Pcdna3

Based on the IKKβ gene (NM_053355), the shRNA sequence was designed (Table I) for the IKKβ gene and a negative control was designed that was verified by BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to have no interference effect on other genes. Then based on the small interfering (si)RNA sequences, complementary single-stranded DNA was designed (Table II).
The lentivirus vector plasmids used in this study are pcDNA3.1+ and pLKO.1 (Tiangen Biotech Co., Ltd), among which the interference and overexpression sequence were constructed in pLKO.1 and in pcDNA3.1+ plasmid respectively. Plasmids were amplified in Escherichia coli., followed by the lentivirus packaging. RLE-6TN cells (1×10⁶/well, 20 µl of virus solution per well) were infected with lentivirus which carried IKKβ-shRNA and IKKβ overexpression, by which the stable expression of sh-IKKβ and IKKβ overexpression was obtained. RLE-6TN cells infected by the virus alone was used as control.

SOX17-expressing plasmids and empty vectors (mock, pcDNA 3.1), and short hairpin RNA (sh)SOX17 and a scramble control were purchased from Tiangen Biotech Co., Ltd. Human SOX17 was cloned by reverse transcription-PCR (RT-PCR) from cDNA derived from adjacent normal tissue samples of patients with endometrial cancer (forward, 5′-GTTCGGATCCGCCATGAGCAGCCCGGATGCG-3′; reverse, 5′-ATGTGAATTCCACGTCAGGATAGTTGCAGTA-3′), and its sequence was confirmed by direct sequencing of PCR products. Sequencing analysis was carried out using the BigDye Terminator v1.1 sequencing kit, according to manufacturer's protocol (Applied Biosystems; Thermo Fisher Scientific, Inc.). To generate human SOX17 expression constructs, the entire encoding region of its cDNA was subcloned in frame into pcDNA 3.1 (Invitrogen; Thermo Fisher Scientific, Inc.). Suppression of SOX17 expression was induced using shSOX17: 5′-CGCACGGAAUUCGAACAGUAU-3′. Negative control shRNA (5′-ACCGAGCAGUACAACGGGAAC-3′; Applied Biosystems; Thermo Fisher Scientific, Inc.) was used as a control.

The cell growth-inhibitory activity was assessed by an MTT assay. HKULC4 cells were seeded in a 96-well plate (2,000 cells/well) and exposed to fresh medium with ABCG1-expressing plasmids or empty vectors (pcDNA 3.1; both Tiangen Biotech Co., Ltd.) for 48 h. Subsequently, 20 µl MTT solution (5 mg/ml; Sigma-Aldrich; Merck-Millipore, Darmstadt, Germany) was added. Following incubation for 4 h at 37°C, the medium was discarded and the formed formazan crystals were dissolved in DMSO (150 µl/well). The absorbance, which is directly proportional to the number of viable cells, was measured at a wavelength of 490 nm using a multilabel counter microplate reader (Safire; Tecan Austria GmbH, Grödig, Austria).