[Multi-gene mutations in ultrasound-guided fine-needle aspiration specimens of thyroid micronodules and diagnostic value in thyroid microcarcinomas using next-generation sequencing].

Objective: To explore the detection of BRAF, RAS, TERT promoter, and TP53 gene mutations in solitary thyroid micronodule (TMN) specimens obtained by ultrasound-guided fine-needle aspiration (US-FNA) using next-generation sequencing (NGS) technology and assess its diagnostic value in thyroid microcarcinomas (TMC). Methods: On-site recruitment of 428 patients with single suspicious TMC who underwent thyroid ultrasound examination, US-FNA, and NGS from September 2018 to July 2021 at Beijing Tongren Hospital affiliated to Capital Medical University. A total of 147 patients were finally included. NGS was used to detect mutations in the BRAF, RAS, TERT promoter, and TP53 genes in the US-FNA specimens. Comparisons were made between patients with and without gene mutations in terms of age, gender, and the maximum diameter of nodules. The diagnostic efficiency of BRAF mutation for TMC was calculated using the receiver operating characteristic (ROC) curve, with postoperative pathology as the gold standard. Results: The age [M (Q1, Q3)] of the 147 patients was 43.0 (32.0, 51.0) years, and 37 were male (25.2%). Among the 147 US-FNA specimens, 97 (66.0%) were detected with BRAF gene mutations, all of which were p.V600E point mutation; 6 (4.1%) were detected with RAS gene mutation, and no TERT promoter or TP53 gene mutations were detected. Postoperative pathology confirmed that 136 cases were TMC, all of which were papillary thyroid microcarcinomas (PTMC); 11 cases (7.5%)were benign. Among 136 TMC samples, BRAF gene mutations were detected in 97 cases (71.3%). There were no statistically significant differences in age, gender, and maximum nodule diameter between patients with and without BRAF gene mutations (all P>0.05). The sensitivity and specificity of BRAF gene mutation in diagnosing TMC were 71.3% and 100.0%, respectively, with an area under the ROC curve (AUC) (95%CI) of 0.857 (0.789-0.925). For nodules classified as Bethesda Ⅲ-Ⅴ, the sensitivity and specificity were 63.0% and 100.0%, respectively, with an AUC (95%CI) of 0.815 (0.680-0.950). Conclusions: NGS technology can successfully detect multiple gene mutations in US-FNA specimens from TMN patients, especially BRAF gene mutation, and BRAF gene mutation has certain value in diagnosing TMC.

[Ultrasound-guided percutaneous thermal ablation assisted by artificial ascites and soft tissue edema in the treatment of special-region hepatic tumors].

Objective: To explore the application value and operation skills of ultrasound-guided percutaneous thermal ablation assisted by artificial ascites or/and soft tissue edema in the treatment of special hepatic tumors located nearby the diaphragm, heart, stomach, gastrointestinal tract, gall bladder, kidney, and other organs. Methods: The clinical data of 132 patients with special-region hepatic tumors treated with ultrasound-guided percutaneous thermal ablation aided by artificial ascites and/or artificial soft tissue edema were retrospectively analyzed. Intraoperative contrast-enhanced ultrasound was used to guide ablation when necessary. During the operation, the ablation needle was lifted or pressed down, or the direction of the needle handle was changed to protect vital organs. The technical success rate of artificial ascites and/or soft tissue edema formation, the complete in activation rate of the tumor, and the complications were observed. Results: There were 74 patients (108 lesions) treated with radiofrequency ablation and 58 patients (82 lesions) treated with microwave ablation. Among them, 81 cases was successfully injected artificial abdominal ascites, with a water volume of (1 301±685) ml; artificial soft tissue edema was successfully formed for 19 patients, with a water volume of (534±258) ml. Both artificial ascites and artificial soft tissue edema were built for 30 patients. The success rate of this hydro-isolation technique was 98.5% (130/132). 129 patients successfully completed the treatment, and the complete inactivation rate of the tumor was 92.5% (172/186). The average postoperative hospital stay was three days. No patient had serious complications, such as surface tumor rupture, gastrointestinal injury, or diaphragm perforation. Conclusions: For hepatic tumors located adjacent to other organs such as the diaphragm, heart, gastrointestinal tract, gallbladder, and kidney, the application of artificial ascites and/or artificial soft tissue edema can reduce the damage to these organs, as well as reduce the possibility of tumor rupture and diaphragm perforation. These methods are safe and effective in ultrasound-guided percutaneous thermal ablation.

