Abi 7900 system

For qRT–PCR, total RNA was extracted and dissolved in reverse‐transcribed RNA into cDNA with a Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland) according to the instructions. qRT–PCR was performed using an ABI 7900 System (Thermo Fisher Scientific, Massachusetts, USA). Primers were synthesised by Tsingke Biological Technology (Beijing, China), and the sequences of primers were as follows: GAPDH: forward 5′‐GGAGCGAGATCCCTCCAAAAT‐3′, reverse: 5′‐GGCTGTTGTCATACTTCTCATGG‐3′, SLAMF8: forward 5′‐CTGATGGTGGATACAAGGG‐3′, reverse SLAMF8: 5′‐GGAAATGGACGTAACGGA‐3′. GAPDH was used as the endogenous control.
A total of 19 GC and 20 CRC tissues were successfully used for RNA‐sequencing analysis. RNA isolation and construction of the RNA‐sequencing library and sequencing were performed by the Shanghai Sangon Biological Engineering Technology Company (Shanghai, China) following the manufacturer’s protocols. We validated the performance of the CCGs and the tag gene using the RNA‐sequencing data by dimension reduction analysis and correlation analysis. Macrophages transfected with a pCMV‐SLAMF8 plasmid or negative control were used for total RNA isolation and RNA sequencing by the Shanghai Sangon Biological Engineering Technology Company (Shanghai, China).

All the RNAs were extracted from AML or normal bone marrow tissues and cells with the TRIzol reagent (Invitrogen, USA). After quantifying and assessing the RNAs with NanoDrop 2000 (Thermo Fisher Scientific, USA), the All-in-One miRNA qRT-PCR Detection Kit (GeneCopoeia, China) was utilized to examine miR-4458 expression. As for the measurement of LINC00665 and DOCK1 mRNA expression, the PrimeScriptVR RT reagent Kit (Takara, Japan) was applied to reverse-transcribe PCR and generate cDNA. Then, the SYBR Premix Ex Taq (Takara, Japan) was used qRT-and subjected to qRT-PCR was conducted through ABI 7900 system (Thermo Fisher Scientific, USA). The relative expression of LINC00665 and DOCK1 were subsequently normalized by GAPDH, while that of miR-4458 was normalized by U6. The 2^−ΔΔCT method was used to calculate their expression levels. The primer sequences are illustrated in Table 2.Table 2

The primer sequences for RT-qPCR.

GENE	Primer sequences (5′–3′)
miR-4458	Forward: AGAGGTAGGTGTGGAAGAA
	Reverse: GCGAGCACAGAATTAATACGAC
U6	Forward: CTCGCTTCGGCAGCACA
	Reverse: AACGCTTCACGAATTTGCGT
LINC00665	Forward: GGTGCAAAGTGGGAAGTGTG
	Reverse: CGGTGGACGGATGAGAAACG
DOCK1	Forward: CCGCCGCAAACTTTTTCCTC
	Reverse: AGATGTGCACAGTGTCTCCG
GAPDH	Forward: AGCCACATCGCTCAGACAC
	Reverse: GCCCAATACGACCAAATCC

The RNA was extracted from LC tissues and cells via TRIzol (Invitrogen), and then reversely transcribed to complementary DNA (cDNA) through the PrimeScript™ strand cDNA synthesis kit (Takara, Dalian, China). The reaction of qRT-PCR was carried out by a Power SYBRTM Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) on an ABI 7900 system (Thermo Fisher Scientific). U6 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as the internal controls for miRNA and circRNA or mRNA, respectively. Primer sequences were listed as follows: circ_0001421, F: 5′-TCTCTGGAGTTCTGATTGCAGGTGG-3′ and R: 5′-TGCTAGGTAAATGGGGTGATTCTGG-3′; miR-4677-3p, F: 5′-CTGTGAGACCAAAGAACTACTCGC-3′ and R: 5′-CTCTACAGCTATATTGCCAGCCAC-3′; CDCA3, F: 5′-GGACCCTGAGACTCCCAGAT-3′ and R: 5′-GCCGCTTACCCTGTCGTAG-3′; GAPDH, F: 5′-CCATTTGCAGTGGCAAAG-3′ and R: 5′-CACCCCATTTGATGTTAGTG-3′; and U6, F: 5′-GCTTCGGCAGCACATATACTAAAAT-3′ and R: 5′-TACTGTGCGTTTAAGCACTTCGC-3′.