Hsa-miR-125a Facilitates Cell Apoptosis Via Wild Type P53 And Bcl-w

IntroductionMicroRNAs(miRNA) are an extensive class of non-coding endogenous RNAs, about 21-24 nucleotides in length. miRNA through completely complementary or partially complementary way recognition target gene 3'-UTR, degrades target mRNA directly or suppresses its translation to inhibit translation gene expression, and regulates target cell apoptosis, proliferation and differentiation. Therefore, miRNA is playing the vital role in the tumor occurrence and the development.MiR-125a is a member of the miRNA family, there are two kinds of precursors and mature forms of body, its mature body with biological activity. At present discovered that it has two kinds of mature body miR-125a-3p and miR-125a-5p.Our group discovered that miR-125a-3p/5p were lower expression in lung cancer cells compared with HBE. Transfection of sense miR-125a-3p/5p can increase miR-125a-3p/5p content in A549 cells and promote A549 cells apoptosis,and the expression of wild-type p53 also will increase. While transfection of antisense miR-125a-3p/5p can make the result opposite,but its regulation mechanism is unclear. So we believe that miR-125a-3p/5p may be through the regulation of wild-type p53 to promote apoptosis in A549 cells.In the miRNA database through the target gene prediction was found that cancer gene MDM2 was miR-125a-3p/5p common target gene,it can inhibit wild-type p53 expression. Bcl-w may be miR-125a-3p/5p common target gene, Bcl-2 may be only the miR-125a-5p target genes. p53 is currently studied quite clearly of apoptosis pathway; Bcl-w, Bcl-2 are Bcl-2 family members, both for apoptosis inhibitory factor. Therefore, we speculate that miR-125a-3p/5p may be through MDM2/p53, Bcl-w, Bcl-2 to regulate lung cancer cells apoptosis. The experiment will through transfection sense or antisense miR-125a-3p/5p to increase or decrease its level in cells, study of lung cancer cells MDM2/p53, Bcl-w, Bcl-2 effect, further reveals the regulation for the molecular mechanism of apoptosis, for the treatment of lung cancer provides a new way.Materials and Methods1.Cell linesHuman lung adenocarcinomal cell lines A549,H1299 Mature miRNA sequences:sense-miR-125a-3p:5'-ACA GGU GAG GUU CUU GGG AGC C-3';antisense-miR-125a-3p:5'-GGC UCC CAA GAA CC U CAC CUG U-3';scramble-miR-1:5'-GGU CGG UGC UCG AUG CAG GUA A-3';sense-miR-125a-5p:5'-UCC CUG AGA CCC UUU AAC CUG UG-3';antisense-miR-125a-5p:5'-CAC AGG UUA AAG GGU CUC AGG GA-3';scramble-miR-2:5'-GGA CGG CGA UCA GAU AAG AGU UC-3';Every sequence contains 2'-oMe modifications at every base without fluorescencent marker.2.Cell cultureCells werecultured in DMEM or RPMI-1640 containing 10%FBS at 37℃,5% CO2.3.Real time PCRTotal RNA was extracted using TRIZOL and reverse transcribed using TaqMan(?) MicroRNA Reverse Transcription Kit.Real-time PCR were performed using TaqMan(?) MicroRNA Assays HAS-MIR-125A-3P/5P and U18 as an internal standard according to a standard TaqMan(?) PCR protocol on an Applied Biosystems 7900HT Fast Real-Time PCR System.All reations were run in triplicate.4.Transient transfection Cells in subcontiguous condition were transfected with sense-mir-125a-3p/5p (20μM),antisense-mir-125a-3p/5p(20μM)and scramble-mir-125a-3p/5p(20μM)with appropriate LipofectamineTM 2000 according to the manufacturer's instruction.Cells were cultured in37℃,5%CO2 for 24 to 72 hours according the need of the assay.5.Reverse Transcription-Polymerase Chain Reaction(RT-PCR)Total RNA was isolated from cells in the logarithmic growth phase using TRIZOL(Invitrogen). RT-PCR procedures followed the protocol(takara).6.Western blotProteins were detected with coomassie brilliant blue method.And proteins were visualized by Super ECL Plus Reagent according to the manufacturer's protocol,β-actin as an internal control.7.Flow cytometry(Annexin V-FITC)Every group was divided into four subgroups:blank control,only Annexin V,only FITC and Annexin V+FITC.Flow cytometry performed on BD FACSaliburTM Flow Cytometer.8.Statistical analysisSPSS13.0 software was employed to analysis the data using t test.Results1.Transfected with sense or antisense miR-125a-3p/5p in A549 cells miR-125a-3p/5p expression level and apoptosis influence.In the A549 cells transfected with sense or antisense miR-125a-3p/5p, with scramble group and idling group for the control group,24h after transfection with Real-time PCR to detect expression of miR-125a-3p/5p and cells apoptosis using flow cytometry. The results showed that:sense miR-125a-3p/5p transfected cells in miR-125a-3p/5p expression and cells apoptosis rate were higher than scramble group and idle group (P<0.01); antisense miR-125a-3p/5p transfected cells in miR-125a-3p/5p expression and cells apoptosis rate were lower than scramble group and idle group (P<0.01); scramble group and idle group compared to untreated group, miR-125a-3p/5p expression and cells apoptosis rate have no significant change (P> 0.05).2.Transfected with sense or antisense miR-125a-3p/5p in A549 cells the expression of MDM2, wild-type p53, Bcl-w and Bcl-2In the A549 cells transfected with sense or antisense miR-125a-3p/5p 24h,though RT-PCR and Western Blot to detect of MDM2, wild-type p53, Bcl-w and Bcl-2 mRNA and protein expression, The results showed that:transfection with sene miR-125a-3p/5p increased p53 mRNA and protein expression, Bcl-w mRNA and protein expression decreased, MDM2 mRNA remained unchanged and protein decreased, Bcl-2 mRNA and protein did not change; but transfection with antisense miR-125a-3p/5p decreased p53 mRNA and protein expression,Bcl-w mRNA and protein expression increased, MDM2 mRNA remained unchanged and protein expression increased, Bcl-2 mRNA and protein remained unchanged.3.After blocking p53 or Bcl-w, miR-125a-3p/5p on the A549 cells apoptosis influenceIn order to clarify whether miR-125a-3p/5p through p53 and/or Bcl-w to regulate apoptosis, while transfected with sense miR-125a-3p/5p and blocking with a monoclonal antibody of wild-type p53 24h; and transfected with antisense miR-125a-3p/5p and blocking with a monoclonal antibody Bcl-w 24h, then detect apoptosis by flow cytometry. The results showed that:the apoptosis inhibition of blocking the wild-type p53 for sense miR-125a-5p is more than miR-125a-3p(P<0.05); the influence of Blocking Bcl-w for antisense miR-125a-3p is more than antisense miR-125a-5p(P<0.05).We also chose p53-negative lung cancer cell lines H1299, transfected sense and antisense miR-125a-3p/5p 24h, using flow cytometry to detect cell apoptosis. The results showed that:the cells apoptosis induced by sense miR-125a-3p is higher than sense miR-125a-5p(P<0.01); and the cells apoptosis inhibited by antisense miR-125a-3p is more than antisense miR-125a-5p(P<0.05).Conclusion1.MiR-125a-3p/5p facilitate cells apoptosis via inhibit MDM2, upregulate the wild type p53, inhibit Bcl-w.2.MiR-125a-5p primarily through upregulate the wild type p53 to promote cells apoptosis,but miR-125a-3p mainly through inhibition of Bcl-w to promote cells apoptosis.