Agents
Primers were ordered from Genewiz Company (Beijing, China). Antibodies, including anti-CENPF (Ag29688), anti-GAPDH (10494–1-AP, 1:5,000), and HRP sheep anti-rabbit/mouse (1:5,000) were ordered from PTG Company (Bellevue, WA, USA); Active-Caspase3 (#ab32042, 1:1,000), Bcl-2 (ab32124, 1:1,000), Bax (ab32503, 1:1,000), Cyclin D1 (ab134175, 1:1,000), p-AKT (ab38449, 1:1,000), and p-mTOR (ab109268, 1:1,000) were purchased from Abcam (Cambridge, United Kingdom).
Cell culture and transfection
Human MM cell lines (A375 and M14) were purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Science (Shanghai, China) and were cultured in 10% fetal bovine serum (FBS), streptomycin (100 μg/mL), and penicillin (100 U/mL) DMEM medium at 37℃ with 5% CO2. The cells were divided into three groups at the logarithmic growth stage, then transfected with miR-383-5p mimics (miR-383-5p), miR-NC (NC) and miR-383-5p mimics+pcDNA 3.1-CENPF (miR-383-5p+CENPF) by using Lipofectamine 2000 (TMO, Waltham, MA, USA) according to the manufacturer’s guidelines. miR-383-5p mimics sequence is 5'-AGAUCAGAAGGUGAUUGUGGCU-3'.
Reverse transcription quantitative PCR (qPCR)
Ultrapure RNA Kit (CwBio, Beijing, China) was performed to isolate the total RNA after 48 h of transfection and cDNA was synthesized using HiFiScript cDNA Synthesis Kit (CwBio, Beijing, China). The expression level of miR-383-5p was calculated by 2−△Ct method.
Reverse transcription primer of miR-383-5p: 5'-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACAGCCAC-3'.
The PCR primers for miR-383-5p are listed as follows:
Forward: 5'-GGGAGATCAGAAGGTGATTGTGGCT-3'
Reverse: 5'-CAGTGCGTGTCGTGGAGT-3'
The primer sequence of U6 as internal reference is as follows:
Forward: 5'-CTCGCTTCGGCAGCACA-3'
Reverse: 5'-AACGCTTCACGAATTTGCGT-3'
Cell growth assays
Cell Counting Kit-8 (CCK8) (Solarbio, Beijing, China) assay and colony formation assay were carried out to evaluate the proliferation and viability ability of A375 and M14 cells. About 3000 cells were seeded into each well of 96-well-plate. OD values were detected every 24 hours. 10 μl CCK8 solution was added to the well two hours before the detection. Then the growth carve was drawn according the OD values. For the colony formation assay, 500 cells were planted in each well of a 6-well plate, and were cultured for 10 days. Then colonies were fixed with 10% neutral formalin for 1 h and dyed with crystal violet (Beyotime, Haimen, China). The cells were photographed under a microscope (Olympus, Tokyo, Japan).
Cell apoptosis assay
Flow cytometer (BD FACSC anto II, BD Biosciences, San Jose, CA, USA) was used to detect the apoptosis of A375 and M14 cells. After 24 hours of transfection, cells were culture in serum-free medium for 24 hours and collected. The apoptosis rate was analyzed by annexin V/FITC (4A Biotech Company, Beijing, China) according to the instructions. Annexin V/FITC and propidium iodide were used to evaluate the percentage of apoptosis. The flow results were analyzed and processed by Flowjo software.
Cell invasion and migration assays
The invasion and migration ability of A375 and M14 cells were evaluated with transwell chamber (Millipore, Billerica, MA, USA). For invasion assay, the frozen matrigel (356234, BD, Franklin Lakes, NJ, USA) was diluted to 1:6 in serum-free medium, and then the 40 μl matrigel was applied to the upper chamber. The 150 μl serum-free medium and 1×104 cells were added to the upper chamber, 500 μl complete medium was added to the lower chamber. After 24 hours of incubation, the surface of each upper chamber was wiped gently with a cotton swab. The cells of lower chamber were washed with PBS and fixed with 4 % PFA for 30 min, then stained with 0.1 % crystal violet for 20 min. For migration assay, there are no matrigel coating and the rest was the same as the invasion assay.
Luciferase reporter assay
Based on bioinformatics, we predicted a complementary relationship between miR-383-5p and 3'UTR of CENPF mRNA. The mutation vector of CENPF 3'UTR was constructed by point mutation. miR-383-5p mimics and CENPF 3'UTR wild type (WT), miR-NC and CENPF 3'UTR wild type (WT), miR-383-5p mimics and mutant vector (Mut), miR-NC and mutant vector (Mut) were transfected into A375 and M14 respectively. 48 hours later, the cells were collected and the luciferase activity was detected with the doul-luciferase reporter gene assay kit (Beyotime, China) according to the instructions.
Western blot assays
The protein of cells was extracted with RIPA (protease inhibitor, CwBio, Beijing, China) buffer. 20 μg protein samples were added into each lane containing 10% SDS-PAGE gel. After transfer, the polyvinylidene fluoride (PVDF) membrane was sealed with 5 % skimmed milk for 1 hour and probed with primary and secondary antibodies. After washing the membrane, ECL developer (B500024, Proteintech, Chicago, IL, USA) was added and Quantity One Software was used to assess the results.
Statistical analyses
The data were analyzed using SPSS software. Each experiment was repeated at least three times. The results were presented as mean ± SD. Differences between two groups were tested by Student's t-test, differences among more than two groups were tested by one-way ANOVA. P<0.05 was considered significant difference.