| Part One BRAF gene mutation in ameloblastomaObjective: Ameloblastoma is a frequently odontogenic epithelial tumor, involving in mandibular and maxillary bones as well as gingiva. V-raf murine sarcoma viral oncogene homolog B1(BRAF) is one of the most important human proto-oncogene, but it is less known the relationship between the molecular pathology of ameloblastoma and its biologic behaviour. In the present study, modified high performance liquid chromatography analysis(DHPLC) and DNA sequencing analysis were used to detect BRAF gene mutation in ameloblastoma, and thus to discuss the relationship between BRAF gene mutation and the clinical pathology and biological behavior of ameloblastoma, in order to provide an important reference to the molecular pathology and targeted therapy.Methods: 1 Tumor specimens and clinical data: Speciments were surgically removed from 30 cases of ameloblastoma patients. Some specimens were fixed in 10% buffered formalin and embedded in paraffin. The tissue blocks were sliced into 4-?m-thick sections for pathological diagnosis. The left specimens were immediately frozen in liquid nitrogen, and stored in-80?C refrigerator. All the cases had complete clinical data, with 10 cases of normal gingival mucosa being included in this study as the normal control. The study was approved by the local ethics committee. 2 DNA extraction: Genomic DNA was extracted by using a QIAamp DNA Mini Kit according to the manufacturer's instructions. 3 The mismatch of primers(BRAF M) was designed according to the BRAF V600 E mutation, which contained a mismatch locus on 3'-terminal, and wild type DNA can't be amplified. Thromboxane A synthase 1(TBXAS1) gene was taken as control for DHPLC detection, which differentiated from BRAF V600 E mutation product size. Exon 15 primers of BRAF gene(BRAF N) were also designed for Sanger sequencing analysis. 4 Polymerase chain reaction(PCR) amplification: Multiple PCR reaction system was as follows: BRAF gene and TBXAS1 were amplified in a multiple PCR system. Go Taq green Master Mix 12.5?l, BRAF M primer F 0.5?l(0.2?M), BRAF M primer R 0.5?l(0.2?M), TBXAS1 primer F 0.5?l(0.2?M), TBXAS1 primer R 0.5?l(0.2?M), DNA template 1?l(50ng), and nuclease-free H2 O 9.5?l. The reaction was amplified with a programmable thermocycler by using the following protocol: pre-denaturation at 95?C for 5 min followed by 30 cycles at 94?C for 30 s, 54?C for 30 s, 72?C for 30 s and a final extension at 72?C for 10 min. Conventional PCR reaction system was as follows: Go Taq green Master Mix 25?l, BRAF N primer F 1.5?l(0.3?M), BRAF N primer R 1.5?l(0.3?M), DNA template 5?l(250ng), and nuclease-free H2 O 17?l. The PCR circulations were as follows: predenaturation 2 min at 95?C, 95?C for 30 s, 58?C for 30 s, 72?C for 30 s, 30 cycles, and a final extension at 72?C for 10 min. The PCR products were loaded in 2% agarose gel electrophoresis for 2.5 hours at 60 V. 5 DHPLC detection: PCR products were denatured at 95?C for 10 min, followed by slow reduction(at a rate of-0.5?C/20s) to room temperature. The PCR product 5?l was then injected into DNASep? of the WAVEDNA Fragment Analysis System(Transgenomic, Omaha, NE, USA) and equilibrated by 0.1mol/L triethylammonium acetate in a DHPLC column. DNA was removed from the column at a constant flow rate of 0.9ml/min by a linear acetonitrile gradient, achieved by mixing buffer A with buffer B. Buffer A and B contained 0.1M triethylammonium acetate and 25% acetonitrile, respectively. The temperature for separation of the V600 E heteroduplex from the homoduplex was 50?C. The eluted DNA was detected spectrophotometrically at a UV absorbance of 260 nm within 10 min. All results were analyzed by using WAVEMAKERTM software(Transgenomic). 6 DNA sequencing: The PCR products were purified by using the SK1131 Gel Extraction kit(Sangon Biotech Co, Ltd. Shanghai) according to the manufacturer's instruction. Mixed 1?l PCR gel recycling product, 1?l Big Dye Mix, 1?l sequencing primer, and 2?l nuclease-free H2 O were amplified as follows: predenaturation 2 min at 98?C, 96?C for 10 s, 50?C for 5s, 60?C for 4 min, 25 cycles and ended at 4?C. The products were purified with 25?l sodium acetate/ethanol, votexing, put on ice for 10 min, and centrifuging 12000 rpm 30 min at 4?C, and the supernatant was discarded, followed by 50?l 70% ethanol washing of sediment twice, centrifuging 12000 rpm at 4?C for 5 min, supernatant was discarded, and vacuum drying for 15 min. Then 12?l TSR was added, votexed thoroughly to dissolve the DNA sediment, after centrifuging briefly, the sample was heated to 95?C for 2 min, snap-cooled on ice and loaded onto the sequencing gel. An automated DNA electrophoresis system was used to detect and analyze the sequencing ladder. Following the loading of samples, electrophoresis was carried out at 1.2 k V constant voltage 25 min, 7.5 k V 2h. The PCR products were sequenced directly from both forward and reverse directions. Data collection and image analysis were performed by using the software supplied with the model 3730 DNA sequencer. 