Nc sirna

BMs extracted from Magnetospirillum gryphiswaldense MSR-1 were presented kindly by professor Li Ying and Jiang Wei (Department of Microbiology, China Agricultural University). STAT 3 siRNA and siRNA (NC) were purchased from GenePharma Co., Ltd. (Shanghai, China), siRNA (NC) was used as the negative control of STAT 3 siRNA without homology. Branched PEI (MW: 25 KD) was purchased from Sigma Aldrich (USA). The cervical carcinoma cell line (HeLa cells), was obtained from China Academy Typical Culture Preservation Committee Cell Library (Shanghai, China). Cell culture medium was composed of Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The cells were incubated in humidified air maintained at 37 °C with 5% CO₂.

Three siRNAs targeting AC005592.2 (siRNA-78 sense strand: GGAAGCUAGUAGAAGAUUUTT and antisense strand: AAAUCUUCUACUAGCUUCCTT; siRNA-273 sense strand: GAAUGGCACUUUGGACAAUTT and antisense strand: AUUGUCCAAAGUGCCAUUCTT; siRNA-402 sense strand: GGAGUAGGCUGACCAGUUATT and antisense strand: UAACUGGUCAGCCUACUCCTT) and scrambled negative control siRNA (siRNA-NC, siRNA-NC sense strand: UUCUCCGAACGUGUCACGUTT and antisense strand: ACGUGAGCACUUCGGAGAATT) were synthesized and purchased from GenePharma (Shanghai, China). The Lipofectamine RNAiMAX kit (Invitrogen, USA) was used to transfect siRNA into CRC cells according to the manufacturer’s instructions. Two of the three siRNA sequences were selected for further studies based on the knockdown efficiency, as confirmed by qRT-PCR.

MC3T3-E1 cells were grown in DMEM/F12 media (Hyclone, UT, USA, #SH30023.01B) with 10% FBS (Gibco, GI, USA, #10099141) and 1% penicillin/streptomycin (Hyclone, UT, USA, #SV30010). Transfection of agomiR-6979-5p, agomiR-NC, antagomiR-6979-5p, antagomiR-NC, lncRNA Rhno1, silncRNA Rhno1, and siRNA-NC (GenePharma, Shanghai, China) at 200uM was conducted using Lipofectamine 3000 (ThermoFisher Scientific, MA, USA, #L3000001) based on provided directions. Lipofectamine 3000 was also used to transfect cells with miRNAs or siRNA oligos. BMP2 siRNA, silncRNA Rhno1, and siRNA-NC (GenePharma, Shanghai, China) were transfected at 50 nM.

Cox15, Bcl6, SIRT1, HIF1-α, HMGB1, Tet2-specific siRNA and non-specific control (NC) siRNAs were purchased from Genepharma. Cells were seeded into 6-well plates at a density of 1 × 10⁵ cells/well in 2 ml complete medium. After overnight incubation, the media was removed and replaced with 500 μl serum-free media and the Lipofectamine RNAiMAX (Thermo Scientific, USA) /siRNA complexes were added to each well. After 4 h, the media was replaced with 2 ml fresh medium containing 10% FBS, and incubated for another 24 h or 48 h at 37 °C. All transfection experiments were performed in triplicate.
The constitutively active Akt or Tet2 plasmids or empty plasmids were purchased from Addgene. Bcl6- or SIRT1-expressing plasmids were purchased from GeneChem. Cells were seeded into 6-well plate the day before transfection. The myr-Akt1 or empty plasmids were transfected with Lipofectamine 2000 (Invitrogen, USA) according to the protocol suggested by the manufacture. After 48 h of transfection, the positive clones were selected.

SiRNAs against SOX5 (SOX5-siRNAs), DNMT1 (DNMT1-siRNAs), p21 (p21-siRNAs) and negative control siRNA (NC-siRNAs) were synthesized by Genepharma (Shanghai, China), and the sequences were shown in
Table 2.

Table 2 The sequences of siRNAs used in the study

Name	Sequence (5′→3′)
siSOX5-1	sense	GACCAUGAUGCUGUCACCAAGGCAA
antisense	UUGCCUUGGUGACAGCAUCAUGGUC
siSOX5-2	sense	GAGAAGUACCCUGACUAUAAGUACA
antisense	UGUACUUAUAGUCAGGGUACUUCUC
siSOX5-3	sense	CAAGACAGCAGCAGCAGCTTCTACA-3′
antisense	TGTAGAAGCTGCTGCTGCTGTCTTG
siDNMT1-1	sense	GCCUCAUCGAGAAGAAUAUTT
antisense	AUAUUCUUCUCGAUGAGGCTT
siDNMT1-2	sense	GGGACUGUGUCUCUGUUAUTT
antisense	AUAACAGAGACACAGUCCCTT
siDNMT1-3	sense	CAGTCCCGAGTATGCGCCCATATTT
antisense	AAATATGGGCGCATACTCGGGACTG
sip21-1	sense	GCGAUGGAACUUCGACUUUTT
antisense	AAAGUCGAAGUUCCAUCGCTT
sip21-2	sense	GAUGGAACUUCGACUUUGUTT
antisense	ACAAAGUCGAAGUUCCAUCTT
sip21-3	sense	GACCAUGUGGACCUGUCACTT
antisense	GUGACAGGUCCACAUGGUCTT
siNC	sense	UUCUCCGAACGUGUCACGUTT
antisense	ACGUGACACGUUCGGAGAATT

The sequences of
SOX5,
DNMT1 and
p21 were synthesized by Generay (Shanghai, China) and inserted into the pCDNA3.1 vector. The plasmids were transfected into the cells using Lipofectamine-2000 (Invitrogen, Carlsbad, USA) according to the manufacturer’s instructions. After 6 h of transfection, the medium containing the transfection reagent was replaced with fresh medium. Forty-eight hours after transfection, the cells were collected for further study.