The GAL/GALR2 axis promotes the perineural invasion of salivary adenoid cystic carcinoma via epithelial-to-mesenchymal transition.

BACKGROUND: Perineural invasion (PNI) is a typical pathological characteristic of salivary adenoid cystic carcinoma (SACC) and other neurotrophic cancers. The mechanism of the neural microenvironment controlling tumor progression during the PNI process is unclear. In the present study, we investigated the role and molecular mechanisms of nerve-derived neuropeptide galanin (GAL) and its receptor (GALR2) in the regulation of PNI in SACC. METHODS: Immunohistochemistry staining and clinical association studies were performed to analyze the expression of GAL and GALR2 in SACC tissues and their clinical value. Dorsal root ganglion or SH-SY5Y cells were co-cultured with SACC cells in vitro to simulate the interactions between the neural microenvironment and tumor cells, and a series of assays including transcriptome sequencing, Western blot, and Transwell were performed to investigate the role and molecular mechanism of GAL and GALR2 in the regulation of SACC cells. Moreover, both the in vitro and in vivo PNI models were established to assess the potential PNI-specific therapeutic effects by blocking the GAL/GALR2 axis. RESULTS: GAL and GALR2 were highly expressed in SACC tissues, and were associated with PNI and poor prognosis in SACC patients (p < 0.05). Nerve-derived GAL activated GALR2 expression in SACC cells and induced epithelial-to-mesenchymal transition (EMT) in SACC cells. Adding human recombinant GAL to the co-culture system promoted the proliferation, migration, and invasion of SACC cells significantly, but inhibited the apoptosis of SACC cells. Adding M871, a specific antagonist of GALR2, significantly blocked the above effects (p < 0.05) and inhibited the PNI of SACC cells in vitro and in vivo (p < 0.05). CONCLUSIONS: This study demonstrated that nerve-derived GAL activated GALR2 expression, and promoted EMT in SACC cells, thereby enhancing the PNI process. Interruption of the GAL/GALR2 axis might be a novel strategy for anti-PNI therapy for SACC.

Deep learning to diagnose Hashimoto's thyroiditis from sonographic images.

Hashimoto's thyroiditis (HT) is the main cause of hypothyroidism. We develop a deep learning model called HTNet for diagnosis of HT by training on 106,513 thyroid ultrasound images from 17,934 patients and test its performance on 5051 patients from 2 datasets of static images and 1 dataset of video data. HTNet achieves an area under the receiver operating curve (AUC) of 0.905 (95% CI: 0.894 to 0.915), 0.888 (0.836-0.939) and 0.895 (0.862-0.927). HTNet exceeds radiologists' performance on accuracy (83.2% versus 79.8%; binomial test, p < 0.001) and sensitivity (82.6% versus 68.1%; p < 0.001). By integrating serologic markers with imaging data, the performance of HTNet was significantly and marginally improved on the video (AUC, 0.949 versus 0.888; DeLong's test, p = 0.004) and static-image (AUC, 0.914 versus 0.901; p = 0.08) testing sets, respectively. HTNet may be helpful as a tool for the management of HT.

An animal experiment study on the application of indocyanine green angiography in the harvest of multi-angiosome perforator flap.

Background: This study sought to explore the application value of indocyanine green angiography (ICGA) in the harvest of multi-angiosome perforator flap and the effect of low molecular weight heparin (LMWH) on the survival of postoperative flap. Methods: Twenty-four SD male rats were selected to construct a three-angiosome perforator flap model with the unilateral iliolumbar artery perforator. They were randomly divided into two groups: the control group was injected with indocyanine green (ICG) into the femoral vein during the operation, and the fluorescence signal was collected and quantitatively analyzed using Real-Time Image Guided System to determine the intraoperative fluorescence imaging length. The experimental group was injected subcutaneously with LMWH (400 U/kg) after 0.5 h postoperatively, and the control group was injected with the same amount of normal saline. The injection was repeated at the same time each day from 0 to 7 days postoperatively. After the flap was sutured in situ, ICGA was performed at 0, 1, 3, 5, and 7 days postoperatively to observe the vascular structure of the two groups of flaps. The flap survival length of the control group was counted at 7 days postoperatively, and the correlation between the intraoperative fluorescence imaging length and the survival length at 7 days postoperatively was calculated. The proportion of distal necrosis of the flaps between the two groups was compared at 7 days postoperatively. Results: The average length of intraoperative fluorescence imaging in the control group was 6.29±0.50 cm, and the survival length of the flap at 7 days postoperatively was 8.24±0.52 cm. The actual survival length was higher than the intraoperative fluorescence imaging length, with a ratio of 1.31±0.08. The difference was statistically significant (P<0.05). At 7 days postoperatively, the flap necrosis ratio of experimental group and control group were 10.92%±1.30% and 19.11%±1.19%, and the flap necrosis ratio of experimental group was lower than that of control group (P<0.001). Conclusions: ICGA can locate the position of perforator, and can be used to predict and observe the length of distal survival of multi-angiosome perforator flap postoperatively. LMWH can promote the distal survival of flap and reduce flap necrosis.