[Risk factors of occult lymph node metastasis of levels â ¢ and â £ in papillary thyroid carcinoma].

Objective: To investigate the potential risk factors for occult lateral cervical lymph node metastasis (LNM) to levels Ⅲ and Ⅳ in patients with papillary thyroid carcinoma (PTC) and the necessity of super-selective lateral lymph node dissection for patients harboring these metastases. Methods: This prospective study included PTC patients who were operated by the same surgeon in the Department of Head and Neck Surgery of Cancer Hospital, Chinese Academy of Medical Sciences from October 2015 through October 2019. Preoperative ultrasound and enhanced Computer Tomography (CT) did not denote suspected enlarged lymph nodes in the lateral neck. All patients underwent lymph node dissection in levels Ⅲ and Ⅳ on the basis of original thyroid collar incision after LNM to level Ⅵ was confirmed by preoperative fine needlebiopsy or intraoperative frozen pathology. Results: Of all 143 patients, 74 (51.7%) had occult LNM in levels Ⅲ and Ⅳ confirmed by postoperative pathology. The average number of metastasized lymph nodes in levels Ⅲ and Ⅳ was 2.64±1.80, and that in level Ⅵ was 3.77±3.27. There was a significant linear positive correlation between the number of metastasized lymph nodes in level Ⅵ and that in levels Ⅲ and Ⅳ (r=0.341, P<0.001). That the metastasized lymph nodes in level Ⅵ equals three was the best predictor of occult lateral LNM to levels Ⅲ and Ⅳ. Univariate analysis showed that age <55 years, tumor size ≥2.0 cm, number of metastasized lymph nodes in level Ⅵ ≥3, and percentage of metastasized lymph nodes in the total number of dissected lymph nodes in level Ⅵ >50% were associated with occult LNM in levels Ⅲ and Ⅳ (P<0.05). Multivariate analysis showed that number of metastasized lymph nodes in level Ⅵ≥3 was an independent risk factor for occult LNM in levels Ⅲ and Ⅳ (P=0.006). Conclusions: Age, tumor size and LNM in level Ⅵ were associated with occult lateral LNM in PTC patients. Lymph node dissection in levels Ⅲ and Ⅳ could be considered for selective patients, since it will help to avoid secondary operation for residual tumor or recurrence resulted from insufficient treatment without increasing the incidence of complications or affecting patients' appearances.

[Histogram analysis of based on two-dimensional ultrasound images to differentiate medullary thyroid carcinoma and thyroid adenoma].

Objective: To investigate the feasibility and value of histogram analysis based on two-dimensional gray-scale ultrasonography in the differential diagnosis of medullary thyroid carcinoma (MTC) and thyroid adenoma (TA). Methods: The preoperative ultrasound images of 86 newly diagnosed MTC patients and 100 TA patients treated in the Cancer Hospital of Chinese Academy of Medical Sciences from January 2015 to October 2021 were collected. Histograms were performed based on the regions of interest (ROIs) delineated manually by two radiologists, thereafter, mean, variance, skewness, kurtosis, percentiles (1st, 10th, 50th, 90th, 99th) were generated. The histogram parameters between the MTC group and the TA group were compared, and the independent predictors were screened by multivariate logistic regression analysis. Receiver operating characteristic (ROC) analysis was used to compare the individual diagnostic efficacy and joint diagnostic efficacy of independent predictors. Results: Multivariate regression analysis showed that mean, skewness, kurtosis and 50th percentile were independent factors. The skewness and kurtosis in the MTC group were significantly higher than those in the TA group, and the mean and 50th percentile were significantly lower than those in the TA group. The area under the individual ROC curve of mean, skewness, kurtosis and 50th percentile is 0.654-0.778. The area under the combined ROC curve is 0.826. Conclusion: Histogram analysis based on two-dimensional gray-scale ultrasonography is a promising tool to distinguish MTC from TA, in which the joint diagnosis value of mean, skewness, kurtosis and 50th percentile is the highest.

