LncRNA FGD5-AS1靶向调节miR-129-5p对口腔鳞状细胞癌细胞增殖、凋亡、迁移和侵袭的影响
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济宁医学院附属滕州市中心人民医院

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济宁医学院校级科研项目,NO.JY2017FS043


Effects of LncRNA FGD5-AS1 targeting and regulating miR-129-5p on the proliferation, apoptosis, migration and invasion of oral squamous cell carcinoma cells
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    摘要:

    目的 探讨长链非编码RNA(LncRNA)FGD5-AS1通过靶向调节miR-129-5p对口腔鳞状细胞癌(OSCC)细胞增殖、凋亡、迁移和侵袭的影响。方法 利用在线数据库分析FGD5-AS1在OSCC中的表达。以在我院收集的30例OSCC患者的肿瘤组织、正常组织和体外培养的人口腔黏膜细胞(HOK)和OSCC细胞(SCC-9、HSC-4、SCC-25、CAL-27)为研究对象,采用qRT-PCR法检测FGD5-AS1和miR-129-5p表达。将FGD5-AS1表达最高的CAL-27细胞系随机分成Control组(正常培养,不转染)、si-NC组(转染si-NC)、si-FGD5-AS1组(转染si-FGD5-AS1)、si-FGD5-AS1 NC inhibitor组(共转染si-FGD5-AS1和NC inhibitor)和si-FGD5-AS1 miR-129-5p inhibitor组(共转染si-FGD5-AS1和miR-129-5p inhibitor),CCK-8法和克隆形成实验检测CAL-27细胞增殖能力;流式细胞术检测CAL-27细胞凋亡水平;划痕愈合实验检测CAL-27细胞迁移能力;Transwell小室检测CAL-27细胞侵袭能力;双荧光素酶报告实验验证FGD5-AS1与miR-129-5p的靶向关系;western blot检测高迁移率族蛋白B1(HMGB1)蛋白表达。构建体内异种移植瘤模型,并分为sh-NC组、sh-FGD5-AS1组、miR-129-5p inhibitor组和sh-FGD5-AS1 miR-129-5p inhibitor组,检测肿瘤体积和肿瘤;qRT-PCR检测移植瘤组织FGD5-AS1、miR-129-5p表达;免疫组化检测移植瘤组织HMGB1、Ki67表达。结果 数据库分析显示,OSCC肿瘤组织中FGD5-AS1的表达水平是正常组织的4倍,且FGD5-AS1表达与OSCC患者分级较差相关。与正常组织或人口腔黏膜细胞相比,肿瘤组织和OSCC细胞系中FGD5-AS1表达明显升高,miR-129-5p表达明显降低(P<0.05),选择FGD5-AS1表达水平最高的CAL-27细胞进行转染实验。与Control组和si-NC组比较,si-FGD5-AS1组细胞凋亡率明显上升,OD值(48 h、72 h、96 h)、划痕愈合率及侵袭细胞数显著降低(P<0.05)。miR-129-5p是FGD5-AS1的靶基因,抑制miR-129-5p表达可逆转干扰FGD5-AS1对OSCC细胞增殖、凋亡、迁移和侵袭的影响,从而恢复FGD5-AS1的促癌作用。干扰FGD5-AS1后,通过显著增强miR-129-5p的表达,进而下调HMGB1的表达(P<0.05)。体内实验显示,沉默FGD5-AS1明显抑制移植瘤生长和HMGB1、Ki67表达(P<0.05),抑制miR-129-5p则相反;抑制miR-129-5p可逆转沉默FGD5-AS1对肿瘤生长和HMGB1、Ki67表达的抑制作用(P<0.05)。结论 FGD5-AS1在OSCC细胞中上调,干扰FGD5-AS1可通过靶向调控miR-129-5p/HMGB1轴,抑制OSCC细胞增殖、迁移、侵袭,促进凋亡。

    Abstract:

    Objective To investigate the effects of long non-coding RNA (LncRNA) FGD5-AS1 on the proliferation, apoptosis, migration and invasion of oral squamous cell carcinoma (OSCC) cells through targeted regulation of miR-129-5p. Methods The expression of FGD5-AS1 in OSCC was analyzed by online database. The tumor tissues, normal tissues, and the human oral mucosal cells (HOK) and OSCC cells (SCC-9, HSC-4, SCC-25, CAL-27) cultured in vitro from 30 OSCC patients collected in our hospital were used as research subjects, qRT-PCR method was performed to detect the expression of FGD5-AS1 and miR-129-5p. The CAL-27 cell line with the highest FGD5-AS1 expression was randomly separated into Control group (normal culture, no transfection), si-NC group (transfected with si-NC), si-FGD5-AS1 group (transfected with si-FGD5-AS1), si-FGD5-AS1 NC inhibitor group (co-transfected with si-FGD5-AS1 and NC inhibitor) and si-FGD5-AS1 miR-129-5p inhibitor group (co-transfected with si-FGD5-AS1 and miR -129-5p inhibitor), CCK-8 method and clone formation assay were used to detect the proliferation ability of CAL-27 cells; the apoptosis level of CAL-27 cells was detected by flow cytometry; the migration ability of CAL-27 cells was detected by wound-healing assay; Transwell chamber was used to detect the invasion ability of CAL-27 cells; and dual luciferase reporter experiment verified the targeting relationship between FGD5-AS1 and miR-129-5p; the expression of high mobility group protein B1(HMGB1) was detected by Western blot. In vivo xenograft tumor model was constructed and divided into sh-NC group, sh-FGD5-AS1 group, miR-129-5p inhibitor group, and sh-FGD5-AS1 miR-129-5p inhibitor group. Tumor volume and tumor were detected. QRT-pcr was used to detect the expression of FGD5-AS1 and miR-129-5p in transplanted tumor tissues. The expression of HMGB1 and Ki67 was detected by immunohistochemistry. Results Database analysis showed that the expression level of FGD5-AS1 in OSCC tumor tissues was 4 times higher than that in normal tissues, and FGD5-AS1 expression was associated with poor grade in OSCC patients. Compared with normal tissues or human oral mucosal cells, the expression of FGD5-AS1 in tumor tissues and OSCC cell lines was significantly increased, and the expression of miR-129-5p was significantly decreased (P<0.05), the CAL-27 cells with the highest expression level of FGD5-AS1 were selected for transfection experiments. Compared with the Control group and the si-NC group, the apoptosis rate of the si-FGD5-AS1 group was significantly increased, and the OD value (48 h, 72 h, 96 h), scratch healing rate and the number of invaded cells were significantly reduced (P<0.05). MiR-129-5p was the target gene of FGD5-AS1. Inhibiting the expression of miR-129-5p was able to reverse the effects of interference FGD5-AS1 on the proliferation, apoptosis, migration and invasion of OSCC cells, thereby restoring the cancer-promoting effect of FGD5-AS1. After FGD5-AS1 was disrupted, HMGB1 expression was down-regulated by significantly enhancing miR-129-5p expression (P<0.05). In vivo experiments showed that FGD5-AS1 silencing significantly inhibited the growth and expression of HMGB1 and Ki67 (P<0.05), inhibition of miR-129-5p was the opposite; Inhibition of miR-129-5p reversed the inhibition of FGD5-AS1 on tumor growth and expression of HMGB1 and Ki67 (P<0.05). Conclusions FGD5-AS1 is up-regulated in OSCC cells. Interfering with FGD5-AS1 can inhibit proliferation, migration and invasion of OSCC cells and promote apoptosis by targeting miR-129-5p / HMGB1 axis.

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  • 收稿日期:2022-12-23
  • 最后修改日期:2023-07-12
  • 录用日期:2023-09-05
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