LINC01123 promotes immune escape by sponging miR-214-3p to regulate B7-H3 in head and neck squamous-cell carcinoma.

Numerous studies have shown that long noncoding RNAs (LncRNAs) are involved in the development and immune escape of head and neck squamous-cell carcinoma (HNSCC). However, the specific regulatory mechanisms by which LINC01123 regulates HNSCC and its correlation with immunity remain unclear. Therefore, this study's primary purpose was to explore the mechanisms by which LINC01123 regulates the immune escape and progression of HNSCC. This study confirmed that LINC01123 is competitively bound to miR-214-3p, and miR-214-3p specifically targets B7-H3. The effects of LINC01123, B7-H3, and miR-214-3p on tumor progression, CD8+T-cell-mediated immune response, and the tumorigenicity of HNSCC in vitro and in vivo were examined through the downregulation or upregulation of LINC01123, B7-H3, and miR-214-3p. Our results indicated that LINC01123 and B7-H3 were highly expressed in HNSCC and are associated with poor prognosis in patients. Notably, overexpression of LINC01123 or B7-H3 or downregulation of miR-214-3p inhibited the function of CD8+T cells and promoted the progression of HNSCC. Therefore, LINC01123 acts as a miR-214-3p sponge to inhibit the activation of CD8+T cells and promote the progression of HNSCC by upregulating B7-H3.

Improved diagnosis of thyroid cancer aided with deep learning applied to sonographic text reports: a retrospective, multi-cohort, diagnostic study.

OBJECTIVE: Large volume radiological text data have been accumulated since the incorporation of electronic health record (EHR) systems in clinical practice. We aimed to determine whether deep natural language processing algorithms could aid radiologists in improving thyroid cancer diagnosis. METHODS: Sonographic EHR data were obtained from the EHR database. Pathological reports were used as the gold standard for diagnosing thyroid cancer. We developed thyroid cancer diagnosis based on natural language processing (THCaDxNLP) to interpret unstructured sonographic text reports for thyroid cancer diagnosis. We used the area under the receiver operating characteristic curve (AUROC) as the primary metric to measure the performance of the THCaDxNLP. We compared the performance of thyroid ultrasound radiologists aided with THCaDxNLP vs. those without THCaDxNLP using 5 independent test sets. RESULTS: We obtained a total number of 788,129 sonographic radiological reports. The number of thyroid sonographic data points was 132,277, 18,400 of which were thyroid cancer patients. Among the 5 test sets, the numbers of patients per set were 439, 186, 82, 343, and 171. THCaDxNLP achieved high performance in identifying thyroid cancer patients (the AUROC ranged from 0.857-0.932). Thyroid ultrasound radiologists aided with THCaDxNLP achieved significantly higher performances than those without THCaDxNLP in terms of accuracy (93.8% vs. 87.2%; one-sided t-test, adjusted P = 0.003), precision (92.5% vs. 86.0%; P = 0.018), and F1 metric (94.2% vs. 86.4%; P = 0.007). CONCLUSIONS: THCaDxNLP achieved a high AUROC for the identification of thyroid cancer, and improved the accuracy, sensitivity, and precision of thyroid ultrasound radiologists. This warrants further investigation of THCaDxNLP in prospective clinical trials.

