YY1-mediated PTEN dephosphorylation antagonizes IR-induced DNA repair contributing to tongue squamous cell carcinoma radiosensitization.

Ionizing radiation (IR) confers a survival advantage in tongue squamous cell carcinoma (TSCC), however, IR resistance limits its efficacy. Although Yin Yang 1 (YY1) has been reported to play a role in genotoxic drug resistance by accelerating DNA repair, its role in TSCC radioresistance remains unclear. In this study, we examined YY1 mRNA and protein expression in human tongue cancer samples using qRT-PCR and western blotting, respectively. DNA array data identified YY1 mRNA expression in IR sensitivity or resistance cell lines and tissues. Tongue carcinoma primary cells and CAL27 cells with YY1 stably overexpressed or knocked-down were exposed to IR and evaluated for cell proliferation and apoptosis by CCK8-assay and caspase-3 assay, respectively. We also examined DNA damage- or repair-related indicators, such as YY1, p-H2AX, nuclear PTEN, p-PTEN, and Rad51 through Western blot analysis. Additionally, we explored the mechanism of IR-induced PTEN nuclear translocation by introducing a series of PTEN phosphorylation site mutations and co-IP assay. We observed that YY1 mRNA and protein are highly expressed in TSCC tissues, which was correlated with worse overall survival. Moreover, higher expression of YY1 and Rad51 was observed in radioresistant cells and tissues, overexpression of YY1 led to IR resistance in TSCC cells, whereas YY1 knockdown sensitized TSCC cells to IR. The underlying mechanism showed that the overexpression of YY1 upregulated nuclear PTEN and Rad51 expression, which is essential for DNA repair. IR upregulated YY1, nuclear PTEN, and Rad51; thus, knockdown of YY1 completely blocked IR-induced upregulation of nuclear PTEN/Rad51. IR upregulated PTEN phosphorylation, and mutation of the phosphorylation site of Ser380 nearly completely blocked IR-induced PTEN nuclear translocation. Furthermore, the phosphatase PP2A negatively regulated pS380-PTEN, and knockdown of YY1 completely blocked IR-induced pS380-PTEN through PP2A. In conclusion, knockdown of YY1 enhanced TSCC radiosensitivity through PP2A-mediated dephosphorylation of PTEN Ser380; thus, antagonizing the IR-induced nuclear PTEN/Rad51 axis and targeting YY1 may reverse IR resistance in TSCC.

Epigallocatechin-3-gallate inhibits proliferation and induces apoptosis in odontogenic keratocyst keratinocytes.

OBJECTIVE: The aim of this study was to investigate the effects of epigallocatechin-3-gallate on the proliferation and apoptosis of odontogenic keratocyst (OKC) keratinocytes in vitro. MATERIALS AND METHODS: Keratinocytes isolated from the epithelial lining of the OKC were cultured in keratinocyte serum-free medium and identified by CK10, CK14, pan-cytokeratin and vimentin immunofluorescence staining. The cells were exposed to EGCG at different concentrations, and proliferation inhibition was measured by cell counting kit 8 assay. Cell cycle and apoptosis were assessed by flow cytometry, and expression of the WNT signalling pathway-related proteins FZD3 and JNK3 was detected by quantitative real-time PCR and Western blotting. Human oral keratinocytes (HOKs) were used as the control. RESULTS: The OKC keratinocytes were successfully cultured. The primary cells were tile-like and expressed the epithelial biomarkers CK10, CK14 and pan-cytokeratin. Epigallocatechin-3-gallate inhibited cell proliferation in a dose- and time-dependent manner, arrested cell cycle in the G1 phase and induced apoptosis of OKC keratinocytes. FZD3 and JNK3 were overexpressed in OKC keratinocytes compared with HOKs and were downregulated by epigallocatechin-3-gallate treatment. CONCLUSION: Epigallocatechin-3-gallate inhibited proliferation and induced apoptosis in OKC keratinocytes, possibly by suppressing the WNT/JNK signalling pathway. It may thus be potentially used for OKC treatment.

Effect of platelet-rich plasma and latissimus dorsi muscle flap on osteogenesis and vascularization of tissue-engineered bone in dogs.

