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. 2022 Mar;11(6):1454-1464.
doi: 10.1002/cam4.4569. Epub 2022 Feb 6.

Downregulation of NCL attenuates tumor formation and growth in HeLa cells by targeting the PI3K/AKT pathway

Jun Ying  1   2 , Ruowang Pan  3 , Zhouhao Tang  1   4 , Jiayin Zhu  1   5 , Ping Ren  1 , Yang Lou  1   4 , Enyong Zhang  3 , Dadao Huang  3 , Penghong Hu  3 , Dong Li  1 , Qiyu Bao  1 , Peizhen Li  1   4
Affiliations

Downregulation of NCL attenuates tumor formation and growth in HeLa cells by targeting the PI3K/AKT pathway

Jun Ying et al. Cancer Med. 2022 Mar.

Abstract

Background: Nucleolin (NCL, C23) is a multifunctional phosphoprotein that plays a vital role in modulating the survival, proliferationand apoptosis of cancer cells. However, the effects of NCL on cervical cancer and the underlying mechanisms behind this are poorly understood.

Methods: Lentiviral transfection technology was used to construct NCL knockdown cell lines. MTT, colony formation assays, and tumorigenic assays in vivo were performed to observe cell proliferation. HOECHST 33342 staining, flow cytometry, and caspase activity assay were used to test cell apoptosis. RNA-Seq, Western blotting, and RT-PCR were conducted to investigate the specific molecular mechanism.

Results: NCL knockdown inhibited cell proliferation and promoted apoptosis both in vivo and in vitro. Mechanistic studies revealed that NCL knockdown inhibited the PI3K/AKT pathway by upregulating FGF, ITGA, TNXB, VEGF, Caspase 3, and Bax, as well as by downregulating AKT, GNB4, CDK6, IL6R, LAMA, PDGFD, PPP2RSA and BCL-2. In addition, the expression levels of apoptosis-related genes after using a PI3K inhibitor LY294002 were consistent with shRNA studies, while treatment with a 740Y-P agonist showed the opposite effect.

Conclusions: Our findings indicate that downregulation of NCL may be a novel treatment strategy forcervical cancer.

Keywords: HeLa cells; PI3K/AKT pathway; RNA-Seq; apoptosis; molecular mechanism; nucleolin.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

FIGURE 1
FIGURE 1
RNA interference inhibited NCL expression levels in HeLa cells. (A) NCL protein expression was detected by Western blotting. (B) Cell proliferation was detected by MTT analysis at 1, 2, 3, 4, 5, and 6 days. (C) Caspase‐3 activity. 1. NC‐HeLa, 2. sh‐NT‐HeLa, 3. sh‐NCL‐HeLa. Mean ± SD from three individual experiments. *p < 0.05, **p < 0.01, versus the sh‐NT‐HeLa group. MTT, 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2, 5‐diphenyltetrazolium bromide; NCL, nucleolin
FIGURE 2
FIGURE 2
Detection of proliferation and apoptosis of HeLa cells after NCL interference. (A) Colony formation assay. Cells were inoculated into plates at a density of 500 cells per well and grown at 37°C for 14 days. Cell colonies were stained with 0.1% crystal violet (Left). Cell colonies were quantified, **p < 0.01 (right). (B) NCL knockdown induced apoptosis morphological features as shown by Hoechst. Hypercoagulable nuclei and semilunar apoptotic bodies represent typical apoptosis morphology (see the red arrow; fluorescence staining ×400). (C) Cell apoptosis was detected using flow cytometry. Representative flow cytometry analysis of Annexin V‐APC/7AAD staining (left). The apoptosis rate was quantified (right). Mean ± SD from three individual experiments. *p < 0.05, versus the sh‐NT‐HeLa group. NCL, nucleolin
FIGURE 3
FIGURE 3
NCL knockdown regulates tumor formation in mice injected with HeLa cells. (A) Tumor volume was measured every 4 days, and the results were presented as a growth curve. (B) Representative images of tumors in nude mice after different treatments. (C) Tumor weight was measured on Day 28. (D) H&E staining (×200). (E) DAPI and TUNEL staining (×200). Results are presented as mean ± SD (n = 5). **p < 0.01, versus the sh‐NT‐HeLa group. DAPI, 4′,6‐diamidino‐2‐phenylindole; NCL, nucleolin
FIGURE 4
FIGURE 4
NCL knockdown induces apoptosis by inhibiting the PI3K/AKT pathway. (A) A heat map of differentially expressed genes in all samples. Rows represent genes, whereas columns represent samples. (B) Pathway enrichment analysis of transcriptome profiling. (C) Enrichment of differentially expressed genes in the PI3K/AKT pathway. (D) Diagram showing the fold‐change of several typical genes using RT‐PCR. **p < 0.01, versus the sh‐NT‐HeLa group. NCL, nucleolin
FIGURE 5
FIGURE 5
Protein levels of PI3K, AKT, Bcl‐2, Bax, and cleaved‐Caspase 3 were detected by Western blotting. (A) Representative Western blot brands. (B) Quantitative analyses of phospho‐PI3K/PI3K, phospho‐AKT/AKT, Bcl‐2/Bax, cleaved‐Caspase 3. Mean ± SD from three individual experiments. *p < 0.05, **p < 0.01, versus the sh‐NT‐HeLa group
FIGURE 6
FIGURE 6
HeLa cells with repressed NCL levels were treated with the PI3K inhibitor LY294002 or the PI3K activator 740Y‐P. (A) Cell proliferation was determined using an MTT assay. (B) The expression levels of the apoptosis‐related genes were measured using RT‐PCR. *p < 0.05, **p < 0.01, versus the sh‐NT‐Hela group. # p < 0.05, ## p < 0.01, versus the sh‐NCL‐Hela group, n = 3. MTT, 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2, 5‐diphenyltetrazolium bromide; NCL, nucleolin
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