Table 1.
Sequences of PCR primers.
Fig 1.
Inhibition of SNAI2 expression enhances anchorage-independent growth of hepatocellular carcinoma (HCC) cells.
(A-D) MHCCLM3 cells were infected with lentivirus expressing shControl or shSNAI2s (shSNAI234/237) and selected by puromycin. (A) The expression of SNAI2 was detected by q-PCR (left) and Western blot (right). (B-D) Tumor sphere culture (B, up) and soft agar colony formation assay (B, down) were performed as described in Materials and Methods. Representative images of tumor spheres or soft agar colonies were shown (B), and the number of spheres (C) or colonies (D) (>100 μm in diameter) were counted and calculated by GraphPad Prism 6.0 software. (E-G) SMMC-7721 cells were infected with lentivirus expressing shControl or shSNAI2 and selected by puromycin. (E) The expression of SNAI2 was detected by q-PCR and Western blot. (F/G) SMMC-7721 cells were cultured under tumor sphere culture condition as in MHCCLM3 cells. Representative images of tumor spheres were shown (F) and the sphere number (>100 μm in diameter) were counted and analyzed by GraphPad Prism 6.0 software (G). (C, D, G) All values were represented as mean with bar as SD of three independent experiments and the P values were shown between two linked groups.
Fig 2.
Alteration of SNAI2 expression has no effect on metastatic properties of HCC cells.
(A-B) MHCCLM3 cells stably expressing shSNAI2 or shControl were used to test scratch wound healing activity (A), and relative migration distance was measured (B) and calculated by GraphPad Prism 6.0 software. (C-G) Metastatic abilities of SMMC-7721 cells expressing shSNAI2 or shControl were detected. Scratch wound healing ability was tested (C) and relative migration distance was measured and calculated by GraphPad Prism 6.0 software (D). Migration ability was investigated by xCELLigence RTCA assays (E) and in vitro transwell migration assay (F/G). Representative images of migration cells (F) were shown, and cell numbers were counted and analyzed by GraphPad Prism 6.0 software (G). All values were represented as mean with bar as SD of three independent experiments. All experiments were repeated at least three times with similar results.
Fig 3.
SNAI2 knockdown promotes the efflux of Hoechst 33342 and increases ATP binding cassette (ABC) transporter and CSC gene expression of MHCCLM3 cells.
(A) Q-PCR was applied to detect mRNA levels of indicated genes in MHCCLM3 cells expressing shSNAI2/shControl shRNAs. Relative expression of indicated genes was normalized with GAPDH mRNA. (B-D) FACS was utilized to sorting cell populations of MHCCLM3 cells expressing shControl or shSNAI2 with low (black frame) and high (red frame) Hoechst 33342 staining (B), followed by Western blot for SNAI2 expression (C). Percentage of Hoechst low or high cell population was shown and compared (D). (E) Q-PCR was applied to detect mRNA levels of ABC transporter genes in MHCCLM3 cells expressing shSNAI2/shControl. Relative expression of ABC transporter genes was normalized with GAPDH mRNA. All values were represented as mean with bar as SD of three independent experiments, and the P values were measured between two linked groups. All experiments were repeated at least three times with similar results. (F) A diagram of the promoter of ABCB1 gene that drives luciferase reporter plasmid pGL3-ABCB1-luc. Empty circle and black ovals represent the transcriptional start point of ABCB1 gene and predicated binding sites of SNAI2 respectively. Sequences of predicated binding sites are shown in the text box below. (G) SMMC-7721 cells expressing shSNAI2 or shControl were transfected with luciferase reporter plasmid pGL3-ABCB1-luc and pRLSV40-Renilla. Luciferase Assay was conducted as described in Materials and Methods. (H) Western blot was applied to measure expression of indicated proteins in SMMC-7721 cells expressing shSNAI2 or shControl.
Fig 4.
Inhibition of SNAI2 expression decreases the sensitivity to chemotherapy drugs of HCC cells.
MHCCLM3 or SMMC-7721 cells were infected with lentivirus expressing shControl or shSNAI2 and selected by puromycin. (A-C) MHCCLM3 cells were treated with different concentrations of Camptothecin (CPT, A) Doxorubicin (Dox, B) and Epirubicin (Epi, C) for 48 hours, cell proliferation was measured by CCK-8 assay (right) and IC50 values (left) were calculated by GraphPad Prism 6.0 software. (D-G) SMMC-7721 cells were incubated with different concentrations of CPT (D), Dox (E), Epi (F), and Sorafenib (G) for 48 hours, cell growth (right) and IC50 values (left) were assessed as in MHCCLM3 cells. All values were represented as mean with bar as SD of three independent experiments and the P values were shown between two linked groups. (H/I) SMMC-7721 cells expressing shControl or shSNAI2 were treated with 10 μM CPT (H) or 0.5 mg/mL Dox (I) for different hours as shown, and Western blot was used to detect indicated proteins.
Fig 5.
Verapamil, inhibitor of ABC transporter, recovers the sensitivity of MHCCLM3 to chemotherapy drugs.
MHCCLM3 cells expressing shSNAI234 (A-B) or shSNAI237 (C-D) were pretreated with 1 μM Verapamil, then CPT (A/C) or Epi (B/D) was added at different concentrations for 48 hours, cell growth was tested by CCK-8 assay (right), and IC50 values (left) of CPT/Epi were calculated by GraphPad Prism 6.0 software. All experiments were repeated three times with similar results. All values were represented as mean with bar as SD of three independent experiments, and the P values were measured between two linked groups.