敲低TXNDC9基因对结直肠癌细胞凋亡和自噬的促进作用及其对裸鼠移植瘤的影响

doi:10.13481/j.1671-587X.20260508

摘要/Abstract

摘要：

目的探讨敲低含硫氧还蛋白结构域蛋白9（TXNDC9）基因对结直肠癌（CRC）细胞凋亡和自噬的影响，并阐明其基于p53依赖途径的潜在分子机制。方法选用人源性结肠上皮细胞系NCM460细胞以及结肠癌细胞系HCT116、SW620、SW480和LoVo细胞。将结肠癌细胞HCT116分为NC组（转染阴性对照si-NC）、si-TXNDC9组（转染si-TXNDC9）和p53信号通路抑制剂PTF-α组（PTF-α组，转染si-TXNDC9并用5 μmol·L^-1 PTF-α处理24 h）。采用平板克隆实验和Transwell小室实验检测各组细胞克隆形成数及侵袭细胞数，Western blotting法检测各组细胞中自噬、凋亡和p53通路相关蛋白表达水平，末端脱氧核苷酸转移酶dUTP缺口末端标记（TUNEL）法检测各组细胞凋亡率，透射电子显微镜（TEM）观察各组细胞自噬小体数。构建裸鼠移植瘤模型，随机分为对照组（接种si-NC转染的HCT116细胞）、敲低组（接种si-TXNDC9转染的HCT116细胞）和抑制剂组（接种si-TXNDC9转染的HCT116细胞并腹腔注射PTF-α），每组5只。每7 d测量并记录各组裸鼠肿瘤体积，于第28天处死各组裸鼠后剥离瘤体并称质量。结果细胞实验，与NCM460细胞比较，HCT116、SW620、SW480和LoVo细胞中TXNDC9蛋白表达水平均明显升高（P<0.05）。与NC组比较，si-TXNDC9组HCT116细胞克隆形成数和侵袭细胞数明显减少（P<0.05），细胞凋亡率明显升高（P<0.05），细胞中B细胞淋巴瘤2（Bcl-2）蛋白和B细胞淋巴瘤特大蛋白（Bcl-XL）表达水平明显降低（P<0.05），Bcl-2相关X蛋白（BAX）表达水平明显升高（P<0.05），自噬小体数明显增加（P<0.05），自噬相关蛋白7（ATG7）和Beclin-1蛋白表达水平及微管相关蛋白1轻链3（LC3）-Ⅱ/LC3-Ⅰ比值均明显升高（P<0.05），P62蛋白表达水平明显降低（P<0.05），P53和P21蛋白表达水平明显升高（P<0.05）。与si-TXNDC9组比较，PTF-α组HCT116细胞克隆形成数和侵袭细胞数明显增加（P<0.05），细胞凋亡率明显降低（P<0.05），细胞中Bcl-2和Bcl-XL蛋白表达水平明显升高（P<0.05），BAX蛋白表达水平明显降低（P<0.05），自噬小体数明显减少（P<0.05），细胞中ATG7和Beclin-1蛋白表达水平及LC3-Ⅱ/LC3-Ⅰ比值明显降低（P<0.05），P62蛋白表达水平明显升高（P<0.05），P53和P21蛋白表达水平明显降低（P<0.05）。动物实验，与对照组比较，敲低组裸鼠肿瘤质量和各时间点肿瘤体积均明显降低（P<0.05）；与敲低组比较，抑制剂组裸鼠肿瘤质量和各时间点肿瘤体积均明显升高（P<0.05）。结论敲低TXNDC9可通过调节p53依赖途径，促进CRC细胞凋亡和自噬，进而抑制其增殖和侵袭能力。

关键词: 结直肠癌, 含硫氧还原蛋白结构域蛋白9, P53蛋白, 细胞凋亡, 细胞自噬

Abstract:

Objective To discuss the effect of thioredoxin domain-containing protein 9 （TXNDC9） gene knockdown on the apoptosis and autophagy of colorectal cancer （CRC） cells， and to clarify its potential molecular mechanism based on the p53-dependent pathway. Methods The human normal colonic epithelial cell line NCM460 and colon cancer cell lines HCT116， SW620， SW480， and LoVo were selected. The HCT116 cells were divided into NC group （transfected with negative control si-NC）， si-TXNDC9 group （transfected with si-TXNDC9）， and p53 pathway inhibitor PTF-α group （PTF-α group， transfected with si-TXNDC9 and treated with 5 μmol·L^-1 PTF-α for 24 h）. Plate colony formation assay and Transwell chamber assay were used to detect the numbers of clone formation and invasion cells in various groups. Western blotting method was used to detect the expression levels of autophagy， apoptosis， and p53 pathway-related proteins in the cells in various groups. Terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） assay was used to detect the apoptotic rates of the cells in various groups.Transmission electron microscope （TEM） was used to observe the numbers of autophagosomes in the cells in various groups. The xenograft tumor models in nude mice were established， and the mice were randomly divided into control group （inoculated with si-NC-transfected HCT116 cells）， knockdown group （inoculated with si-TXNDC9-transfected HCT116 cells）， and inhibitor group （inoculated with si-TXNDC9-transfected HCT116 cells and intraperitoneally injected with PTF-α）（n=5）. The tumor volumes of nude mice in various groups were measured and recorded every 7 days， and the mice were sacrificed on the 28th day， and the tumors were stripped and weighed. Results In cell experiments， compared with the NCM460 cells， the expression levels of TXNDC9 protein in the HCT116， SW620， SW480， and LoVo cells were significantly increased （P<0.05）. Compared with NC group， the numbers of clone formation and invasion cells of HCT116 cells in si-TXNDC9 group were significantly decreased （P<0.05）， the apoptotic rate of the cells was significantly increased （P<0.05）， the expression levels of B-cell lymphoma 2（Bcl-2） and B-cell lymphoma-extra large （Bcl-XL） proteins in the cells were significantly decreased （P<0.05）， the expression level of Bcl-2-associated X protein （BAX） was significantly increased （P<0.05）， the number of autophagosomes was significantly increased （P<0.05）， the expression levels of autophagy-related protein 7 （ATG7） and Beclin-1 proteins and the ratio of microtubule-associated protein 1 light chain 3 （LC3）-Ⅱ/LC3-Ⅰ were significantly increased （P<0.05）， the expression level of P62 protein was significantly decreased （P<0.05）， and the expression levels of P53 and P21 proteins were significantly increased （P<0.05）. Compared with si-TXNDC9 group， the numbers of clone formation and invasion cells of HCT116 cells in PTF-α group were significantly increased （P<0.05）， the apoptotic rate of the cells was significantly decreased （P<0.05）， the expression levels of Bcl-2 and Bcl-XL proteins in the cells were significantly increased （P<0.05）， the expression level of BAX protein was significantly decreased （P<0.05）， the number of autophagosomes was significantly decreased （P<0.05）， the expression levels of ATG7 and Beclin-1 proteins and the LC3-Ⅱ/LC3-Ⅰ ratio in the cells were significantly decreased （P<0.05）， the expression level of P62 protein was significantly increased （P<0.05）， and the expression levels of P53 and P21 proteins were significantly decreased （P<0.05）. In animal experiments， compared with control group， the tumor weights and tumor volumes at each time point in knockdown group were significantly decreased （P<0.05）； compared with knockdown group， the tumor weights and tumor volumes at each time point in inhibitor group were significantly increased （P<0.05）. Conclusion TXNDC9 knockdown can promote apoptosis and autophagy in the CRC cells by regulating the p53-dependent pathway， thereby inhibiting their proliferation and invasion abilities.

Key words: Colorectal cancer, Thioredoxin domain-containing protein 9, P53 protein, Apoptosis, Autophagy

中图分类号:

R735.3

张育,李曙光,曹铭富,黄迪. 敲低TXNDC9基因对结直肠癌细胞凋亡和自噬的促进作用及其对裸鼠移植瘤的影响[J]. 吉林大学学报(医学版), 2026, 52(5): 1263-1272.

Yu ZHANG,Shuguang LI,Mingfu CAO,Di HUANG. Promotion effect of TXNDC9 gene knockdown on apoptosis and autophagy of colorectal cancer cells and its influence in xenograft tumor in nude mice[J]. Journal of Jilin University(Medicine Edition), 2026, 52(5): 1263-1272.