[Active surveillance for thyroid micro-malignant nodules].

Objective: To evaluate the effect of ultrasound diagnosis of thyroid micro-malignant nodules and accumulate practical experience for the management of active surveillance for them, so as to avoid overtreatment. Methods: A total of 949 patients who were diagnosed with thyroid malignant nodules using ultrasonography, with the nodules being less than 1 cm in size and without regional lymph node metastasis or distant metastasis, were included. They were treated by the same surgeon of the Department of Head and Neck Surgery, Cancer Hospital, Chinese Academy of Medical Sciences from February 2014 to December 2020. 112 patients chose immediate surgery. The rest patients were asked to accept ultrasound examination every 6 months to 1 year. Follow-up endpoints: tumor size growth of 3 mm, tumor volume increase greater than 50%, lymph node metastasis or distant metastasis. Results: The median follow-up time was 19 months. 713 patients underwent surveillance for more than 6 months. Of the 713 patients, 570 (79.9%) were women, with mean age at 43.5 years old. Tumor progression was observed in 47 (6.6%) patients with a cumulative incidence of 2.7% (1 year), 7.2% (2 years) and 9.5% (3 years). In multivariate analysis, patient age [HR=0.508, 95%CI: 0.275-0.939, P=0.031], lesion number [HR=2.945, 95%CI: 1.593-5.444, P=0.001] and tumor size [HR=2.245, 95%CI: 1.202-4.192, P=0.011] at the beginning of observation were independent risk factors for tumor progression in patients with minimal thyroid malignant nodules during follow-up. During a median (range) active surveillance of 19 (6-80) months, 74 patients chose surgery during the surveillance. Among the 186 patients who underwent surgery, only 3 patients were diagnosed with fibrotic nodules in pathology, while the rest were papillary thyroid carcinoma. The ultrasound accuracy reached 98.4%(183/186). Conclusions: Ultrasonography is an effective method of diagnosing malignant thyroid nodules. Thyroid micro-malignant nodules progress slowly. As a result, it is safe to observe them instead of taking immediate surgery. Patient age, lesion number and tumor size at the beginning of observation are independent risk factors for the tumor progression of malignant nodules.

[Establishment of a preoperative prediction model for axillary lymph node burden in patients with early breast cancer].

Objective: To explore the method of predicting high lymph node load in patients with early breast cancer to avoid unnecessary sentinel lymph node biopsy. Methods: The clinicopathological and thoracic multi-slice spiral CT (MSCT) data of 2620 patients with early (cT1~2N0M0) breast cancer treated in the Affiliated Cancer Hospital of Zhengzhou University from January 1, 2014 to August 1, 2018 were collected. According to the postoperative pathological results, the patients were divided into the group with axillaryhigh lymph node burden (HNB) and the non-HNB group. The influencing factors of axillary lymph node burden in patients with early breast cancer were determined by univariate and multivariate analysis, and the diagnostic model of MSCT to HNB was established. The best cutoff value for the diagnosis of HNB was determined through analyzing the receiver operative characteristic (ROC) curve, and the consistency between MSCT diagnosis and pathological diagnosis was evaluated by Kappa test. Results: Among the 2 620 patients, 168 were diagnosed of HNB. Univariate analysis showed that the tumor size, the status of human epidermal growth factor receptor 2 (HER-2), the number of abnormal lymph nodes showed in MSCT, the ratio of the length to the diameter of the maximum abnormal lymph node as shown in MSCT, the condition of the maximum abnormal lymph node door, and the parenchyma of the maximum abnormal lymph node were related to axillary lymph node burden in patients with early breast cancer (P<0.05). Multivariate analysis showed that the number of abnormal lymph nodes showed in MSCT was an independent influencing factor of axillary HNB in patients with early breast cancer. Compared with patients without abnormal lymph nodes, the OR values of patients with 1, 2, 3 or more abnormal lymph nodes displayed by MSCT and in axillary HNB status were 3.305, 9.379, 126.163 and 780.953, respectively. Using 3 or more abnormal lymph nodes detected by MSCT to predict the area under the ROC curve of axillary HNB in patients with early breast cancer, the area was 0.928, the sensitivity was 82.1%, the specificity was 95.4%, and the accuracy was 94.5%. Kappa test showed that the consistency between MSCT diagnosis and pathological diagnosis was relatively high (Kappa=0.629, P<0.001). Conclusions: The number of abnormal lymph nodes showed in MSCT is an independent influencing factor of axillary HNB in patients with early breast cancer. Taking 3 or more abnormal lymph nodes showed in MSCT as the threshold can help to predict the axillary HNB status of early breast cancer patients and exempt some of them from unnecessary sentinel lymph node biopsy.

