5p,and KIF18 B expression in HCC tissue specimens were determined and the relationship between circ-0070934 and clinicopathological characteristics was analyzed.Methods:(1)The protein mass spectrometry and Co-immunoprecipitation assay were performed to find and identify the interaction between KIF18 B and other proteins.(2)The regulatory effect of circ-0070934 on the interaction between KIF18 B and p53 was verified by Western Blot and proximity ligation assay.(3)The influence of circ-0070934 on the cell localization of p53 in cells was verified by protein isolation assay.(4)This study performed gain-of-function experiments to verify whether p53 can rescue the influence of circ-0070934 on the proliferation of HCC cell lines.(5)Circ-0070934 overexpressing or circ-0070934 knockdown HCC cell lines were constructed by transfecting an overexpressing lentivirus and sh RNA,respectively and screening by puromycin selection.(6)A subcutaneous xenograft tumor model in nude mice,pulmonary metastasis tumor model,and spleen-hepatic metastasis model were established to analyze the influence of circ-0070934 overexpression and knockdown on the effect on the growth of xenograft tumors and outgrowth of metastases of HCC cells in vivo.(7)IHC was performed to analyze the effect of circ-0070934 expression levels on KIF18 B,EMT-related,and MMPs-related proteins in xenograft tumors.(8)The clinical significance of the circ-0070934 parental gene LARP1 B and the underlying signaling pathways of circ-0070934 were analyzed using bioinformatics.(9)The expression levels of circ-0070934 were detected in applied 56 HCC tissues by q RT-PCR and analyzed the correlation between the expression of circ-0070934 and clinicopathological characteristics.Results:(1)KIF18B interacted with p53.(2)Circ-0070934 facilitated the interaction between KIF18 B and p53.(3)Circ-0070934 favored the nuclear export of p53.(4)p53 partially reversed the effect of circ-0070934 on the proliferation of HCC cell lines.(5)Stable cell lines with circ-0070934 knockdown or overexpression were cultured in a high concentration of puromycin containing medium.(6)The promotion of tumor growth and metastasis by circ-0070934 overexpression was verified in mice xenograft model and spleen-hepatic metastasis model compared than the control group,which was reversed by miR-1247-5p.This study also found that circ-0070934 knockdown markedly inhibited the tumor growth and lung metastases compared than the control group.(7)IHC examination of xenograft tumor indicated that circ-0070934 markedly altered the expression levels of KIF18 B,EMT-related,and MMPs-related proteins.(8)Differences in the tissue expression of circ-0070934 parental gene LARP1 B were not associated with clinical staging and patient prognosis of HCC.Circ-0070934 was highly expressed in HCC tissues of GSE97332 chip,which might be related to cellular transcription,autophagy and TGF-β signaling pathways.(9)Circ-0070934 was highly expressed in HCC tissues and higher circ-0070934 expression correlated with progressive clinicopathological characteristics.Conclusions: Circ-0070934 was highly expressed in HCC tissues.Circ-0070934 promoted the malignant progression by inhibiting p53-dependent growth arrest.