OBJECTIVE: To use an in vitro model of chondrogenesis to identify microRNAs (miRNAs) with a functional role in cartilage homeostasis. METHODS: The expression of miRNAs was measured in the ATDC5 cell model of chondrogenesis using microarray and was verified using quantitative reverse transcription-polymerase chain reaction. MicroRNA expression was localized by in situ hybridization. Predicted miRNA target genes were validated using 3'-untranslated region-Luc reporter plasmids containing either wild-type sequences or mutants of the miRNA target sequence. Signaling through the Smad pathway was measured using a (CAGA)(12) -Luc reporter. RESULTS: The expression of several miRNAs was regulated during chondrogenesis. These included 39 miRNAs that are coexpressed with miRNA-140 (miR-140), which is known to be involved in cartilage homeostasis and osteoarthritis (OA). Of these miRNAs, miR-455 resides within an intron of COL27A1 that encodes a cartilage collagen. When human OA cartilage was compared with cartilage obtained from patients with femoral neck fractures, the expression of both miR-140-5p and miR-455-3p was increased in OA cartilage. In situ hybridization showed miR-455-3p expression in the developing limbs of chicks and mice and in human OA cartilage. The expression of miR-455-3p was regulated by transforming growth factor β (TGFβ) ligands, and miRNA regulated TGFβ signaling. ACVR2B, SMAD2, and CHRDL1 were direct targets of miR-455-3p and may mediate its functional impact on TGFβ signaling. CONCLUSION: MicroRNA-455 is expressed during chondrogenesis and in adult articular cartilage, where it can regulate TGFβ signaling, suppressing the Smad2/3 pathway. Diminished signaling through this pathway during the aging process and in OA chondrocytes is known to contribute to cartilage destruction. We propose that the increased expression of miR-455 in OA exacerbates this process and contributes to disease pathology.
OBJECTIVE: To use an in vitro model of chondrogenesis to identify microRNAs (miRNAs) with a functional role in cartilage homeostasis. METHODS: The expression of miRNAs was measured in the ATDC5 cell model of chondrogenesis using microarray and was verified using quantitative reverse transcription-polymerase chain reaction. MicroRNA expression was localized by in situ hybridization. Predicted miRNA target genes were validated using 3'-untranslated region-Luc reporter plasmids containing either wild-type sequences or mutants of the miRNA target sequence. Signaling through the Smad pathway was measured using a (CAGA)(12) -Luc reporter. RESULTS: The expression of several miRNAs was regulated during chondrogenesis. These included 39 miRNAs that are coexpressed with miRNA-140 (miR-140), which is known to be involved in cartilage homeostasis and osteoarthritis (OA). Of these miRNAs, miR-455 resides within an intron of COL27A1 that encodes a cartilage collagen. When human OA cartilage was compared with cartilage obtained from patients with femoral neck fractures, the expression of both miR-140-5p and miR-455-3p was increased in OA cartilage. In situ hybridization showed miR-455-3p expression in the developing limbs of chicks and mice and in human OA cartilage. The expression of miR-455-3p was regulated by transforming growth factor β (TGFβ) ligands, and miRNA regulated TGFβ signaling. ACVR2B, SMAD2, and CHRDL1 were direct targets of miR-455-3p and may mediate its functional impact on TGFβ signaling. CONCLUSION: MicroRNA-455 is expressed during chondrogenesis and in adult articular cartilage, where it can regulate TGFβ signaling, suppressing the Smad2/3 pathway. Diminished signaling through this pathway during the aging process and in OA chondrocytes is known to contribute to cartilage destruction. We propose that the increased expression of miR-455 in OA exacerbates this process and contributes to disease pathology.
Authors: Tao Wang; Rui-Qiao Yan; Jun Cao; Ling-Ling Cao; Xuan-Pu Zhang; Xing-Nuan Li; Ping Wu; Xiao-Ou Zhou; Jian-Fang Wu; Xiao-Yuan Xu Journal: Nan Fang Yi Ke Da Xue Xue Bao Date: 2016-02-20
Authors: Antonina Parafioriti; Ingrid Cifola; Clarissa Gissi; Eva Pinatel; Laura Vilardo; Elisabetta Armiraglio; Andrea Di Bernardo; Primo Andrea Daolio; Armando Felsani; Igea D'Agnano; Anna Concetta Berardi Journal: Cell Death Discov Date: 2020-06-10