[Preliminary study of superselective lymph node dissection in regional lateral cervical lymph node metastasis of papillary thyroid carcinoma].

Objective: To explore the applied value of super-selective cervical lymph node dissection in papillary thyroid carcinoma (PTC) patients with clinically suspicious lateral lymph node metastasis (LNM). Methods: We retrospectively analyzed the clinical data of 232 cN1b PTC patients who underwent surgery from September 2013 to May 2018 in the Department of Head and Neck Surgical Oncology, National Cancer Center. Among them, 90 cases received super-selective neck dissection (level Ⅲ and IV) and 142 cases underwent selective neck dissection (level Ⅱ-Ⅳ). The LNM of two groups were analyzed. Results: Postoperative pathological results showed that 173 cases had LNM in the central compartment. The LNM cases of level Ⅱ-Ⅳ were 47, 147 and 130, respectively. Eight patients of super-selective neck dissection and 6 of selective neck dissection had postoperative lymphatic fistulas (P=0.146). No patients in super-selective neck dissection group while 9 patients in the selective lymph node dissection group had postoperatively permanent impairment of shoulder mobility, the difference was statistically significant (P=0.015). In the super-selective neck dissection group, 2 patients had long-term postoperative incision discomfort, and 5 cases had obvious cicatrix after surgery. In the patients with selective neck dissection, 27 cases experienced long-term incision discomfort after surgery, and 26 patients had apparent scar tissue, the differences were statistically significant (P<0.005). There was no recurrence during the follow-up. Conclusions: Super-selective neck dissection is a feasible, safe and effective treatment for cN1b PTC patients. It can improve the quality of postoperative life and avoid the over treatment for patients.

[Ex-vivo experimental study of ultrasound-guided radiofrequency ablation for the treatment of hepatocellular carcinoma].

Objective: To explore the effective mode of ultrasound-guided radiofrequency ablation for the treatment of hepatocellular carcinoma (HCC). Methods: The morphological changes of human liver cancer cells HepG2 and human permanent liver cells LO2 after high temperature treatment were observed. The cell counting kit 8 (CCK-8) array was used to detect cell proliferation ability. The HepG2 cells (ΔHepG2 cells) and LO2 cells (ΔLO2 cells) those were still alive after treatment with 47℃ were collected and subjected to secondary high temperature treatment, and their proliferation ability was tested by the CCK-8 array. The ablation was performed at different locations of the ex-vivo porcine liver. The consistency of the vaporization area of the ultrasound image with that of the actual ablation was compared, the temperature of each area in the ultrasonic image was observed. Results: After incubated water was heated to 50℃, with the extension of the culture time, the number of adherent cells of HepG2 and LO2 cells became less and less, and gradually lost their normal cell morphology. After 50℃ and above, all of the proliferation rates of HepG2 cells, LO2 cells, ΔHepG2 cells and ΔLO2 cells were decreased. Compared with the 37℃ group, the differences were statistically significant (P<0.05), and the cells were irreversible damaged. There was no significant difference between the transverse diameters of the sonographic vaporization zones and the actual ablation zones (P>0.05). Nine minutes was considered to be an effective ablation time point since the temperatures in the hyperechoic areas were all above 53℃. Conclusion: The HCC cells within the vaporization zone observed by ultrasound can be damaged irreversibly when they were effectively ablated for over 9 minutes at 105℃ and 200 W.

[Discussion on diagnosis and treatment of hepatobiliary malignancies during the outbreak of COVID-19].

