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Role of SMC1A overexpression as a predictor of poor prognosis in late stage colorectal cancer.

Wang J, Yu S, Cui L, Wang W, Li J, Wang K, Lao X - BMC Cancer (2015)

Bottom Line: Recent studies have concluded that SMC1A is involved in the pathogenesis of cancer.Functional analysis indicated that SMC1A knockdown by small interfering RNA (siRNA) mediated the significant inhibition of cell proliferation; induced cell cycle arrest and apoptosis via the suppression of CDK4, PCNA and PARP; and blocked the activation of the Erk1/2 and Akt cascades in CRC cells.These results suggest that SMC1A plays an essential role in the development of CRC and may be a predictive factor in patients with CRC.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgical Oncology, Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310009, China. jianweidr@163.com.

ABSTRACT

Background: Structural maintenance of chromosomes 1A (SMC1A) is a member of the cohesion family of proteins that plays crucial roles in cell cycle control. Recent studies have concluded that SMC1A is involved in the pathogenesis of cancer. This study aims to evaluate the functional role of SMC1A in colorectal cancer (CRC) both in vitro and in vivo, and the underlying molecular mechanisms.

Methods: We firstly investigated the expression levels of SMC1A in 427 CRC specimens. Antigen expression was determined by immunohistochemical analysis of SMC1A on tissue microarrays. Stable SMC1A knockdown CRC cell lines were employed. The effects of SMC1A depletion on cell growth in vitro were examined by MTT, colony formation and flow cytometry assays. Tumor forming was evaluated by nude mice model in vivo. To detect the activation of intracellular signaling, pathscan intracellular signaling array and western blotting were performed.

Results: The expression of SMC1A was much stronger in CRC tumor tissues than in adenomas and normal colorectal tissues. High SMC1A expression, indicated as an independent poor prognostic predictor for patients with stage III and stage IV CRC, was correlated with overall survival (OS) (p = 0.008). Functional analysis indicated that SMC1A knockdown by small interfering RNA (siRNA) mediated the significant inhibition of cell proliferation; induced cell cycle arrest and apoptosis via the suppression of CDK4, PCNA and PARP; and blocked the activation of the Erk1/2 and Akt cascades in CRC cells. In addition, SMC1A depletion significantly decreased the growth of subcutaneously inoculated tumors in nude mice.

Conclusions: These results suggest that SMC1A plays an essential role in the development of CRC and may be a predictive factor in patients with CRC. The inhibition of SMC1A may serve as a promising therapeutic strategy for human CRC.

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Related in: MedlinePlus

Functional analysis of SMC1A by shRNA in CRC cells. (A) Expression analysis of SMC1A in five different CRC cell lines by real-time PCR (upper panel) and western blotting (lower panel). (B) Expression analysis of SMC1A in RKO cells after Lv-shSMC1A infection by real-time PCR (upper panel) and western blotting (lower panel). (C) Expression analysis of SMC1A in SW480 cells after Lv-shSMC1A infection by real-time PCR (upper panel) and western blotting (lower panel). β-actin gene and GAPDH protein were used as internal controls. The proliferation levels of RKO (D, E) and SW480 (F) cells after Lv-shSMC1A infection analyzed by the MTT assay. The number of colonies in RKO (G, H) and SW480 (I) cells after Lv-shSMC1A infection analyzed by the colony formation assay. (J) The percentages of RKO cells using three different treatments in different phases (left panel) and the sub-G1 phase (right panel) of the cell cycle. (K) RKO cells stained with Annexin V and 7-AAD analyzed using flow cytometer (left panel). Q1, Annexin V−/7-AAD+; Q2, Annexin V+/7-AAD+; Q3, Annexin V−/7-AAD−; Q4, Annexin V+/7-AAD−. Quantification of the percentage of early apoptotic cells and late apoptotic cells (right panel). *p < 0.05, **p < 0.01, ***p < 0.001 in comparison with non-silencing shRNA infected control.
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Fig2: Functional analysis of SMC1A by shRNA in CRC cells. (A) Expression analysis of SMC1A in five different CRC cell lines by real-time PCR (upper panel) and western blotting (lower panel). (B) Expression analysis of SMC1A in RKO cells after Lv-shSMC1A infection by real-time PCR (upper panel) and western blotting (lower panel). (C) Expression analysis of SMC1A in SW480 cells after Lv-shSMC1A infection by real-time PCR (upper panel) and western blotting (lower panel). β-actin gene and GAPDH protein were used as internal controls. The proliferation levels of RKO (D, E) and SW480 (F) cells after Lv-shSMC1A infection analyzed by the MTT assay. The number of colonies in RKO (G, H) and SW480 (I) cells after Lv-shSMC1A infection analyzed by the colony formation assay. (J) The percentages of RKO cells using three different treatments in different phases (left panel) and the sub-G1 phase (right panel) of the cell cycle. (K) RKO cells stained with Annexin V and 7-AAD analyzed using flow cytometer (left panel). Q1, Annexin V−/7-AAD+; Q2, Annexin V+/7-AAD+; Q3, Annexin V−/7-AAD−; Q4, Annexin V+/7-AAD−. Quantification of the percentage of early apoptotic cells and late apoptotic cells (right panel). *p < 0.05, **p < 0.01, ***p < 0.001 in comparison with non-silencing shRNA infected control.