PURPOSE: The present study evaluated the effects of platelet-rich plasma (PRP) and the latissimus dorsi muscle flap on osteogenesis and vascularization of tissue-engineered bone. MATERIALS AND METHODS: Bone marrow stromal cells (BMSCs) were subcultured, and PRP was obtained from the same dogs. Demineralized bone matrix (DBM) was prepared from homologous bone. The complexes of DBM/BMSCs/PRP were implanted into areas A and B on the left side of the dogs' backs; complexes of DBM/BMSCs without PRP were implanted in areas C and D on the right side of the same dog. The implants in areas A and C were wrapped with a latissimus dorsi muscle flap, and the implants in areas B and D were wrapped with inferior fascia. At 4, 8, and 12 weeks later, the implants were removed for evaluation. RESULTS: The radiographic evaluation, descriptive histologic analysis, and histologic quantitative analysis showed that the PRP/BMSCs/DBM complex was better than the BMSCs/DBM complex in both vascularization and osteogenesis of the ectopic tissue-engineered bones, and the complex wrapped with the latissimus dorsi muscle flap was better than that packed with superficial fascia without blood vessels. CONCLUSIONS: The PRP and blood vessels in the latissimus dorsi muscle could cooperatively promote osteogenesis and vascularization in tissue-engineered bone.

Gene mutations in the D-loop region of mitochondrial DNA in oral squamous cell carcinoma.

The present study aimed to investigate gene mutations in the displacement­loop (D­loop) region of mitochondrial DNA (mtDNA) in patients with oral squamous cell carcinoma (OSCC) in order to examine the role of gene mutation in mtDNA in OSCC tumorigenesis. mtDNA was obtained from cancer tissues, paracancerous tissues and normal mucosal tissues of thirty patients with OSCC. The D­loop region of the mtDNA was amplified using polymerase chain reaction, sequenced and then analyzed by Chromas software and BLAST to identify the mutation sites. Mutations in the D­loop region were observed in the cancer tissue samples of eight out of thirty cases with OSCC, with a mutation rate of 27%. There were nine mutations in total, including one point mutation, two base deletions, three insertion mutations and three heterozygous mutations. In these mutations, base deletions were different from each other and heterozygous mutations did not have the same mutation form; however, the three insertion mutations were the same, consisting of an insertion of a C base. One case contained a T/A heterozygous mutation as well as base insertion of C. The eight cases with mutations in the D­loop region consisted of three cases of tongue cancer, two cases of soft palate cancer, one case of floor of the mouth cancer, one case of oropharyngeal cancer and one case of lip cancer. This study demonstrated mutations in the mtDNA D­loop region in OSCC cells; however, the association between occurrences of OSCC and mtDNA mutations requires further investigation.

[Expression and clinical significance of the genes of Wnt signaling pathway in keratocystic odontogenic tumor of the jaw bones].

PURPOSE: To study the role of genes of Wnt signaling pathway in keratocystic odontogenic tumor (KCOT) of the jaw bones. METHODS: Fresh specimens of KCOT and the same patient 's normal oral mucosa were obtained. Then RNA was extracted. Gene chip was used to detect the genes of Wnt signaling pathway. RESULTS: Compared to normal oral mucosa, there were 5 genes of Wnt signaling pathway in KCOT changed, including CAMK2A down-regulated, FZD3, MAPK10, PRKX and WNT5a up-regulated. CONCLUSIONS: There are abnormal expressions of genes of Wnt pathway in KCOT. Genes of Wnt pathway plays certain roles in KCOT.

[The effect evaluation of suction drainage to prevent fistula after superficial parotidectomy].

PURPOSE: To evaluate the clinical value of suction drainage to prevent fistula after parotidectomy, and seek the best opportunity to remove the drainage according to the draining output and duration. METHODS: One hundred and ninety-four patients with parotid diseases after superficial parotidectomy were assigned into pressure dressing group and suction drainage group. Pressure dressing was used after suction drainage tube was removed in the pressure group, while suction drainage tube was fixed through the process in the suction group. Postoperative salivary fistula occurrence between the 2 groups was analyzed with Pearson chi-square test, and the contribution of the output and duration resulting in salivary fistula was analyzed by Fisher's exact test with SPSS 19.0 software package. RESULTS: The occurrence of salivary fistula in the pressure dressing group and suction group was 11.6% and 15.5%, respectively in the suction group. No significance difference was found between the 2 groups (P>0.05). In the suction drainage group, significant correlation of the draining duration and salivary fistula was not found (P>0.05). However, the draining output less than 20 mL resulted in lower salivary fistula rate compared with the draining output of 20-30 mL. CONCLUSIONS: According to our findings, suction drainage can be used as a substitute for pressure dressing after parotidectomy in preventing salivary fistula, and the best timing of drainage extubation is when the draining output is less than 20 mL within 24 hours.

