2022 Volume 45 Issue 11 Pages 1653-1659
Runt-related transcription factor 2 (RUNX2), a specific transcription factor of osteocytes, has been confirmed to be involved in the malignant biological behavior of various tumor cells, including renal cell carcinoma. However, the mechanism of action of RUNX2 in renal cell carcinoma cells is not yet fully understood. In this study, RUNX2-negative A498 cells and strongly positive ACHN cells were selected as the study subjects. An invasion chamber assay was used to detect the invasive ability of the cells. The expression of each protein was detected by Western blotting or immunofluorescence assays. The invasive ability of A498 cells was enhanced after the expression of RUNX2 protein was upregulated, whereas ACHN cells decreased after the expression of RUNX2 protein was silenced. The expression of calcium-activated neutral protease 2 (Calpain2) and fibronectin (FN) proteins was upregulated in A498 cells overexpressing RUNX2 protein, whereas it was downregulated after the downregulation of RUNX2 protein expression in ACHN cells. It was found that Calpain2 small interfering RNA (siRNA) or calpain inhibitor calpeptin could inhibit the expression of FN in ACHN and A498 cells overexpressing RUNX2. Calpain2 siRNA or calpeptin inhibited the invasion of A498 cells overexpressing RUNX2. Similarly, in ACHN cells, Calpain2 siRNA or calpeptin inhibited cell invasion. RUNX2 upregulates FN protein expression via Calpain2, thereby mediating renal cell carcinoma invasion.
Renal cell carcinoma, a common urinary system tumor, originates from the renal tubular epithelial cells.1) The World Cancer Report 2020, predicted that there will be approximately 19.29 million new cancer cases worldwide in 2020, with China accounting for 23.7% of cases. Globally, there are 431000 new cases of renal cell carcinoma, and China accounts for 16.9% of these.2) The incidence of renal cell carcinoma is lower than the overall incidence of cancer. However, due to China’s large population base, the incidence of renal cell carcinoma in China ranks first worldwide. Generally, early-stage renal cell carcinoma has a favorable prognosis, with a five-year survival rate as high as 90 percent.3,4) However, advanced renal cell carcinoma tends to invade adjacent organs and even develop distant metastasis, a significant cause of death in patients with renal cell carcinoma.3,4)
Runt-related transcription factor 2 (RUNX2), a specific transcription factor for osteocytes, plays an essential role in the formation and reconstruction of bone tissue.5,6) Clinical data have shown that RUNX2 is abnormally expressed in some tumors.7–9) Studies have also confirmed that RUNX2 plays a role in promoting cancer in malignant tumors and can be involved in promoting the occurrence of various tumors,10) invasion and metastasis,11) drug resistance,12) and cell stemness.13) Additionally, RUNX2 can participate in renal cell carcinoma malignant biological behaviors such as cell proliferation, migration, and invasion.14,15) However, its mechanism of action is not yet fully understood.
Calcium-activated neutral protease 2 (Calpain2) is a Ca2+-dependent cysteine protease widely expressed in various tissues that can catalyze a variety of substrate-restricted hydrolyses.16) Initial studies found that Calpain2 and other calpain family members are mainly involved in regulating skeletal muscle growth and muscle tenderness.17) However, with increasing research, Calpain2 has been found to be closely related to the occurrence and development of tumors. It has been found to be involved in various physiological processes such as cell proliferation and invasion, cytoskeleton remodeling, cell cycle regulation, apoptosis, and signal transduction.18–20) The activity of Calpain2 is regulated by Ca2+ and the endogenous inhibitory protein calpastatin.19) Ca2+ can activate Calpain2, and calpastatin binds to adjacent Calpain2 and reduces its activity.19,21) However, whether Calpain2 mediates the biological effect of RUNX2 in renal cell carcinoma remains unclear and requires further study. In our study, we observed the effect of RUNX2 on the invasion of renal cell carcinoma cells and the mediating effect of Calpain2 by altering the expression of RUNX2 in renal cell carcinoma cells. This study aimed to provide a theoretical basis for exploring the mechanisms of action of RUNX2.