7 Statistical analysis: Fisher's exact test was used to analyze the relationship between BRAFV600 E mutation status and clinicopathological features, and a P value<0.05 was considered significant. Statistical analysis was performed with SPSS 19.0(SPSS, Inc., Chicago, IL).Results:1 Clinicopathological features: A total of 30 cases of ameloblastoma were olled in the study, including 16 males and 14 females, age ranged from 15 to 60 year old, with an average age of 33.8. A total of 27 cases were of mandible, 3 cases of maxilla; primary cases were 19, and recurrent cases 11. Histolgically, the 30 cases of ameloblastoma were included 10 of follicular type, 9 of plexiform types, and 11 of unicystic type. Seven of 30 cases(23.3%) showed tumor cell concentrative and actively proliferative, and 5 cases(16.7%) showed tumor cell intra-capsular invasion. 2 The same band was detected in the PCR amplification products of ameloblastoma and gingiva by electrophoresis gel. The curve V600 E BRAF gene mutation showed two elution peaks with DHPLC: the first peak was TBXAS1 gene amplification product, 100 bp, and the second was BRAF mutated gene amplification product, 126 bp, besides, positive control and negative control were set. BRAF V600 E mutation was detected in 18 out of the 30 cases(60%) by DHPLC. 3 A DNA sequence was obtained from all specimens by Sanger sequencing. Out of the 30 ameloblastoma specimens, 18 cases were detected the V600 E mutaton. The wild type was found in the rest of the cases, none of which was detected the V600 E mutation in normal gingiva. The BRAFV600 E mutation is a T1799 A transversion that results in the substitution of valine by glutamic acid at codon 600(V600E). With Poly Phen2 and SIFT software the mutation was predicted to probably damage the function of BRAF protein. 4 The correlation between BRAF V600 E mutation and clinicopathological features: No statistically significant correlation was found between V600 E mutation and age, gender, location, histologic type, capsule invasion of the tumor. However, V600 E mutation was closely related with active proliferation and clinical recurrence. BRAF mutation rate of the active proliferation(100%) was significantly higher than that of non-active proliferation(47.8%)(P<0.05). BRAF mutation rate in clinical recurrence(90.9%) was significantly higher than that of non-recurrence(42.1%)(P<0.05).Part Two SMO gene mutation in ameloblastomaObjective: Ameloblastoma is a benign odontogenic neoplasm but locally infiltrative with high risk of recurrence. Sonic hedgehog(SHH) signaling pathway plays a critical role in tooth development. And smoothened(SMO) gene was considered as the signal converter in SHH pathways. In the present study, polymerase chain reaction-single strand conformation polymorphism and DNA sequencing analysis were used to detect SMO gene mutation in ameloblastoma, thus to discuss the relationship between SMO gene mutation and the clinical pathology and biological behavior of ameloblastoma.Methods: 1 Tumor specimens and clinical data were the same as Part One. 2 DNA extraction: Genomic DNA was extracted by using a QIAamp DNA Mini Kit according to the manufacturer's instructions. 3 The 12 exons of SMO gene primers were designed according to the previous report and Primer Premier 5.0(Premier Biosoft Inc., Canada). 4 PCR amplification: Go Taq green Master Mix 25?l, SMO N primer F 1.5?l(0.3?M), SMO N primer R 1.5?l(0.3?M), DNA template 5?l(250ng), and nuclease-free H2 O 17?l. The PCR circulations are as follows: predenaturation 2 min at 95?C, 95?C for 30 s, 58?C for 30 s, 72?C for 30 s, 30 cycles, and a final extension at 72?C for 10 min. The PCR products were loaded in 2% agarose gel electrophoresis for 2.5 hours at 60 V. 5 PCR-SSCP detection: PCR product 5?l was mixed with SSCP loading buffer dye 5?l(95% formamide, 0.05% xylene cyanole FF, 0.05% bromophenol blue, 0.5M EDTA). The mixture was denatured at 95?C for 10 min and snap-frozen on ice for 5 min until loaded onto 10% polyacrylamide gels. Gels were run at 250 V for 5 min and then 60 V for 12 hours in a buffer containing 1×TBE. After electrophoresis, the gels were stained by silver nitrate. Silver Staining for SSCP consisted of fixation in 10% alcohol and 0.5% glacial acetic acid for 6 min, washing twice with distilled water for 10 s, 0.1% silver reaction for 20 min, washing 5 times with distilled water for less than 1 min, developing(0.4% formaldehyde, 10%, 1.5% sodium hydroxide) for 10 min, stopping in 0.75% sodium carbonate for 10 min, and DNA bands could be visualized on a light box. 6 DNA sequencing. 