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. 2023 Feb 28;14(1):287-301.
doi: 10.21037/jgo-23-10.

Silencing proline-rich coiled-coil 2C inhibit the proliferation and metastasis of liver cancer cells

Kai Zhang  1 , Jiaming Xu  1 , Ran Chen  2
Affiliations

Silencing proline-rich coiled-coil 2C inhibit the proliferation and metastasis of liver cancer cells

Kai Zhang et al. J Gastrointest Oncol. .

Abstract

Background: Proline-rich coiled-coil 2C (PRRC2C) is located in the chromosome region lq where hepatocellular carcinoma (HCC) frequently undergoes genomic fragment amplification, but its role in HCC is unknown. In this study, we aimed to explore the correlation of PRRC2C with HCC diagnosis and progression, as well as its influence on the biological behavior of HCC cells.

Methods: The Cancer Genome Atlas (TCGA) RNA-sequencing datasets of 371 cases of primary liver cancer and 50 normal liver tissue specimens were obtained to analyze correlation between PRRC2C expression and HCC staging, grades, and overall survival. After confirming expression of PRRC2C in HCC cells, PRRC2C silencing was performed. Celigo cell counting, cell clone formation, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and Flow cytometry were used to detect the cell proliferation and apoptosis; wound healing and Transwell assays were used to detect the invasion abilities of cells. Xenograft transplantation in nude mice was performed to investigate the impact of PRRC2C knockdown on tumorigenic capabilities. In addition, the expression levels of EMT (epithelial-mesenchymal transition)-related genes, including E-cadherin, N-cadherin, Twistl, Snail, Slug, and Smad2/3/4, were detected.

Results: Analysis of TCGA data sets revealed that patients with high PRRC2C expression had significantly shorter overall survival. PRRC2C was abundantly expressed in four human hepatocarcinoma cell lines. After knockdown PRRC2C, the proliferation of HCC cells were suppressed and the numbers of apoptotic cells increased. Migration and invasion ability of HCC cells were inhibited by PRRC2C knockdown. Meanwhile, PRRC2C silencing inhibited the tumor formation (indicated by reduced tumor volume and weight compared to the control group) in BALB/c (Bagg Albino Laboratory-bred strain) nude mice. The expressions of EMT-related genes N-cadherin and Vimentin were significantly lower in the PRRC2C knockdown group than in the control group.

Conclusions: PRRC2C promotes the proliferation and metastasis of liver cancer cells and inhibited apoptosis, potentially through upregulation of EMT related N-cadherin and Vimentin.

Keywords: Apoptosis; PRRC2C; hepatocellular; neoplasm metastasis; proliferation.

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Conflict of interest statement

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jgo.amegroups.com/article/view/10.21037/jgo-23-10/coif). The authors have no conflicts of interest to declare.

Figures

Figure 1
Figure 1
Correlations between proline-rich coiled-coil 2C (PRRC2C) expression with LIHC (liver hepatocellular carcinoma) staging, grade, and overall survival. RNA-sequencing datasets of 371 liver cancer samples and 50 control samples were obtained from The Cancer Genome Atlas (TCGA) database portal (http://cancergenome.nih.gov/). Differences in PRRC2C expression levels were analyzed between cancer and control (A), as well as among different TNM stage (B) and pathological grades (C). The cancer samples were divided into high and low expression groups and overall survival (OS) were compared between the groups using Kaplan-Meier survival analysis (D). Real-time reverse transcription polymerase chain reaction (RT-PCR) was performed to detect PRRC2C mRNA levels in BEL-7402, BEL-7404, SMMC-7721, and Hep G2 cell lines (E). DNA segments containing shRNA constructs targeting PRRC2C (shPRRC2C) or non-specific control (shCtrl) were cloned into transfer vector and then introduced into cells via lentivirus infection. The mRNA levels of PRRC2C were tested using RT-PCR and compared between cells infected with different shRNA constructs (F). Western blot was used to detect PRRC2C knockdown efficiency, representative result was shown (G). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as an internal control. n=3. **P<0.01. ****P<0.0001.
Figure 2
Figure 2
Proline-rich coiled-coil 2C (PRRC2C) knockdown inhibits cell viability/proliferation and promotes apoptosis. Viability/proliferation of cells transfected with shPRRC2C or shCtrl was examined with: Celigo cell count (A), MTT assay (B), and Clone formation assay (Crystal violet staining) (C). Apoptosis of transfected cells were detected using Annexin-V and PI staining followed by flow cytometry (D). **P<0.01. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
Figure 3
Figure 3
Proline-rich coiled-coil 2C (PRRC2C) knockdown inhibits cell migration and invasion. Capability of cell migration was detected using wound healing assay (100×) (A). Cell invasion was examined using Transwell assay (Crystal violet staining, 100×) (B). **P<0.01.
Figure 4
Figure 4
Proline-rich coiled-coil 2C (PRRC2C) knockdown inhibits tumor formation in vivo cells transfected with shPRRC2C (KD) or shCtrl (NC) were inoculated into BALB/c nude mice to induce xenograft tumor. The growth of tumor was monitored by fluorescence detection (A). Tumors were isolated from experimental animals at different time points. Sizes and growth rate were quantified and compared between cells transfected with shCtrl and shPRRC2C constructs (B).
Figure 5
Figure 5
Effects of proline-rich coiled-coil 2C (PRRC2C) knockdown on EMT related gene expression. RT-PCR was used to detect the mRNA levels of genes coding N-cadherin (CDH2), E-cadherin (CDH1), TWIST1, SMAD2/3/4 and SNAI1/2. Vimentin and N-cadherin were detected by Western blotting. ***P<0.001. ****P<0.0001. RT-PCR, reverse transcription-polymerase chain reaction.
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