Early Pregnancy Exposure to Rare Earth Elements and Risk of Gestational Diabetes Mellitus: A Nested Case-Control Study.

Objective: The extensive use of rare earth elements (REEs) in many technologies was found to have effects on human health, but the association between early pregnancy exposure to REEs and gestational diabetes mellitus (GDM) is still unknown. Methods: This nested case-control study involved 200 pregnant women with GDM and 200 healthy pregnant women from the Peking University Birth Cohort in Tongzhou. We examined the serum concentrations of 14 REEs during early pregnancy and analyzed their associations with the risk of GDM. Results: When the elements were considered individually in the logistic regression model, no significant associations were found between REEs and GDM, after adjusting for confounding variables (P > 0.05). In weighted quantile sum (WQS) regression, each quartile decrease in the mixture index for REEs resulted in a 1.67-fold (95% CI: 1.12-2.49) increased risk of GDM. Neodymium (Nd), Praseodymium (Pr), and Lanthanum (La) were the most important contributors in the mixture. Conclusion: The study findings indicated that early pregnancy exposure to lower levels of REE mixture was associated with an increased risk of GDM, and Nd, Pr, and La exhibited the strongest effects in the mixture.

Toughening of Poly(l-Lactide) with Branched Polycaprolactone: Effect of Chain Length.

In this work, a series of branched polycaprolactone (BPCL) samples with different ε-caprolactone (CL) chain lengths were synthesized and used to toughen poly (lactic acid) (PLA). The spherical structure increased the free volume, facilitating the free movement of the PLA chain segment and increasing the ductility. In addition, the hydrogen bonds between the multi-terminal hydroxyl group of BPCL x and PLA improved the interaction between them. The glass-transition temperatures (T g) and crystallization temperatures (T c) of the blends were significantly lower than those of PLA, and these temperatures increased with the chain length of polycaprolactone. BPCL x increased the crystallization rate of PLA through heterogeneous nucleation. A longer chain length of CL increased the mutual entanglement in the blends, reduced the hydrogen bonding between BPCL x and PLA, and increased the entanglement of BPCL x chains. When the chain length of CL was 6, the impact strength and elongation at break of the PLA/BPCL blends exhibited an increase of 151.72 and 465.8%, respectively, as compared with PLA.

Embolization and sclerotherapy of maxillofacial arteriovenous malformations with the use of fibrin glue combined with pingyangmycin.

OBJECTIVE: The objectives of this study were to document the results of using fibrin glue (FG) combined with pingyangmycin (PYM) for the embolism and sclerotherapy of maxillofacial arteriovenous malformations (AVMs). STUDY DESIGN: We reviewed the associated clinical data from December 2012 to June 2017 for 25 patients with maxillofacial AVMs. The major treatment method was direct percutaneous puncture and injection of FG combined with PYM. Treatment outcomes were assessed through physical examination, Doppler ultrasonography, computed tomography, and 3-dimensional computed tomography angiography scans. Follow-up time ranged from 12 months to 3 years after the last treatment (mean 21 months). RESULTS: Of the 25 lesions, 80% showed greater than 90% reduction, 12% showed greater than 75% reduction, and 8% showed greater than 50% reduction. Superficial skin necrosis or mucous ulcer occurred in 3 patients and healed without intervention. Regrowth was observed in 3 patients with extensive lesions involving multiple anatomic regions. CONCLUSIONS: These data suggest that embolization and sclerotherapy with the use of FG combined with PYM are safe and effective for the treatment of small- to medium-sized, locally dilated maxillofacial AVMs. For AVMs involving multiple anatomic regions, combined application of this approach with other options should be considered.

Sympathetic innervation contributes to perineural invasion of salivary adenoid cystic carcinoma via the ß2-adrenergic receptor.

