Smad3 promotes pancreatic cancer progression by regulating autophagy and apoptosis through p38/MAPK signaling pathway

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Smad3 promotes pancreatic cancer progression by regulating autophagy and apoptosis through p38/MAPK signaling pathway

Basic Medicine | 更新时间：2026-01-09

- Smad3 promotes pancreatic cancer progression by regulating autophagy and apoptosis through p38/MAPK signaling pathway
- Journal of Army Medical University Vol. 44, Issue 19, Pages: 1968-1978(2022)
- 作者机构：
  
  1.710021 西安，西安医学院临床医学院中尼友好拉吉姆医学实验室
  2.710077 西安，西安医学院第一附属医院妇产科
- 作者简介：
- 基金信息：
  
  the Young Talents Support Project of Shaanxi Provincial Colleges and Universities and Commission of Science and Technology(20200314);the General Program of Shaanxi Provincial Department of Science and Technology(2021JM-495);and the International (Regional) Cooperation and Exchange Project of National Natural Science Foundation of China(81550110256)
- DOI：10.16016/j.2097-0927.202205059
  CLC： R394.2;R730.23;R735.9
- Received：11 May 2022，
  
  Revised：2022-07-04，
  
  Published：15 October 2022
- 稿件说明：
移动端阅览
Xudong ZHANG, Hao'ang LIU, Zhihao WANG, et al. Smad3 promotes pancreatic cancer progression by regulating autophagy and apoptosis through p38/MAPK signaling pathway[J]. Journal of Army Medical University, 2022, 44(19): 1968-1978.
DOI：

Xudong ZHANG, Hao'ang LIU, Zhihao WANG, et al. Smad3 promotes pancreatic cancer progression by regulating autophagy and apoptosis through p38/MAPK signaling pathway[J]. Journal of Army Medical University, 2022, 44(19): 1968-1978. DOI： 10.16016/j.2097-0927.202205059.

摘要

目的

2

观察Smad3对胰腺癌细胞Panc-1、BxPC-3凋亡、自噬的影响，初步探讨Smad3表达水平影响胰腺癌进程的可能机制。

方法

2

基于GEPIA2数据库进行生物信息学分析，探究Smad3与胰腺癌发生、发展及生存的相关性。利用CRISPR/Cas9系统以及短发卡RNA(ShRNA)敲除及敲减Smad3的表达，采用Real-Time quantitative PCR(qPCR)和Western blot检测细胞中Smad3 mRNA和蛋白水平变化。实验组转入ShSmad3-1、ShSmad3-2序列，将不靶向任何基因的Sh-Scramble序列作为对照组(Scr)。将转入Smad3 guide RNA(gRNA)序列作为CRISPR/Cas9的实验组，转入空载质粒的作为CRISPR/Cas9的对照组。采用Western blot检测细胞中LC3、p62、p38以及p-p38蛋白变化情况，同时在敲减Smad3的胰腺癌细胞用PI-Annexin V试剂盒染色并进行流式分析。利用pLVX-Smad3质粒进行回补实验，并用p38抑制剂SB203580处理细胞检测相关蛋白的变化情况。

结果

2

GEPIA2数据库及临床标本分析结果显示，Smad3在胰腺癌组织中高表达(

P

<

0.05)。在胰腺癌细胞BxPC-3中转入ShSmad3-1、ShSmad3-2后，Smad3的mRNA和蛋白水平均显著降低(

P

<

0.05)。同时在Panc-1中转入Smad3 guide RNA筛选到2个Smad3敲除细胞系。与对照组相比，敲减Smad3胰腺癌细胞凋亡显著增加(

P

<

0.05)。Western blot实验发现：Smad3敲减及敲除后，p-p38的蛋白水平显著增加(

P

<

0.05)，LC3、p62水平显著降低(

P

<

0.05)。进一步通过回补实验及利用SB203580处理胰腺癌细胞，发现p62、LC3蛋白水平能回复(

P

<

0.01)。

结论

2

干扰Smad3促进胰腺癌细胞过度自噬，进而导致细胞凋亡增加，可能与激活p38/MAPK信号通路有关。

Abstract

Objective

2

To investigate the effect of Smad3 on the apoptosis and autophagy of pancreatic cancer cells Panc-1 and BxPC-3

in order to preliminarily verify the effect of Smad3 expression in the progression of pancreatic cancer.

Methods

2

Bioinformatics analysis was performed based on GEPIA2 database to explore the correlation of Smad3 with the occurrence

development

and survival of pancreatic cancer. The CRISPR/Cas9 system and short hairpin RNA (sh-RNA) were used respectively to knock out or reduce the expression of Smad3. Real-time quantitative PCR (qPCR) and Western blotting were subsequently adopted to detect the changes of Smad3 at mRNA and protein levels. The sh-RNA experimental groups were transferred with ShSmad3-1 and ShSmad3-2 sequences

and the CRISPR/Cas9 experimental group were transferred with Smad3 guide RNA (gRNA) sequence

using Sh-Scramble sequence (Scr) and empty plasmid as the corresponding control groups

respectively. Western blotting was applyed to determine the protein changes of LC3

p62

p38 and p-p38 in the cells. Meanwhile

Smad3 knockdown pancreatic cancer cells were stained with PI-Annexin V kit and then analyzed by flow cytometry. To further prove the effect of Smad3 on pancreatic cancer cells

the pLVX-Smad3 plasmid was used to perform a rescue experiment

and in another assay

the cells were treated with p38 inhibitor SB203580 to observe the changes of related proteins.

