Cw2569

Total RNA extraction from SK-Hep-1 cells, HCC-LM3 cells, and tumor tissues was performed using Trizol reagent (15,596,026, Thermo, USA). cDNA was obtained by reverse transcription using a reverse transcription kit (CW2569, CWBIO, China). Subsequently, proceed with the steps of RNA library preparation, sequencing, and data analysis.

Total RNA was extracted from cells or tumor tissues using TRIzol reagent (15596026, Thermo). cDNA was synthesized using an mRNA reverse transcription kit (CW2569, CWBIO) and miRNA reverse transcription kit (CW2141, CWBIO). The expression levels of target genes were analyzed by UltraSYBR Mixture (CW2601, CWBIO) and the 2^−ΔΔCT method. Primer sequences are shown in Table 1.

Total RNAs were extracted with TRIzol (15,596,026, Thermo), and reversely transcribed to prepare cDNAs using a cDNA synthesis kit (CW2569, CWBIO). The relative expression of targets was performed using the UltraSYBR Mixture kit (CW2601, ConWin) on QuantStudio 1 Real-Time PCR (Thermo). The following experimental parameters were applied for PCR amplification: 95 °C for 30 s, and 40 cycles of 95 °C for 5 s and 60 °C for 15 s. Targets were normalized with reference to β-actin. The primers are listed in Table 1.
Table 1

Primer sequences

Targets	F (5’-3’)	R (5’-3’)
AR	GCCCAGTAACTACCCGAGCAT	TCCTGATTCCCATGACCCCTT
PSA	CTGCTCGTGGGTCATTCTGA	TAGACAGGTCGGTGGGACAA
PI3K	TGCGTCTACTAAAATGCATGG	AACTGAAGGTTAATGGGTCA
Akt1	AGCCCTGGACTACCTGCACTCG	CTGTGATCTTAATGTGCCCGTCCT
mTOR	CCAAAGGCAACAAGCGATCCCGAA	CTCCAAGTTCCACACCGTCCA
HIF-1α	TGGTATTATTCAGCACGACT	GCCAGCAAAGTTAAAGCATC
c-Myc	CACTAACATCCCACGCTCTGA	AAACCGCATCCTTGTCCTGT
hnRNPs	AGACGAAGACTGAGCGGTTG	AGCCGAAAACAAGAAGGGGA
VEGF	TGCTCTACTTCCCCAAATCACT	ACTCACTTTGCCCCTGTCG
HK2	GTGAATCGGAGAGGTCCCAC	GCTAACTTCGGCCACAGGAT
PFK1	AATCTGCAAGAAAGCAGCGG	TACCAACTCGAACCACAGCC
PKM2	CGTCATTCATCCGCAAGGCAT	CACGAGCCACCATGATCCCA
β-actin	ACCCTGAAGTACCCCATCGAG	AGCACAGCCTGGATAGCAAC

Total RNA was extracted by Trizol (15,596,026, Thermo). Reverse transcription of cDNA was performed by using mRNA (CW2569, CWBIO, China) kit. The sequences of target genes were searched on NCBI. Primers were designed by primer5 software. Primers were synthesized by Beijing Muscularidae. The target genes were detected by UltraSYBR Mixture (CW2601, CWBIO, China) and fluorescence quantitative RCP instrument (PIKOREAL96, Thermo). β-actin as internal reference. The relative expression of target genes (Table 1) in each individual sample compared to the reference gene was calculated using 2^−ΔΔCt algorithm. ANOVA and Tukey’s post-test were used to analyze the differences in gene expressions between groups.
Table 1

Primer sequence

Gene	Primer sequence	Length
IDOL	FGAGAAACCGGATCTCCCAGC	216 bp
	RTCTCCAAACTTGGTCTGGGC
FMO3	FAGGTTACCATGGGGAAGAAAG	137 bp
	RAAATTTCCACAGGCCCCCAA
SREBP-2	FTGAGCCAGGAAGCCCTCTAT	137 bp
	RGGGGGTTAAAGGAGAGGCAC
LDLR	FACCAATCTCTAAGCCAAACCC	188 bp
	RCAGATCATTTCCGACGCCAT
HMGB1	FCTATATTACGGTTTGCCCCTT	209 bp
	RACTGGCACTTTAAGAAAACGAT
COX-2	FCTCTATCACTGGCATCCCCTT	169 bp
	RCATTCCTACCACCAGCAACCC
IL-6	FGCAATAACCACCCCTGACCCAA	154 bp
	RGCTACATTTGCCGAAGAGCC
E-selectin	FTATGGCTGAAACCGCAACACC	140 bp
	RATCCTTTCCCTTCATTAGCCAAC
ICAM1	FTCTTCCTCGGCCTTCCCATA	152 bp
	RAGGTACCATGGCCCCAAATG
SRBI	FACTTTCCAGGCATGTTCCCCTT	151 bp
	RTCAACCTTGCTCAGCCCGTTC
H-actin	F ACCCTGAAGTACCCCATCGAG	224 bp
	R AGCACAGCCTGGATAGCAAC

Total RNA was isolated from the DRG tissues in each group using TRIzol® reagent (15596026; Thermo Fisher, USA). cDNA was synthesized using an mRNA reverse transcription kit (CW2569; CWBIO, China). UltraSYBR Mixture (CW2601; CWBIO, China) was used for PCR. The fluorescent quantitative PCR system used was a Thermo Fisher (PIKOREAL96). β-Actin was used as the internal reference. The relative expression levels were calculated using the 2^-ΔΔCt method. The primer sequences (Sangon Biotech, Shanghai, China) are listed in Table 2.

The total RNA was isolated from lung tissue homogenate by TRIzol reagents. Then, the total RNA was used to synthesize cDNA using reverse transcription kits (CW2569, CWBIO). Thereafter, quantitative PCR (qPCR) was conducted by SYBR Green qPCR kits (11201ES08, YE SEM, China) and carried out on real-time PCR instruments (LightCycler 96, Roche, Germany). The relative mRNA expressions were calculated as the ratio of GAPDH and analyzed by the 2^−ΔΔCt method. The qPCR primer sequences designed in the experiment are presented in Table 1.

QRT-PCR was performed to detect PTPRZ1, p120, β-catenin, RhoA, Rac1, CDC42, cyclin D1, and c-myc expression levels. To put it simply, total RNA was extracted by the Trizol method, RNA was reversely transcribed into cDNAs in accordance with the instruction of a reverse transcription kit (CW2569, CWBIO, China). SYBR Green qPCR mix (Invitrogen) was performed to test genes relative expression in ABI 7900 system. The relative level of the gene was calculated by the 2^–ΔΔCt method with GAPDH as the internal gene. The primer sequence used in this study is shown in Table 1. Each group was tested three times.