KHSRP通过激活JAK/STAT信号通路对结直肠癌细胞生物学行为的影响

摘要/Abstract

摘要：

目的探讨KH型剪切调节蛋白（KHSRP）激活Janus激酶（JAK）/信号转导与转录激活因子（STAT）信号通路对结直肠癌（CRC）恶性生物学行为的影响，并阐明其可能的作用机制。方法选取64例CRC患者的CRC组织和癌旁正常组织，体外培养人CRC HT29、SW620、SW480、DLD-1、LOVO和RKO细胞及人正常结直肠黏膜FHC细胞，提取CRC组织和组细胞中总RNA，采用实时荧光定量PCR（RT-qPCR）法检测CRC组织癌旁正常组织和各种细胞中KHSRP mRNA表达水平。将HT29和SW620细胞分为sh-NC组（无相关性的核苷酸序列插入慢病毒质粒）和sh-KHSRP组（转染敲降KHSRP慢病毒）；SW480和DLD-1细胞分为oe-NC组（无相关性的核苷酸序列插入慢病毒质粒）和oe-KHSRP组（转染过表达KHSRP慢病毒）。采用免疫组织化学（IHC）染色法分析CRC组织和癌旁正常组织中KHSRP蛋白表达情况，细胞计数试剂盒8（CCK-8）法检测各组CRC细胞增殖活性，Transwell小室实验检测各组CRC细胞的迁移细胞数和侵袭细胞数，Western blotting法检测各组CRC细胞中KHSRP、JAK1、磷酸化JAK1（p-JAK1）、JAK2、磷酸化JAK2（p-JAK2）、STAT1、STAT2、STAT3和STAT5蛋白表达水平，裸鼠皮下移植瘤实验检测各组小鼠肿瘤质量和肿瘤体积。结果与癌旁正常组织比较，CRC组织中KHSRP mRNA表达水平升高（P<0.01）；与人正常结直肠黏膜FHC细胞比较，CRC细胞中KHSRP mRNA表达水平均升高（P<0.05），后续实验选择HT29和SW620细胞进行KHSRP敲降，选择SW480和DLD-1细胞进行 KHSRP过表达。Western blotting法检测，CRC组织和细胞中KHSRP蛋白表达量高于癌旁正常组织和FHC细胞。IHC染色法分析，与癌旁正常组织比较，CRC组织中KHSRP蛋白表达水平升高（P<0.01）。RT-qPCR法检测，与sh-NC组比较，sh-KHSRP组CRC HT29和SW620细胞中KHSRP mRNA表达水平降低（P<0.01）；与oe-NC组比较，oe-KHSRP组SW480和DLD-1细胞中KHSRP mRNA表达水平升高（P<0.01），提示细胞转染成功。CCK-8法检测，与sh-NC组比较，敲降KHSRP后，sh-KHSRP组HT29和SW620细胞增殖活性降低（P<0.05或P<0.01）；与oe-NC组比较，过表达KHSRP后，oe-KHSRP组SW480和DLD-1细胞增殖活性升高（P<0.05或P<0.01）。与sh-NC组比较，敲降KHSRP后，sh-KHSRP组HT29和SW620细胞的迁移细胞数和侵袭细胞数减少（P<0.05）；与oe-NC组比较，过表达KHSRP后，oe-KHSRP组SW480和DLD-1细胞的迁移细胞数和侵袭细胞数增多（P<0.05）。敲降KHSRP后，oe-KHSRP组小鼠肿瘤体积和质量小于sh-NC组（P<0.05或P<0.01）；过表达KHSRP后，oe-KHSRP组小鼠肿瘤体积和质量大于oe-NC组（P<0.05或P<0.01）。与sh-NC组比较，sh-KHSRP组CRC细胞中JAK1、p-JAK-1和STAT3蛋白表达水平明显降低（P<0.05或P<0.01）；与oe-NC组比较，oe-KHSRP组CRC细胞中JAK1、p-JAK-1和STAT3蛋白表达水平明显升高（P<0.05或P<0.01）。结论 KHSRP高表达可促进CRC细胞增殖、迁移和侵袭，促进小鼠CRC细胞皮下移植瘤的生长，其机制可能与其激活JAK1/STAT3信号通路有关联。

关键词: 结直肠肿瘤, KH型剪切调节蛋白, Janus激酶, 信号转导与转录激活因子, 恶性进展, 细胞增殖, 细胞侵袭, 细胞迁移

Abstract:

Objective To discuss the effect of KH-type splicing regulatory protein（KHSRP） on the malignant biological behaviors of colorectal cancer （CRC） by activating the Janus kinase（JAK）/signal transducer and activator of transcription（STAT） signaling pathway， and to clarify its possible mechanism. Methods The CRC tissue and adjacent normal tissue from 64 CRC patients were selected. The human CRC cells （HT29， SW620， SW480， DLD-1， LOVO， and RKO） and normal human colorectal mucosal FHC cells were cultured in vitro. The total RNA from CRC tissue and cells were extracted， real-time fluorescence quantitative PCR （RT-qPCR） was used to detect the expression levels of KHSRP in the CRC tissue， adjacent normal tissue and all kinds of cells. The HT29 and SW620 cells were divided into sh-NC group （lentiviral plasmid inserted with non-targeting nucleotide sequence） and sh-KHSRP group （transfected with KHSRP knockdown lentivirus）. The SW480 and DLD-1 cells were divided into oe-NC group （lentiviral plasmid inserted with non-targeting nucleotide sequence） and oe-KHSRP group （transfected with KHSRP overexpression lentivirus）. Immunohistochemistry （IHC） staing in method was used to analyze the expressions of KHSRP in CRC tissue and adjacent normal tissue； CCK-8 method was used to detect the proliferation activities of the CRC cells in various groups； Transwell assay was used to detect the numbers of migration and invasion cells in various groups； Western blotting method was used to detect the expression levels of KHSRP， JAK1， phosphorylated JAK1（p-JAK1）， JAK2， phosphorylated JAK2（p-JAK2）， STAT1， STAT2， STAT3， and STAT5 proteins in the CRC cells in various groups. The subcutaneous xenograft tumor models in the nude mice were used to measure the tumor volumes and weights of the mice in various groups. Results Compared with adjacent normal tissue， the expression level of KHSRP in the CRC tissue was increased （P<0.01）. Compared with FHC cells， the expression levels of KHSRP in the CRC cells were increased （P<0.05）. Therefore， the HT29 and SW620 cells were selected for knockdown of KHSRP， while the SW480 and DLD-1 cells were selected for over-expression of KHSRP. The Western blotting results showed that the expression amounts of KHSRP protein in the CRC tissue and cells were higher than those in adjacent normal tissue and FHC cells. The IHC results showed that compared with adjacent normal tissue， the expression level of KHSRP protein in CRC tissue was increased （P<0.01）. The RT-qPCR results showed that compared with sh-NC group， the expression levels of KHSRP mRNA in the HT29 and SW620 cells in sh-KHSRP group were decreased （P<0.01）； compared with oe-NC group， the expression levels of KHSRP mRNA in the SW480 and DLD-1 cells in oe-KHSRP group were increased （P<0.01）， indicating successful transfection. The CCK-8 results showed that compared with sh-NC group， the proliferation activities of the HT29 and SW620 cells in sh-KHSRP group after knockdown of KHSRP were decreased （P<0.05 or P<0.01）； compared with oe-NC group， the proliferation activities of the SW480 and DLD-1 cells in oe-KHSRP group after over-expression of KHSRP were increased （P<0.05 or P<0.01）. Compared with sh-NC group， the numbers of migration and invasion cells in the HT29 and SW620 cells in sh-KHSRP group after knockdown of KHSRP were decreased （P<0.05）； compared with oe-NC group， the numbers of migration and invasion cells in the CRC cells in oe-KHSRP group after over-expression of KHSRP were increased （P<0.05）. After knockdown of KHSRP， the tumor volume and weight in sh-KHSRP group were smaller than those in sh-NC group （P<0.05 or P<0.01）， while the tumor volume andweight in oe-KHSRP group were larger than those in oe-NC group after over-expression of KHSRP （P<0.05 or P<0.01）. Compared with sh-NC group， the expression levels of JAK1， p-JAK1， and STAT3 proteins in the CRC cells in sh-KHSRP group were significantly decreased （P<0.05 or P<0.01）； compared with oe-NC group， the expression levels of JAK1， p-JAK1， and STAT3 proteins in the CRC cells in oe-KHSRP group were significantly increased （P<0.05 or P<0.01）. Conclusion High expression of KHSRP promotes the proliferation， migration， and invasion of the CRC cells and enhances the growth of subcutaneous xenograft tumors in the mice； its mechanism may be associated with its activation of the JAK1/STAT3 signaling pathway.

Key words: Colorectal neoplasms, KH-type splicing regulatory protein, Janus kinase, Signal transducer and activator of transcription, Malignant progression, Cell proliferation, Cell invasion, Cell migration

中图分类号:

R735.3

李宏丽,王梦瑶,刘洋洋,张卉,李丽,韦海涛. KHSRP通过激活JAK/STAT信号通路对结直肠癌细胞生物学行为的影响[J]. 吉林大学学报(医学版), 2025, (): 1-11.

Hongli LI,Mengyao WANG,Yangyang LIU,Hui ZHANG,Li LI,Haitao WEI. Effect of KHSRP on biological behavior of colorectal cancer cells through activation of JAK/STAT signaling pathway[J]. Journal of Jilin University(Medicine Edition), 2025, (): 1-11.