Results

2

The results of GEPIA2 database and clinical specimen analysis showed that Smad3 was highly expressed in pancreatic cancer tissues (

P

<

0.05). After transfection of ShSmad3-1 and ShSmad3-2 into pancreatic cancer cells BxPC-3

both the mRNA and protein levels of Smad3 were significantly decreased (

P

<

0.05). Meanwhile

2 Smad3 knockout cell lines were screened out after transferring gRNA into Panc-1. As compared with the control group

Smad3 knockdown remarkably increased the apoptosis of pancreatic cancer cells (

P

<

0.05)

and Western blotting also indicated that after Smad3 knockdown or knockout

the protein level of p-p38 was up-regulated (

P

<

0.05)

while those of LC3 and p62 were down-regulated (

P

<

0.05). Further study found that the protein levels of p62 and LC3 were restored by the rescue assay as well as by the treatment of pancreatic cancer cells with SB203580 (

P

<

0.01).

Conclusion

2

Interference in Smad3 promotes excessive autophagy in pancreatic cancer cells and leads to increased apoptosis

possibly through activation of p38/MAPK signaling pathway.

关键词

Keywords

references

MILLER K D, NOGUEIRA L, MARIOTTO A B, et al . Cancer treatment and survivorship statistics, 2019[J ] . CA Cancer J Clin, 2019, 69(5): 363-385. DOI: 10.3322/caac.21565.

ZEESHAN M S, RAMZAN Z. Current controversies and advances in the management of pancreatic adenocarcinoma[J]. World J Gastrointest Oncol, 2021, 13(6): 472-494. DOI: 10.4251/wjgo.v13.i6.472.

YANG A N, HERTER-SPRIE G, ZHANG H K, et al . Autophagy sustains pancreatic cancer growth through both cell-autonomous and nonautonomous mechanisms[J ] . Cancer Discov, 2018, 8(3): 276-287. DOI: 10.1158/2159-8290.CD-17-0952.

PENG L, YANG C W, YIN J W, et al . TGF-β2 induces Gli1 in a Smad3-dependent manner against cerebral ischemia/reperfusion injury after isoflurane post-conditioning in rats[J ] . Front Neurosci, 2019, 13(1): 636. DOI: 10.3389/fnins.2019.00636.

ITOH Y, SAITOH M, MIYAZAWA K. Smad3-STAT3 crosstalk in pathophysiological contexts[J]. Acta Biochim Biophys Sin (Shanghai), 2018, 50(1): 82-90. DOI: 10.1093/abbs/gmx118.

SHENG J Y, WANG L, TANG P M K, et al . Smad3 deficiency promotes beta cell proliferation and function in db/db mice via restoring Pax6 expression[J ] . Theranostics, 2021, 11(6): 2845-2859. DOI: 10.7150/thno.51857..

JIANG Z, GUO Y, MIAO L F, et al . SMAD3 silencing enhances DNA damage in radiation therapy by interacting with MRE11-RAD50-NBS 1 complex in glioma[J ] . J Biochem, 2019, 165(4): 317-322. DOI: 10.1093/jb/mvy110..

SEO H Y, LEE S H, LEE J H, et al . Clusterin attenuates hepatic fibrosis by inhibiting hepatic stellate cell activation and downregulating the Smad3 signaling pathway[J ] . Cells, 2019, 8(11): 1442. DOI: 10.3390/cells8111442.

TANG P M K, ZHOU S, MENG X M, et al . Smad3 promotes cancer progression by inhibiting E4BP 4 -mediated NK cell development[J ] . Nat Commun, 2017, 8(2): 14677. DOI: 10.1038/ncomms14677..

HUANG Y T, CHENG A C, TANG H C, et al . USP7 facilitates SMAD3 autoregulation to repress cancer progression in p53-deficient lung cancer[J ] . Cell Death Dis, 2021, 12(10): 880. DOI: 10.1038/s41419-021-04176-8.

MUÑOZ M D, DE LA FUENTE N, SÁNCHEZ-CAPELO A. TGF-β/Smad3 signalling modulates GABA neurotransmission: implications in Parkinson's disease[J]. Int J Mol Sci, 2020, 21(2): 590. DOI: 10.3390/ijms21020590.

FALCICCHIA C, TOZZI F, ARANCIO O, et al . Involvement of p38 MAPK in synaptic function and dysfunction[J ] . Int J Mol Sci, 2020, 21(16): 5624. DOI: 10.3390/ijms21165624.

