The Cancer Genome Atlas database
Correlation between GNA14 and KLF7 was analyzed in normal samples and UCEC samples from The Cancer Genome Atlas (TCGA, http://cancergenome.nih.gov) database.
Human UCEC cells KLE, Hec-1-A and Hec-1-B cells were obtained from the American Type Culture collection (ATCC). All the cells were maintained in RPMI 1640 (Invitrogen), which was supplied with 10% fetal bovine serum (FBS, Gibco) and 1% antibiotics solution (Corning), at 37oC with 5% CO2.
GNA14, KLF7 and HAS2 interference
siRNA was used to interfere GNA14, KLF7 and HAS2 in UCEC cells. siRNAs against negative control, GNA14, KLF7 and HAS2 were purchased from GenePharma and were transfected into KLE, Hec-1-A and Hec-1-B cells by RNAiMAX (Invitrogen). 48 hours later, the cells were subjected to immunoblotting analysis of knockdown efficiency and functional experiments. For in vivo experiments, lentivirus-mediated KLF7 knockdown was performed to establish stable cell lines. siRNA sequences were as follow: siGNA14, 5’-CTACAGATACAGACAATAT-3’; siKLF7-1: 5’-CCGGCUACUUCUCAGCUUU-3’; siKLF7-2; 5’-GGUGAGGACUUGGACUGUU-3’, and siHAS2; 5’-CCAGUAUCAGUUUGGUUUA-3’.
GNA14 and KLF7 overexpression
Lentivirus was used to overexpress GNA14 and KLF7 in UCEC cells. The coding sequence of GNA14 or KLF7 was cloned into pCDH vectors. Lentivirus was packaged in 293FT cells and were concentrated using PEG6000. The virus was harvested to infect UCEC cells and the overexpression efficiency was detected by immunoblotting. Then the cells were subjected to functional experiments.
Total proteins were extracted from cells using RIPA buffer (Beyotime, Shanghai, China), containing protease inhibitor cocktail and phosphatase inhibitor cocktail (Roche). Protein concentration was detected by BCA kit (Beyotime). Subsequently, equal amount of total proteins was separated on 10-15% SDS-PAGE gels and transferred onto PDVF membranes. After incubating with 5% skim milk, primary, and secondary antibodies, the protein abundance was measured on a chemiluminescence detector. Primary antibody against GNA14 was from Abnova (Taiwan). HAS2 antibody was from Invitrogen. KLF7 and GAPDH antibody, and all the secondary antibodies were from SantaCruz.
Quantitative Real Time PCR (qRT-PCR)
Total RNA was extracted from UCEC cells using Trizol regent, following to the manufacturer’s instructions. RNA was reversely transcribed using M-MLV reverse transcriptase (Promega). The abundance of cDNA was detected by SYBR master mixture. Primer sequences were as follow: HAS forward, 5’-TCCTGGATCTCATTCCTCAGC-3’ , and reverse, 5’-TGCACTGAACACACCCAAAATA-3’; GAPDH forward, 5’-TGACTTCAACAGCGACACCCA -3’, and reverse, 5’-CACCCTGTTGCTGTAGCCAAA-3’.
Cell proliferation was detected by CCK8 assay. A total of 2000 UCEC cells were in triplicate seeded into 96-well plates, which contained 100 ul culture medium in each well. At indicated time, 10 ul of CCK8 regent was added into each well and the plates were shocked for 30 seconds, followed by incubation at 37oC for 3 hours. Then OD value at 450 nm was measured on the microplate. Cell viability was normalized to the OD450 of day 1 (OD450 value of the cells 8 hours after seeding).
UCEC cells were seeded into 6-well plates at equal concentration. 8-10 days later, colonies were formed and washed by PBS for three times. Then, they were fixed by methyl alcohol and stained by crystal violet. Lastly, the colonies were washed by clear water and dried at room temperature.
Cell cycle was detected by PI staining and was analyzed on flow cytometry. Indicated cells were seeded in triplicate in 6-well plates. The cells were harvested and washed by PBS for two times. Then the cells were stained by PI and cell cycle was analyzed on flow cytometry.
Apoptosis was detected by PI/Annexin V staining and was analyzed on flow cytometry. UCEC cells were seeded in triplicate in 6-well plates. The cells were trypsinized by EDTA-free trypsin and washed by PBS for two times. Then the cells were stained by PI/Annexin V and apoptosis was analyzed on flow cytometry.
In vivo tumorigenesis
6-week-old immunodeficient nude mice (female, BALB/c) were obtained from Charles River (Beijing, China). Equal number of shCtrl and shKLF7 Hec-1-B cells were subcutaneously implanted into the mice, which were randomly divided into 5 mice per group. Tumors were formed and the mice were euthanatized. Mice were sacrificed by using carbon dioxide euthanasia method according to the protocols. Then tumors were collected for photographing and tumor weighting. All animal experiments were compliant with ethic regulations and approved by the First Hospital of Lanzhou University. The experiments were carried out according to to Institutional Animal Care and Use Committee guidelines of the First Hospital of Lanzhou University.
Migration was assessed by transwell assay. Equal amount of siCtrl, siKLF7-1 and siKLF7-2 Hec-1-B cells in 200 ul FBS-free culture medium were seeded onto the upper layer of transwell chamber. The lower chamber contained 500 ul 10% FBS culture medium. 24 hours later, the cells on the upper layer were removed and the cells on the lower layer were fixed by methyl alcohol and stained by crystal violet. Migrated cells were photographed under the microscope.
Migration was assessed by wound healing assay. The cells were seeded into 6-well plates. After reaching 90% confluence, 200 ul pipettes were used to create wounds with similar breadth in each well. Cell supernatants were removed and fresh culture medium was added into each well. Cells were photographed at 0 hour and 48 hours later under microscope.
Statistical difference was analyzed by GraphPad prism software. Student's t test was applied to compare the difference between two groups and one-way ANOVA analysis was used to analyze the difference among multiple groups. Statistical significance was considered when p<0.05.