Human osteosarcoma cell lines, including U-2OS, Saos-2, MG-63, MNNHG/HOS, and human osteoblast hFOB1.19, were purchased from American Type Culture Collection (ATCC) (VA, USA) and cultured according to the instructions from the supplier (www.atcc.org). U-2OS and Saos-2 cells were cultured in 90% McCoy’s 5A (modified) medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA). MG-63 and MNNG/HOS cells were grown in 90% Minimum Essential Medium α (MEM α) (Gibco, USA) supplemented with 10% FBS (Gibco, USA). hFOB1.19 cells were incubated in 90% Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (DMEM/F12) supplemented with 10% FBS (Gibco, USA). All cells were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO₂. After being cultured, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays were performed to determine Lnc-KASRT expression in the hFOB1.19, U-2OS, Saos-2, MG-63, and MNNHG/HOS cell lines, with hFOB1.19 serving as a control.

Overexpression plasmids, including the Lnc-KASRT overexpression plasmid and control overexpression plasmid, were constructed with pEX-2 by Guangzhou RiboBio Co., Ltd. (Guangzhou, China). Knockdown plasmids, including the Lnc-KASRT knockdown plasmid and control knockdown plasmid, were constructed with pRNAT-U6.1/Neo by Guangzhou RiboBio Co., Ltd. (Guangzhou, China). The constructed plasmids were transfected into U-2OS cells and Saos-2 cells using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions and a previously reported method (21). Following transfection, the cells in each cell line (U-2OS and Saos-2) were divided into four groups: Lnc-KASRT overexpression cells (KASRT(+) group); control overexpression cells (NC(+) group); Lnc-KASRT knockdown cells (KASRT(-) group); and control knockdown cells (NC(-) group). In the above groups, the determination of Lnc-KASRT expression was performed by RT-qPCR assays at 24 h post transfection. The cell viability assessment was carried out with the use of Cell Counting Kit-8 (CCK-8) (Dojindo, Japan) at 0, 24, 48, and 72 h. Cell apoptosis was measured by Annexin V/propidium iodide (AV/PI) assay with an Annexin V-FITC apoptosis detection kit (R&D, USA) at 48 h. The CCK-8 assay and AV/PI assay were completed following the manufacturer’s instructions. The assessment of migration ability and invasion ability was performed at 48 h through wound healing assays and Transwell assays with Matrigel basement membrane matrix (BD, USA)-coated chambers (Corning, USA), and all procedures were performed according to the methods described in previous studies (22, 23). Moreover, the expression of serine- and arginine-rich splicing factor 1 (SRSF1), Kruppel-like factor 6 (KLF-6) transcript variant D (also known as KLF-6-SV1), and KLF-6 wild type (KLF-6-WT) was detected by RT-qPCR and Western blot analysis at 48 h. Notably, in U-2OS cells after transfection, chemosensitivity to cisplatin, methotrexate, and doxorubicin was detected by coculture with 0.0–6.4 μM cisplatin, 0.0–6.4 μM methotrexate, and 0–32 nM doxorubicin.

The KLF-6-SV1 knockdown plasmid and control knockdown plasmid were constructed by Guangzhou RiboBio Co., Ltd. (Guangzhou, China) using pRNAT-U6.1/Neo. KLF-6-SV1 knockdown plasmid and control knockdown plasmid were transfected into NC(+) group cells and KASRT(+) group cells using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions and to the method previously reported (21), which generated another four groups: NC(+)&KLF-6-SV1(-) group, NC(+)&NC(-) group, KASRT(+)&KLF-6-SV1(-) group, and KASRT(+)&NC(-) group. KLF-6-SV1 expression was detected by performing RT-qPCR and Western blot experiments at 24 h post transfection, and Lnc-KASRT expression, cell viability, cell apoptosis, migration ability, and invasion ability were determined according to the methods described above. Additionally, the expression of SRSF1, P21, and Cyclin D1 (CCND1) was detected by RT-qPCR and Western blot assays at 48 h post transfection.