TIAN X D, TRAUB B, XIE X H, et al . Opposing oncogenic functions of p38 mitogen-activated protein kinase alpha and beta in human pancreatic cancer cells[J ] . Anticancer Res, 2020, 40(10): 5545-5556. DOI: 10.21873/anticanres.14567.

SHEN T, HUANG Z H, SHI C F, et al . Pancreatic cancer-derived exosomes induce apoptosi s of T lymphocytes through the p38 MAPK-mediated endoplasmic reticulum stress[J ] . FASEB J, 2020, 34(6): 8442-8458. DOI: 10.1096/fj.201902186R.

KLIONSKY D J, PETRONI G, AMARAVADI R K, et al . Autophagy in major human diseases[J ] . EMBO J, 2021, 40(19): e108863. DOI: 10.15252/embj.2021108863.

KOCATURK N M, AKKOC Y, KIG C, et al . Autophagy as a molecular target for cancer treatment[J ] . Eur J Pharm Sci, 2019, 134(1): 116-137. DOI: 10.1016/j.ejps.2019.04.011.

ONORATI A V, DYCZYNSKI M, OJHA R, et al . Targeting autophagy in cancer[J ] . Cancer, 2018, 124(16): 3307-3318. DOI: 10.1002/cncr.31335.

LI J B, CHEN X, KANG R, et al . Regulation and function of autophagy in pancreatic cancer[J ] . Autophagy, 2021, 17(11): 3275-3296. DOI: 10.1080/15548627.2020.1847462.

YAMAMOTO K, VENIDA A, YANO J, et al . Autophagy promotes immune evasion of pancreatic cancer by degrading MHC-I[J ] . Nature, 2020, 581(7806): 100-105. DOI: 10.1038/s41586-020-2229-5.

LI W M, ZHU J Q, DOU J, et al . Phosphorylation of LAMP2A by p38 MAPK couples ER stress to chaperone-mediated autophagy[J ] .Nat Commun, 2017, 8(1): 1763. DOI: 10.1038/s41467-017-01609-x.

MODI S, KIR D, BANERJEE S, et al . Control of apoptosis in treatment and biology of pancreatic cancer[J ] . J Cell Biochem, 2016, 117(2): 279-288. DOI: 10.1002/jcb.25284.

ANSARI D, TINGSTEDT B, ANDERSSON B, et al . Pancreatic cancer: yesterday, today and tomorrow[J ] . Future Oncol, 2016, 12(16): 1929-1946. DOI: 10.2217/fon-2016-0010.

MCGUIGAN A, KELLY P, TURKINGTON R C, et al . Pancreatic cancer: a review of clinical diagnosis, epidemiology, treatment and outcomes[J ] . World J Gastroenterol, 2018, 24(43): 4846-4861. DOI: 10.3748/wjg.v24.i43.4846.

LIU N, FENG J, LU X Y, et al . Isorhamnetin inhibits liver fibrosis by reducing autophagy and inhibiting extracellular matrix formation via the TGF-β1/Smad3 and TGF-β1/p38 MAPK pathways[J ] . Mediators Inflamm, 2019, 2019: 1-14. DOI: 10.1155/2019/6175091..

JI J, YU Q, DAI W Q, et al . Apigenin alleviates liver fibrosis by inhibiting hepatic stellate cell activation and autophagy via TGF-β1/Smad3 and p38/PPARα pathways[J ] . PPAR Res, 2021, 2021: 1-15. DOI: 10.1155/2021/6651839..

YANG C, CHEN X C, LI Z H, et al . SMAD3 promotes autophagy dysregulation by triggering lysosome depletion in tubular epithelial cells in diabetic nephropathy[J ] . Autophagy, 2021, 17(9): 2325-2344. DOI: 10.1080/15548627.2020.1824694.

CAO Q Q, YOU X, XU L J, et al . PAQR3 suppresses the growth of non-small cell lung cancer cells via modulation of EGFR-mediated autophagy[J ] . Autophagy, 2020, 16(7): 1236-1247. DOI: 10.1080/15548627.2019.1659654..

DENG S, SHANMUGAM M K, KUMAR A P, et al . Targeting autophagy using natural compounds for cancer prevention and therapy[J ] . Cancer, 2019, 125(8): 1228-1246. DOI: 10.1002/cncr.31978.

WANG R C, WEI Y J, AN Z Y, et al . Akt-mediated regulation of autophagy and tumorigenesis through beclin 1 phosphorylation[J ] . Science, 2012, 338(6109): 956-959. DOI: 10.1126/science.1225967.

阚月一, 王娅杰, 李琦, 等. 肿瘤发展过程中自噬与凋亡的相互作用[J]. 中国比较医学杂志, 2018, 28(2): 106-112. DOI: 10.3969.j.issn.1671-7856.2018.02.019.

KAN Y Y, WANG Y J, LI Q, et al . Interaction between autophagy and apoptosis during tumor development[J ] . Chin J Comp Med, 2018, 28(2): 106-112. DOI:10.3969.j.issn.1671-7856.2018.02.019.

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