Regulation of Renal Fibrosis by miR-4709-3p

The HK-2 cells transfection was done using miR-4709-3p mimics or inhibitors for 6 hours and later exposed to TGF-β1 (5 ng/ml) for 48 hours. The total RNA was then extracted from HK-2 cells using TRIzol reagent (Invitrogen/Thermo Fisher Scientific) following the manufacture’s guideline. cDNAs processing was done by the reverse transcription of RNA using the GoScriptTM Reverse Transcription kit (Promega). Later, qPCR was analyzed using the SYBR® Premix Ex Taq™ II (Takara, Japan). GAPDH and U6 were utilized as the analysis normalization for the abundances of miR-4709-3p and other mRNAs, respectively, as used elsewhere^{36 (link)}. The primers’ sequences are described in Table 2. All experiments were carried out in triplicates, and mRNA expressions were determined by the 2^−ΔΔCt method.Table 2.

List of primers used in this study

List of Primers used in this study
Gene	Forward Primer	Reverse Primer
Hsa-α-SMA	AAGAGCATCCCACCCTGC	TAGCCACATACATGGCTGGG
Hsa-FN	ACAACACCGAGGTGACTGAG	GGACACAACGATGCTTCCTGA
Hsa-Col1	GTGGATACGCGGACTTTG	TCCATCATACTGAGCAGCA
Mmu-α-SMA	CCAACCGGGAGAAAATGA	CAGACGCATGATGGCAT
Mmu-FN	GTCTCCTGGGAGAGGAGC	TGATCAGCATGGACCACT
Mmu-Col1	GGTCCTGATGGCAAAAC	TCCATCTTTGCCAGCAGGA
LATS2	AGGCCAAAGACTTTTCCTGC	CACGTACACAGGCTGGCAGC
Mmu-β-Actin	CAGCTGAGAGGGAAATCGTG	CGTTGCCAATAGTGATGACC
Hsa-β-Actin	ACCATTGGCAATGAGCGGTTC	GGTCTTTGCGGATGTCCACGT
miR-4709-3p	Qiagen (Cat#MS00039914)
snRNA RNU6B	Qiagen (Cat#MS00033740)