7 Statistical analysis: Fisher's exact test was used to analyze the relationship between SMO mutation status and clinicopathological features, and a P value<0.05 was considered significant. Statistical analysis was performed with SPSS 19.0(SPSS, Inc., Chicago, IL).Results:1 Clinicopathological features were the same as Part One. 2 The same band was detected in the PCR amplification products of ameloblastoma and gingiva by electrophoresis gel. Of the 30 cases, 9 cases abnormal SSCP migration bands were detected in exon 3, 10 cases in exon 5, 12 cases in exon 6, 5 cases in exon 10. There was no altered SSCP band detected in other exons and normal gingiva. 3 A DNA sequence was obtained from all specimens by Sanger sequencing. A synonymous mutation from A to G was located at nucleotide position 808 of exon 2, and did not result in an amino acid change at E176 E. E194 E mutation from A to G was detected in 9 cases, and located at nucleotide position 862 of exon 3. Of the 11 mutations in exon 5, 5 cases were identified missense mutation from C to A, position1371, T364 N, and 6 cases synonymous mutation from G to A, position 1417, A379 A. A total of 12 mutations from A to G were located at nucleotide position 1444 of exon 6, and did not result in an amino acid change at G383 G. In exon 10, 6 cases were identified missense mutation from G to C, position2049, S590 T. The T364 N mutation was predicted to possibly damage the function of SMO protein with Poly Phen2 and SIFT software, while S590 T probably damaged SMO protein function with Poly Phen2 but tolerated with SIFT. 4 The correlation between SMO mutation and clinicopathological features: No statistically significant correlation was found between SMO mutation and gender, location, histologic type or clinical recurrence of patients. However SMO mutation was closely related with age, active proliferation and capsule invasion. SMO mutation rate of the 20 years old or younger(100%) was significantly higher than that of over 20 years old(29.6%)(P<0.05). SMO mutation rate of the active proliferation(85.7%) was significantly higher than that of non-active proliferation(25.7%)(P<0.05). SMO mutation rate in capsule invasion(80.0%) was significantly higher than that of non-capsule invasion(28.0%)(P<0.05).Part Three RECK gene mutation and protein expression in ameloblastomaObjective: Multiple factors and multiple stages were involved in the tumourigenesis and progressive process of tumors. MMP-9 promotes tumor invasion and progression by destroying the extracelluar matrix. RECK gene, as a new tumor suppressor gene, has been highlighted because of its correlation with metastasis and invasiveness by inhibiting MMP-9. The aim of this study was to detect RECK gene mutation by DNA sequencing analysis in ameloblastoma, and to characterize the relationship between RECK protein and MMP-9 protein expression and the clinical pathology and biological behavior of ameloblastoma.Methods: 1 Tumor specimens and clinical data were the same as Part One. 2 DNA extraction: Genomic DNA was extracted by using a QIAamp DNA Mini Kit according to the manufacturer's instructions. 3 Exon 1, 8, 9, 11, 13 and 15 of RECK gene primers were designed according to the previous report and Primer Premier 5.0(Premier Biosoft Inc., Canada). 4 PCR amplification: Go Taq green Master Mix 25?l, RECK N primer F 1.5?l(0.3?M), RECK N primer R 1.5?l(0.3?M), DNA template 5?l(250ng), and nuclease-free H2 O 17?l. The PCR circulations are as follows: predenaturation 2 min at 95?C, 95?C for 30 s, 58~60?C for 30 s, 72?C for 30 s, 30 cycles, and a final extension at 72?C for 10 min. The PCR products were loaded in 2% agarose gel electrophoresis for 2.5 hours at 60 V. 5 Western blot analysis: Tissue total protein was extracted by using RIPA buffer(Solarbio, Beijing, China). Protein concentrations were determined by the BCA protein Assay Kit(Bioworld, USA). Equivalent amounts of protein(50mg for RECK blot and MMP-9 blot) were resolved by SDS-PAGE and transferred to polyvinylidene fluoride membranes(Millipore, USA). The membranes were blocked for 1h in 5% non-fat milk and incubated overnight at 4?C with the following primary antibodies: anti-RECK(mouse monoclonal antibody, Santa, USA) at 1:1000 and anti-MMP-9(rabbit monoclonal antibody, Abcam, China) at 1:2000. Expression of ?-actin was used as a control detected with anti-?