Objective: From December 2019, the new coronavirus pneumonia (COVID-19) broke out in Wuhan, Hubei, and spread rapidly to the nationwide. On January 20, 2020, the National Health Committee classified COVID-19 pneumonia as one of B class infectious diseases and treated it as class A infectious disease. During the epidemic period, the routine diagnosis and treatment of tumor patients was affected with varying degrees. In this special period, we performed the superiority of the multi-disciplinary team of diagnosis and treatment, achieved accurate diagnosis and treatment of patients with hepatobiliary malignant tumors, provided support for these patients with limited medical resources, and helped them to survive during the epidemic period.On the basis of fully understanding the new coronavirus pneumonia, the treatment strategy should be changed timely during the epidemic, and more appropriate treatment methods should be adopted to minimize the adverse effect of the epidemic on tumor treatment.

[Comparison of antibiotics and saline irrigation with tube drainage in the treatment of postoperative abdominal and pelvic infectious effusion].

Objective: To compare the efficiency of saline irrigation, antibiotics irrigation and high-concentration antibiotics perfusion with tube drainage in the treatment of infectious effusion. Methods: Clinical and sonographic features of abdominal and pelvic infectious effusion of 64 patients with malignant tumor collected from September 2013 to September 2017 were retrospectively analyzed. The changes of effusion size and temperature, catheterization time were evaluated in saline irrigation group and antibiotics irrigation group. The catheterization time was compared between antibiotics irrigation group and high-concentration antibiotics perfusion group. Results: The effective rates of the saline irrigation group and the antibiotics irrigation group were 68.6% and 86.8%, respectively, and the times of catheterization were 11.9±8.4 days and 7.8±4.8 days, respectively, with significantly statistical difference (both P<0.05). However, the effective rates of the antibiotics irrigation group and the high concentration antibiotic perfusion group were 86.8% and 100.0%, respectively (P=0.067), while the times of catheterization were 7.8±4.8 days and 3.6±3.1 days, respectively (P<0.001). Conclusion: The antibiotic irrigation with tube drainage, especially the high concentration perfusion is more effective than saline in the treatment of abdominal and pelvic infection effusion.

[Efficacy and safety of vandetanib on advanced medullary thyroid carcinoma: single center result from a phase â ¢ study].

Objective: There is no effective therapy for patients with advanced medullary thyroid carcinoma (MTC). Vandetanib,a novel multitargeted receptor tyrosine kinase inhibitor, has previously shown antitumor activity in phase Ⅱ studies of patients with advanced MTC. This study was to evaluate the efficacy and the safety of vandetanib on advanced MTC. Methods: This study was an open, international multi-center phase Ⅲ clinical trial and the study number was NCT01298323. The single-center study was a sub-group analysis of the international study, which was conducted on 9 pathologically confirmed advanced MTC patients by Cancer Hospital Chinese Academy of Medical Sciences between March 2012 and October 2017. Vandetanib (300 mg) was orally administered daily till death or withdrawal. The efficacy was evaluated according to RECIST criteria and the adverse events were evaluated according to NCI criteria. Results: The objective response rate was 3/9,and the disease control rate was 4/9. The median progression-free survival was 44 months. All patients who had the elevated levels of calcitonin (CTN) and carcino-embryonic antigen (CEA) before treatment began to show the decreases in the level of CTN and CEA after 3 months and later showed again the increases in the levels of both tumor markers with tumor progression. By ROC curve analysis, CTN was of statistically significance(P<0.05, 95%CI 0.558-0.834), but CEA was not(P>0.05). Adverse events were generally mild (grade 1 or 2),including hypertension (9 cases),skin rash (9 cases), and diarrhea (6 cases). Two patients developed grade 3 elevation of serum glutamate pyruvate transaminase and one patient developed grade 3 elevation of drug-related bowel disease. No grade 4 drug-related adverse event occurred. Conclusions: Vandetanib is effective and well tolerated for patients with locally advanced or metastatic MTC who have no chance for surgery. This indicates the increase of CTN is clinically relevant to disease progression, but the number of patients are extremely low, and, therefore further research is needed. Long-term use of vandetanib may cause resistance.

[The value of ultrasound classification in BI-RADS category 4 of breast complex cystic masses].