Human clear renal cell carcinoma cells 786-O were purchased from the Shanghai Cell Bank, Chinese Academy of Sciences (China). Human renal cell carcinoma cell lines, A498 and CAKI-2, were purchased from the BeNA Culture Collection (China). ACHN cells were purchased from the Kunming Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The 786-O cells were cultured in RPMI-1640 medium (GIBCO BRL, Grand Island, NY, U.S.A.) supplemented with 10% fetal bovine serum (FBS; GIBCO BRL). A498 cells were cultured in minimum essential medium (MEM) (GIBCO BRL) supplemented with 10% FBS. ACHN cells were cultured in MEM medium (GIBCO BRL) supplemented with 10% FBS, 1% non-essential amino acids (NANJING COBIOER BIOSCIENCES CO., LTD., China), and 1 mM sodium pyruvate (GIBCO BRL). CAKI-2 cells were cultured in McCoy’s 5a medium (GIBCO BRL) supplemented with 10% FBS. All cells were cultured with 5% CO2 in a cell incubator at 37 °C.
RUNX2 Plasmid TransfectionThe plasmid pEX-3 vector containing RUNX2 was synthesized by GenePharma Inc. (China). A498 cells were seeded in six-well plates and transfected using the GenJet Plus DNA In Vitro Transfection Kit according to the manufacturer’s instructions. The cells were then transfected with RUNX2-plasmid pEX-3 vector, plasmid pEX-3 empty vector, and control.
Small Interfering RNA (siRNA) TransfectionThe siRNA was synthesized by GenePharma Inc. (Shanghai, China). ACHN cells were seeded in 6 cm dishes (NEST, China), and RUNX2 siRNA and negative controls were transfected into the ACHN cells using Lipofectamine 2000 (Thermo Fisher, Waltham, MA, U.S.A.) according to the manufacturer’s instructions. Simultaneously, untransfected cells were used as control.
Invasion AssayInvasion chambers (8 µm; Corning Incorporated, Corning, NY, U.S.A.) coated with matrigel (BD Biosciences, San Jose, CA, U.S.A.) were prepared. The desired cells were resuspended, 2 × 104 cells were pipetted into the upper chamber, and a medium containing 30% FBS was added to the lower chamber. After necessary treatment, the cells were fixed and stained according to the manufacturer’s instructions and photographed under a CKX41-A21PHP inverted microscope (Olympus Corporation, Tokyo, Japan). Invasion rate (%) = number of invaded cells in the experimental group/number of invaded cells in the control group ×100%.
Western BlotCells that required protein extraction were washed with ice-cold phosphate-buffered saline (PBS) (HyClone, Logan, UT, U.S.A.). The cells were collected in 1.5 mL centrifuge tubes, and Radio-Immunoprecipitation Assay (RIPA) Lysis Buffer (MedChem Express, Monmouth Junction, NJ, U.S.A.) was added to lyse the cells. After the lysates were centrifuged at 4 °C and 12000 rpm, the supernatant was aspirated for protein quantification, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) samples were prepared. The electrophoretic samples were separated from total proteins by SDS-PAGE gel electrophoresis. The separated proteins were then transferred from the gel to a polyvinylidene difluoride (PVDF) membrane (Millipore Corp., Billerica, MA, U.S.A.). The PVDF membrane was blocked with a 5% bovine serum albumin (BSA; Solarbio Life Sciences, China) blocking solution and incubated at 4 °C with RUNX2 (1 : 1000), calpastatin (1 : 1000), fibronectin (FN) (1 : 1000), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1 : 1000) (Cell Signaling Technology, Beverly, MA, U.S.A.) or Calpain2 (1 : 2000) (Santa Cruz, CA, U.S.A.) overnight. The next day, the PVDF membrane was washed with primary antibody and then incubated with goat anti-rabbit immunoglobulin G (IgG)-HRP or goat anti-mouse IgG-HRP secondary antibody (Absin, China) for 1 h. After washing off the secondary antibody, the PVDF membrane was developed using enhanced chemiluminescence (ECL) solution (Millipore Corp.) to develop the protein using the ChemiDoc XRS+ gel imaging system (Bio-Rad, Hercules, CA, U.S.A.).
ImmunofluorescenceFirst, sterile coverslips were plated in six-well plates (NEST). The desired cells were then resuspended and seeded into the six-well plates at a density of 3 × 105. After 24 h of culture, the cells were fixed with 4% paraformaldehyde (Solarbio life sciences) for 10 min, and if necessary, the cells were treated with 5% Triton X-100 (absin) for 10 min. Subsequently, the cells were blocked with goat serum (absin) for 30 min and then incubated with Calpain2 (1 : 500) or FN (1 : 300) antibodies at 4 °C overnight. Furthermore, the primary antibody was washed and incubated with the corresponding fluorescent secondary antibody (1 : 250) for 45 min. The cells were then capped with a 4′,6-diamidino2-phenylindole (DAPI)-containing fluorescence quencher (Absin). Finally, the cells were observed and photographed using a fluorescence microscope.
