Background/Purpose:

Non-template addition of nucleotides on the 3’ termini of mRNAs acts as a mechanism that controls mRNA stability. ZCCHC11 is a member of the non-canonical poly A polymerase (ncPAP) family. It has been implicated in post-transcriptional regulation of various genes, including some cytokines, by modification of their poly A tails or by modification of microRNAs that target these mRNAs. Role of ZCCHC11 in chondrocyte physiology and in the pathogenesis of osteoarthritis (OA) has not been studied. In the present study we determined the expression of ZCCHC11 in damaged and undamaged cartilage obtained from OA patients. We also studied the effect of IL-1b on the expression of ZCCHC11 in primary human chondrocytes. We further investigated the effect of gene silencing of ZCCHC11 on the secreted levels of several cytokines.

Methods:

Primary human chondrocytes were isolated from the deep zone of the cartilage obtained from OA patients who underwent total knee joint replacement surgery. Silencing of ZCCHC11 gene was carried out by using ON-TARGETplus siRNA pool (Thermo Fisher Scientific, Waltham, MA). Protein levels of 40 cytokines in culture supernatants after siRNA transfections were analyzed simultaneously using a glass-based human cytokine microarray (RayBio Human Cytokine Array). Poly A tail length analysis was performed by reverse transcription followed by PCR using an adaptor reverse primer and a forward primer specific for IL-6 mRNA. The PCR products were run on 2% agarose gel and visualized by EtBr staining. mRNA levels of ZCCHC11 and IL-6 genes were quantitated by using the TaqMan assays (Applied Biosystems, Carlsbad, CA). Data were derived using Origin 6.1 software and P<0.05 was considered significant.

Results:

There was higher expression of ZCCHC11 mRNA in damaged cartilage as compared to unaffected smooth cartilage obtained from OA patients. mRNA expression of ZCCHC11 was significantly up-regulated by treatment of human primary chondrocytes with IL-1b. Cytokine protein array analysis revealed a significant decrease in secreted levels of IL-6 and a subset of cytokines upon siRNA mediated silencing of ZCCHC11 gene. Quantitative PCR analysis showed that mRNA expression of IL-6 gene was also suppressed in human chondrocytes with depleted  ZCCHC11 mRNA. Poly A tail length analysis showed a significant shortening of IL-6 mRNA poly A tail upon silencing of ZCCHC11 gene indicating that ZCCHC11 is required for IL-6 mRNA stability.

Conclusion:

The data presented here for the first time show the expression of ZCCHC11 in human cartilage. Differential expression of ZCCHC11 in damaged and undamaged cartilage and its stimulation by IL-1b, a cytokine up-regulated during OA, point to a role of this enzyme in osteoarthritis. Furthermore, our gene silencing studies show the effect of ZCCHC11 on secretion of IL-6, an important cytokine involved in the pathogenesis of OA, via modifying its mRNA at the 3’ end. Taken together, the present study reveals a novel RNA modifying enzyme ZCCHC11 as an important player in OA pathophysiology.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/terminal-uridyltransferase-enzyme-zcchc11-regulates-interleukin-6-expression-in-primary-human-osteoarthritis-chondrocytes/