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Tumor-suppressor activity of RRIG1 in breast cancer.

Zhang G, Brewster A, Guan B, Fan Z, Brown PH, Xu XC - BMC Cancer (2011)

Bottom Line: Reverse-transcription polymerase chain reaction and western blot assays were used to detect the changes in gene expression.The immunohistochemical data showed that RRIG1 expression was reduced in breast cancer tissues compared with normal and atypical hyperplastic breast tissues.Expression of RRIG1 also reduced phosphorylated Erk1/2 and Akt levels; c-Jun, MMP9, and Akt expressions; and RhoA activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Clinical Cancer Prevention, The University of Texas M D Anderson Cancer Center, Houston, Texas 77030, USA.

ABSTRACT

Background: Retinoid receptor-induced gene-1 (RRIG1) is a novel gene that has been lost in several types of human cancers. The aim of this study was to determine whether RRIG1 plays a role in breast cancer, such as in the suppression of breast cancer cell growth and invasion.

Methods: Immunohistochemistry was used to detect RRIG1 expression in breast tissue specimens. Gene transfection was used to restore or knock down RRIG1 expression in breast cancer cell lines for analysis of cell viability, colony formation, and migration/invasion potential. Reverse-transcription polymerase chain reaction and western blot assays were used to detect the changes in gene expression. The RhoA activation assay was used to assess RRIG1-induced inhibition of RhoA activity.

Results: The immunohistochemical data showed that RRIG1 expression was reduced in breast cancer tissues compared with normal and atypical hyperplastic breast tissues. RRIG1 expression was inversely correlated with lymph node metastasis of breast cancer but was not associated with the status of hormone receptors, such as estrogen receptor, progesterone receptor, or HER2. Furthermore, restoration of RRIG1 expression inhibited proliferation, colony formation, migration, and invasion of breast cancer cells. Expression of RRIG1 also reduced phosphorylated Erk1/2 and Akt levels; c-Jun, MMP9, and Akt expressions; and RhoA activity. In contrast, knockdown of RRIG1 expression promoted breast cancer cell proliferation, colony formation, migration, and invasion potential.

Conclusion: The data from the current study indicated that RRIG1 expression was reduced or lost in breast cancer and that restoration of RRIG1 expression suppressed breast cancer cell growth and invasion capacity. Future studies will determine the underlying molecular mechanisms and define RRIG1 as a tumor-suppressor gene in breast cancer.

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Tumor cell migration and invasion assays. All experiments were performed in triplicate and repeated twice. A, Migration assay. The RRIG1 sense and antisense-transfected MDA-MB-231 and MDA-MB-435 cells, respectively, were grown in G418-containing medium and then subjected to cell migration assay in Boyden chambers without Matrigel for 24 h. The cells that migrated were stained with 1% crystal violet solution and then counted and summarized. B, Invasion assay. These gene-transfected breast cancer cells were grown in G418-containing medium and then subjected to the cell invasion assay in Boyden chambers with Matrigel for 48 h. The invaded cells were stained with 1% crystal violet solution and then counted and summarized. *p < 0.05 vs. the control.
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Figure 3: Tumor cell migration and invasion assays. All experiments were performed in triplicate and repeated twice. A, Migration assay. The RRIG1 sense and antisense-transfected MDA-MB-231 and MDA-MB-435 cells, respectively, were grown in G418-containing medium and then subjected to cell migration assay in Boyden chambers without Matrigel for 24 h. The cells that migrated were stained with 1% crystal violet solution and then counted and summarized. B, Invasion assay. These gene-transfected breast cancer cells were grown in G418-containing medium and then subjected to the cell invasion assay in Boyden chambers with Matrigel for 48 h. The invaded cells were stained with 1% crystal violet solution and then counted and summarized. *p < 0.05 vs. the control.

Mentions: We then detected expression of RRIG1 mRNA in 6 different breast cancer cell lines using semiquantitative RT-PCR and found that MDA-MB-435 and T47D expressed high levels of RRIG1 mRNA and that MCF-7 and SK-Br3 expressed low levels of RRIG1 mRNA. In contrast, MDA-MB-231 and ZR75-1 did not express RRIG1 mRNA (Figure 2A). Given these findings, we chose MDA-MB-231 and MDA-MB-435 for modulation of RRIG1 expression to assess the changed cell behaviors and gene expression. We transiently transfected RRIG1 sense and antisense cDNAs into the MDA-MB-231 and MDA-MB-435 cell lines, respectively. After gene transfection, RRIG1 expression was knocked down by RRIG1 antisense vector in MDA-MB-435 cells, while RRIG1 cDNA transfection restored RRIG1 expression in MDA-MB-231 cells (Figure 2B). Next, we used the MTT assay to detect the changed cell viability by RRIG1 and found that restoration of RRIG1 expression reduced cell viability in MDA-MB-231 cells, but knockdown of RRIG1 expression induced cell viability in MDA-MB-435 cells after 5 d of cultures (Figure 2C). Furthermore, RRIG1 expression decreased the number of colonies in MDA-MB-231 cells, and knockdown of RRIG1 expression in MDA-MB-435 cells increased the numbers of colonies in soft agar (Figure 2D). In addition, our data also showed that RRIG1 decreased migration and invasion capacity of MDA-MB-231 cells but increased the migratory and invasion capacity of MDA-MB-435 cells (Figure 3).


