Figures & data
Table 1. The correlation between the lncRNA DARS-AS1 expression level and the Pathological factors of HCC patients
Figure 1. DARS-AS1 expression in HCC tissues and cells and its correlation with HCC prognosis
A. The DARS-AS1 expression in 50 HCC tissues and non-tumor tissues was examined by RT-qPCR. *** indicatedP< 0.001. B.Kaplan-Meier analysis was adopted to assess the correlation between the DARS-AS1 level and the overall survival rate of HCC patients. C. The DARS-AS1 profile in normal liver cell line L-02 and HCC cell lines (Huh-7 and HCC-LM3) was determined by RT-qPCR. **, *** indicatedP< 0.01, P< 0.001 compared with the L-02 group. N= 3.
Figure 2. DARS-AS1 subserved HCC proliferation, invasion and EMT
A. DARS-AS1 overexpression models were constructed in Huh-7 and HCC-LM3, respectively, and the DARS-AS1 expression was tested by RT-qPCR. B and C. CCK-8 experiment was implemented to verify Huh-7 and HCC-LM3 cell proliferation. D. Cell proliferation was examined by the colony formation assay. E. Cell apoptosis was monitored by the TUNEL assay. F. The expression of Caspase3, Bax and Bcl2 in HCC cells was compared by WB. G. Transwell assay was performed to test cell invasion. H. The expression of E-cadherin, N-cadherin and Vimentin in HCC cells was compared by WB. NS, **, *** representedP> 0.05,P< 0.01,P< 0.001, compared with the Vector group. N = 3.
Figure 3. DARS-AS1 overexpression accelerated the growth and metastasis of HCC cells
Huh7 cells were transfected with DARS-AS1 overexpression plasmids and negative control vectors. The xenograft tumor model was constructed in Huh7 cells. A. On day 28, the nude mice were killed, and the tumors were isolated. B. Tumor volume from day 7 to day 28. C. Tumor weight at day 28. N = 5. D. Ki67-positive cells were counted by IHC; N = 5. E. HE staining exhibited Huh7 metastasis to lung tissues; N = 10. F. The expression of E-cadherin and Vimentin in tumor tissues was compared by IHC. *** indicatedP< 0.001 compared to the Vector group. N= 5.
Figure 4. DARS-AS1 promoted the CKAP2 expression
A and B. The CKAP2 profile in HCC tissues was examined by RT-qPCR (A) and WB (B). C. Linear regression was further used to analyze the correlation between the levels of CKAP2 and DARS-AS1 in HCC tissues, which confirmed that the two were positively correlated. D. GEPIA database revealed that CKAP2 and DARS-AS1 were positively correlated in HCC tissues. E-G. The CKAP2 profile in HCC cells overexpressing DARS-AS1 was monitored by RT-qPCR, WB and cell immunofluorescence, respectively. H-I. The CKAP2 expression in LIHC tissues and non-tumor tissues was analyzed by the GEPIA database, and the relationships between the CKAP2 level and HCC patients’ overall survival and disease-free survival were analyzed. *, *** indicated P< 0.05, P< 0.001. N= 3.
Figure 5. Overexpressing CKAP2 heightened the proliferation, invasion and EMT of HCC cells
A CKAP2 overexpression and knockdown models were constructed in Huh-7 cells, respectively. WB was implemented to verify the CKAP2 expression. B. Huh-7 cell proliferation was examined by CCK-8. C. Cell proliferation was gauged by the colony formation assay. D. Transwell assay was conducted to test cell invasion. E-F. WB was applied to compare the expression of E-cadherin, N-cadherin and Vimentin in HCC cells. G The FAK/ERK profile in HCC cells transfected with DARS-AS1 overexpression plasmids was determined by WB. H WB was utilized to test the FAK/ERK expression in HCC cells transfected with CKAP2 overexpression plasmids or sh-CKAP2. NS, **, *** indicated P> 0.05,P< 0.01,P< 0.001. N= 3.
Figure 6. DARS-AS1 targeted miR-3200-5p, and the latter targeted CKAP2
A-B. The potential miRNAs binding to DARS-AS1 and CKAP2 were predicted by the Starbase database. As a result, miR-3200-5p had a common binding site with DARS-AS1 and CKAP2. C-D. The wild-type or mutated dual-luciferase reporter gene vectors of DRAS-1 and CKAP2 and miR-3200-5p mimics were transfected into Huh7 cells. E. The miR-3200-5p expression in DARS-AS1-overexpressed HCC cells was examined by RT-qPCR. F. HCC cells overexpressing miR-3200-5p were constructed by transfecting the HCC cells with miR-3200-5p mimics. G. RT-qPCR was applied to test the mRNA level of CKAP2 in the miR-3200-5p overexpression model. NS, *** indicated P> 0.05,P< 0.001. N = 3.
Figure 7. DARS-AS1 weakened the tumor-suppressive effect mediated by miR-3200-5p overexpression
miR-3200-5p mimics or DARS-AS1 overexpression plasmids were transfected into Huh7 cells. A-D. RT-qPCR and WB were utilized to check DARS-AS1 (A), miR-3200-5p (B), CKAP2 (C) and FAK/ERK (D) levels. E. CCK-8 was performed to verify Huh-7 and HCC-LM3 cell proliferation. F. Cell proliferation was examined by the colony formation assay. G. Transwell assay was implemented to test cell invasiveness. H. The expression of E-cadherin, N-cadherin and Vimentin in HCC cells was compared by WB. NS, *, **, *** indicated P> 0.05,P< 0.05,P< 0.01,P< 0.001. N= 3.
Availability of data and material
The data sets used and analyzed during the current study are available from the corresponding author on reasonable request.