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.
OBJECTIVE
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.
Department of Biotechnology, University of Kashmir, Srinagar, 190006, India.
Raihana
R
Maqbool
Department of Biotechnology, University of Kashmir, Srinagar, 190006, India.
Rabiya
R
Rashid
Department of Biotechnology, University of Kashmir, Srinagar, 190006, India.
Rehana
R
Ismail
Department of Biotechnology, University of Kashmir, Srinagar, 190006, India.
Saif
S
Niaz
Department of General and Minimal Invasive Surgery, SKIMS, Srinagar, Soura, India.
Nisar Ahmad
NA
Chowdri
Department of Biotechnology, University of Kashmir, Srinagar, 190006, India. mahboob@kashmiruniversity.ac.in.
Mahboob Ul
MU
Hussain
D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D018031:Connexin 43 / Q000378:metabolism*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology*; D006801:Humans; D061986:MCF-7 Cells; D035683:MicroRNAs / Q000378:metabolism*; D060787:Neoplasm Grading; D060888:Real-Time Polymerase Chain Reaction; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis*
D018031:Connexin 43; C500216:GJA1 protein, human; C531888:MIRN125 microRNA, human; D035683:MicroRNAs; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53
Breast cancer; Connexin43; miR-125b; p53