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参考文献 23

1	XI Y， XU P F. Global colorectal cancer burden in 2020 and projections to 2040［J］. Transl Oncol， 2021， 14（10）： 101174.
2	XIA C F， DONG X S， LI H， et al. Cancer statistics in China and United States， 2022： profiles， trends， and determinants［J］. Chin Med J （Engl）， 2022， 135（5）： 584-590.
3	SUNG H， FERLAY J， SIEGEL R L， et al. Global cancer statistics 2020： GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries［J］. CA Cancer J Clin， 2021， 71（3）： 209-249.
4	MORGAN E， ARNOLD M， GINI A， et al. Global burden of colorectal cancer in 2020 and 2040： incidence and mortality estimates from GLOBOCAN［J］. Gut， 2023， 72（2）： 338-344.
5	XU Y Q， BAO Y X， QIU G Z， et al. METTL3 promotes proliferation and migration of colorectal cancer cells by increasing SNHG1 stability［J］. Mol Med Rep， 2023， 28（5）： 217.
6	TANIUCHI K， OGASAWARA M. KHSRP-bound small nucleolar RNAs associate with promotion of cell invasiveness and metastasis of pancreatic cancer［J］. Oncotarget， 2020， 11（2）： 131-147.
7	CAIAZZA F， OFICJALSKA K， TOSETTO M， et al. KH-type splicing regulatory protein controls colorectal cancer cell growth and modulates the tumor microenvironment［J］. Am J Pathol， 2019， 189（10）： 1916-1932.
8	JOHNSON H M， NOON-SONG E， AHMED C M. Noncanonical IFN signaling， steroids， and STATs： a probable role of V-ATPase［J］. Mediators Inflamm， 2019， 2019： 4143604.
9	XUE C， YAO Q F， GU X Y， et al. Evolving cognition of the JAK-STAT signaling pathway： autoimmune disorders and cancer［J］. Signal Transduct Target Ther， 2023， 8（1）： 204.
10	HANAHAN D. Hallmarks of cancer： new dimensions［J］. Cancer Discov， 2022， 12（1）： 31-46.
11	XU J Y， WANG D S， MA H L， et al. KHSRP combines transcriptional and posttranscriptional mechanisms to regulate monocytic differentiation［J］. Blood Sci， 2022， 4（3）： 103-115.
12	CHEN L， ZHAO T J. Identification of KHSRP-regulated RNAs in esophageal cancer by integrated bioinformatics analysis［J］. Cancer Biother Radiopharm， 2021， 36（5）： 412-424.
13	HUANG J G， SACHDEVA M， XU E， et al. The long noncoding RNA NEAT1 promotes sarcoma metastasis by regulating RNA splicing pathways［J］. Mol Cancer Res， 2020， 18（10）： 1534-1544.
14	YAN M X， SUN L， LI J， et al. RNA-binding protein KHSRP promotes tumor growth and metastasis in non-small cell lung cancer［J］. J Exp Clin Cancer Res， 2019， 38（1）： 478.
15	HE L， TIAN L. Downregulation of miR-409-3p suppresses LPS-induced inflammation in human bronchial epithelial cells through SOCS3/JAK1/STAT3 signaling： The implication for bronchopneumonia［J］. Mol Med Rep， 2021， 23（3）： 190.
16	TANG J J， HAO T D K， LIM J J， et al. JAK/STAT signaling in hepatocellular carcinoma［J］. Hepat Oncol， 2020， 7（1）： HEP18.
17	LIU C R， FENG H Q， SONG L H， et al. Synergistic effects of thalidomide and cisplatin are mediated via the PI3K/AKT and JAK1/STAT3 signaling pathways in cervical cancer［J］. Oncol Rep， 2022， 48（4）： 169.
18	SARAPULTSEV A， GUSEV E， KOMELKOVA M， et al. JAK-STAT signaling in inflammation and stress-related diseases： implications for therapeutic interventions［J］. Mol Biomed， 2023， 4（1）： 40.
19	LAMICHHANE S， MO J S， SHARMA G， et al. microRNA 452 regulates IL20RA-mediated JAK1/STAT3 pathway in inflammatory colitis and colorectal cancer［J］. Inflamm Res， 2021， 70（8）： 903-914.
20	JIANG Y， XU C H， ZHAO Y， et al. LINC00926 is involved in hypoxia-induced vascular endothelial cell dysfunction via miR-3194-5p regulating JAK1/STAT3 signaling pathway［J］. Eur J Histochem， 2023， 67（1）： 3526.
21	BROOKS A J， PUTOCZKI T. JAK-STAT signalling pathway in cancer［J］. Cancers （Basel）， 2020， 12（7）： 1971.
22	WANG F， WANG X C， LI J R， et al. CircNOL10 suppresses breast cancer progression by sponging miR-767-5p to regulate SOCS₂/JAK/STAT signaling［J］. J Biomed Sci， 2021， 28（1）： 4.
23	CHAN S， LIU Z X， CHEN Y Y， et al. The JAK-STAT signaling-related signature serves as a prognostic and predictive biomarker for renal cell carcinoma immunotherapy［J］. Gene， 2024， 927： 148719.

Gene		Primer sequences（5'-3'）
KHSRP	R	AATGAGTACGGATCTCGGATTGG
	F	CCGTCATCTTGCTTGAACTGTA
JAK1	R	ACGCTCTGGGAAATCTGCTA
	F	ATGATGGCTCGGAAGAAAGG
STAT3	R	CTGGCCTTTGGTGTTGAAAT
	F	AAGGCACCCACAGAAACAAC
GAPDH	R	GAAGGTGAAGGTCGGAGTC
	F	GAAGATGGTGATGGGATTTC

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