lncRNA GK-IT1通过调控醛缩酶A影响非小细胞肺癌细胞的恶性进展

doi:10.3969/j.issn.1674-8115.2022.05.006

摘要/Abstract

摘要：

目的·探讨长链非编码RNA（long non-coding RNA，lncRNA）GK-IT1通过调控醛缩酶A（aldolase A，ALDOA）对非小细胞肺癌（non-small-cell lung cancer，NSCLC）细胞A549及H358恶性进展的影响。方法·应用实时荧光定量PCR（quantitative real-time PCR，qRT-PCR）检测42对NSCLC组织和癌旁组织以及NSCLC细胞系中GK-IT1的表达。荧光原位杂交（fluorescence in situ hybridization，FISH）实验检测GK-IT1在A549及H358中的亚细胞定位。RNA免疫共沉淀（RNA immunoprecipitation，RIP）检测GK-IT1与ALDOA的相互作用关系。以2条GK-IT1的小干扰RNA序列（Si-GK-IT1 #1和Si-GK-IT1 #2）及空白对照（Si-NC）转染A549及H358细胞，再以Si-GK-IT1 #2和ALDOA过表达质粒共转染上述细胞系。设置Si-NC组、Si-GK-IT1 #1及Si-GK-IT1 #2组，采用CCK-8及EdU实验检测细胞增殖能力，Transwell侵袭实验与细胞划痕实验检测细胞侵袭能力及迁移能力。Western blotting检测各实验组内波形蛋白（vimentin）、E钙黏蛋白（E-cadherin）、N钙黏蛋白（N-cadherin）及ALDOA的表达水平。结果·与正常组织相比，NSCLC组织中GK-IT1的表达量明显升高；与正常支气管上皮细胞BEAS-2B相比，H358、A549细胞中GK-IT1的表达量明显升高（均P<0.05）。FISH实验表明GK-IT1主要位于细胞质；RIP实验及生物信息学分析表明GK-IT1可能与ALDOA蛋白结合；CCK-8实验结果显示，Si-GK-IT1 #1及Si-GK-IT1 #2组较Si-NC组细胞增殖能力明显受到抑制（均P<0.05）；EdU实验结果显示，Si-GK-IT1 #1及Si-GK-IT1 #2组较Si-NC组细胞EdU阳性率显著降低（均P<0.05）；Transwell侵袭实验结果显示，与对照组相比，Si-GK-IT1 #1及Si-GK-IT1 #2组细胞侵袭能力明显受到抑制（均P=0.000）；细胞划痕实验结果显示，Si-GK-IT1 #1及Si-GK-IT1 #2组较Si-NC组细胞迁移率明显降低（均P=0.000）。敲减GK-IT1后，ALDOA、vimentin表达水平降低，E-cadherin表达水平升高。过表达ALDOA可逆转敲低GK-IT1对NSCLC细胞增殖、侵袭、迁移及蛋白表达水平的影响（均P<0.05）。结论·GK-IT1可通过调控ALDOA蛋白促进NSCLC细胞的增殖、侵袭与迁移能力。

关键词: 长链非编码RNA, GK-IT1, 醛缩酶A, 非小细胞肺癌, 细胞侵袭, 细胞迁移

Abstract:

Objective·To investigate the effects of long non-coding RNA (lncRNA) GK-IT1 on the carcinogenesis of non-small-cell lung cancer (NSCLC) A549 and H358 cells by regulating aldolase A (ALDOA).

