Lv nc

A lentiviral vector containing human lncSSBP1 was purchased from GenePharma (Suzhou, China) and used to overexpression lncSSBP1 (referred to as Lv-lncSSBP1). The negative control lentivirus (Lv-NC) was also purchased from GenePharma. An shRNA lentivirus vector containing the target sequence of lncSSBP1 (5′-GGCGACAAGCAACAACAATCA-3′) was also used to knock down lncSSBP1 expression. The sequence was cloned into pGLVH1 (GenePharma, SuZhou, China) to generate Sh-lncSSBP1. A negative control lentivirus containing a non-targeting shRNA sequence (5′-TTCTCCGAACGTGTCACGT-3′; referred to as sh-NC) was used as a control. All cloned sequences were verified by automated sequencing (GenePharma, SuZhou, China).
All lentiviral vectors, including Lv-lncSSBP1, Lv-NC, sh-lncSSBP1 and sh-NC, were transfected into 293FT cells for packaging. The virus particles were harvested 72 h after transfection of 293FT cells. For stable transfection, BEAS-2B cells were grown in six-well plates to 50% confluence, and 1 mL of viral supernatant was added with 1 μL polybrene. The interference efficiency of the template was verified by RT-PCR analysis.

The lentivirus system (LV-NC, LV-circ_0027885, short hairpin (sh) -circ_0027885, sh-NC) was used to infect hBMSCs. These products were purchased from Shanghai Genechem. LV-NC, LV-circ_0027885, sh-NC and sh-circ_0027885 transfected hBMSCs were harvested after incubation with 1 mg/ml puromycin for 72 h. NC mimics, miR-203-3p mimics, NC inhibitor and miR-203-3p inhibitor were obtained from Shanghai GenePharma. At about 70% confluence, hBMSCs were transfected with NC mimics, miR-203-3p mimics, NC inhibitor and miR-203-3p inhibitor by Lipofectamine™2000 (Invitrogen) to perform follow-up experiments.

Human LDOC1 cDNA was amplified, purified, and inserted into a lentiviral vector encoding enhanced GFP [27 (link), 28 (link)]. The recombinant lentiviral vector expressing LDOC1 (Lv-LDOC1) and the empty vector (Lv-NC; negative control, encoding GFP alone) were constructed by Shanghai GenePharma Co., Ltd. (Shanghai, China) and verified by sequencing. Packaging, purification, and titer determination of the recombinant lentiviruses were implemented in HEK293T cells as previously described [29 (link), 30 (link)]. In two precise preparations, the titers of the recombinant lentiviruses were 2 × 10⁸ and 5 × 10⁸ infectious units/mL, respectively.
TPC-1 cells were cultured in 24-well plates and infected with the lentivirus at a multiplicity of infection (MOI) of 20, 50, or 100 for 24 h. Flow cytometry analysis revealed that approximately 95 % of the cells were infected at an MOI of 50. To obtain stable LDOC1 expression, cells were selected using puromycin (Sigma-Aldrich, St. Louis, MO, USA), and LDOC1 expression in the infected cells was validated by means of qRT-PCR and western blotting.