Nucleozol reagent

At the endpoint, the transfected cells were subjected to total RNA isolation by using NucleoZOL Reagent (Takara Bio USA, Mountain View, CA) according to the manufacturer’s introduction. For qPCR analysis of mRNA transcripts, total RNA was used for reverse transcription with the hexamer and M-MuLV (NEB). The cDNA products were diluted as templates for qPCR. The qPCR primers were designed by Primer3 Plus program [48 (link)]. For assessing the miR expression levels mediated by the three expression systems, the reverse transcription reactions were carried out by using miR-specific reverse primers that were complementary with the 3′-end six nucleotides of mature miR-5p and/or miR-3p, preceded with a 44-nt artificial stem-loop sequence. SYBR green-based quantitative real-time PCR analysis was performed by following our previously optimized TqPCR protocol [49 (link)]. The qPCR reactions were done in triplicate. All expression values were normalized to the reference gene GAPDH expression by using the 2^–ΔΔCt method [50 (link)–53 (link)]. The sequences of the qPCR primers are listed in Supplemental Table 1.

Total RNA was extracted from endometrial tissues, HEC-1A, and Ishikawa cells using NucleoZOL reagent (Takara) according to the manufacturer’s instructions. Complementary DNA was synthesized using Moloney murine leukemia virus reverse transcriptase (MMLV-RT, Promega) and random hexamers (BioFact). Quantitative real-time reverse transcription PCR (qRT-PCR) analysis was performed in triplicates using the SYBR green real-time PCR master mix reagent (Biofact) and QuantStudio 3 Real-time PCR System (Life Technologies Inc.). Relative mRNA expression was calculated from the comparative threshold cycle (C_t) values relative to human GAPDH. The following primers were used—GAPDH: forward 5′-TTGCCATCAATGACCCCTTCA-3′, reverse 5′-CGCCCCACTTGATTTTGGA-3′; Cyclophilin: forward 5′-GCAAAGTGAAAGAAGGCA-3′, reverse 5′-CCATTCCTGGACCCAAAG-3′; SESN2: forward 5′-ACTGCGTCTTTGGCATCAG-3′, reverse 5′-CTTCTCTGAGTGGCGGAAGT-3′; MKI67: forward 5′-ACGCCTGGTTACTATCAAAAG-3′, reverse 5′-CAGACCCATTTACTTGTGTTGGA-3′; CDKN1A: forward 5′-TGTCCGTCAGAACCCATGC-3′, reverse 5′-AAAGTCGAAGTTCCATCGCTC-3′; and CDKN1B: forward 5′-AACGTGCGAGTGTCTAACGG-3′, reverse 5′-CCCTCTAGGGGTTTGTGATTCT-3′.

CK was provided by Zhejiang Hongguan Bio-pharma Co., Ltd., and dissolved in DMSO, and diluted in PBS. Modified Eagle’s Medium of Alpha (α-MEM), Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12), fetal bovine serum (FBS), and penicillin/streptomycin were purchased from Gibco (United States). Beta-glycerolphosphate, dexamethasone, ascorbic acid phosphate, Safranine O, and Fast Green were purchased from Sigma (United States). Alizarin Red S was purchased from Solarbio (Beijing, China). Cell Counting Kit-8 (CCK-8) was purchased from Beyotime (Beijing, China). NucleoZOL reagent, Reverse Transcription Kit and SYBR-Green Master Mix were supplied by Takara (Japan). Hematoxylin-eosin (H&E) was purchased from biosharp (China). Primary antibodies against CD31, β-catenin, and DAPI were supplied by Santa Cruz Biotechnology (United States); Primary antibodies anti-CTSK, anti-ALP, anti-OPG, anti-RANKL, anti-Runx2, and anti-OPN were purchased from Bioss (China); Anti-GAPDH, and DAPI were obtained from Abcam (United States). Secondary antibodies HRP-conjugated Goat Anti-Rabbit IgG, Goat anti-Mouse IgG (H + L), Rabbit Anti-Rabbit IgM/Cy3 and Rabbit Anti-Mouse IgM/FITC were obtained from Bioss (China). Dual-Luciferase Reporter Assay System was supplied by Promega Company (United States). Matrigel was purchased from Becton Dickinson (United States).

Subconfluent cells were treated with various conditions for 48h. Total RNA was isolated by using the NucleoZOL reagent (Takara Bio USA Inc.), and subjected to reverse transcription (RT) reactions as previously described [63 (link)–66 (link)]. The RT cDNA products were used as PCR templates. The primers for the genes of interest were designed by using Primer3 Plus program (Supplementary Table 1). TqPCR reactions were carried out by using SYBR Green-based Forget-Me-Not™ qPCR Master Mix (Biotium Inc., Hayward, CA) on a CFX-Connect unit (Bio-Rad Laboratories, Hercules, CA) as described [67 (link)–70 (link)]. Relative gene expression was normalized to GAPDH by using the 2^−∆∆Ct method. All qPCR reactions were done in triplicate.