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Up-Regulated CCDC34 Contributes to the Proliferation and Metastasis of Hepatocellular Carcinoma

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Up-Regulated CCDC34 Contributes to the Proliferation and Metastasis of Hepatocellular Carcinoma

Zhibin Lin et al. Onco Targets Ther.

Abstract

Background: Coiled-coil domain-containing protein 34 (CCDC34), which belongs to the CCDCs family, has been recently reported to be up-regulated in various kinds of tumors. However, its role in the development of hepatocellular carcinoma (HCC) still remains unclear.

Materials and methods: In this study, real-time polymerase chain reaction (RT-PCR) and Western blot analysis were performed to measure the mRNA and protein levels of CCDC34 in clinical samples. Kaplan-Meier method was used to analyze the relationship between CCDC34 and the prognosis in HCC patients. CCK-8 and colony formation assays were conducted to investigate CCDC34's effect on the cell proliferation, and Transwell assays were used to detect CCDC34's effect on the cell metastasis. Moreover, subcutaneous xenograft tumor model and lung metastasis model were applied to confirm the impact of CCDC34 on the HCC development. Lastly, RNA sequencing and Western blot analysis were performed to probe the underlying mechanism of CCDC34's effect on HCC.

Results: CCDC34 was significantly induced in HCC tissues, and the overexpression of CCDC34 predicted the poor outcomes among HCC patients. It was verified by the in vitro and in vivo experiments that CCDC34-knockdown potently inhibited the proliferation and metastasis of HCC cells. Subsequent results indicated that CCDC34 inhibition can affect the activation of protein kinase B (PKB or AKT) as well as epithelial-mesenchymal transition (EMT) process.

Conclusion: CCDC34 is significantly associated with HCC. It will become a promising prognostic biomarker and therapeutic target against HCC.

Keywords: CCDC34; CCND1; EMT; HCC; PI3K/AKT; proliferation.