-actin mouse monoclonal antibody, ZSGB-BIO, China). The membrane was incubated with Horse-radish peroxidase-conjugated antimouse and anti-rabbit immunoglobulin G secondary antibody(ZSGB-BIO, China) at 1:2000. Bands were detected by enhanced chemiluminescence(ECL, advansta, USA). The excess detection reagent was drained, and the membrane was wrapped with plastic wrap. The protein side of the membrane was exposed the to X-ray film. 6 DNA sequencing. 7 Statistical analysis: The data of protein expressions of RECK and MMP-9 were analysed by one-way ANOVA, by using the StudentNewman-Keuls test. Fisher's exact test was used to analyze the relationship between RECK mutation status and clinicopathological features, and a P value<0.05 was considered significant. Statistical analysis was performed with SPSS 19.0(SPSS, Inc., Chicago, IL).Results:1 Clinicopathological features were the same as Part One. 2 A DNA sequence was obtained from all specimens by Sanger sequencing. The same band was detected in the PCR amplification products of ameloblastoma and gingiva by electrophoresis gel. The missense mutation in 11 tumors was found to occur in exon 9 with G to A transversion at 909 leading to a valine to isoleucine substitution at codon 275(V275I), another missense mutation in 3 tumors was found to occur in exon 11 with A to G transversion at 1269 leading to an isoleucine to valine substitution at codon 395(I395V). A total of 12 mutations from A to G, were located at nucleotide position 1646 of exon 13, and the mutation did not result in an amino acid change at P520 P. A synonymous mutation from T to A in 11 tumors was located at nucleotide position 1961 of exon 15, and did not result in an amino acid change at R625 R. The V275 I mutation was predicted to possibly damage the function of RECK protein with Poly Phen2 and SIFT software, while I395 V tolerated RECK protein function with Poly Phen2 with SIFT. 4 The correlation between RECK mutation and the expressions of RECK protein and MMP-9 protein: Gene Tool from Syngene software was performed in this study. The results showed that the relative expressions of RECK protein was 0.43±0.08, 0.66±0.06 and 0.87±0.06 in RECK mutation group, non-mutation group and normal gingival group, respectively. The relative expression of MMP-9 protein protein was 0.83±0.07, 0.63±0.05 and 0.49±0.05 in RECK mutation group, non-mutation group and normal gingiva group respectively. Statistical analysis showed that RECK protein expression in RECK mutation group was lower than that of RECK non-mutation group(P<0.05), and RECK protein expression in RECK mutation group and non-mutation group was lower than that of normal gingival group(P<0.05). MMP-9 protein expression in RECK mutation group was higher than that of RECK non-mutation group(P<0.05), and MMP-9 protein expressions in RECK mutation group and RECK non-mutation group were higher than those of normal gingival group(P<0.05). 5 The correlation between RECK mutation and clinicopathological features: No statistically significant correlation was found between RECK mutation and age, gender, location or histologic type of the tumor. However, RECK mutation was closely related with active proliferation, capsule invasion and clinical recurrence. RECK mutation rate of the active proliferation(85.7%) was significantly higher than that of non-active proliferation(34.8%)(P<0.05). RECK mutation rate in capsule invasion(100%) was significantly higher than that of non-capsule invasion(36.0%)(P<0.05). RECK mutation rate in clinical recurrence(81.8%) was significantly higher than that of non-recurrence(26.3%)(P<0.05).Conclusions: 1 BRAF gene V600 E mutation was found in 60% of the ameloblastoma. 2 BRAF gene V600 E mutation was closely related with active proliferation and clinical recurrence of ameloblastoma. 3 SMO gene mutations detected were distributed on exon 2, 3, 5, 6 and 10, and showed single base substitution. The mutational hot points of SMO gene were on codon 364 and codon 590, and T364 N had major effects on protein expression. 4 The high incidence of SMO gene mutation was detected in young patients, and was closely related to the tumor active proliferation and capsular invasion of ameloblastoma. 5 RECK gene mutations detected were distributed on exon 9, 11, 13 and 15, and showed single base substitution. The mutational hot points of RECK gene were on codon 275 and codon 395, and V275 I had major effects on protein expression. 6 RECK gene mutation was closely related to the active proliferation, capsular invasion and clinical recurrence of ameloblastoma. RECK protein expression was closely related to RECK gene mutation. |
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