Pcdna3.1 plasmid

To construct plasmids expressing HAX1, the full-length human HAX1 sequence was synthesized by Guangzhou RiboBio Co., Ltd. and ligated into the pcDNA3.1 plasmid (cat. no. V79020; Thermo Fisher Scientific, Inc.). For cell transfection, 2 mg/ml HAX1 vector, 50 pmol/ml miR-125a mimics (cat. no. miR10000443-1-5; Guangzhou RiboBio Co., Ltd.), 50 pmol/ml mimics negative control (mimics NC; cat. no. miR1N0000001-1-5; Guangzhou RiboBio Co., Ltd.), 50 pmol/ml miR-223 mimics (cat. no. miR10000280-1-5; Guangzhou RiboBio Co., Ltd.), 50 pmol/ml miR-125a inhibitor (cat. no. miR20000443-1-5; Guangzhou RiboBio Co., Ltd.), 50 pmol/ml inhibitor NC (cat. no. miR2N0000001-1-5; Guangzhou RiboBio Co., Ltd.), small interfering RNA (siRNA) against HAX1 (siHAX1; cat. no. siG000010456A-1-5; Guangzhou RiboBio Co., Ltd.) and siRNA negative control (siNC; cat. no. siN0000002-1-5, Guangzhou RiboBio Co., Ltd.) were transfected into SK-Hep1 and SNU-387 cells. Cell transfection was conducted using Lipofectamine™ 2000 (cat. no. 11668; Invitrogen; Thermo Fisher Scientific, Inc.). In addition, untreated cells were used as control. SNU-387 cells transfected with pcDNA3.1 empty plasmid were used as the NC group, while SK-Hep1 cells transfected with siRNA negative control used as the siNC group.

LNCaP cells in the androgen-dependent logarithmic growth phase were digested with trypsin and inoculated in a medium containing 10% non-androgen (activated charcoal-treated) FBS and cultured at 37°C with 5% CO₂. After 10 generations of culture, bicalutamide (1.0 µM) was added to the culture medium for 20 generations, and then the concentration of bicalutamide was increased to 5.0 µM to continue subculture. By 6 months of subculture, hormone-resistant prostate cancer cells had been constructed successfully.
The ARV7 overexpression vector was based on the PCDNA3.1 plasmid (Guangzhou RiboBio Co., Ltd.). The primers were designed and the RNA fragment of the ARV7 gene was obtained using the cell genome cDNA as a template. The fragment was inserted into multiple cloning sites downstream of the plasmid PCDNA3.1 to obtain the wild-type ARV7-overexpression vector. The PCDNA3.1-ARV7 plasmid and DH5α competent cells mixed culture PCDNA3.1-ARV7. The constructed PCDNA3.1-ARV7 vector and PCDNA3.1 vector were introduced into LNCaP-B cells, respectively, using Lipofectamine 2000 reagent according to the manufacturer's instructions, and ARV7 protein expression was verified using western blotting.