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The carboxy-terminal domain of connexin 43 (CT-Cx43) modulates the expression of p53 by altering miR-125b expression in low-grade human breast cancers

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Abstract

Purpose

Connexin 43 (Cx43) is a widely expressed gap junction protein. It can also regulate various gap-junction independent processes, including cellular proliferation. The latter regulatory functions have been attributed to its carboxy-terminal domain, CT-Cx43. CT-Cx43 has been found to be expressed independent of full-length Cx43 in various cell types. Its nuclear localization has additionally raised the possibility that it may regulate the expression of particular genes, including miRNAs, known play a role in the regulation of cellular proliferation. Here, we set out to uncover the molecular mechanism(s) underlying CT-Cx43 mediated gene (de-)regulation in human breast cancer.

Methods

Western blotting and quantitative real time PCR were carried to assess the expression of CT-Cx43 and miR-125b in a panel of 60 primary human breast cancer tissues and its paired normal adjacent tissues. In addition, CT-Cx43 was exogenously expressed in the breast cancer-derived cell line MCF-7 and its effect on the expression of miR-125b and its downstream target p53 were evaluated, as well as its effect on cellular proliferation and death using MTT and LDH assays, respectively.

Results

We found that CT-Cx43, but not full-length Cx43, was down-regulated in low grade human breast cancers. In addition, we found that the tumor suppressor protein p53 exhibited a decreased expression in the CT-Cx43 down-regulated samples. Interestingly, we found that miR-125b, a negative regulator of p53, exhibited an inverse expression relationship with CT-Cx43 in the breast cancer samples tested. This inverse relationship was confirmed by exogenous expression of CT-Cx43 in MCF-7 cells. In addition, we found that CT-Cx43 up-regulation and subsequent miR-125b down-regulation resulted in a decreased proliferation of MCF-7 cells.

Conclusions

Our data suggest a mechanism by which CT-Cx43 may regulate cell proliferation. Targeting of CT-Cx43 and/or miR-125b may be instrumental for therapeutic intervention in human breast cancer.

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Acknowledgments

The junior research fellowships to RM (UGC-JRF 19-06/2011 Ci EU-IV), SN (ICMR JRF-2012/HRD-29) and project fellowships to RI (BT/PR14336/MED/30/493/2010) and RR (SR/WOS-A/LS-661/2012) are highly acknowledged. The present work was supported by project grant (BT/PR11917/MED/30/181/2009) from the Department of Biotechnology (DBT), New Delhi, to MUH.

Conflict of interest

All authors declare no conflict of interest

Author information

Authors and Affiliations

  1. Department of Biotechnology, University of Kashmir, Srinagar, 190006, India

    Raihana Maqbool, Rabiya Rashid, Rehana Ismail, Saif Niaz & Mahboob Ul Hussain

  2. Department of General and Minimal Invasive Surgery, SKIMS, Srinagar, Soura, India

    Nisar Ahmad Chowdri

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  1. Raihana Maqbool
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Correspondence to Mahboob Ul Hussain.

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Raihana Maqbool and Rabiya Rashid contributed equally to the work.

Electronic supplementary material

Supplementary Fig. 1

Total RNA was isolated from the normal and breast cancer samples as described in material and methods. To check the integrity of the RNA, 5 μl of each sample was loaded on formaldehyde agarose gel. After electrophoresis, the agarose gel was incubated for 5–10 min in MOPS buffer containing ethidium bromide. The gel was visualized in gel-documentation apparatus. As shown in suppl. Fig. 1, intact and prominent bands, corresponding to 28S and18S rRNA can be detected. Moreover, RNA bands corresponding to small RNAs can also be detected. Thus, the presence of intact RNA bands established the integrity of the RNA obtained from the tissue samples. (GIF 181 kb)

High Resolution Image (TIFF 69.5 kb)

