Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Apr 11;22(1):150.
doi: 10.1186/s12935-022-02555-5.

RPL15 promotes hepatocellular carcinoma progression via regulation of RPs-MDM2-p53 signaling pathway

Rui Shi  1 , Zirong Liu  2
Affiliations

RPL15 promotes hepatocellular carcinoma progression via regulation of RPs-MDM2-p53 signaling pathway

Rui Shi et al. Cancer Cell Int. .

Abstract

Backround: RPL15 has been found to participate in human tumorigenesis. However, its function and regulatory mechanism in hepatocellular carcinoma (HCC) development are still unclear. Current study investigated the effects of RPL15 in HCC.

Methods: The expression of RPL15 in clinical tissues and cell lines of HCC was detected by RT-qPCR, Western blotting, and Immunohistochemistry (IHC). Colony formation, CCK-8, flow cytometry, Wound healing and Transwell invasion assays, were used to detect the carcinoma progression of HCC cells with RPL15 overexpression or knockdown in vitro. A xenograft model was constructed to assess the effect of RPL15 knockdown on HCC cells in vivo. The expression of CDK2 and Cyclin E1 related to cell cycles, Bax and Bcl-2 related to cell apoptosis, E-cadherin, N-cadherin and Vimentin related to epithelial-mesenchymal transition (EMT), p53 and p21 related to p53 signaling pathway, were detected by Western blotting. The connection between p53, MDM2 and RPL5/11 affected by RPL15 was analyzed using immunoprecipitation and Cycloheximide (CHX) chase assay.

Results: Elevated RPL15 was identified in HCC tissues, which was not only a prediction for the poor prognosis of HCC patients, but also associated with the malignant progression of HCC. RPL15 silencing arrested HCC cell cycle, suppressed HCC cell colony formation, proliferation, invasion, and migration, and induce cell apoptosis. On the contrary, RPL15 upregulation exerted opposite effects. Results also indicated that HCC cell invasion and migration were associated with EMT, and that the RPs-MDM2-p53 pathway was implicated in RPL15-mediated oncogenic transformation. In addition, RPL15 knockdown significantly suppressed HCC xenografts growth.

Conclusions: RPL15 played crucial roles in HCC progression and metastasis, serving as a promising candidate for targeted therapies.

Keywords: Hepatocellular carcinoma; MDM2; RPL15; Tumor progression; p53.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no conflicts of interest.