The pLV-CMV-IRES-Puro vector (Hanbio, China) was used to construct the Lnc-KASRT overexpression plasmid, and the pLV-U6-RFP-T2A-Puro vector (Hanbio, China) was used to construct the Lnc-KASRT knockdown plasmid according to the methods described previously (24). To generate overexpression and knockdown lentiviruses, plasmid and envelope helper vectors were cotransfected into 293T cells using Lipofectamine 2000 (Invitrogen, USA). Control lentivirus was provided by Hanbio Biotechnology Co., Ltd. (Shanghai, China). After harvesting, the constructed lentivirus was used to infect Saos-2 cells, and then the stably infected Saos-2 cells were screened with 2 μg/mL puromycin (Sigma, USA). These cells were marked as KASRT(+) cells, KASRT(-) cells, and NC cells depending on the infected lentivirus (Lnc-KASRT overexpression lentivirus, Lnc-KASRT knockdown lentivirus, or control lentivirus).

All animal experiments were approved by the Institutional Animal Care and Use Committees (IACUC) of our institution and were performed in accordance with institutional guidelines and ethical standards. A tumor xenograft model was constructed according to methods described previously (25). Male BALB/C nude mice (5–6 weeks of age, 16–18 g) were maintained under specific pathogen-free conditions. Approximately 2 × 10⁶ KASRT(+) cells, KASRT(-), cells and NC cells were suspended in 100 μl PBS and then injected subcutaneously into the right side of the posterior flank of nude mice. The mice were divided into the KASRT(+) group, KASRT(-) group, and NC group. Tumor volumes were examined every 7 days using a Vernier caliper (Mitutoyo, Japan). Tumor volumes were calculated using the equation: Volume = a*b²/2 (mm³), where a is the largest diameter and b is the perpendicular diameter. Four weeks after cell injection, mice were sacrificed, and tumors were harvested. After weighing, tumors were stored properly for further analysis. For tumors placed in 10% formalin and embedded in paraffin, hematoxylin-eosin (HE) staining was performed for histological analysis, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay was carried out to detect cell apoptosis, and immunochemistry (IHC) was performed to detect protein expression of SRSF1, KLF-6-SV1, KLF-6-WT, MMP-1, and MMP-9. For tumors stored at -80°C, SRSF1, KLF-6-SV1, KLF-6-WT, MMP-1, and MMP-9 mRNA expression was evaluated by RT-qPCR assays.

Total RNA was extracted by TRIzol™ Reagent (Invitrogen, USA), and then reverse transcription and PCR were performed with the PrimeScript™ RT reagent Kit (Takara, Japan) and TB Green™ Fast qPCR Mix (Takara, Japan), respectively. Gene expression was calculated by the 2^-△△Ct method with GAPDH as the internal reference according to a previously reported method (26). Primer sequences are listed in Supplementary Table 1 .

Western blotting was performed by standard procedures according to previous studies (22, 23). Protein extraction and quantification were performed using RIPA buffer (Sigma, USA), and a bicinchoninic acid kit was used for protein determination (Sigma, USA). Furthermore, NuPAGE™ 4–20% Tris-Acetate Midi Protein Gels (Thermo, USA), XCell SureLock™ Mini-Cell (Invitrogen, USA), Pierce™ECL Plus Western Blotting Substrate (Invitrogen, USA), X-ray film (Kodak, USA), and Gel Imager (Thermo, USA) were also used. Antibody information is presented in Supplementary Table 2 .

HE staining, TUNEL assay, and IHC experiments were performed according to the methods described previously (26, 27). The formalin-fixed and paraffin-embedded tumor specimens were sliced into 4 μm sections. Then, a hematoxylin and eosin staining kit (Beyotime, China) was used for HE staining, which was performed in accordance with the kit’s standard protocol. The TUNEL assay was performed using a TumorTACS in situ apoptosis kit (R&D, USA) according to the manufacturer’s manual. Furthermore, IHC was completed according to the standard operating protocol of The Early Detection Research Network (EDRN), and the antibodies used for the IHC assays are listed in Supplementary Table 2 . Images were obtained using an Olympus BX41 microscope (Olympus, Japan).