Conflict of interest statement

The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
The overexpression of CCDC34 in HCC tissues. Notes: (A) The expression of CCDC34 in the HCC tissues and adjacent normal tissues from TCGA database (n=50). (B) The overall survival rate of patients with high or low CCDC34 expression in TCGA database (P=0.004). (C) The qRT-PCR assays for the CCDC34’s mRNA expression in the HCC tissues and adjacent normal tissues (n=21). **P<0.01. (D) Representative Western blot assays for the CCDC34 protein expression in the HCC tissues (T) and adjacent normal tissues (N). Abbreviations: HCC, hepatocellular carcinoma; TCGA, the Cancer Genome Atlas; qRT-PCR, quantitative real-time polymerase chain reaction.
Figure 2
Figure 2
HCC cell proliferation and clonogenicity in vitro facilitated by CCDC34. Notes: (A) The knockdown efficiency of shCCDC34 in the MHCC97-H and SMMC-7721 cell lines measured by the qRT-PCR and Western blot. (B and C) The proliferation ability of the MHCC97-H and SMMC-7721 cells transfected with shCCDC34 or NC lentivirus were detected by the CCK-8 assays (B) and the colony formation assays (C). The “sh” represents the shCCDC34 group and NC stands for the negative control group. (D) The overexpression efficiency of CCDC34 in the BEL-7404 and SMMC-7721 cell lines were measured by the qRT-PCR and Western blot (E and F). The proliferation ability of the BEL-7404 and SMMC-7721 cells transfected with the CCDC34-OE or NC lentivirus were detected by the CCK-8 assays (E) and the colony formation assays (F). The CCDC34 represents the CCDC34-OE group and NC stands for the negative control group. *P<0.05; **P<0.01; ***P<0.001. Abbreviations: HCC, hepatocellular carcinoma; qRT-PCR, quantitative real-time polymerase chain reaction; NC, negative control; CCK-8, Cell Counting Kit-8; OE, overexpression.
Figure 3
Figure 3
HCC cell migration and invasion in vitro regulated by CCDC34. Notes: (A and B) The migration and invasion ability of the MHCC97-H (A) and SMMC-7721 (B) cells transfected with the shCCDC34 or NC lentivirus were measured by the transwell assays. (C and D). The migration and invasion ability of the BEL-7404 (C) and SMMC-7721 (D) cells transfected with the CCDC34-OE or NC lentivirus were measured by the transwell assays. Scale bar = 100 μm. **P<0.01; ***P<0.001. Abbreviations: HCC, hepatocellular carcinoma; NC, negative control; OE, overexpression.
Figure 4
Figure 4
HCC cell growth and metastasis in vivo suppressed by CCDC34-KD. Notes: (A) The MHCC97-H cells transfected with shCCDC34 or NC lentivirus were subcutaneously injected into the nude mice (n=8). The top tumors were produced by MHCC97-H-NC cells, while the bottom by MHCC97-H-shCCDC34 cells. (B) The volume and weight of tumors harvested from the nude mice were measured. (C) Representative H&E staining in lung sections obtained from the nude mice injected with the MHCC97-H-shCCDC34 or NC cells through the tail veins (n=7). Scale bar = 500 μm (left) and 50 μm (right). The top tumors were produced by MHCC97-H-NC cells, while the bottom by MHCC97-H-shCCDC34 cells. (D) The number of metastatic tumors per lung was counted. **P<0.01; ***P<0.001. Abbreviations: KD, knockdown; HCC, hepatocellular carcinoma; NC, negative control.
Figure 5
Figure 5
The activation of AKT pathways and EMT process hampered by silencing of CCDC34. Notes: (A) A scatter plot shows the number of the DEGs analyzed by the RNA sequencing in the BEL-7404-CCDC34-OE cells (fold change >2.0 and P-value <0.05). (B) The KEGG pathway enrichment analysis of all the DEGs. (C) A heatmap consisting of the DEGs extracted from the five HCC-related modules (red) of Figure 5B. (D) Protein-protein interaction (PPI) network analysis for the DEGs extracted from the five HCC-related modules (red) of Figure 5B. The size of the nodes indicates the number of neighboring nodes directly connected to the node. (E) Representative Western blot assays for the protein level of CCDC34, AKT, p-AKT, P21, CCND1, N-cadherin and E-cadherin. Abbreviations: EMT, epithelial-mesenchymal transition; DEGs, differentially expressed genes; OE, overexpression; KEGG, Kyoto Encyclopedia of Genes and Genomes; PPI, protein-protein interaction.

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References

    1. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics, 2012. CA Cancer J Clin. 2015;65(2):87–108. doi:10.3322/caac.21262 - DOI - PubMed
    1. Yang JD, Hainaut P, Gores GJ, Amadou A, Plymoth A, Roberts LR. A global view of hepatocellular carcinoma: trends, risk, prevention and management. Nat Rev Gastroenterol Hepatol. 2019;16(10):589–604. doi:10.1038/s41575-019-0186-y - DOI - PMC - PubMed
    1. Xia F, Wu LL, Lau WY, et al. Adjuvant sorafenib after heptectomy for barcelona clinic liver cancer-stage C hepatocellular carcinoma patients. World J Gastroenterol. 2016;22(23):5384–5392. doi:10.3748/wjg.v22.i23.5384 - DOI - PMC - PubMed
    1. Burkhard P, Stetefeld J, Strelkov SV. Coiled coils: a highly versatile protein folding motif. Trends Cell Biol. 2001;11(2):82–88. doi:10.1016/S0962-8924(00)01898-5 - DOI - PubMed
    1. Hu X, Zhao Y, Wei L, et al. CCDC178 promotes hepatocellular carcinoma metastasis through modulation of anoikis. Oncogene. 2017;36(28):4047–4059. doi:10.1038/onc.2017.10 - DOI - PubMed
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