Supplementary Fig. 2

Total protein was extracted from the tumor and adjacent normal breast samples as described in material and methods. 30 μg of protein was loaded on SDS-PAGE and electrophoresis was performed. After PAGE, Western blot was performed using anti-Cx43 antibody as described previously. As shown in suppl. Fig. 2, anti-Cx43 detected FL-Cx43 and CT-Cx43 in the normal samples (N). However, the tumor samples (T) showed decreased expression of FL-Cx43 as well as CT-Cx43. (GIF 169 kb)

High Resolution Image (TIFF 61.3 kb)

Supplementary Fig. 3

To investigate whether CT-Cx43 has the potential to bind the miR-125b promoter, we expressed CT-Cx43 as GST fusion protein in bacterial system. The GST-CT-Cx43 was purified from the bacterial lysate using Gluthatione-agarose beads. Additionally, 200 bps of miR-125b promoter (upstream of transcriptional site containing the Tata box; Gene ID: 406,911) was PCR amplified using the forward primers 5`-TTTGAAAAGACACTAAATCCT-3` and the reverse primer 5’ACAATGTTTTCTTTCTTGAGA-3`. Prior to PCR, the primers were 32p labeled using T4 polynucleotide kinase and gamma phosphate (32P) labeled ATP. The 200 bp DNA probe and purified GST-CT-CX43 were incubated together and gel-shift assay was performed. As shown in suppl. Fig. 3, lane II, addition of CT-Cx43 did not show any binding to the miR-125 DNA. Moreover, addition of anti-CT-Cx43 did not show and supershift of miR-125b probe (lane III). Thus, our initial data indicate that CT-Cx43 does not bind the proximal promoter element of miR-125b. Further upstream of 200 bp needs to further screened for any binding of CT-Cx43. (GIF 62.9 kb)

High Resolution Image (TIFF 34.2 kb)

Supplementary Fig. 4

In order study the effect of CT-Cx43 on the expression of various growth related miRNAs, we transiently transfected MCF-7 cells with either CT-Cx43 plasmid or with empty plasmid. After 36 h transfection, RNA was isolated and using stem-loop primers strategy (for details consult material methods of the main manuscript) many miRNAs were PCR amplified (here denoted as “a, b, c, d, e, f, g, h, I, j), including the miR-125b. As shown in suppl Fig. 4, various miRNAs showed differential regulation between empty plasmid and CT-Cx43 transfected MCF-7 cells (compare “a & a`; e & e`; f & f`). Interestingly, miR-125b showed highly significant up-regulation in CT-Cx43 transfected MCF-7 cells. (GIF 291 kb)

High Resolution Image (TIFF 79.5 kb)

Supplementary Fig. 5

In order to establish the possibility of nuclear localization of CT-Cx43 in breast samples, we isolated the nuclear and cytosolic fraction from the normal and tumor breast cancer samples using nuclear extraction kit (Sigma adrich). 30 μgs of each fraction was run on 12 % SDS-PAG and Western blot was performed using anti-Cx43 antibody as described in material and methods. As shown in Suppl. Fig. 5, anti-Cx43 antibody detected FL-Cx43 in the cytosolic fraction (Cyt) fraction of normal (N) as well as in the tumor (T) samples. Moreover, CT-Cx43 was detected in the cytosolic (Cyt.) and nuclear fractions (Nuc.) of the normal samples (N). However, very less/or no signal of CT-Cx43 was detected from the nuclear fraction (Nuc.) of tumor samples (T). Hence, it may be concluded that CT-Cx43 has the potential to localize in cell nucleus when it is expressed in the cells. (GIF 122 kb)

High Resolution Image (TIFF 56.2 kb)

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Maqbool, R., Rashid, R., Ismail, R. et al. The carboxy-terminal domain of connexin 43 (CT-Cx43) modulates the expression of p53 by altering miR-125b expression in low-grade human breast cancers . Cell Oncol. 38, 443–451 (2015). https://doi.org/10.1007/s13402-015-0240-x

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