Methods·The expression of GK-IT1 in 42 pairs of NSCLC tissues and adjacent tissues, and NSCLC cell lines were tested by quantitative real-time PCR (qRT-PCR). The subcellular localization of GK-IT1 in A549 and H358 cells was tested by fluorescence in situ hybridization (FISH). RNA immunoprecipitation (RIP) was used to assess the interaction of GK-IT1 and ALDOA. A549 and H358 cells were transfected with GK-IT1 small interfering RNA sequences (Si-GK-IT1 #1 and Si-GK-IT1 #2) and negative control (Si-NC), and then the two cell lines were also cotransfected with Si-GK-IT1 #2 and ALDOA overexpression plasmid. The Si-NC, Si-GK-IT1 #1, Si-GK-IT1 #2 groups were set. CCK-8 and EdU assay were used to detect cell proliferation. Transwell invasion and scratch assay were used to observe cell invasion and migration. Western blotting was carried out to verify the expression of vimentin, E-cadherin, N-cadherin and ALDOA in each experimental group.

Results·Compared with the normal tissues, the relative expression of GK-IT1 was significantly higher in NSCLC tissues. Compared with BEAS-2B cells, the expression of GK-IT1 in H358 and A549 cells was significantly higher (P<0.05). FISH assay indicated that GK-IT1 was mainly located in the cytoplasm. RIP and bioinformatics analysis suggested that GK-IT1 might interact with ALDOA. Compared with the Si-NC group, the results of CCK-8 assay showed that the cell proliferation of the Si-GK-IT1 #1 and Si-GK-IT1 #2 group was significantly inhibited (P<0.05). The results of EdU assay showed that the ratio of EdU positive cells in the Si-GK-IT1 #1 and Si-GK-IT1 #2 group was significantly lower than that in the Si-NC group (P<0.05). Compared with the Si-NC group, the results of Transwell invasion assay indicated that the invasion of the Si-GK-IT1 #1 and Si-GK-IT1 #2 group was significantly inhibited (all P=0.000). The results of cell scratch assay showed that the healed wound ratio in the Si-GK-IT1 #1 and Si-GK-IT1 #2 group was remarkably lower than that in Si-NC group (all P=0.000). GK-IT1 knockdown significantly decreased the expression of ALDOA and vimentin, while the expression of E-cadherin was increased. Overexpression of ALDOA reversed the effects of GK-IT1 silencing on cell proliferation, invasion, migration and protein expression of NSCLC cells (all P<0.05).

Conclusion·GK-IT1 could promote the proliferation, invasion and migration of NSCLC cells via regulating ALDOA.

Key words: long non-coding RNA (lncRNA), GK-IT1, aldolase A (ALDOA), non-small cell lung cancer (NSCLC), cell invasion, cell migration

中图分类号:

R734.2

刘子杨, 王小文, 陈力. lncRNA GK-IT1通过调控醛缩酶A影响非小细胞肺癌细胞的恶性进展[J]. 上海交通大学学报（医学版）, 2022, 42(5): 591-601.

LIU Ziyang, WANG Xiaowen, CHEN Li. lncRNA GK-IT1 influences the carcinogenesis of non-small cell lung cancer cells through regulating aldolase A[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(5): 591-601.

图/表 9

图1 NSCLC组织中 GK-IT1 及 ALDOA 的表达Note： A. GK-IT1 expression in NSCLC tissues and normal tissues. B. ALDOA expression in NSCLC tissues and normal tissues. ①P=0.001, ②P=0.003. C. GK-IT1 expression in different T stage groups. ③P=0.009.

Fig 1 Expression of GK-IT1 and ALDOA in NSCLC tissues

图2 GK-IT1 在NSCLC细胞系中的表达及其敲减效果Note： A. Comparison of GK-IT1 expression in NSCLC cell lines. ①P=0.000, ②P=0.003, compared with the BEAS-2B group. B. The knockdown efficiency of GK-IT1 in H358 cells. C. The knockdown efficiency of GK-IT1 in A549 cells. ③P=0.002, ④P=0.001, ⑤P=0.000, compared with the Si-NC group.

Fig 2 GK-IT1 expression and knockdown effect in NSCLC cell lines

图3 敲减 GK-IT1 对NSCLC细胞增殖的影响Note： A/B. The proliferation of H358 cells (A) and A549 cells (B) after GK-IT1 silencing was assessed by CCK-8 assay. C. Statistical analysis of EdU assay in H358 and A549 cells. ①P=0.018, ②P=0.001, ③P=0.008, ④P=0.002, ⑤P=0.000, ⑥P=0.001, ⑦P=0.005, compared with the Si-NC group.

