Patient selection and human tissues
Group 1 (for miRNA array)
Three patients have been clarified as stage IIB-IVA according to International Federation of Gynecology and Obstetrics (FIGO),, who had received radiotherapy in our hospital between 2000 and 2008. According to the 2013 NCCN cervical cancer clinical practice guidelines, patients received standard radiotherapy, pelvic radiotherapy and branch radiotherapy, with a total dose of 70 Gy at point A . Two pathologists evaluated histology independently. According to the hospital's internal review and ethics committee, all samples were collected with informed consent.
Group 2 for QPCR. 84 cervical tumors were obtained from our department, between January 2002 and June 2011. None of themreceived any treatment before surgery. All patients have been confirmed HPV infection. Two patients without HPV infection were chosen as normal controls.
Mirnas Real Time Polymerase Chain Reaction Array
After isolation total RNA from samples, Universal cDNA synthesis kit (Exiqon) was used. MiRNAs were detected by the miRCURY LNA™ Universal real time microRNA polymerase chain reaction system(Exiqon, kangchen, China).
HeLa and SiHa were purchased from China Academic Sinica Cell Repository, Shanghai, China. They were in MEM medium(Gibco, Los Angeles, CA, USA) with 10% fetal bovine serum (Gibco,Australia)in an incubator under humidity at 37℃with 5% CO2.
Mir-100-5p mimics purchased from Genepharma (Shanghai, China), were transient transfected into cells with Lipofectamine™ 2000 (Invitrogen). Mock-transfected condition was made by negative control mimics with fluorescence. All the cells were collected at 48h post-transfection, and the levels of mir-100 were elevated by RT-PCR assay as follow. The sequences of miR-100-5p mimics and negative control were shown below:
Negative control(forward 5’-UUCUCCGAACGUGUCACGUTT -3’
Reverse 5’-ACGUGACACGUUCGGAGAATT -3’);
Mirna Expression Analysis And Rt-pcr Assay
The RNAs were collected from cells by Trizol extraction (Invitrogen), and cDNAs were acquired using an RNA reverse transcription amplification kit (Takara). RT-PCR assays were performed by SYBR Green Real-time PCR Universal Reagent (GenePharma Co.,Ltd.) and analyzed by BIO-RAD fluorescence quantitative PCR machine. Primers were as follows:
u6 was used as control for miRNA, GAPDH was used as control for mRNA. Both of them was calculated with the 2 − ΔΔCT method.
After cultured (1×104 cells/well) in separate 96-well plates for 24h, cells were transfected with miR-100-5p mimics. At day 1, day 2, day 3, day 4 after transfection, cells activation were evaluated by Cell Counting Kit-8 assay, the absorbance were read at 450 nm.
Cell Apoptosis Assay
After digestion with EDTA-free trypsin, incubation with Annexin-V and PI for 10 min at20°C, flow cytometry was performed (FACSCalibur, Becton Dickinson, Franklin Lakes, NJ, USA)
Siha cells (2×106) were transfected with miR-100 or Negative Control for 2 days following animal experiments institutional ethical guidelines. Cells were digested and resuspended in 200µL PBS .PBS with cells or without cells were injected into female BALB/c athymic nude mouse(four-week-old). Six nude mice were used. Mice were anesthetized with Isoflurane (Forene, Abbott GmbH & Co.KG, Wiesbaden, Germany) with 1 L*min-1 O2 flow for euthanasia.
miRanda,TargetScan were chosen for predicting targetgene.
After embedded by paraffin- and fixed with formalin, tissue was cut into 5µm section, deparaffinization, rehydration, quenche, and retrieve.Incubation at 4°C overnight with Anti-mTOR (phospho S2448) antibody (HRP) (ab196914) (Abcam).
Establishment Of Radioresistant (Rr) Cell Lines
Cells were exposed to two Gy 3 times, four Gy 3 times, six Gy 3 times, eight Gy 2 times and ten Gy 2 times at 300 cGy/min with a linear accelerator (SIMEN, Germany). [16–17].
HeLa and SiHa cells(3×105cells/well) were cultured in 6-well plates overnight, then 10µM DAC(5-aza-2-deoxycytidine) or 10µM DMSO (dimethyl sulfoxide) was separately added to cells per 24h. Cells were harvested after 5 days, and RNAs were extracted for detecting the level of mir-100 as mentioned above.
Bisulfite Sequencing Pcr(Bsp)
Following extraction ( QIAamp DNA Mini Kit),Genomic DNA was subjected to EpiTect® Plus DNA Bisulfite Kit. After purifirationt, modified DNA was chosen as nested PCR reactions template. The primers were: outer primers, 5’-ATTCGAATTTAGTGGAATTAGAATC-3′ (forward) and 5′-AACCTACAACAACAACAACAACG-3′(reverse); nested primers, 5′-TTAGTA ATTTTAGGTTAGAGGGTTATCG-3′ (forward) and 5′-ACTCCAAAAACCC ATAACTAACCG-3′ (reverse). The second-round PCR products were subjected to electrophoresis on agarose gels, excised, and inserted into the TOPO cloning vector (Invitrogen, K4600-01).Clones were used for DNA sequencing randomly.
Protein was extracted from cells with ice-cold radioimmunoprecipitation lysis solution, then centrifugated to remove cell debris. N-cadherin (ab18203) and E-cadherin (ab194982) from Abcam(Cambridge, MA, USA) were chosen as primary antibodies and HRP-conjugated secondary antibody was purchased from Sangon (Shanghai, China).GAPDH and β-actin were employed as loading controls.
CircRNA expression profiles in CC
We enriched the contained circRNAs and digested them with RNase A, then reversely transcribed them to RNA by fluorescent reagents and random primers. We used Human circRNA Arrays (8 × 15 K, Arraystar, Rockville, MD, USA) to determine the circRNA profile. Those circRNAs with fold change ≥ 2 were identified as differentially expression circRNAs. The data were analyzed by using R software limma and Arraystar program (Arraystar).
Biotin-coupled probe pull-down assay
Hela and Siha cells were transfected by biotinylated miR-100-5p mimics or its mutant (Genepharma, Suzhou, China). Then cells underwent harvest, lysis, sonicate, and incubation with magnetic beads at 4℃ overnight. The RNA mix bound to the magnetic beads was eluted and treated with Trizol for further qRT-PCR.
Florescent in situ hybridization
Fluorescent in situ hybridization probes for circCASC15, miR-100, and 18S RNA were designed and synthesized by Synbio Technologies (Suzhou, China). 18S RNA was used as a positive control. The signals of the probe were examined by the Florescent in Situ Hybridization Kit (RiboBio, Guangzhou, China).
All data were based onthree independent replicates. Statistical analyses were analyzed by SPSS 13.0. Data were presented as the mean values ± standard error. P<0.05 was considered statistically significant.