PURPOSE: Perineural invasion (PNI) is reported to correlate with local recurrence and poor prognosis of salivary adenoid cystic carcinoma (SACC). However, the pathogenesis of PNI remains unclear. The aims of this study were to investigate the correlation between sympathetic innervation and SACC PNI and to elucidate how the sympathetic neurotransmitter norepinephrine (NE) regulates the PNI process. MATERIALS AND METHODS: Sympathetic innervation and ß2-adrenergic receptor (ß2-AR) expression in SACC tissues were evaluated by immunohistochemistry. The NE concentrations in SACC tissues and dorsal root ganglia (DRG) coculture models were measured by ELISA. ß2-AR expression in SACC cells was detected by performing quantitative real-time polymerase chain reaction (qRT-PCR) and immunofluorescence assay. SACC cells were treated with NE, the nonselective α-AR blocker phentolamine, the ß2-AR antagonist ICI118,551, or were transfected with ß2-AR small interfering RNA (siRNA). Proliferation was evaluated in methyl thiazolyl tetrazolium assay, and migration was evaluated in Transwell assay and wound-healing assay. PNI was tested through both Transwell assay and a DRG coculture model. The expressions of epithelial-mesenchymal transition (EMT) markers and matrix metalloproteinases (MMPs) were measured by performing qRT-PCR and Western blot assay. RESULTS: Sympathetic innervation and ß2-AR were highly distributed in SACC tissues and correlated positively with PNI (P=0.035 and P=0.003, respectively). The sympathetic neurotransmitter NE was overexpressed in SACC tissues and DRG coculture models. Exogenously added NE promoted proliferation, migration, and PNI of SACC cells via ß2-AR activation. NE/ß2-AR signaling may promote proliferation, migration, and PNI by inducing EMT and upregulating MMPs. However, ß2-AR inhibition with either an antagonist or siRNA abrogated NE-induced PNI. CONCLUSION: Collectively, our findings reveal the supportive role of sympathetic innervation in the pathogenesis of SACC PNI and suggest ß2-AR as a potential therapeutic target for treating PNI in SACC.

Socioeconomic and geographic variations in the prevalence, awareness, treatment and control of dyslipidemia in middle-aged and older Chinese.

BACKGROUND AND AIMS: Unevenly socioeconomic development and nutrition transition might bring large variations in the epidemiology of dyslipidemia. We aimed to estimate the prevalence, awareness, treatment and control of dyslipidemia in different socioeconomic statuses and geographic regions in China, and to assess the associated factors and comorbidities of dyslipidemia. METHODS: We included participants aged 45 years and above from a nationally representative investigation: the China Health and Retirement Longitudinal Study 2011. Dyslipidemia was defined based on the 2016 guideline of Chinese Prevention and Treatment of Dyslipidemia in adults. Multivariable logistic regression was adopted to assess the potentially associated factors and commodities of dyslipidemia. RESULTS: In 2010, the prevalence of dyslipidemia was 42.84% among people aged 45 years and above. Low level of high-density lipoprotein cholesterol (HDL-C) was the most common type of dyslipidemia. The awareness, treatment and control rates among dyslipidemic subjects were 20.27%, 14.41% and 4.94%, respectively. In dyslipidemic patients who were under treatment, the control rate was 34.26%. People aged 50-59 years were at a significantly higher risk of dyslipidemia than those aged 45-49 years. Male gender, living in North China, overweight, obesity, central obesity, hypertension, diabetes and hyperuricemia were significantly associated with a higher risk of dyslipidemia. Current alcohol drinking and underweight were linked to a lower prevalence of dyslipidemia. CONCLUSIONS: Our study revealed a high prevalence of dyslipidemia among middle-aged and older Chinese. The awareness, treatment and control rates were far from ideal and geographic inequality was highlighted. More efforts are needed to prevent and manage dyslipidemia in China.

Diagnosis of thyroid cancer using deep convolutional neural network models applied to sonographic images: a retrospective, multicohort, diagnostic study.

