Cancer Res Treat.  2021 Apr;53(2):576-583. 10.4143/crt.2020.801.

Knockdown of EMMPRIN (OX47) in MRMT-1 Carcinoma Cells Inhibits Tumor Growth and Decreases Cancer-Induced Bone Destruction and Pain

Affiliations
  • 1Department of Cell Biology and Genetics and Key Laboratory of Environment and Genes Related to Diseases, Xi’an Jiaotong University, Xi’an, China
  • 2Shaanxi Key Laboratory of Brain Disorders and Institute of Basic and Translational Medicine, Xi’an Medical University, Xi’an, China
  • 3Department of Obstetrics, The First Affiliated Hospital, Xi’an Jiaotong University, Xi’an, China

Abstract

Purpose
Bone destruction and pain caused by cancer is one of the most devastating complications of cancer patients with bone metastases, and it seriously affects the quality of patients’ life. Extracellular matrix metalloproteinase inducer (EMMPRIN) is a cell adhesion molecule with increased expression in a variety of tumors. This study focused to clarify the specific function of EMMPRIN in bone metastasis of breast cancer.
Materials and Methods
Adenovirus with shRNA-EMMPRIN was transfected into MRMT-1 rat breast carcinoma cells, and the MRMT-1 cells with different expression levels of EMMPRIN were implanted into the bone marrow cavity of rat tibia. Next, the effect of down-regulation of EMMPRIN was evaluated as follows: bone damage was detected by X-ray radiological and tartrate-resistant acid phosphatase staining; the tumor burden was evaluated by hematoxylin and eosin staining; the test of pain-related behaviors was assessed used the bilateral paw withdrawal mechanical threshold; and the levels of secretory factors in tumor conditioned medium were determined by using enzyme-linked immunosorbent assay.
Results
We found that down-regulation of EMMPRIN in tumor cells can simultaneously reduce tumor burden, relieve cancer-induced bone destruction and pain.
Conclusion

Materials and Methods
EMMPRIN is expected to be a therapeutic target for relieving bone metastasis of breast cancer and alleviating cancerinduced bone destruction and pain. The method of targeting EMMPRIN may be a promising strategy for the treatment of cancer in the future.

Keyword

EMMPRIN (OX47); Breast neoplasms; Bone destruction; Pain

Figure

  • Fig. 1 Down-regulation of extracellular matrix metalloproteinase inducer (OX47) inhibited growth of MRMT-1 rat breast carcinoma cells in vitro and in vivo. Real-time quantitative reverse transcription–polymerase chain reaction (qRT-PCR) (A) and western blot (B) analyses of OX47 expression in MRMT-1 cells transfected with control-shRNA virus and OX47-shRNA virus. For qRT-PCR analyses, glyceraldehyde 3-phosphate dehydrogenase was used as an internal control; For western blot analyses, tubulin was used as an internal control. For qRT-PCR study, each experiment was repeated three times and the standard deviation is denoted using error bars. shRNA control vs. shRNA OX47; **p < 0.01. (C) Growth curve of MRMT-1 cells transduced with control-shRNA (solid circle) and OX47-shRNA (open circle) by MTT assay. Standard deviation is denoted using error bars (n=6). *p < 0.05. (D) Representative H&E staining results of the tumor-bearing tibia in sham operation group (left) (receiving implantation of heat-killed tumor cells), control-shRNA group (middle) and OX47-shRNA group (right) on the 28th day after MRMT-1 cells injections. (E) Statistical analysis of the percentage tumor area in tibia (n=6) were measured by examining six different visual fields under a microscope at 200× magnification in a blinded manner. Multiple comparisons were performed with one-way ANOVA, ****p < 0.0001.

  • Fig. 2 Down-regulation of extracellular matrix metalloproteinase inducer (OX47) alleviated cancer-induced bone destruction. Representative radiographs images (A) and quantification (B) of bone destruction in control-shRNA group and OX47-shRNA group on the 14th and 21st day after MRMT-1 cells injection, shRNA control vs. shRNA OX47; **p < 0.01. (C, D) Representative tartrate-resistant acid phosphatase staining photomicrographs (C) and quantification (D) of osteoclasts in control-shRNA group (n=6) and OX47-shRNA group (n=6) on the 21st day after MRMT-1 cells injection, shRNA control vs. shRNA OX47; **p < 0.01. (E) Knockdown of OX47 increased the pH value of tumor conditioned medium (TCM) which collected from MRMT-1 cells incubating for 12–72 hours, shRNA control vs. shRNA OX47; *p < 0.05. (F) Enzyme-linked immunosorbent assay quantitative results of solvable factors in TCM, shRNA control vs. shRNA OX47; *p < 0.05, **p < 0.01.

  • Fig. 3 Down-regulation of extracellular matrix metalloproteinase inducer (OX47) relieved cancer-induced pain. Ipsilateral (A) and contralateral (B) hind paw withdrawal responses to von Frey hair stimulation in MRMT-1 cells (OX47 is knocked down or not) injected rats, shRNA control vs. shRNA OX47; *p < 0.05, **p < 0.01; shRNA control vs. Sham; #p < 0.01. PWMT, paw withdrawal mechanical threshold.

  • Fig. 4 Immunohistochemical analysis of c-Fos (an immediate-early gene for assessing sensory neuron activity) and glial fibrillary acidic protein (GFAP; a glial marker). (A, B) Representative photomicrographs of c-Fos and GFAP immunostaining in the dorsal horn within spinal L4 segments of sham rats and tumor-bearing rats (A). GFAP and c-Fos positive cells were counted by examining six different visual fields under a microscope at 200× magnification in a blinded manner. Multiple comparisons were performed with one-way ANOVA, ****p < 0.0001 (B).


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