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参考文献 32

[1]	WU Y J， HE S Y， CAO M D， et al. Comparative analysis of cancer statistics in China and the United States in 2024［J］. Chin Med J， 2024， 137（24）： 3093-3100.
[2]	赵英楠，高君妍，贺子轩，等. 中国结直肠癌早筛早诊早治高质量发展的思考［J］. 中国实用内科杂志， 2025， 45（5）： 373-380.
[3]	HE D， CHEN S L， WANG X Y， et al. Icaritin represses autophagy to promote colorectal cancer cell apoptosis and sensitized low-temperature photothermal therapy via targeting HSP90-TXNDC9 interactions［J］. Adv Sci， 2025， 12（20）： 2412953.
[4]	SHAO B. Autophagy-dependent apoptosis induction by oridonin are mediated by ROS-dependent AMPK-mTOR-ULK1 pathway in colon cancer［J］. Am J Cancer Res， 2025， 15（4）： 1902-1918.
[5]	ALAM M， ALAM S， SHAMSI A， et al. Bax/Bcl-2 cascade is regulated by the EGFR pathway： therapeutic targeting of non-small cell lung cancer［J］. Front Oncol， 2022， 12： 869672.
[6]	TAUCHER E， MYKOLIUK I， FEDIUK M， et al. Autophagy， oxidative stress and cancer development［J］. Cancers， 2022， 14（7）： 1637.
[7]	EL-KHOULY D， THABET N A， SAYED-AHMED M， et al. Promotion of autophagy and apoptosis in colorectal cancer exposed to imatinib and thymoquinone［J］. J Biochem Mol Toxicol， 2025， 39（4）： e70238.
[8]	ZHOU W J， FANG C， ZHANG L W， et al. Thioredoxin domain-containing protein 9 （TXNDC9） contributes to oxaliplatin resistance through regulation of autophagy-apoptosis in colorectal adenocarcinoma［J］. Biochem Biophys Res Commun， 2020， 524（3）： 582-588.
[9]	张仙宏，李乐. p53调控肿瘤代谢的研究进展［J］. 中国科学：生命科学， 2023， 53（4）： 431-448.
[10]	ZHENG T T， CHEN K K， ZHANG X， et al. Knockdown of TXNDC9 induces apoptosis and autophagy in glioma and mediates cell differentiation by p53 activation［J］. Aging， 2020， 12（18）： 18649-18659.
[11]	JIANG Z S， SUN X G， LI Y， et al. Anlotinib induced ferroptosis through the p53/xCT/GPX4 pathway in non-small cell lung cancer［J］. Transl Oncol， 2025， 53： 102289.
[12]	王洛伟，杜奕奇，柏愚，等. 中国消化道肿瘤的早诊早治现状及挑战［J］. 中国实用内科杂志， 2025， 45（5）： 353-356.
[13]	XU W L， TANG Y J， YANG Y， et al. Depletion of CPNE7 sensitizes colorectal cancer to 5-fluorouracil by downregulating ATG9B expression［J］. J Cell Mol Med， 2024， 28（8）： e18261.
[14]	WANG J， PAN X T， LI J， et al. TXNDC9 knockdown inhibits lung adenocarcinoma progression by targeting YWHAG［J］. Mol Med Rep， 2022， 25（6）： 203.
[15]	YANG Q C， HAO N， LI R H， et al. High level of TXNDC9 predicts poor prognosis and contributes to the NF-κB-regulated metastatic potential in gastric cancer［J］. Neoplasma， 2022， 69（1）： 103-112.
[16]	LU A G， WANGPU X， HAN D P， et al. TXNDC9 expression in colorectal cancer cells and its influence on colorectal cancer prognosis［J］. Cancer Investig， 2012， 30（10）： 721-726.
[17]	OPYDO M， MLYCZYŃSKA A， MLYCZYŃSKA E， et al. Synergistic action of MCL-1 inhibitor with BCL-2/BCL-XL or MAPK pathway inhibitors enhances acute myeloid leukemia cell apoptosis and differentiation［J］. Int J Mol Sci， 2023， 24（8）： 7180.