BACKGROUND: The incidence of thyroid cancer is rising steadily because of overdiagnosis and overtreatment conferred by widespread use of sensitive imaging techniques for screening. This overall incidence growth is especially driven by increased diagnosis of indolent and well-differentiated papillary subtype and early-stage thyroid cancer, whereas the incidence of advanced-stage thyroid cancer has increased marginally. Thyroid ultrasound is frequently used to diagnose thyroid cancer. The aim of this study was to use deep convolutional neural network (DCNN) models to improve the diagnostic accuracy of thyroid cancer by analysing sonographic imaging data from clinical ultrasounds. METHODS: We did a retrospective, multicohort, diagnostic study using ultrasound images sets from three hospitals in China. We developed and trained the DCNN model on the training set, 131 731 ultrasound images from 17 627 patients with thyroid cancer and 180 668 images from 25 325 controls from the thyroid imaging database at Tianjin Cancer Hospital. Clinical diagnosis of the training set was made by 16 radiologists from Tianjin Cancer Hospital. Images from anatomical sites that were judged as not having cancer were excluded from the training set and only individuals with suspected thyroid cancer underwent pathological examination to confirm diagnosis. The model's diagnostic performance was validated in an internal validation set from Tianjin Cancer Hospital (8606 images from 1118 patients) and two external datasets in China (the Integrated Traditional Chinese and Western Medicine Hospital, Jilin, 741 images from 154 patients; and the Weihai Municipal Hospital, Shandong, 11 039 images from 1420 patients). All individuals with suspected thyroid cancer after clinical examination in the validation sets had pathological examination. We also compared the specificity and sensitivity of the DCNN model with the performance of six skilled thyroid ultrasound radiologists on the three validation sets. FINDINGS: Between Jan 1, 2012, and March 28, 2018, ultrasound images for the four study cohorts were obtained. The model achieved high performance in identifying thyroid cancer patients in the validation sets tested, with area under the curve values of 0·947 (95% CI 0·935-0·959) for the Tianjin internal validation set, 0·912 (95% CI 0·865-0·958) for the Jilin external validation set, and 0·908 (95% CI 0·891-0·925) for the Weihai external validation set. The DCNN model also showed improved performance in identifying thyroid cancer patients versus skilled radiologists. For the Tianjin internal validation set, sensitivity was 93·4% (95% CI 89·6-96·1) versus 96·9% (93·9-98·6; p=0·003) and specificity was 86·1% (81·1-90·2) versus 59·4% (53·0-65·6; p<0·0001). For the Jilin external validation set, sensitivity was 84·3% (95% CI 73·6-91·9) versus 92·9% (84·1-97·6; p=0·048) and specificity was 86·9% (95% CI 77·8-93·3) versus 57·1% (45·9-67·9; p<0·0001). For the Weihai external validation set, sensitivity was 84·7% (95% CI 77·0-90·7) versus 89·0% (81·9-94·0; p=0·25) and specificity was 87·8% (95% CI 81·6-92·5) versus 68·6% (60·7-75·8; p<0·0001). INTERPRETATION: The DCNN model showed similar sensitivity and improved specificity in identifying patients with thyroid cancer compared with a group of skilled radiologists. The improved technical performance of the DCNN model warrants further investigation as part of randomised clinical trials. FUNDING: The Program for Changjiang Scholars and Innovative Research Team in University in China, and National Natural Science Foundation of China.

Early hemoperfusion for emergency treatment of carbamazepine poisoning.

OBJECTIVES: To investigate the clinical value of early hemoperfusion (HP) in emergency treatment of carbamazepine (CBZ) poisoning. METHODS: 104 patients with acute CBZ poisoning treated from August 2004 to October 2015 in the Emergency Department were reviewed. Patients were categorized into three groups: group A, who received HP treatment in the Emergency Department; group B, who received HP treatment in the blood purification room; and group C, who did not received HP treatment. Pharmacokinetic parameters of CBZ and remission of complications were compared among the three groups. RESULTS: Both groups A and B had lower time to peak, area under curve and maximum concentration values than group C (P<0.05), and these kinetics indexes were significantly lower in group A than in group B (P<0.05). The mean retention times were 0.85±0.08, 1.20±0.15 and 2.52±0.29days in the three groups, respectively, and were significantly lower value in group A than in group B (P<0.05). The incidences of respiratory depression and seizure in group A were significantly lower than those of groups B and C (P<0.05). Group A had significantly higher Glasgow coma scale (GCS) scores at 4h after admission than the other two groups (P<0.05), and group B had significantly higher GCS scores than group C at 6h after admission (P<0.05). CONCLUSIONS: Initiation of HP in the early treatment stage of CBZ poisoning upon admission to an emergency department can significantly reduce the plasma concentration and retention period of CBZ, relieve the symptoms and shorten the overall treatment period.

