Cell culture, plasmids and probes/primers
Human PCa cell lines PC3, Du145, LNCaP and a non-malignant prostate cell line RWPE1 were obtained from the American Type Culture Collection (ATCC) (Manassas, VA) and grown according to ATCC protocol. These human-derived cell lines were authenticated by DNA short-tandem repeat analysis by ATCC. The experiments with cell lines were performed within 6 months of their procurement/resuscitation. Plasmids pEZX-MT01 miRNA 3′UTR target expression clones for Src (HmiT017696-MT01), AKT (HmiT004995-MT01) and miRNA Target clone control vector for pEZX-MT01 (CmiT000001-MT01) (GeneCopoeia, Rockville, MD) were purchased. TaqMan probes for hsa-miR-23b (miR-23b) and negative control pre-miR were purchased from Applied Biosystems (Foster City, CA). siRNA duplexes ((Src (Human)-3 unique 27mer siRNA duplexes (SR304574)) were purchased from Origene (Origene Technologies, Inc., Rockville, MD).
Quantitative real-time PCR
Tissue samples from radical prostectomy were obtained from the Veterans Affairs Medical Center, San Francisco, CA, USA. Total RNA was extracted and assayed for mature miRNAs and mRNAs using the TaqMan MicroRNA Assays and Gene Expression Assays, respectively, in accordance with the manufacturer’s instructions (Applied Biosystems). All RT reactions were run in a 7500 Fast Real Time PCR System (Applied Biosystems). Relative expression was calculated using the comparative Ct.
Methylation analysis of miR-23b by quantitative methylation-specific PCR (qMSP)
To investigate the mechanism involved in reduced levels of miR-23b in PCa we performed methylation analysis in the 1.0 kb upstream sequence of miR-23b. Two CpG islands-CG-1 (−120 to −3bp) and CG-2 (−820 to −650bp) were located in 1.0 kb upstream sequence and further analyzed for methylation status in cell lines and tissue samples. DNA was available only for 38 (19 pairs) of laser captured microdisected tissue samples and these samples were from the same cohort of 118 samples for which RNA expression was available. DNA was bisulfite converted using EZ DNA Methylation-Gold Kit (Zymo Research, Orange, CA, USA) according to the manufacturer’s protocol. The converted DNA was amplified by PCR with 400 pM of either primer set F1/R1, or F2/R2, and HotStar Taq Plus DNA Polymerase (Qiagen, Valencia, CA, USA). PCR was performed by denaturation at 95°C for 5 minutes, followed by 15 cycles of 94°C for 30 seconds, 56°C for 30 seconds and 72°C for 30 seconds. 2μl of the PCR product was added to 40 μl solution containing 20 μl TaqMan Fast Universal PCR Master Mix (2X) (Applied Biosystems), 500 pM primers F1/R1 or F2/R2. The mixed solution was aliquoted evenly into two tubes, and was added 1 μl 5μM probe for methylation reaction (PM) for Methylation (M) reaction and 1μl 5μM probe for unmethylaiton reaction (PU) probe for Unmethylation (U) reaction, respectively. Methylation in CGI-1 and CGI-2 was measured by realtime quantitative PCR with an Applied Biosystems 7500 Fast Sequence Detection. For each sample, the percent of methylation was calculated by the difference of Ct in M reaction (Ct-M) and Ct in U reaction (Ct-U).
In Situ Hybridization
hybridization was performed as described previously (22
). Briefly cell lines and tissues were stained using DIG-labeled locked nucleic acid (LNA)-based probes specific for mir-23b and U6 following the manufacturer’s protocol (Exiqon, Inc Woburn, MA) and detected using anti-DIG-Fluorescein, Fab Fragments (Roche Applied Science, Indianapolis, IN) (for cell lines) and BM Purple AP Substrate (Roche Applied Science) (for tissues). In situ hybridization (ISH) results for tissue array were graded according to quick score (percent cells stained × intensity of stain) and normalized to U6 levels.