Statistical AnalysisStatistical data analysis was performed using SPSS17.0 for windows, and the data were expressed as mean ± standard error of the mean (S.E.M.). All data conform to the normal distribution by Shapiro–Wilk test. The data between multiple groups were analyzed using one-way ANOVA, and a least significant difference (LSD) t-test was used for multiple pairwise comparisons. Statistical significance was set at p < 0.05.
To select a suitable cell model for this study, we first observed the expression levels of RUNX2 in four renal cell carcinoma cell lines. Among the four cell lines, A498 cells hardly expressed RUNX2 protein, while ACHN cells expressed the highest RUNX2 protein (Fig. 1A). Therefore, we selected A498 and ACHN cells for further experiments. A498 and ACHN cells were transfected with a RUNX2 plasmid and RUNX2 siRNA to alter RUNX2 protein expression levels. The results showed that A498 cells overexpressed RUNX2 with enhanced cell invasion ability (Figs. 1B, D). However, after silencing RUNX2 protein expression, the invasive ability of ACHN cells was reduced (Figs. 1C, E).
A: The expression of RUNX2 protein in A498, ACHN, 786-O and CAKI-2 cells was detected by Western blot. N = 3. B: A498 cells were transfected with RUNX2 plasmid and empty control, and the expression of RUNX2 protein was detected by Western blot experiment. ** p < 0.01. N = 3. C: ACHN cells were transfected with RUNX2 siRNA and negative control, and the expression of RUNX2 protein was detected by Western blot. ** p < 0.01. N = 3. D: Changes in cell invasion ability of A498 cells transfected with RUNX2 plasmid. ** p < 0.01. N = 5. E: Changes in cell invasion ability of ACHN cells transfected with RUNX2 siRNA. ** p < 0.01. N = 5.
Considering that Calpain2 plays a role in malignant cell invasion, we examined the effect of RUNX2 on Calpain2 protein expression. Western blotting and immunofluorescence staining showed that the expression of Calpain2 protein was upregulated in A498 cells overexpressing RUNX2 protein (Figs. 2A, B). However, Calpain2 protein expression was downregulated after ACHN cells downregulated RUNX2 protein expression (Figs. 2C, D). Concurrently, we found that the expression of calpastatin protein was downregulated in A498 cells overexpressing the RUNX2 protein (Fig. 2A), whereas calpastatin protein expression was upregulated after downregulation of RUNX2 protein expression in ACHN cells (Fig. 2C).
A, B: Western blot and immunofluorescence (100×) experiments were used to detect the expression of Calpain2 or/and Calpastatin protein in A498 cells transfected with RUNX2 plasmid, respectively. ** p < 0.01. N = 3. C, D: Western blot and immunofluorescence (100×) experiments were used to detect the protein expression changes of Calpain2 or/and Calpastatin in ACHN cells transfected with RUNX2 siRNA. ** p < 0.01. N = 3.
The effect of RUNX2 on FN protein expression was observed; Western blotting and immunofluorescence staining showed that FN protein expression was upregulated in A498 cells overexpressing RUNX2 (Figs. 3A, B). Calpain2 protein expression was downregulated after the downregulation of RUNX2 protein expression in ACHN cells (Figs. 3C, D).
A, B: Western blot and immunofluorescence (100×) experiments detected FN protein expression changes in A498 cells transfected with RUNX2 plasmid. ** p < 0.01. N = 3. C, D: Western blot and immunofluorescence (100×) experiments detected FN protein expression changes in ACHN cells transfected with RUNX2 siRNA. ** p < 0.01. N = 3.
To explore the role of Calpain2 in RUNX2-induced changes in FN protein expression, we silenced or inhibited Calpain2 expression/activity, respectively, and then observed the effect of RUNX2 on FN protein expression. The results showed that both Calpain2 siRNA and calpain inhibitor calpeptin could inhibit the downregulation of FN expression in A498 cells overexpressing RUNX2 (Fig. 4A). Similarly, either Calpain2 siRNA or calpain inhibitor calpeptin also inhibited the downregulation of FN expression in ACHN cells (Fig. 4B).
A: After A498 cells transfected with Vector and RUNX2 plasmids were treated with Calpain2 siRNA or Calpain inhibitor Calpeptin, the changes of FN protein expression were detected by Western blot. ** p < 0.01. N = 3. B: ACHN cells were treated with Calpain2 siRNA or Calpain inhibitor Calpeptin, and the changes of FN protein expression were detected by Western blot. ** p < 0.01. N = 3.