Fig 3 E?ects of GK-IT1 knockdown on the proliferation of NSCLC cells

图4 敲减 GK-IT1 对NSCLC细胞侵袭及迁移能力的影响Note： A/B. Cell invasion was evaluated by Transwell invasion assay after knockdown of GK-IT1 in H358 cells (A) and A549 cells (B). C. Statistical analysis of Transwell invasion assays in H358 and A549 cells. D/E. Wound scratch assay was used to detect the migration of A549 cells (D) and H358 cells (E) after knockdown of GK-IT1. F. Statistical analysis of scratch assays in H358 and A549 cells ①P=0.000, compared with the Si-NC group.

Fig 4 Effects of GK-IT1 knockdown on the invasion and migration of NSCLC cells

图5 GK-IT1 的亚细胞定位

Fig 5 Subcellular localization of GK-IT1

图6 敲减 GK-IT1 对NSCLC细胞中ALDOA及EMT相关蛋白表达水平的影响Note： A. Western blotting for detecting expression levels of ALDOA, E-cadherin, N-cadherin, and vimentin after knocking down GK-IT1 in A549 cells. B. Western blotting was applied to verify the expression levels of ALDOA, E-cadherin, N-cadherin, and vimentin after silencing GK-IT1 in H358 cells. ①P=0.007, ②P=0.004, ③P=0.002, ④P=0.001, ⑤P=0.000, ⑥P=0.003, compared with the Si-NC group.

Fig 6 Effects of GK-IT1 knockdown on the expression of ALDOA and EMT-related proteins in NSCLC cells

图7 GK-IT1 与ALDOA的相互作用Note：A. Bioinformatics analysis for predicting interaction probabilities of GK-IT1 and ALDOA. B. RIP showed that GK-IT1 was significantly enriched with anti-ALDOA antibody compared with anti-IgG in A549 and H358 cells. ①P=0.000, compared with the anti-IgG group.

Fig 7 Interaction between GK-IT1 and ALDOA

图8 过表达 ALDOA 对敲除 GK-IT1 的NSCLC细胞增殖、侵袭和迁移的影响Note： A/B. CCK-8 assay revealed that the proliferation was significantly increased after the overexpression of ALDOA in GK-IT1-knockdown H358 cells (A) and A549 cells (B). C. Statistical analysis of EdU assay. D/E. Transwell invasion assay indicated that the invasion of Si-GK-IT1 #2 transfected H358 cells (D) and A549 cells (E) was significantly enhanced after the overexpression of ALDOA. F. Statistical analysis of Transwell invasion assay. G/H. Wound scratch assay showed that the migration was significantly promoted after the overexpression of ALDOA in GK-IT1-knockdown H358 cells (G) and A549 cells (H). I. Statistical analysis of wound scratch assay. ①P=0.000, ②P=0.009, ③P=0.013, ④P=0.001, ⑤P=0.011, ⑥P=0.006, ⑦P=0.002, ⑧P=0.017, compared with the Si-GK-IT1 #2 group.

Fig 8 Effects of ALDOA overexpression on the proliferation, invasion and migration of GK-IT1-knockdown NSCLC cells

图9 过表达 ALDOA 对敲除 GK-IT1 的NSCLC细胞ALDOA及EMT相关蛋白表达的影响Note： A/B. Western blotting results of H358 cells (A) and A549 cells (B). ①P=0.000, ②P=0.002, ③P=0.003, ④P=0.010, ⑤P=0.001, compared with the Si-GK-IT1 #2 group.

Fig 9 Effects of ALDOA overexpression on the expression of ALDOA and EMT-related proteins in GK-IT1-knockdown NSCLC cells

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