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Integrin α6A splice variant regulates proliferation and the Wnt/β-catenin pathway in human colorectal cancer cells.

Groulx JF, Giroux V, Beauséjour M, Boudjadi S, Basora N, Carrier JC, Beaulieu JF - Carcinogenesis (2014)

Bottom Line: The α6A silencing was also found to be associated with a significant repression of a number of Wnt/β-catenin pathway end points.Moreover, it was accompanied by a reduction in the capacity of these cells to develop tumours in xenografts.Taken together, these results demonstrate that the α6A variant is a pro-proliferative form of the α6 integrin subunit in CRC cells and appears to mediate its effects through the Wnt/β-catenin pathway.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Intestinal Physiopathology, Department of Anatomy and Cell Biology and Department of Medicine, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Quebec J1H 5N4, Canada.

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Knockdown of the α6A subunit in human CRC cells. (A) Representative gel showing the results of a competitive reverse transcription–polymerase chain reaction for the detection of α6A and α6B transcripts in stably expressing shctl and shα6A Caco-2/15, DLD-1, T84 and HT29 cells. (B) qPCR using probes specific for total α6, α6A and α6B confirming the specificity of abolition of α6A variant expression by shα6A relative to shctrl; *P ≤ 0.05, **P ≤ 0.01, ANOVA, n = 3. (C) Representative WB for detection of α6A and α6B subunits in shctl- and shα6A-infected Caco-2/15, DLD-1, T84 and HT29 cells. Keratin 18 (K18) was used as loading control. Densiometric analysis of α6A and α6B protein levels in shctl- versus shα6A-infected cell populations; *P ≤ 0.05, **P ≤ 0.01, t-test, n = 3.
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Figure 2: Knockdown of the α6A subunit in human CRC cells. (A) Representative gel showing the results of a competitive reverse transcription–polymerase chain reaction for the detection of α6A and α6B transcripts in stably expressing shctl and shα6A Caco-2/15, DLD-1, T84 and HT29 cells. (B) qPCR using probes specific for total α6, α6A and α6B confirming the specificity of abolition of α6A variant expression by shα6A relative to shctrl; *P ≤ 0.05, **P ≤ 0.01, ANOVA, n = 3. (C) Representative WB for detection of α6A and α6B subunits in shctl- and shα6A-infected Caco-2/15, DLD-1, T84 and HT29 cells. Keratin 18 (K18) was used as loading control. Densiometric analysis of α6A and α6B protein levels in shctl- versus shα6A-infected cell populations; *P ≤ 0.05, **P ≤ 0.01, t-test, n = 3.

Mentions: To investigate the involvement of the α6A variant in CRC cell behaviour, four well-characterized CRC cell lines—Caco-2/15, DLD-1, T84 and HT29 cells—were infected with an shRNA targeting the α6A variant. The α6A shRNA was designed in order to recognize a unique sequence of the short exon 25 (119pb) specific to the α6A mRNA transcript. Specificity of knockdown of α6A was first confirmed by competitive PCR using primers that amplify both variants of α6. The results revealed a decrease in α6A variant mRNA expression in shα6A versus shctl-treated CRC cells in all four cell lines tested (Figure 2A). Further analysis by qPCR revealed that knocking down α6A led to a decrease of ~50% of total α6 mRNA levels (Figure 2B). Analysis of splice variants showed an 80–90% decrease in α6A transcript levels in shα6A cells compared with controls, without affecting mRNA levels of α6B (Figure 2B). WB analysis of α6A and α6B performed to extend our observations at the protein level confirmed the specific abolition of the α6A variant, without significantly affecting α6B in all CRC cell lines (Figure 2C).


Integrin α6A splice variant regulates proliferation and the Wnt/β-catenin pathway in human colorectal cancer cells.

Groulx JF, Giroux V, Beauséjour M, Boudjadi S, Basora N, Carrier JC, Beaulieu JF - Carcinogenesis (2014)

Knockdown of the α6A subunit in human CRC cells. (A) Representative gel showing the results of a competitive reverse transcription–polymerase chain reaction for the detection of α6A and α6B transcripts in stably expressing shctl and shα6A Caco-2/15, DLD-1, T84 and HT29 cells. (B) qPCR using probes specific for total α6, α6A and α6B confirming the specificity of abolition of α6A variant expression by shα6A relative to shctrl; *P ≤ 0.05, **P ≤ 0.01, ANOVA, n = 3. (C) Representative WB for detection of α6A and α6B subunits in shctl- and shα6A-infected Caco-2/15, DLD-1, T84 and HT29 cells. Keratin 18 (K18) was used as loading control. Densiometric analysis of α6A and α6B protein levels in shctl- versus shα6A-infected cell populations; *P ≤ 0.05, **P ≤ 0.01, t-test, n = 3.
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Figure 2: Knockdown of the α6A subunit in human CRC cells. (A) Representative gel showing the results of a competitive reverse transcription–polymerase chain reaction for the detection of α6A and α6B transcripts in stably expressing shctl and shα6A Caco-2/15, DLD-1, T84 and HT29 cells. (B) qPCR using probes specific for total α6, α6A and α6B confirming the specificity of abolition of α6A variant expression by shα6A relative to shctrl; *P ≤ 0.05, **P ≤ 0.01, ANOVA, n = 3. (C) Representative WB for detection of α6A and α6B subunits in shctl- and shα6A-infected Caco-2/15, DLD-1, T84 and HT29 cells. Keratin 18 (K18) was used as loading control. Densiometric analysis of α6A and α6B protein levels in shctl- versus shα6A-infected cell populations; *P ≤ 0.05, **P ≤ 0.01, t-test, n = 3.
Mentions: To investigate the involvement of the α6A variant in CRC cell behaviour, four well-characterized CRC cell lines—Caco-2/15, DLD-1, T84 and HT29 cells—were infected with an shRNA targeting the α6A variant. The α6A shRNA was designed in order to recognize a unique sequence of the short exon 25 (119pb) specific to the α6A mRNA transcript. Specificity of knockdown of α6A was first confirmed by competitive PCR using primers that amplify both variants of α6. The results revealed a decrease in α6A variant mRNA expression in shα6A versus shctl-treated CRC cells in all four cell lines tested (Figure 2A). Further analysis by qPCR revealed that knocking down α6A led to a decrease of ~50% of total α6 mRNA levels (Figure 2B). Analysis of splice variants showed an 80–90% decrease in α6A transcript levels in shα6A cells compared with controls, without affecting mRNA levels of α6B (Figure 2B). WB analysis of α6A and α6B performed to extend our observations at the protein level confirmed the specific abolition of the α6A variant, without significantly affecting α6B in all CRC cell lines (Figure 2C).

Bottom Line: The α6A silencing was also found to be associated with a significant repression of a number of Wnt/β-catenin pathway end points.Moreover, it was accompanied by a reduction in the capacity of these cells to develop tumours in xenografts.Taken together, these results demonstrate that the α6A variant is a pro-proliferative form of the α6 integrin subunit in CRC cells and appears to mediate its effects through the Wnt/β-catenin pathway.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Intestinal Physiopathology, Department of Anatomy and Cell Biology and Department of Medicine, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Quebec J1H 5N4, Canada.

Show MeSH
Related in: MedlinePlus