[Screening of the gene mutation in D-loop region of mitochondrial DNA in oral squamous cell carcinoma].

OBJECTIVE: To investigate the gene mutation in D-loop region of mitochondrial DNA (mtDNA) in oral squamous cell carcinoma (OSCC) tissue and to explore the role of the gene mutation in D-loop region in the OSCC tumorigenesis. METHODS: mtDNA was obtained from cancer, paracancerous and normal mucosa tissues of thirty patients with OSCC. The D-loop regions of mtDNA were amplified with PCR, sequencing and then analyzed by Chromas software and BLAST to identify the mutation site. RESULTS: Mutation in the D-loop region was found in eight cases, with the mutation rate of 27%. There were nine mutations totally, including one point mutation, two base deletions, three insertion mutations, three heterozygous mutations. In these mutations, base deletions were different from each other and heterozygous mutations had no same mutation form, while the three insertion mutations were same, the insertion of base C. One case had T/A heterozygous mutation and base C insertion at the same time. CONCLUSIONS: There were mutations in mtDNA D-loop in OSCC, but the relationship between occurrence of OSCC and mutation of mtDNA needs further study.

[An experimental study of endothelial progenitor cells on bone formation in tissue-engineered bone in dogs].

PURPOSE: To observe the effects of endothelial progenitor cells (EPCs) on bone formation of tissue engineered bone in dogs. METHODS: Bone marrow stromal cells (BMSCs) and EPCs were derived from dog bone marrow and cultured in different medium in vitro. They were seeded on demineralized bone matrix (DBM) to build tissue engineered bone, then the construct was implanted into the fasciae of latissimus dorsi muscle, the degree of bone formation was analyzed with imaging and histological methods at different time points. SPSS13.0 software package was used for statistical analysis. RESULTS: 4, 8, 12 weeks after operation, X-ray film showed bone mineral density (BMD) in the EPCs group was significantly higher than the control group, and there was significant difference between the two groups(P<0.05); Histological examination revealed that the degree of bone formation in the EPCs group was higher than the control group, the new bone area and blood vessel area between the two groups were significantly different(P<0.05). CONCLUSION: EPCs can promote bone formation and accelerate new bone formation in tissue-engineering bone.

[The mutations of germline succinate dehydrogrnase subunit B (SDHB) in sporadic paragangliomas].

PURPOSE: The purpose of this study was to investigate the somatic mutations of human mitochondria succinate dehydrogenase subunit B (SDHB) in sporadic paragangliomas. METHODS: Eight exons of SDHB gene in 8 sporadic paragangliomas cases were amplified by PCR and sequenced, respectively. The sequences were analyzed to find mutations compared with human homology sequence in Genebank and SNP database. RESULTS: Nine sequence variations were found in 8 cases, in which one mutation was found in one case (1/8, 12.5%). The mutation was identified as the sixty four base pair in exon 2 of SDHB(c.136C>T), resulting in a change from a arginine to a stop codon (p.Arg90X). The left 8 variations were polymorphisms. CONCLUSIONS: The mutation of SDHB exists in sporadic paragangliomas patients and it might play a significant role in paragangliomas tumorigenesis.

[The mutations of the D-loop hypervariable region II and hypervariable region III of mitochondrial DNA in oral squamous cell carcinoma].

OBJECTIVE: To investigate the frequency of mitochondrial DNA (mtDNA) D-loop hypervariable region II (HVR II) and hypervariable region III (HVR III) mutations in oral squamous cell carcinoma (OSCC) and their correlation to provide the new targets for the prevention and treatment of OSCC. METHODS: The D-loop HVR II and HVR III regions of mtDNA in seven cases with OSCC tissues, matched with paracancerous tissues and normal mucosa tissues from the same case, were amplified by polymerase chain raction (PCR), then were detected by direct sequencing to find the mutantsites after the comparison of all sequencing results with the mtDNA Cambridge sequence in the GenBank database. RESULTS: 82 (56 species) nucleotide changes, with 51(26 species) nucleotide polymorphism, were found after the comparison of all sequencing results with the mtDNA Cambridge sequence in the GenBank database. 31(30 species) mutations, with 21 located within the HVR II and HVR III regions, were found in 3 tumor tissue samples, their paracancerous and normal mucosa tissue were found more polymorphic changes but no mutation. The mtDNA D-loop HVR II and HVR III regions mutation rate was 42.9% (3/7) in OSCC. CONCLUSION: The mtDNA D-loop HVR II and HVR III regions were highly polymorphic and mutable regions in OSCC. It suggested that the D-loop HVR II and HVR III regions of mtDNA might play a significant role in the tumorigenesis of OSCC. It may become new targets for the gene therapy of OSCC by regulating the above indexes.