Tumor-suppressor activity of RRIG1 in breast cancer.

Zhang G, Brewster A, Guan B, Fan Z, Brown PH, Xu XC - BMC Cancer (2011)

Tumor cell migration and invasion assays. All experiments were performed in triplicate and repeated twice. A, Migration assay. The RRIG1 sense and antisense-transfected MDA-MB-231 and MDA-MB-435 cells, respectively, were grown in G418-containing medium and then subjected to cell migration assay in Boyden chambers without Matrigel for 24 h. The cells that migrated were stained with 1% crystal violet solution and then counted and summarized. B, Invasion assay. These gene-transfected breast cancer cells were grown in G418-containing medium and then subjected to the cell invasion assay in Boyden chambers with Matrigel for 48 h. The invaded cells were stained with 1% crystal violet solution and then counted and summarized. *p < 0.05 vs. the control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 3: Tumor cell migration and invasion assays. All experiments were performed in triplicate and repeated twice. A, Migration assay. The RRIG1 sense and antisense-transfected MDA-MB-231 and MDA-MB-435 cells, respectively, were grown in G418-containing medium and then subjected to cell migration assay in Boyden chambers without Matrigel for 24 h. The cells that migrated were stained with 1% crystal violet solution and then counted and summarized. B, Invasion assay. These gene-transfected breast cancer cells were grown in G418-containing medium and then subjected to the cell invasion assay in Boyden chambers with Matrigel for 48 h. The invaded cells were stained with 1% crystal violet solution and then counted and summarized. *p < 0.05 vs. the control.
Mentions: We then detected expression of RRIG1 mRNA in 6 different breast cancer cell lines using semiquantitative RT-PCR and found that MDA-MB-435 and T47D expressed high levels of RRIG1 mRNA and that MCF-7 and SK-Br3 expressed low levels of RRIG1 mRNA. In contrast, MDA-MB-231 and ZR75-1 did not express RRIG1 mRNA (Figure 2A). Given these findings, we chose MDA-MB-231 and MDA-MB-435 for modulation of RRIG1 expression to assess the changed cell behaviors and gene expression. We transiently transfected RRIG1 sense and antisense cDNAs into the MDA-MB-231 and MDA-MB-435 cell lines, respectively. After gene transfection, RRIG1 expression was knocked down by RRIG1 antisense vector in MDA-MB-435 cells, while RRIG1 cDNA transfection restored RRIG1 expression in MDA-MB-231 cells (Figure 2B). Next, we used the MTT assay to detect the changed cell viability by RRIG1 and found that restoration of RRIG1 expression reduced cell viability in MDA-MB-231 cells, but knockdown of RRIG1 expression induced cell viability in MDA-MB-435 cells after 5 d of cultures (Figure 2C). Furthermore, RRIG1 expression decreased the number of colonies in MDA-MB-231 cells, and knockdown of RRIG1 expression in MDA-MB-435 cells increased the numbers of colonies in soft agar (Figure 2D). In addition, our data also showed that RRIG1 decreased migration and invasion capacity of MDA-MB-231 cells but increased the migratory and invasion capacity of MDA-MB-435 cells (Figure 3).

Bottom Line: Reverse-transcription polymerase chain reaction and western blot assays were used to detect the changes in gene expression.The immunohistochemical data showed that RRIG1 expression was reduced in breast cancer tissues compared with normal and atypical hyperplastic breast tissues.Expression of RRIG1 also reduced phosphorylated Erk1/2 and Akt levels; c-Jun, MMP9, and Akt expressions; and RhoA activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Clinical Cancer Prevention, The University of Texas M D Anderson Cancer Center, Houston, Texas 77030, USA.

ABSTRACT

Background: Retinoid receptor-induced gene-1 (RRIG1) is a novel gene that has been lost in several types of human cancers. The aim of this study was to determine whether RRIG1 plays a role in breast cancer, such as in the suppression of breast cancer cell growth and invasion.

Methods: Immunohistochemistry was used to detect RRIG1 expression in breast tissue specimens. Gene transfection was used to restore or knock down RRIG1 expression in breast cancer cell lines for analysis of cell viability, colony formation, and migration/invasion potential. Reverse-transcription polymerase chain reaction and western blot assays were used to detect the changes in gene expression. The RhoA activation assay was used to assess RRIG1-induced inhibition of RhoA activity.

Results: The immunohistochemical data showed that RRIG1 expression was reduced in breast cancer tissues compared with normal and atypical hyperplastic breast tissues. RRIG1 expression was inversely correlated with lymph node metastasis of breast cancer but was not associated with the status of hormone receptors, such as estrogen receptor, progesterone receptor, or HER2. Furthermore, restoration of RRIG1 expression inhibited proliferation, colony formation, migration, and invasion of breast cancer cells. Expression of RRIG1 also reduced phosphorylated Erk1/2 and Akt levels; c-Jun, MMP9, and Akt expressions; and RhoA activity. In contrast, knockdown of RRIG1 expression promoted breast cancer cell proliferation, colony formation, migration, and invasion potential.

Conclusion: The data from the current study indicated that RRIG1 expression was reduced or lost in breast cancer and that restoration of RRIG1 expression suppressed breast cancer cell growth and invasion capacity. Future studies will determine the underlying molecular mechanisms and define RRIG1 as a tumor-suppressor gene in breast cancer.

Show MeSH
Related in: MedlinePlus