MORC2, a novel oncogene, is upregulated in liver cancer and contributes to proliferation, metastasis and chemoresistance

Abstract

Microrchidia 2 (MORC2) is important in DNA damage repair and lipogenesis, however, the clinical and functional role of MORC2 in liver cancer remains to be fully elucidated. The aim the present study was to clarify the role of MORC2 in liver cancer. Expression profile analysis, immunohistochemical staining, reverse transcription-quantitative polymerase chain reaction analysis and western blot analysis were performed to evaluate the levels of MORC2 in liver cancer patient specimens and cell lines; subsequently the expression of MORC2 was suppressed or increased in liver cancer cells and the effects of MORC2 on the cancerous transformation of liver cancer cells were examined in vitro and in vivo. MORC2 was upregulated in liver cancer tissues, and the upregulation was associated with certain clinicopathologic features of patients with liver cancer. MORC2 knockdown caused marked inhibition of liver cancer cell proliferation and clonogenicity, whereas the overexpression of MORC2 substantially promoted liver cancer cell proliferation. In addition, the knockdown of MORC2 inhibited the migratory and invasive ability of liver cancer cells, whereas increased migration and invasion rates were observed in cells with ectopic expression of MORC2. In a model of nude mice, the overexpression of MORC2 promoted tumorigenicity and markedly enhanced pulmonary metastasis of liver cancer. Furthermore, MORC2 regulated apoptosis and its expression level had an effect on the sensitivity of liver cancer cells to doxorubicin, 5-fluorouracil and cisplatin. Mechanically, MORC2 modulated the mitochondrial apoptotic pathway, possibly in a p53-dependent manner, and its dysregulation also resulted in the abnormal activation of the Hippo pathway. For the first time, to the best of our knowledge, the present study confirmed that MORC2 was a novel oncogene in liver cancer. These results provide useful insight into the mechanism underlying the tumorigenesis and progression of liver cancer, and offers clues into potential novel liver cancer therapies.

Figure 1

Gene Expression Omnibus datasets show that the expression of MORC2 is increased in liver cancer tissue. In (A) GSE14520 and (B) GSE22058, the expression level of MORC2 in liver cancer tissues was upregulated, compared with that in adjacent tissues. MORC2, microrchidia 2.

Figure 2

Protein expression level of MORC2 is increased in human liver cancer samples. (A) Expression of MORC2 was upregulated in 72.6% of liver cancer patient samples examined by immunohistochemistry. (B) Representative images of staining of MORC2 protein in three pairs of liver cancer and adjacent tissues (scale bar, 5 µm). MORC2, microrchidia 2.

Figure 3

mRNA expression level of MORC2 is increased in human liver cancer samples and the protein expression level of MORC2 is increased in liver cancer cell lines. (A) Lysates from paired tissues of liver cancer and adjacent tissue were analyzed by reverse transcription-quantitative polymerase chain reaction analysis for the detection of MORC2. β-actin was used as a loading control. (B) Western blot analysis of the expression of MORC2 in the immortalized L02 normal liver cell line and six liver cancer cell lines. MORC2, microrchidia 2.

Figure 4

Higher expression of MORC2 is associated with increased somatic copy number variation in patients with liver cancer. Genome instability (DNA copy number alteration) was detected in patients with liver cancer in The Cancer Genome Atlas. Data are presented as the mean ± standard deviation. MORC2, microrchidia 2.

Figure 5

GSEA results indicate that MORC2 modulates the proliferation of liver cancer cells. GSEA analysis of Gene Ontology terms showed MORC2 may regulate gene sets associated with cell cycle checkpoint (left), cell cycle progress (middle) and mitosis (right). GSEA, gene set enrichment analysis; MORC2, microrchidia 2.

Figure 6

MORC2 modulates proliferation of liver cancer cells in vitro. (A) Knockdown of MORC2 in HepG2 (left), Bel-7402 (middle) and LM3 (right) cell lines by siRNA targeting MORC2 was confirmed by western blot analysis; β-actin was used as a loading control. (B) Effect of MORC2-knockdown on the proliferation of HepG2 (left), Bel-7402 (middle) and LM3 (right) cells was determined using a cell counting kit-8 assay. (C) Effect of MORC2-knockdown on colony numbers was determined using a colony formation assay in HepG2 (left), Bel-7402 (middle) and LM3 (right) cells. ^*P<0.05; ^**P<0.01; ^***P<0.001. MORC2, microrchidia 2; siRNA, small interfering RNA; CON, control; KD, knockdown; NC, negative control.