[18]	PALABIYIK A. The role of Bcl-2 in controlling the transition between autophagy and apoptosis （Review）［J］. Mol Med Rep， 2025， 32（1）： 1-9.
[19]	ASHRAFIZADEH M， AHMADI Z， MOHAMMADINEJAD R， et al. Monoterpenes modulating autophagy： a review study［J］. Basic Clin Pharmacol Toxicol， 2020， 126（1）： 9-20.
[20]	XIE Q Q， LIU Y， LI X F. The interaction mechanism between autophagy and apoptosis in colon cancer［J］. Transl Oncol， 2020， 13（12）： 100871.
[21]	WU Y X， YE H P， PENG B， et al. MiR-643 functions as a potential tumor suppressor in gastric cancer by inhibiting cell proliferation and invasion via targeting TXNDC9［J］. Ann Clin Lab Sci， 2021， 51（4）： 494-502.
[22]	WANG Y M， XU X， TANG J， et al. Apatinib induces endoplasmic reticulum stress-mediated apoptosis and autophagy and potentiates cell sensitivity to paclitaxel via the IRE-1α-AKT-mTOR pathway in esophageal squamous cell carcinoma［J］. Cell Biosci， 2021， 11（1）： 124.
[23]	王蓓蕾，于洋，韩桂玲，等. 肺痿冲剂对肺纤维化小鼠肺纤维化、自噬与上皮间质转化的影响［J］. 郑州大学学报（医学版）， 2025， 60（5）： 613-617.
[24]	DEVIS-JAUREGUI L， ERITJA N， DAVIS M L， et al. Autophagy in the physiological endometrium and cancer［J］. Autophagy， 2021， 17（5）： 1077-1095.
[25]	VOLIK P I， ZAMARAEV A V， EGORSHINA A Y， et al. Ally or traitor： the dual role of p62 in Caspase-2 regulation［J］. Cell Death Dis， 2024， 15： 827.
[26]	SONG C F， XU W， WU H K， et al. Photodynamic therapy induces autophagy-mediated cell death in human colorectal cancer cells via activation of the ROS/JNK signaling pathway［J］. Cell Death Dis， 2020， 11： 938.
[27]	PANATTA E， ZAMPIERI C， MELINO G， et al. Understanding p53 tumour suppressor network［J］. Biol Direct， 2021， 16（1）： 14.
[28]	WEN J， WANG D L. Deciphering the PTM codes of the tumor suppressor p53［J］. J Mol Cell Biol， 2022， 13（11）： 774-785.
[29]	VADDAVALLI P L， SCHUMACHER B. The p53 network： cellular and systemic DNA damage responses in cancer and aging［J］. Trends Genet， 2022， 38（6）： 598-612.
[30]	YOO H S， WON S B， KWON Y H. Luteolin induces apoptosis and autophagy in HCT116 colon cancer cells via p53-dependent pathway［J］. Nutr Cancer， 2022， 74（2）： 677-686.
[31]	CHATTERJEE T， GUHA D， DHAR J， et al. Adenosine dialdehyde， a methyltransferase inhibitor， induces colorectal cancer cells apoptosis by regulating PIMT： p53 interaction［J］. Biochem Biophys Res Commun， 2023， 684： 149134.
[32]	WANG H L， GUO M， WEI H D， et al. Targeting p53 pathways： mechanisms， structures and advances in therapy［J］. Signal Transduct Target Ther， 2023， 8： 92.

Group	Number of clone formation	Number of invasion cells
NC	121.96±15.08	244.81±21.65
Si-TXNDC9	53.23±3.95^*	67.49±5.71^*
PTF-α	95.17±8.89^△	210.35±23.82^△

Group	Tumor weight(m/mg)	Tumor volume(V/mm³)
Group	Tumor weight(m/mg)	（t/d） 7	14	21	28
Control	765.56±55.49	152.01±10.16	373.12±25.19	586.29±46.47	722.29±51.45
Knockdown	401.19±35.64^*	92.31±8.49^*	161.05±18.62^*	263.49±23.64^*	389.16±35.47^*
Inhibitor	605.06±51.19^△	139.94±14.03^△	295.16±19.06^△	432.19±42.31^△	587.46±49.75^△
F	71.770	40.162	128.367	86.730	66.015
P	<0.001	<0.001	<0.001	<0.001	<0.001