Paraquat poisoning induced pulmonary epithelial mesenchymal transition through Notch1 pathway.

Progressive pulmonary fibrosis is the most characteristic feature of subacute PQ poisoning. Epithelial-to-mesenchymal transition (EMT) is reported to be involved in the pulmonary fibrosis after PQ exposure. Recent evidence suggested Notch signaling is required for EMT. In this study, we investigated whether Notch1 and TGF-ß1/Smad3 signaling was involved in EMT caused by PQ. It is demonstrated that A549 cells underwent EMT after treated with PQ at dose of 300 µmol/L for 6 days, charactered by increasing expression of mesenchymal marker α-SMA and decreasing expression of epithelial marker E-cadherin. We found that there was an apparent increased expression of Notch1 and jagged-1 in PQ induced EMT process. EMT could be enhanced by Jagged-1 ligand of Notch1, and be blocked by DAPT, a γ-secretase inhibitor. Our data also showed that the expression of TGF-ß1/Smad3 increased after Notch1 is elevated in EMT caused by PQ. Jagged-1 significantly induced SMA expression, and this induction was completely inhibited by SB431542 in A549 cells. In conclusion, we demonstrated that Notch1 pathway was important in EMT induced by PQ, and TGF-ß1/Smad3 signaling partly plays a role as the downstream of Notch1.

Overexpression of KAI1/CD82 suppresses in vitro cell growth, migration, invasion and xenograft growth in oral cancer.

KAI1/CD82 is a metastatic suppressor gene in human prostate cancer and several other types of cancer in humans. The present study aimed to examine the role of the overexpression of KAI1 in the progression of oral cancer. Human KAI1/CD82 cDNA was transfected into OSCC­15 and 293T cell lines, and its effects on OSCC­15 cell proliferation, invasion and apoptosis were assessed by performing a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay, Matrigel invasion and Annexin V­FITC staining, respectively. In addition, a xenograft model was used to assess the effect of KAI1/CD82 on the in vivo growth of tumors. The overexpression of KAI1/CD82 inhibited the proliferation and invasion of OSCC-15 cells. It also enhanced the apoptotic rate of the OSCC­15 cells. Furthermore, the overexpression of KAI1/CD82 inhibited tumor growth in the xenograft model. The results demonstrated that the overexpression of KAI1/CD82 significantly inhibited the proliferation and invasion of human oral cancer cells, and inhibited tumor growth in the xenograft model. Therefore, KAI1/CD82 may be considered as a potential therapeutic target in oral cancer.

p12CDK2-AP1 interacts with CD82 to regulate the proliferation and survival of human oral squamous cell carcinoma cells.

p12 cyclin-dependent kinase 2 (CDK2)-associating protein 1 (p12CDK2-AP1) has been demonstrated to negatively regulate the activity of CDK2. However, the underlying molecular mechanism remains largely unknown. We aimed to determine the potential binding proteins of p12CDK2-AP1 and to elucidate the role of p12CDK2-AP1 in the regulation of the proliferation, invasion, apoptosis, and in vivo growth of human oral squamous cell carcinoma cells. The protein-protein interaction was predicted using computational decision templates. The predicted p12CDK2­AP1 interacting proteins were overexpressed in human oral squamous cell carcinoma OSCC-15 cells, and the protein binding was examined using co-precipitation (Co-IP). Cell proliferation and invasion were determined via MTT assay and Transwell system, respectively. Cell apoptosis was evaluated using Annexin V-FITC/PI double staining followed by flow cytometric analysis. The in vivo growth of OSCC-15 cells was examined in nude mouse tumor xenografts. We found that overexpression of either p12CDK2-AP1 or CD82 significantly suppressed the proliferation and invasion but promoted the apoptosis of OSCC-15 cells (P<0.05). Importantly, combined overexpression of p12CDK2-AP1 and CD82 showed synergistic antitumor activity compared with the overexpression of a single protein alone (P<0.05). Additionally, the simultaneous overexpression of p12CDK2-AP1 and CD82 significantly suppressed the in vivo tumor growth of OSCC-15 cells in nude mice compared with the negative control (P<0.05). Our findings indicate that p12CDK2-AP1 interacts with CD82 to play a functional role in suppressing the in vitro and in vivo growth of OSCC-15 cells.

