Miscripiirt kit

Total RNA (tissue samples and cells) was extracted with the miRNeasy Mini Kit (Qiagen, San Diego, CA, USA). Nuclear and cytoplasmic RNAs were isolated using the NE-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific) based on the manufacturer’s instructions. For RNase R treatment, total RNA from BC cells was digested with 4 U/μg RNase R (Geneseed) at 70 °C for 10 min. RNA was reversely transcribed with the M-MLV Reverse Transcriptase (Promega, Madison, WI, USA) or miScripIIRT kit (Qiagen). Quantitative-PCR was executed with the SYBR Green PCR Master Mix (Vazyme, Nanjing, China). The data were analyzed by the 2^−ΔΔCt method. The primers utilized in the research were as follows: β-actin (Forward: 5′-CTTCGCGGGCGACGAT-3′); Reverse: 5′-CCACATAGGAATCCTTCTGACC-3′), circ_IRAK3: (Forward: 5′-CTCGGTCATCTGTGGCAGTA-3′; Reverse: 5′-GTGCCCAGGACCAAAGTAAT-3′), IRAK3: (Forward: 5′-AGGATTTCCGCGGTTGTGT-3′; Reverse: 5′-ACTCAACACTGCTCCCAGG-3′), KIF2A: (Forward: 5′-TCGTACCTGCATGATTGCCA-3′; Reverse: 5′-CCACTACCCTGTGAGAAGGG-3′, miR-603: (Forward: 5′-CGCGCACACACTGCAATTAC-3′; Reverse: 5′-AGTGCAGGGTCCGAGGTATT-3′, and U6 small nuclear RNA (U6): Forward: 5′-CTCGCTTCGGCAGCACA-3′; Reverse: 5′-AACGCTTCACGAATTTGCGT-3′. β-actin or U6 was used as an internal control.

Clinical and xenograft tissues were homogenized with a gentle MACSTM Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany), and cultured cells were lysed using QIAzol Lysis Reagent (79306). The concentration and purity of the RNA were determined by using NanoDrop 2000 (Thomer Fisher). For microRNA, the miScripIIRT kit (QIAGEN GmbH, D-40724 Hilden, GERMANY) was used for reverse transcription, and the miScript SYBR® Green PCR kit was used for qRT-PCR with specific primers for miR-193a-5p, and the RNU6b (U6) was used as internal control. For large mRNA analysis, reverse transcription of RNA was performed by using the M-MLV First Strand Kit (Life Technologies). The Platinum SYBR Green qPCR Super Mix UDG Kit (Invitrogen) was used for the qRT-PCR of mRNAs. The real-time PCR experiments were carried on a CFX96™ Real-Time System (Bio-Rad). All data were normalized with GAPDH and analyzed by adopting 2^-ΔΔCt method as described previously [8 (link)].

Total RNA was isolated from cultured cells using QIAzol Lysis Reagent (79,306). To analyze the expression of miR-145, reverse transcription and qRT-PCR were performed using miScripIIRT kit (QIAGEN, Hilden, Germany) and miScript SYBR® Green PCR kit according to the manufacturer’s protocol. U6 or β-actin was used as the internal control. The sequences of primers are listed in Table 2.
Table 2.

Primers for qRT-PCR

Primer name	Primer sequence
miR-145 Forward	5’-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAGGGATTC-3’
miR-145 Reverse	5’-ACACTCCAGCTGGGGTCCAGTTTTCCCAGGA-3’
U6 Forward	5’-CTCGCTTCGGCAGCACA-3’
U6 Reverse	5’-AACGCTTCACGAATTTGCGT-3’
AKT3 Forward	5’-TGTGGATTTACCTTATCCCCTCA-3’
AKT3 Reverse	5’-GTTTGGCTTTGGTCGTTCTGT-3’
β-actin Forward	5’-GACTTAGTTGCGTTACACCCTTTCTTG-3’
β-actin Reverse	5’-GACTGCTGTCACCTTCACCGTTC-3’