[Clinical biological behavior and treatment strategy of hemangioma in oral and maxillofacial region].

Hemangioma can not be prevented until nowadays. Early identification and definite diagnosis are necessary to achieve the best outcome. The clinical biological behaviors of hemangiomas in oral and maxillofacial region are completely investigated and deeply analyzed in this paper. There are two distinct phrase of rapid proliferation of hemangioma, with explicit onset and lasting time. Although there is still controversy over the treatment modality of hemangiomas in oral and maxillofacial region, the only way to success is to treat the lesions in early stage, in particular, the first rapid proliferation phrase is the best opportunity and strategic point to control the growth of hemangioma or even cure the disease. Upon definite diagnosis, laser therapy, sclerotherapy and oral steroids are among the treatment options. The protocol of laser therapy and sclerotherapy is addressed in this paper. Careful planning and appropriate manipulation are the key components to the management of hemangioma in oral and maxillofacial region.

[The value of single photon emission computed tomography-computed tomography in diagnosis of carcinoma invasion of mandible].

OBJECTIVE: To investigate the value of single photon emission computed tomography(SPECT)-computed tomography(CT) in diagnosis of oral carcinoma invasion to mandible. METHODS: Thirty-four patients with oral carcinoma invasion to mandible were divided into two groups, group A (surrounding invasion) and group B (central invasion). The edge of the invasion was evaluated by SPECT-CT, CT and pathological examination. The results of CT and SPECT-CT were analyzed by quantitative methods. RESULTS: In group A, the cancer-invaded area of the mandible exhibited on SPECT-CT was 1.0 cm lager than that on pathological examination, 2.4 cm lager than that on CT. The difference of invaded area shown on CT was 1.4 cm smaller than that of pathological examination. There were significant difference among the three methods. In group B, the affected area on SPECT-CT was 1.2 cm lager than that of pathological examination, 4.2 cm lager than that of CT. The invision area on CT was 3.0 cm smaller than that of pathological examination. There were significant difference among the three methods. CONCLUSIONS: SPECT-CT could find the jaw central tumor earlier than CT and the range of lesion showed by SPECT-CT was the adequate range of bone incision during operation. The range of lesion showed by CT was influenced by the type of tumor and the range of bone incision was determined according to the pathological type. If the false negative result was eliminated, only SPECT-CT or CT was needed to estimate the invasion range of mandible.

[Early laser intervention of hemangioma in facial and neck regions of infant].

OBJECTIVE: The study was to evaluate the method of early laser intervention of hemangioma in facial and neck regions of infant. METHODS: Between January 1999 and December 2006, twelve patients, aged 6 days to 3 months, with cutaneous hemangioma in facial and neck regions, were treated with laser, eight cases with Nd:YAG laser therapy and four cases with Venus laser therapy. Four cases with hemangioma in facial and neck regions of infant treated with oral corticosteroid were as control. The outcome was recorded with 1 to 6 years of follow-up. RESULTS: Total resolution was obtained in twelve patients with laser intervention. Atrophic scars occurred in eight patients with Nd:YAG laser therapy, without other complications, such as ulceration, life-threatening hemorrhage and et al. No scar occurred in four patients with Venus laser therapy. Recurrence was not seen in twelve cases with laser therapy with follow-up. Hemangiomas enlarged continuously in four cases with oral corticosteroid therapy. CONCLUSION: Early laser intervention is an excellent management of cutaneous hemangioma in facial and neck regions of infant.

[A comparison between CT and SPECT in detecting mandibular invasion by lower gingival squamous cell carcinoma].

