Cell culture. The RPE cell line ARPE-19 was obtained from the ATCC (Manassas, VA). And these cells were cultured in the RPMI-1640 medium (Gibco, Gaithersburg, MD, USA) which was added with 10% fetal bovine serum (Gibco, USA). These cells were placed in the 37℃ humid atmosphere with 5% CO2. LPS and ripasudil were added in the culture medium to simulate and treat the RPE cells. The inhibitor of the miR-136-5p (Ribbio, Guangzhou China) was transfected with the lipo-2000. All the operations of the experiment were carried out according to the instructions.
Luciferase reporter assay. 1×105 cells were plated into the 24 well plates. After the attachment of these cells, 0.1µg reporter vector with 3’UTR-ROCK1 or mutant-3’UTR-ROCK1 (Genechem, China), 0.02µg Renilla luciferase vector (Genechem, China) and the miR-136-5p inhibitor or the NC were contransfected into these cells. The targeting effect of miR-136-5p on the ROCK2 was detected in the same way. After 48 hours, the luciferase activity was measured. All the operations in this experiment followed the manufacturer’s recommendations.
Western-blotting. Proteins of these cells were lysed and collected with RIPA (Beyotime, China). Next, the concentration of these proteins was determined with the BCA (Beyotime, China) method. Then these proteins were separated with the 10% SDS-PAGE gel (Beyotime, China). Next, the proteins were transferred to the PVDF membranes (Millipore, USA). The PVDF membranes were blocked with the 5% skim milk powder for 1.5 h. Then the membranes were incubated with the primary antibody at 4℃ overnight. The primary antibodies were ROCK1 (#4035, CST), ROCK2 (#9029, CST), Bcl-2 (#4223, CST), Bax (#5023, CST), Cleaved-caspase3 (#9664, CST), Cleaved-caspase9 (#20750, CST), caspase-3(#14220, CST), caspase-9 (#9502, CST), NLRP-3 (#15101, CST), ASC (#13833, CST), Caspase-1 (#24232, CST), IL-1β (#12703, CST), IL-18 (Abcam, ab191152) and p-65 NF-κB (Abcam, ab16502). On the second day, the membranes were washed with PBST three times. Next, the membranes were incubated with the second antibody (Abcam, ab205718). After that the membranes were washed three times with PBST. Final, the membranes were exposed and the band was observed to clarify the changes of expression of these proteins.
RT-PCR. Total RNA was extracted from the cells by the trizol methods. After that, the mRNA was reverse transcribed into cDNA by the RT reagent Kit (TakaRa, Japan). RT-PCR was performed with the SYBR Green method. The amplification process was performed with the ABI 7500 system (Applied Biosystems, USA). The relative expression of the target genes was calculated by the 2-∆∆Ct method. The following primers were used in this project: miR-136-5p forward primer 5′-ACACTCCAGCTGGGACTCCATTTGTTTT-3′; reverse primer 5′-CCAGTGCAGGGTCCGAGGT-3′; GAPDH forward primer 5′-AATTCCATGGCACCGTCAAG-3′; reverse primer 5′-TGGACTCCACGACGTACTC-3′.
CCK-8 assay. CCK-8 assays were used to detect cell viability after the stimulation of the LPS. The cells were plated into the 96 well plate. After that, the cells were cultured with the medium containing LPS for 24 hours. Then the CCK-8 (Dojindo, Japan) was diluted with the cultured medium and added into the 96 well plate. The 96 well plate was incubated in the incubator for two hours and finally the absorbance was measured with the microplate Reader.
ELISA assay. To determine the changes of TNF-α, IL-6 and IL-1β releasing of RPE cells in response to different kinds of stimulation, the levels of TNF-α, IL-6 and IL-1β in the culture supernatant were determined by the ELISA assay. The ELISA kit was obtained from Abcam and Sigma-Aldrich: TNF-α (Abcam, ab181421), IL-6 (Abcam, ab178013) and IL-1β (Sigma-Aldrich, RAB0273). All the operation of this experiment was carried out according to the instructions.
Apoptosis assay. The different groups of RPE cells were seeded into the six well plates and treated with LPS or ripasudil. After that, these cells were washed with the cold PBS for three times. Then the binding buffer (Beyotime, China) was used to suspend these cells. Next, PI and Annexin V (Beyotime, China) were added into the binding buffer to detect the apoptosis ratios of the RPE cells. The cells were incubated with the PI and Annexin V for 40 minutes. At last, the apoptosis ratios of these cells were analyzed with the flow cytometry (Beckman, USA).
Statistical analysis. The analysis of the data in this paper was performed with the GraphPad Prism 7.0. The comparison between different groups was evaluated by the Student’s t test. All the data in this research were presented as mean ± SD and all the experiments in this paper were repeated three times. The difference between diverse groups was considered as statistically significant difference when the value of P was less than 0.05.