Mentions: As shown in Figure 2A, the expression of SMC1A was observed in all five CRC cell lines. RKO and SW480 cell lines were used to investigate loss of function in the following study. Both cell lines were cultured and successfully infected with Lv-shCon or Lv-shSMC1A with an infection rate greater than 80%. The expression levels of SMC1A were significantly decreased (p < 0.01) in Lv-shSMC1A groups (Figure 2B and C). Similar result was observed in RKO cells treated with another shRNA against SMC1A. These results indicated that the lentivirus-mediated shRNA targeting SMC1A could effectively knock down SMC1A expression in CRC cells.Figure 2


Role of SMC1A overexpression as a predictor of poor prognosis in late stage colorectal cancer.

Wang J, Yu S, Cui L, Wang W, Li J, Wang K, Lao X - BMC Cancer (2015)

Functional analysis of SMC1A by shRNA in CRC cells. (A) Expression analysis of SMC1A in five different CRC cell lines by real-time PCR (upper panel) and western blotting (lower panel). (B) Expression analysis of SMC1A in RKO cells after Lv-shSMC1A infection by real-time PCR (upper panel) and western blotting (lower panel). (C) Expression analysis of SMC1A in SW480 cells after Lv-shSMC1A infection by real-time PCR (upper panel) and western blotting (lower panel). β-actin gene and GAPDH protein were used as internal controls. The proliferation levels of RKO (D, E) and SW480 (F) cells after Lv-shSMC1A infection analyzed by the MTT assay. The number of colonies in RKO (G, H) and SW480 (I) cells after Lv-shSMC1A infection analyzed by the colony formation assay. (J) The percentages of RKO cells using three different treatments in different phases (left panel) and the sub-G1 phase (right panel) of the cell cycle. (K) RKO cells stained with Annexin V and 7-AAD analyzed using flow cytometer (left panel). Q1, Annexin V−/7-AAD+; Q2, Annexin V+/7-AAD+; Q3, Annexin V−/7-AAD−; Q4, Annexin V+/7-AAD−. Quantification of the percentage of early apoptotic cells and late apoptotic cells (right panel). *p < 0.05, **p < 0.01, ***p < 0.001 in comparison with non-silencing shRNA infected control.
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Fig2: Functional analysis of SMC1A by shRNA in CRC cells. (A) Expression analysis of SMC1A in five different CRC cell lines by real-time PCR (upper panel) and western blotting (lower panel). (B) Expression analysis of SMC1A in RKO cells after Lv-shSMC1A infection by real-time PCR (upper panel) and western blotting (lower panel). (C) Expression analysis of SMC1A in SW480 cells after Lv-shSMC1A infection by real-time PCR (upper panel) and western blotting (lower panel). β-actin gene and GAPDH protein were used as internal controls. The proliferation levels of RKO (D, E) and SW480 (F) cells after Lv-shSMC1A infection analyzed by the MTT assay. The number of colonies in RKO (G, H) and SW480 (I) cells after Lv-shSMC1A infection analyzed by the colony formation assay. (J) The percentages of RKO cells using three different treatments in different phases (left panel) and the sub-G1 phase (right panel) of the cell cycle. (K) RKO cells stained with Annexin V and 7-AAD analyzed using flow cytometer (left panel). Q1, Annexin V−/7-AAD+; Q2, Annexin V+/7-AAD+; Q3, Annexin V−/7-AAD−; Q4, Annexin V+/7-AAD−. Quantification of the percentage of early apoptotic cells and late apoptotic cells (right panel). *p < 0.05, **p < 0.01, ***p < 0.001 in comparison with non-silencing shRNA infected control.
Mentions: As shown in Figure 2A, the expression of SMC1A was observed in all five CRC cell lines. RKO and SW480 cell lines were used to investigate loss of function in the following study. Both cell lines were cultured and successfully infected with Lv-shCon or Lv-shSMC1A with an infection rate greater than 80%. The expression levels of SMC1A were significantly decreased (p < 0.01) in Lv-shSMC1A groups (Figure 2B and C). Similar result was observed in RKO cells treated with another shRNA against SMC1A. These results indicated that the lentivirus-mediated shRNA targeting SMC1A could effectively knock down SMC1A expression in CRC cells.Figure 2