We further verified the effect of inhibiting Calpain2 or FN on RUNX2-induced renal cell invasion. The results from the invasion chambers showed that Calpain2 siRNA or calpeptin inhibited the invasion of RUNX2-overexpressing A498 cells (Fig. 5A). Similarly, Calpain2 siRNA or calpeptin inhibited cell invasion in ACHN cells overexpressing RUNX2 (Fig. 5B).
A: After A498 cells transfected with RUNX2 plasmid were treated with Calpain2 siRNA or FN siRNA, the invasion ability of cells was detected by invasion chamber assay. ** p < 0.01. N = 5. B: ACHN cells were treated with Calpain2 siRNA or FN siRNA, and the cell invasion ability was detected by Western blot. ** p < 0.01. N = 5.
It has been confirmed that RUNX2 plays a positive regulatory role in the occurrence and development of malignant tumors.10–13) Recent studies have shown that RUNX2 is abnormally elevated in renal cell carcinoma and is associated with poor prognosis in patients with tumors.22) Additionally, in vitro studies have shown that RUNX2 expression is involved in the proliferation, migration, and invasion of renal cell carcinoma cells.22) Another study showed that the long non-coding RNA SNHG4 acts on miR-204-5p, upregulating RUNX2 to promote renal cell cancer cell invasion.15) However, the role and related mechanisms of action of RUNX2 in renal cells are not yet fully understood. To explore the role of RUNX2, we screened A498, a low RUNX2 expressing cell, and ACHN, a high RUNX2 expressing cell, from four renal cell carcinoma cell liness. We found that exogenous upregulation of RUNX2 protein expression in A498 cells enhanced cell invasive ability while interference with endogenous RUNX2 protein expression in ACHN cells decreased cell invasive ability. Therefore, it was inferred that RUNX2 can promote cell invasive activity in renal cell carcinoma, which is consistent with the previous reports.
Furthermore, we found that RUNX2 could mediate the upregulation of Calpain2 protein expression and the downregulation of calpastatin protein expression in A498 and ACHN renal cell carcinoma cell lines. Silencing of Calpain2 protein inhibited RUNX2-induced cell invasion. A previous study showed that Calpain2 was involved in mediating cell migration and invasion in highly aggressive prostate cancer DU-145 cells.23) A recent study showed that hnRNPK-regulated LINC00263 promoted lung cancer, colorectal cancer, neuroblastoma, and melanoma cell invasion through miR-147a/Calpain2.24) Therefore, we inferred from these findings that RUNX2 may have mediated renal cell carcinoma cell invasion through Calpain2 in our study.
FN, a macromolecular glycoprotein in the cell-matrix, exists widely in animal tissues and tissue fluids and has a variety of biological functions.25) Most FN in the blood circulation are synthesized by hepatocytes; however, various animal body cells can also secrete FN.25,26) Generally, plasma and cellular FN mediate distinct biological behaviors.25,26) Among them, plasma FN is indispensable for early wound healing, whereas cellular FN mediates late wound healing and angiogenesis.25,26) FN has been confirmed to be closely related to inflammation,27) tissue fibrosis28) and malignant tumors.29) Studies have shown that baicalein inhibits breast cancer cell invasion by inhibiting FN-induced epithelial–mesenchymal transition, and Calpain2 is involved in the inhibitory effect of baicalein. Therefore, it has been suggested that Calpain2 is an upstream regulatory protein of FN.30) To explore whether renal cell carcinoma cell invasion mediated by RUNX2 through Calpain2 is related to FN, we first observed the expression of FN and found that RUNX2 induced FN protein expression in A498 and ACHN cells. Further studies found that silencing or inhibiting the expression/activity of Calpain2 could down-regulate FN expression in ACHN and A498 cells overexpressing RUNX2. Simultaneously, by observing the cell invasion activity, it was found that silencing or inhibiting the expression/activity of Calpain2 could also inhibit the invasive ability of the cells. Hence, it was suggested that RUNX2 mediates renal cell carcinoma cell invasion through the upregulation of FN expression by Calpain2.
In conclusion, the results of this study showed that RUNX2 mediates renal cell carcinoma cell invasion by upregulating FN expression, and Calpain2 mediates this effect. Our study demonstrated a part of the mechanism of RUNX2-mediated invasion of renal cells; this can serve as a theoretical support for the clinical treatment of renal cell carcinoma.
This work was supported by the 2020 Medical Science Research Project Plan of Hebei Province (No. 20201100).
The authors declare no conflict of interest.
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