Downregulation of p53 promotes in vitro perineural invasive activity of human salivary adenoid cystic carcinoma cells through epithelial-mesenchymal transition-like changes.

Salivary adenoid cystic carcinoma (SACC) is a malignant tumor that is characterized by perineural invasion (PNI). p53 is an essential tumor-suppressor gene and p53 mutations play a critical role in tumor occurrence and progression (e.g., pancreatic, prostate and head and neck cancer). However, the regulatory role of the p53 gene in SACC and the PNI process remains unknown. In the present study, we employed RNA interference technique to downregulate p53 gene expression in SACC-83 cells to explore the role of p53 in the PNI process. Our results showed that the downregulation of the p53 gene induced significant 'epithelial-mesenchymal transition (EMT)-like changes' in SACC-83 cells, including decreased expression levels of epithelial markers (E-cadherin, EMA and CK5) and increased expression levels of mesenchymal markers (vimentin, N-cadherin and C-cadherin). The downregulation of p53 also caused a lower apoptotic index of Annexin V-FITC/PI and a lower number of SACC-83 cells in the second G0/G1 phase of the cell cycle. Furthermore, the downregulation of the p53 gene resulted in a significant increase in PNI activity in the SACC-83 cells. Thus, our findings revealed that downregulation of p53 promoted in vitro PNI activity through 'EMT-like changes' in SACC-83 cells. The present study suggests the essential regulatory role of p53 in the PNI activity of SACC cells, and implies that p53 may be a new target gene for the clinical treatment of SACC.

The CCL5/CCR5 axis contributes to the perineural invasion of human salivary adenoid cystic carcinoma.

Salivary adenoid cystic carcinoma (SACC) has a unique tendency for perineural invasion (PNI), which results in tumor recurrence and poor prognosis. Recent studies have shown that the chemokine CCL5 and its receptor CCR5 play important roles in tumor invasion and metastasis. However, the role of the CCL5/CCR5 axis in the PNI of SACC has not been studied to date. In the present study, we evaluated the expression of CCL5 and CCR5 in SACC cases and nerve tissues, and performed a series of in vitro assays with the SACC cell line, SACC-83, to indicate the role of the CCL5/CCR5 axis in the PNI of SACC. We found that CCL5 (35.9%; 23/64) and CCR5 (70.3%; 45/64) were positively expressed in SACC cases, and the expression of CCR5 was significantly associated with the PNI of SACC (P<0.05). We also found that SACC-83 cells expressed the functional receptor, CCR5, for the chemokine CCL5, as demonstrated by calcium mobilization and actin polymerization assays. Furthermore, we found that exogenous CCL5 significantly facilitated the migration, invasion and PNI activity of SACC-83 cells in vitro (P<0.05). Further study showed that the CCR5 inhibitor (maraviroc) effectively blocked the migration, invasion and PNI activity of SACC-83 cells with or without CCL5 stimulation (P<0.05). These results indicate that the CCL5/CCR5 axis plays a critical role in the PNI of SACC, and that antagonists against CCR5 may be an effective anti-PNI agent for SACC therapy.

Interaction between p12CDK2AP1 and a novel unnamed protein product inhibits cell proliferation by regulating the cell cycle.

Human p12CDK2AP1 protein is encoded by the cyclin­dependent kinase 2­associated protein 1 (CDK2AP1) gene. This protein suppresses cell growth, differentiation and angiogenesis in numerous types of carcinoma by interacting with certain cell cycle proteins, including CDK2 and DNA polymerase α/primase. p12CDK2AP1 exerts its functions predominantly through protein­protein interactions. Therefore, the identification of other p12CDK2AP1­interacting proteins may clarify its role in cell cycle regulation and carcinogenesis. The aim of this study was to identify additional p12CDK2AP1­interacting proteins. A novel unnamed protein product (UPP, BC006130) was identified through using a yeast two­hybrid system. The interaction of p12CDK2AP1 with the UPP was further verified by glutathione S-transferase pull­down and co­immunoprecipitation experiments in vitro. The qPCR results following overexpression and siRNA assays demonstrated that the expression levels of the UPP were mediated by the CDK2AP1 gene. Furthermore, overexpression of the UPP gene was shown to shorten the length of the G2/M phase of the cell cycle in normal and tumor cell lines in a flow cytometry assay. The results of human tumor xenografts experiments in Balb/c nude mice indicated that stable transfection with the UPP gene was able to inhibit tumor cell proliferation in vivo. Overall, this study identified and characterized a novel interactive protein of p12CDK2AP1, which may inhibit cell proliferation by mediating the cell cycle. It expands the understanding of the mechanisms of p12CDK2AP1 and its potential as a cancer therapeutic target.