Flow cytometry, cell viability, migration, clonability and invasion assays
FACS analysis for cell cycle and apoptosis was done 72 hours post-transfection using nuclear stain DAPI for cell cycle analysis or ANNEXIN V-FITC /7-AAD KIT (Beckman Coulter, Inc. Fullerton, CA) for apoptosis analysis according to the manufacturer’s protocol. Cell viability was determined at 24, 48 and 72 h by using the CellTiter 96 AQueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI) according to the manufacturer’s protocol. For colony formation assay, cells were seeded at low density (1000 cells/plate or 200 cells/plate) and allowed to grow untill visible colonies appeared. Then, cells were stained with Giemsa and colonies were counted. Cytoselect 24-well cell migration and invasion assay kit (Cell Biolabs, Inc) was used for migration and invasion assays according to manufacturer’s protocol.
Immunoblotting and Immunofluorescence
Immunoblotting was performed as described previously (23
). The antibodies used were specific for Src
(2123; Cell Signaling), pSrc
(2101; Cell Signaling), MEK1/2
(4694; Cell Signaling), pMEK1/2(Ser217/221)
(9154; Cell Signaling), p44/42 MAPK (Erk1/2)
(4695; Cell Signaling), p-p44/42 MAPK (Thr2021/Tyr204)
(4370; Cell Signaling), STAT3
(9132; ), p-STAT3 (Tyr705)
(9145; Cell Signaling), Akt
(4685; Cell Signaling), p-Akt(Ser473)
(4060; Cell Signaling), Bad
(9292; Cell Signaling), p-Bad(Ser136)
(4366; Cell Signaling), FAK
(3285; Cell Signaling), c-Jun
(9165; Cell Signaling) and GAPDH
(sc-32233; Santa Cruz Biotechnology, Inc.). Blots were visualized using Western blotting luminal reagent (sc-2048; Santa Cruz Biotechnology, Inc.).
For immunofluorescence, cells were transfected with precursors of miR-23b or cont-miR for 72 hours, washed and fixed with acetone-methanol (1:1) mixture and hybridized with the specific primary antibodies against EMT markers. Cells were washed and hybridized with fluorescein conjugated secondary antibody (1:1000) and mounted with ProLong Gold antifade reagent with DAPI (Invitrogen-Life Technologies).
The complimentary sites in 3′UTR of Src and Akt for miR-23b and mutated sequences are given in Supplemental Table 7
. The Src, Akt and control vectors were purchased from GeneCopoeia and named Src-3′UTR and Akt 3′UTR. Mutated 3′UTR sequences of Src and Akt were cloned and named Mut Src3′UTR and Mut Akt3′UTR. For reporter assays, cells were transiently transfected with wild-type or mutated reporter plasmid and miR-23b or control-miR. Firefly luciferase activities were measured using the Dual Luciferase Assay (Promega, Madison, WI) 18 hr after transfection and the results were normalized with Renilla luciferase. Each reporter plasmid was transfected at least three times and each sample was assayed in triplicate.
In vivo intratumoral delivery of miR-23b and genistein
The antitumor effect of miR-23b was determined by local administration of miR-23b precursor in established tumors. Each mouse was injected sub-cutaneously with 5.0×106 PC3 prostate cancer cells. Once palpable tumors developed (average volume 55mm3), 6.25 μg of synthetic miRNA complexed with 1.6 μl siPORT Amine transfection reagent (Ambion, Austin, TX) in 50 μl PBS was delivered eight times intratumorally at 3-day intervals. Tumor growth was followed for 21 days from the first injection. All animal care was in accordance with the institutional guidelines.
Statistical analyses were performed with StatView for Windows (SAS Institute Inc. NC, USA), GraphPad-Pris-5 and MedCalc. All quantified data represents average of at least triplicate samples or as indicated. Error bars represent standard deviation of mean. All tests were performed two tailed and p-values <0.05 were considered statistically significant. Receiver operating curves (ROC) were calculated to determine potential of miR-23b or its methylation to discriminate between malignant and non-malignant samples. Chi-square tests were performed to determine correlation between miR-23b expression and clinicopathological characteristics. For disease progression analysis Kaplan-Meier approach (log-rank test) and Multiple Regression analysis were performed.