PURPOSE: The purpose of this study is to compare the value of CT and SPECT in diagnosis of lower gingival carcinoma invading the mandible. METHODS: From February 2002 to October 2006, twenty-one patients with lower gingival squamous cell carcinoma were enrolled.The data of CT and SPECT were studied,and compared with histopathological findings. RESULTS: Among the 21 patients,the sensitivity, accuracy, negative predictive value and Youden's index of SPECT were 100.00%, 95.24%, 100.00% and 1.00,respectively. While the sensitivity, accuracy, negative predictive value and Youden's index of CT were 80.00%, 80.95%, 20.00% and 0.80, respectively. There were four false negatives assessments of bone invasion(80.00%) by CT scan, while no false negatives by SPECT. CONCLUSIONS: SPECT is superior to CT, and can be used as a routine screening method to assess lower gingival carcinoma invading the mandible.

[Effect of platelet rich plasma on vascularization of tissue-engineered bone].

OBJECTIVE: To study the effects of platelet rich plasma (PRP) on vascularization of tissue-engineered bone. METHODS: Bone marrow stromal cell (BMSC) were isolated from iliac bone of dogs. PRP was obtained from the same dog and demineralized bone matrix (DBM) was prepared from homologous bone. Twelve dogs were divided into three groups and the back of each dog was divided into four areas. The DBM- BMSC- PRP was implanted in the area A and B; the DBM-BMSC without PRP was implanted in the area C and D. The implants in the areas A and C were wrapped using myo-fascia with blood vessel of latissimus dorsi. The implants in the area B and D were wrapped using superficial fascia of the back without blood vessel. The implants were taken out 4, 8 and 12 weeks later for examination. RESULTS: The degree of calcification and blood vessel formation of the implants was A > B > C > D. CONCLUSIONS: Both PRP and vessels of latissimus dorsi muscle could promote calcification and vascularization in tissue-engineered bone, when used separately or in combination.

[Osteoma in the soft palate: report of one case and review of the literature].

Osteoma is a benign tumor, which is composed of mature differentiated bone tissue and derived from osteoplast in periosteum endothecium. Osteomas is common in the bone in clinic, however, osteoma in the soft tissue is distinctly rare. Clinically, the lesion presents as hard, slowly growing mass with clear border. This article reported a case with soft palate osteoma. The clinical manifestation, differential diagnosis, pathologic diagnosis and treatment were discussed. Surgical resection with postoperative follow-up is suggested as the main treatment for this disease.

[Study of MSCs in vitro cultured on demineralized bone matrix of mongrel].

PURPOSE: To manufacture demineralized bone matrix(DBM) of mongrel and to explore the feasibility of DBM as scaffold of bone tissue engineering. METHODS: Thigh bones of mongrel were degreased, demineralized, deproteined, freezed, dried and sterilized to form DBM. Mesenchymal stem cells(MSCs) were seeded onto the scaffold and their growth were examined by inverted phase contrast microscope and scanning electron microscope. RESULTS: DBM had a three-dimensional mesh structure.The mean pore diameter of DBM was (254.39+/-88.71)microm and the pore rate was about 70%.MSCs could adhere to the surface and inner walls of DBM, proliferated well and secreted a large amount of extracellular matrix. CONCLUSION: DBM has satisfactory biocompatibility.

[Effect of platelet-rich plasma and latissimus dorsi myofascia with blood vessel on vascularization of tissue engineered bone in dogs].

OBJECTIVE: To study the effect of platelet-rich plasma (PRP) and latissimus dorsi myofascia with blood vessel on vascularization of tissue engineered bone in dogs. METHODS: Bone marrow stromal cells (BMSCs) were isolated from iliac bone of dogs. PRP was obtained from the same dog. And demineralized bone matrix (DBM) were prepared from homologuous bone. ABCD 4 areas were divided on the back of dog. PRP/BMSCs/DBM was implanted around the vessels of lattisimus dorsi muscle in the A. PRP/BMSCs/DBM wrapped by superficial fascia in the B. BMSCs/DBM was implanted around vessels of lattisimus dorsi muscle in the C. BMSCs/DBM wrapped by superficial fascia in the D area of the same dog. 4, 8, 12 weeks after implantation, gross specimen and histology examination were made. RESULTS: Osteogenesis and blood vessel formation results were A>B>C>D area. CONCLUSION: The results suggested that the PRP and latissimus dorsi myofascia with blood vessels could promote calcification and vascularization in tissue-engineered bone.