Silencing P12CDK²AP¹ with a lentivirus promotes HaCaT cell proliferation.

The tumor suppressor P12CDK2AP1 negatively regulates cyclin-dependent kinase 2 (CDK2) activities and suppresses DNA replication. Notably, P12CDK2AP1 is known to be downregulated in head and neck squamous cell carcinomas (HNSCCs). Silencing of specific gene expression by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) using expression vectors and retroviruses has become a powerful tool for the genetic analysis of mammalian cells. In the present study, we utilized lentivirus­mediated shRNA for functional gene knockdown in normal human skin keratinocytes (HaCaT) cells in order to assess the potential role of P12CDK2AP1 in HNSCCs. Lentivirus­mediated RNA interference (RNAi) effectively reduced endogenous P12CDK2AP1 expression in HaCaT cells and significantly promoted HaCaT cell proliferation in vitro. Lentiviral vectors have the ability to infect dividing and non-dividing cells as well as to achieve long­term multilineage gene expression. Thus, additional studies are needed to investigate the use of such vectors as a therapeutic tool for the delivery of siRNAs.

HAb18G/CD147 cell-cell contacts confer resistance of a HEK293 subpopulation to anoikis in an E-cadherin-dependent manner.

BACKGROUND: Acquisition of resistance to "anoikis" facilitates the survival of cells under independent matrix-deficient conditions, such as cells in tumor progression and the production of suspension culture cells for biomedical engineering. There is evidence suggesting that CD147, an adhesion molecule associated with survival of cells in tumor metastasis and cell-cell contacts, plays an important role in resistance to anoikis. However, information regarding the functions of CD147 in mediating cell-cell contacts and anoikis-resistance remains limited and even self-contradictory. RESULTS: An anoikis-resistant clone (HEK293ar), derived from anoikis-sensitive parental Human Embryonic Kidney 293 cells, survived anoikis by the formation of cell-cell contacts. The expression of HAb18G/CD147 (a member of the CD147 family) was upregulated and the protein was located at cell-cell junctions. Upregulation of HAb18G/CD147 in suspended HEK293ar cells suppressed anoikis by mediating the formation of cell-cell adhesions. Anoikis resistance in HEK293ar cells also required E-cadherin-mediated cell-cell contacts. Knock-down of HAb18G/CD147 and E-cadherin inhibited cell-cell contacts formation and increased anoikis sensitivity respectively. When HAb18G/CD147 was downregulated, E-cadherin expression in HEK293ar cells was significantly suppressed; however, knockdown of E-cadherin by E-cadherin siRNA or blocking of E-cadherin binding activity with a specific antibody and EDTA had no significant effect on HAb18G/CD147 expression. Finally, pretreatment with LY294002, a phosphoinositide 3-kinase (PI3K/AKT) inhibitor, disrupted cell-cell contacts and decreased cell number, but this was not the case in cells treated with the extracellular signal-regulated kinase (ERK) inhibitor PD98059. CONCLUSIONS: Our results provide new evidence that HAb18G/CD147-mediated cell-cell contact confers anoikis resistance in an E-cadherin-dependent manner; and cell-cell contact mediated resistance to anoikis implicates PI3K pathway in a highly relevant cell model (HEK293ar). Understanding of the role of HAb18G/CD147 cell-cell contacts in anoikis resistance may help in understanding the survival of cells in anchorage-independent growth, such as cells in tumor metastasis and suspension culture produced for biomedical engineering. Our results also contribute to a better understanding of the biology of HEK293 cell spheroids, a major workhorse for producing human therapeutic agents and viral vaccines.