Bar charts and line charts with error bars were used to display a statistical summary of the mean value and standard deviation (SD). Comparison between two independent samples was determined by the unpaired t test; multiple comparisons between a control group and other experimental groups were determined by one-way ANOVA followed by Dunnett’s test; multiple comparisons between each group were determined by one-way ANOVA followed by Tukey’s test. Statistical analysis and graph plotting were performed using GraphPad Prism 7.02 software (GraphPad Software Inc., USA). Significance was defined as a P < 0.05, which is displayed as *P < 0.05; **P < 0.01; ***P < 0.001, and nonsignificant is marked as NS.

Lnc-KASRT was elevated in several osteosarcoma cell lines, such as U-2OS, MG-63, and MNNG/HOS, but was not changed in the Saos-2 cell line compared to the control cell line hFOB1.19 ( Figure 1A ). After transfection in both U-2OS and Saos-2 cells, lnc-KASRT was increased in the KASRT (+) group compared to the NC (+) group but was decreased in the KASRT (-) group compared to the NC (-) group, indicating successful transfection ( Figures 1B, C ). Regarding cell viability, lnc-KASRT overexpression promoted cell proliferation and expression of the antiapoptotic marker BCL-2 and reduced the cell apoptosis rate and expression of the proapoptotic marker C-Caspase3 in both U-2OS ( Figures 2A–D ) and Saos-2 cells ( Figures 2E–H ). In terms of cell mobility, lnc-KASRT overexpression enhanced cell migration and invasion in both U-2OS ( Figures 3A–D ) and Saos-2 cells ( Figures 3E–H ). Moreover, lnc-KASRT knockdown exhibited the opposite effect on these cell functions ( Figures 2A–H ) ( Figures 3A–H ). However, lnc-KASRT had little effect on chemosensitivity in U-2OS cells, except that it had a slight effect on doxorubicin chemosensitivity in U-2OS cells ( Supplementary Figures S1A–C ); therefore, subsequent exploration of chemotherapy sensitivity was not continued.

Lnc-KASRT overexpression upregulated KLF6-SV1 expression but downregulated SRSF1 and KLF6-WT expression in both U-2OS ( Figures 4A–D ) and Saos-2 cells ( Figures 4E–H ). At the same time, lnc-KASRT knockdown decreased KLF6-SV1 expression and increased SRSF1 and KLF6-WT expression in both U-2OS ( Figures 4A–D ) and Saos-2 cells ( Figures 4E–H ).

After transfection with its overexpression plasmid, lnc-KASRT was upregulated, while KLF6-SV1 was downregulated by its knockdown plasmid, indicating successful transfection. Further analyses revealed that lnc-KASRT positively regulated KLF6-SV1, but KLF6-SV1 did not affect lnc-KASRT in either U-2OS ( Figures 5A–C ) or Saos-2 cells ( Figures 5D–F ). Interestingly, KLF6-SV1 knockdown enhanced cell apoptosis while reducing cell proliferation, migration, and invasion, as reflected by AV/PI, CCK-8, wound healing, Transwell assays, and pro/anti-apoptotic marker expression in both U-2OS and Saos-2 cells ( Figures 6A–H , 7A–H ). Importantly, further analyses showed that KLF6-SV1 knockdown impaired the effect of lnc-KASRT on regulating cell apoptosis, proliferation, migration, and invasion in both U-2OS and Saos-2 cells ( Figures 6A–H , 7A–H ).

Lnc-KASRT overexpression downregulated P21 but upregulated CCND1; KLF6-SV1 knockdown increased P21 while reducing CCND1 in both U-2OS ( Figures 8A–C ) and Saos-2 cells ( Figures 8D–F ). Notably, KLF6-SV1 knockdown weakened the effect of lnc-KASRT regulation of P21 and CCND1 in both U-2OS and Saos-2 cells ( Figures 8A–F ).

Lnc-KASRT overexpression facilitated tumor growth ( Figures 9A–D ) and reduced tumor apoptosis ( Figures 9E, F ) in osteosarcoma mice. In contrast, Lnc-KASRT knockdown repressed tumor growth while inducing HE-reflected necrosis and TUNEL-reflected tumor apoptosis in osteosarcoma mice ( Figures 9A–F ). Furthermore, lnc-KASRT overexpression increased KLF6-SV1, MMP1, and MMP9 expression and decreased SRSF1 and KLF6-WT expression in osteosarcoma mice ( Figures 10A–F ), but lnc-KASRT downregulation showed the opposite trend ( Figures 10A–F ).