Figure 7

MORC2 regulates liver cancer cell proliferation via cell cycle and apoptosis. (A) Effects of MORC2-knockdown on the percentage of cells in the S/G2/M-phase were determined by FACS analysis in HepG2 (left), Bel-7402 (middle) and LM3 (right) cells. (B) Following Annexin V-FITC/propidium iodide staining of the indicated cells following treatment without or with H₂O₂ (1 mmol) for 12 h, effects of MORC2-knockdown on HepG2 cell apoptosis were determined by FACS analysis (above) and quantitative analysis of apoptotic cell numbers (below). Data are presented as the mean ± standard deviation of three independent experiments. ^**P<0.01; ^***P<0.001. FACS, fluorescence-activated cell sorting; MORC2, microrchidia 2; si, small interfering RNA; CON, control; KD, knockdown; NC, negative control.

Figure 8

MORC2 modulates migration, invasion and metastasis of liver cancer cells in vitro. (A) Cell migration was assessed using a Transwell assay in HepG2 (left), Bel-7402 (middle) and LM3 (right) cells following transfection of the cells with MORC2 siRNA for 48 h. The cells that migrated into the bottom surface of the filters were stained. Magnification, ×40. (B) Cell invasion was assessed using a Transwell assay with Matrigel in HepG2 (left), Bel-7402 (middle) and LM3 (right) cells following transfection of the cells with MORC2 siRNA for 48 h. The cells that invaded into the lower surface of the filters were stained. Magnification, ×40. (C) Movement ability was detected by scratch wound healing assays in HepG2 (left), Bel-7402 (middle) and LM3 (right) cells following transfection of the cells with MORC2 siRNA for 48 h. Magnification, ×40. Data are presented as the mean ± standard deviation of six independent experiments. ^***P<0.001. MORC2, microrchidia 2; siRNA, small interfering RNA; CON, control; KD, knockdown; NC, negative control.

Figure 9

Overexpression of MORC2 promotes proliferation of SMMC7721 cells in vitro. (A) Overexpression of MORC2 in SMMC7721 cells by lentivirus-mediated packed pLV-MORC2 vector was confirmed by western blot analysis; β-actin was used as a loading control. (B) Effect of overexpression of MORC2 on the proliferation on SMMC7721 cells was determined using a cell counting kit-8 assay. (C) Effect of overexpression of MORC2 on colony numbers was determined by a colony formation assay in SMMC7721 cells. (D) Effect of overexpression of MORC2 on the percentage of cells in the S/G2/M-phase and on (E) SMMC7721 cell apoptosis were determined by fluorescence-activated cell sorting analysis. Data are presented as the mean ± standard deviation of three or six independent experiments. ^*P<0.05; ^**P<0.01; ^***P<0.001. MORC2, microrchidia 2; CON, control.

Figure 10

Overexpression of MORC2 promotes migration, invasion and motility of SMMC7721 cells in vitro. (A) Cell migration was assessed using a Transwell assay in SMMC7721^MORC2 and SMMC7721^vector cells; the cells that migrated to the bottom surface of the filters were stained. Magnification, ×100. (B) Cell invasion was assessed using a Transwell assay with Matrigel in SMMC7721^MORC2 and SMMC7721^vector cells; cells, which migrated to the bottom surface of the filters, were stained. Magnification, ×100. (C) Movement ability was detected using scratch wound healing assays in SMMC7721^MORC2 and SMMC7721^vector cells. Magnification, ×40. Date are presented as the mean ± standard deviation of three or six independent experiments. ^***P<0.001. MORC2, microrchidia; CON, control.

Figure 11

Overexpression of MORC2 promotes malignant phenotypes of SMMC7721 cells in vivo. (A) Representative data showing that the overexpression of MORC2 significantly promoted tumor growth in nude mice xenograft model (n=6). (B) Representative images showing the colonization of SMMC7721^MORC2 and SMMC7721^vector cells in the lung of recipient mice. Magnification, ×100. (C) Incidence and severity of lung metastasis in pulmonary metastasis model with SMMC7721^MORC2 and SMMC7721^vector cells. ^*P<0.05. MORC2, microrchidia.

Figure 12

Dyregulation of MORC2 disrupts several crucial cancer-related pathways. (A) Knockdown of MORC2 activates apoptotic pathways. (B) Based on the Kyoto Encyclopedia of Genes and Genomes database, gene expression profile analysis identified significant different pathways modulated by MORC2. (C) In HepG2 cells, knockdown of MORC2 activated the p53 pathway. (D) In HepG2 cells, knockdown of MORC2 inhibited the Hippo pathway. MORC2, microrchidia; PARP, poly (ADP-ribose) polymerase; Bcl-2, B-cell lymphoma-2; Bax, Bcl-2-associated X protein; PUMAα, p53 upregulated modulator of apoptosis α; p-, phosphorylated; si, small interfering RNA; NC, negative control; YAP1, Yes-associated protein 1; TAZ, transcriptional co-activator with PDZ-binding motif.