Bottom Line: Recent studies have concluded that SMC1A is involved in the pathogenesis of cancer.Functional analysis indicated that SMC1A knockdown by small interfering RNA (siRNA) mediated the significant inhibition of cell proliferation; induced cell cycle arrest and apoptosis via the suppression of CDK4, PCNA and PARP; and blocked the activation of the Erk1/2 and Akt cascades in CRC cells.These results suggest that SMC1A plays an essential role in the development of CRC and may be a predictive factor in patients with CRC.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgical Oncology, Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310009, China. jianweidr@163.com.

ABSTRACT

Background: Structural maintenance of chromosomes 1A (SMC1A) is a member of the cohesion family of proteins that plays crucial roles in cell cycle control. Recent studies have concluded that SMC1A is involved in the pathogenesis of cancer. This study aims to evaluate the functional role of SMC1A in colorectal cancer (CRC) both in vitro and in vivo, and the underlying molecular mechanisms.

Methods: We firstly investigated the expression levels of SMC1A in 427 CRC specimens. Antigen expression was determined by immunohistochemical analysis of SMC1A on tissue microarrays. Stable SMC1A knockdown CRC cell lines were employed. The effects of SMC1A depletion on cell growth in vitro were examined by MTT, colony formation and flow cytometry assays. Tumor forming was evaluated by nude mice model in vivo. To detect the activation of intracellular signaling, pathscan intracellular signaling array and western blotting were performed.

Results: The expression of SMC1A was much stronger in CRC tumor tissues than in adenomas and normal colorectal tissues. High SMC1A expression, indicated as an independent poor prognostic predictor for patients with stage III and stage IV CRC, was correlated with overall survival (OS) (p = 0.008). Functional analysis indicated that SMC1A knockdown by small interfering RNA (siRNA) mediated the significant inhibition of cell proliferation; induced cell cycle arrest and apoptosis via the suppression of CDK4, PCNA and PARP; and blocked the activation of the Erk1/2 and Akt cascades in CRC cells. In addition, SMC1A depletion significantly decreased the growth of subcutaneously inoculated tumors in nude mice.

Conclusions: These results suggest that SMC1A plays an essential role in the development of CRC and may be a predictive factor in patients with CRC. The inhibition of SMC1A may serve as a promising therapeutic strategy for human CRC.

Show MeSH
Related in: MedlinePlus