Table_1_LncRNA KASRT Serves as a Potential Treatment Target by Regulating SRSF1-Related KLF6 Alternative Splicing and the P21/CCND1 Pathway in Osteosarcoma: An In Vitro and In Vivo Study.docx

PurposeLong non-coding RNA KLF6 alternative splicing regulating transcript (lnc-KASRT) locates within the intronic region of SRSF1, possessing the potential to regulate KLF6 alternative splicing to promote carcinogenicity. Then, the current in vitro and in vivo study aimed to investigate the effect of lnc-KASRT on regulating tumor malignant behaviors, and the implication of its interaction with KLF6 alternative splicing in osteosarcoma.
MethodsLnc-KASRT overexpression or knockdown plasmid was transfected into U-2OS and Saos-2 cells. Then, KLF6-SV1 knockdown plasmid with or without lnc-KASRT overexpression plasmid was transfected into these cells for compensative experiments. In vivo, lnc-KASRT overexpression or knockdown Saos-2 cells were injected in mice for tumor xenograft construction.
ResultsLnc-KASRT expression was increased in most osteosarcoma cell lines compared to control cell line. Lnc-KASRT overexpression promoted cell viability, mobility, and anti-apoptotic marker expression, while reducing apoptosis rate and pro-apoptotic marker expression; meanwhile, it regulated SRSF1, KLF6 alternative splicing (increased KLF6-splice variant 1 (KLF6-SV1), decreased KLF6-wild type (KLF6-WT)), and followed P21/CCND1 pathway in U-2OS/Saos-2 cells. The lnc-KASRT knockdown exhibited opposite trends. Subsequent compensative experiments disclosed that KLF6-SV1 knockdown attenuated most of the tumor-promoting effects of lnc-KASRT overexpression in U-2OS/Saos-2 cells. In vivo experiments further validated that lnc-KASRT enhanced tumor growth and reduced tumor apoptosis; meanwhile, it also increased tumor KLF6-SV1, MMP-1, and MMP-9 expressions but decreased tumor SRSF1 and KLF6-WT expressions in xenograft mice.
ConclusionLnc-KASRT serves as a potential treatment target via regulating SRSF1-related KLF6 alternative splicing and following P21/CCND1 pathway in osteosarcoma.

目的：长链非编码RNA KLF6可变剪接调控转录本（long non-coding RNA KLF6 alternative splicing regulating transcript，简称lnc-KASRT）定位于剪接因子SRSF1的内含子区域，具备调控KLF6可变剪接以促进致癌进程的潜力。本研究通过体外及体内实验，旨在探究lnc-KASRT对肿瘤恶性生物学行为的调控效应，及其与KLF6可变剪接的相互作用在骨肉瘤中的作用机制。
方法：将lnc-KASRT过表达质粒或敲低质粒转染至U-2OS与Saos-2细胞中。随后，分别联合或不联合lnc-KASRT过表达质粒，将KLF6剪接变体1（KLF6-splice variant 1, KLF6-SV1）敲低质粒转染至上述细胞以开展补偿实验。体内实验中，将过表达或敲低lnc-KASRT的Saos-2细胞接种至小鼠体内，构建异种移植瘤模型。
结果：与对照细胞系相比，大多数骨肉瘤细胞系中lnc-KASRT的表达水平显著升高。在U-2OS及Saos-2细胞中，过表达lnc-KASRT可促进细胞活力、细胞迁移能力与抗凋亡标志物的表达，同时降低细胞凋亡率与促凋亡标志物的表达；此外，其可调控SRSF1及KLF6的可变剪接（上调KLF6剪接变体1（KLF6-SV1），下调KLF6野生型（KLF6-wild type, KLF6-WT）），并激活P21/CCND1通路。敲低lnc-KASRT则呈现相反的效应。后续补偿实验结果显示，敲低KLF6-SV1可削弱过表达lnc-KASRT所介导的大部分促肿瘤效应。体内实验进一步验证，lnc-KASRT可促进肿瘤生长并抑制肿瘤细胞凋亡；同时，在异种移植瘤小鼠模型中，lnc-KASRT可上调肿瘤组织中KLF6-SV1、基质金属蛋白酶1（matrix metalloproteinase 1, MMP-1）与基质金属蛋白酶9（matrix metalloproteinase 9, MMP-9）的表达，下调SRSF1与KLF6-WT的表达。
结论：lnc-KASRT可通过调控SRSF1相关的KLF6可变剪接及下游P21/CCND1通路，成为骨肉瘤潜在的治疗靶点。