Figures

Fig. 1
Fig. 1
RPL15 was upregulated in human HCC tissues and cells. A, B, C Results of RT-qPCR, western blot and immunohistochemical analysis showed that RPL15 was upregulated in human HCC tissues. D Increased RPL15 levels were also detected in HCC cells. Data are presented as the mean ± SD, n = 3; *P < 0.05, compared to the control group
Fig. 2
Fig. 2
RPL15 unregulated in HCC predicted poor prognosis of HCC patients. A The expression of RPL15 was unregulated in T (tumor tissue) compared to N (negative para-carcinoma tissue) in TCGA HCC dataset. B, C Kaplan–Meier analysis indicated that HCC patients with high RPL15 expression displayed a significantly shorter overall survival (OS), but not obvious disease-free survival (DFS), the dotted lines mean 95% Confidengce Interval. *P < 0.05, compared to the control group
Fig. 3
Fig. 3
RPL15 downregulation could inhibit HCC cells proliferation in vivo and in vitro. A The efficiency of RPL15 overexpression in Hep3B and knockdown in HCCLM3 cells was confirmed by western blot. B CCK-8 assays showed that Hep3B cells with RPL15 overexpression displayed more active proliferative capacity while the RPL15-silenced HCCLM3 cells had weaker proliferative ability. C Results of colony formation assays showed that RPL15 knockdown significantly reduced while RPL15 overexpression increased the number of colonies. D Representative tumor image of xenograft mice. E, F Tumor volumes and weights of HCC cell xenograft model in RPL15-knockdown group were significantly reduced. Data are presented as the mean ± SD, n = 3; *P < 0.05, compared to the control group. NC means negative control; RPL15-KD, RPL15 stable knockdown
Fig. 4
Fig. 4
RPL15 knockdown blocked cell cycle progression in HCC cells. A RPL15 knockdown in HCCLM3 cells induced the cell cycle arrest in G1 phase while RPL15 overexpression in Hep3B cells induced the converse effects. B Western blot analysis revealed that RPL15 silencing decreased CDK2 and Cyclin E1 expressions, while RPL15 overexpression promoted their expressions. Data are presented as the mean ± SD, n = 3; *P < 0.05, compared to the control group
Fig. 5
Fig. 5
RPL15 knockdown enhanced HCC cells apoptosis. A RPL15 knockdown promoted HCCLM3 cell apoptosis, and RPL15 overexpression prevented Hep3B cell apoptosis. B Western blot analysis revealed that RPL15 knockdown upregulated the expression of Bax and downregulated the expressions of Bcl-2, and RPL15 overexpression induced the converse effects. Data are presented as the mean ± SD, n = 3; *P < 0.05, compared to the control group
Fig. 6
Fig. 6
RPL15 silencing inhibited HCC cells migration and invasion. A Wound-healing assay indicated that RPL15 silencing reduced HCCLM3 cell migration while RPL15 overexpression enhanced Hep3B cell migration. B Transwell assay revealed that RPL15 knockdown decreased the invasion capability of HCCLM3 cells while overexpression of RPL15 in Hep3B cells demonstrated the opposite effect. C The expressions of EMT-related genes were detected after RPL15 was silenced or overexpressed in HCC cells via western blot. Data are presented as the mean ± SD, n = 3; *P < 0.05, compared to the control group
Fig. 7
Fig. 7
RPL15 involved in RPs-MDM2-p53 signaling pathway. A Western blot analysis revealed that the levels of p21 and p53 were significantly increased in HCCLM3 cells with RPL15 knockdown. B Western blotting from 0–8 h after cycloheximide (CHX) treatment to stop protein synthesis showed more stability of p53 in HCCLM3 cells with RPL15 knockdown. C RPL15 silencing suppressed the interaction of MDM2 with p53, the binding was determined by immunoprecipitation. D Effect of RPL15 on the binding of MDM2 with RPL11 and RPL5 was determined by immunoprecipitation, RPL15 silencing suppressed the interaction
Fig. 8
Fig. 8
RPL15 mediated HCC progression via the RPs-MDM2-p53 signaling pathway. A The efficiency of p53 knockdown in HCCLM3 cells was confirmed by western blot. B CCK-8 assays showed that si-p53 reversed the inhibitory function of proliferation in HCCLM3 cell with RPL15 knockdown. C Wound-healing assay indicated that si-p53 reversed the inhibitory function of migration in HCCLM3 cell with RPL15 knockdown. D Transwell assay revealed that si-p53 reversed the inhibitory function of invasion in HCCLM3 cell with RPL15 knockdown. Data are presented as the mean ± SD, n = 3; *P < 0.05, compared to the control group; #P < 0.05, compared to the si-RPL15 group
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 Countries. Cancer J Clin. 2021;71(3):209–249. - PubMed
    1. Shi JJ, Dang SS. Recent advances in molecular targeted therapy of hepatocellular carcinoma. World Chin J Dig. 2018;26(34):2008–2017. doi: 10.11569/wcjd.v26.i34.2008. - DOI
    1. Kang TW, Rhim H. Recent advances in tumor ablation for hepatocellular carcinoma. Liver Cancer. 2015;4:176–187. doi: 10.1159/000367740. - DOI - PMC - PubMed
    1. Hannan KM, Sanij E, Rothblum LI, Hannan RD, Pearson RB. Dysregulation of RNA polymerase I transcription during disease. Biochim Biophys Acta. 2013;1829(3–4):342–360. doi: 10.1016/j.bbagrm.2012.10.014. - DOI - PMC - PubMed
    1. Orsolic I, Jurada D, Pullen N, Oren M, Eliopoulos AG, Volarevic S. The relationship between the nucleolus and cancer: current evidence and emerging paradigms. Semin Cancer Biol. 2016 doi: 10.1016/j.semcancer.2015.12.004. - DOI - PubMed