Objective: To analyze the feature of breast complex cystic masses and to classify it at ultrasonography (US), which applied to the Breast Imaging Reporting and Data System (BI-RADS) categories 4a to 4c with pathological results as the golden standards. Methods: The ultrasonographic data and clinical features of 78 patients with complex cystic masses confirmed by pathology in Cancer Hospital from July 2014 to June 2017 were retrospectively reviewed. The complex cystic breast masses were divided into four classes on the basis of their US features: type 1 [thick wall and (or) thick septa (> 0.5 mm)], type 2 (one or more intra-cystic masses), type 3 (mixed cystic and solid components with cystic components more than 50%) and type 4 (mixed cystic and solid components with solid components more than 50%). Positive values (PPVs) were calculated for each type. Multiple linear regression analysis was used to analyze the ultrasonographic features of the masses (lesion size, margins, blood flow resistance index, calcification, and axillary lymph nodes, etc.) with malignant correlation. Results: There were 81 lesions in 78 patients. Among the 81 masses based on US appearance, 14 (17.3%) were classified as type Ⅰ, 18 (22.2%) as type Ⅱ, 18 (22.2%) as type Ⅲ, and 31 (38.3%) as type Ⅳ. The positive predictive values of the malignant lesions of type Ⅰ, type Ⅱ, Ⅲ and Ⅳ were 7.1%, 16.7%, 61.1% and 48.3%, respectively (P=0.040). In all the 81 masses, 14 were BI-RADS categories 4a, 18 were BI-RADS categories 4b and 49 were BI-RADS categories 4c. Masses with maximum diameter equal to or larger than 2.0 cm, unclear margins, RI≥0.7 and presence of abnormal axillary nodes assessment had a high probability of malignancy (P=0.030, 0.038, <0.001 and 0.025, respectively). Conclusion: Ultrasound typing is helpful for differentiating benign and malignant breast complex cysts and classifying BI-AIDS 4a to 4c, thus providing clearer treatment for clinical practice.

[Dye-tattooing under ultrasound guidance in preoperative localization of neck recurrences from thyroid cancer].

Objective: To evaluate the efficacy and safety of the application of dye-tattooing under ultrasound guidance in preoperative localization of neck recurrences from thyroid cancer. Methods: Between October 2014 to September 2016, 25 patients with 34 lesions were enrolled. There were 22 cases of papillary thyroid carcinoma and three cases of medullary thyroid carcinoma, all of which could not be detected by computed tomography. Surgeons located the recurrent lesions using dye-tattooing under ultrasound guidance along with radiologist three days before the operation. Results: All lesions were successfully located (100%), 32 of which were located directly and two of which were located indirectly. Postoperative pathological examination confirmed 25 metastases of papillary thyroid carcinoma, two metastases of medullary thyroid carcinoma, and seven cases of false positives. The accuracy of ultrasound diagnosis was 79.4%. After 15 months of follow-up, neither tumor residual nor recurrences was detected according to imaging tests. Conclusions: Dye-tattooing under ultrasound guidance represents a reliable and safe method for localization of neck recurrences from thyroid cancer. The cooperation between experienced surgeons and radiologists will be crucial to successful location.

[Effect of Rab11 gene expression on the invasion and migration of cervical cancer cell line SiHa in hypoxia].

Objective: To explore the expression of Ras-related protein 11 (Rab11) in hypoxia, the effect of Rab11 on the invasion and migration of cervical cancer cell line SiHa and its possible mechanism. Methods: SiHa cells were divided into 4 groups, the normoxic blank group (normal culture in normoxia), the hypoxic blank group (normal culture in hypoxia), the negative control group [transfection of negative control small interfering RNA (siRNA) in hypoxia], the Rab11-siRNA group (transfection of Rab11 siRNA in hypoxia). Western blot was used to examine the expression of Rab11, integrin α5, integrin ß3, phosphorylated focal adhesion kinase (p-FAK), phosphorylated phosphatidylinositol 3 kinase (p-PI3K) protein, together with the expression of Ras correlative C3 creotoxin substrate 1 (Rac1), which was critical in regulating cell invasion. The mRNA expression of Rab11 in the 4 groups was detected by realtime-qPCR. The cell invasion was detected by matrigel assay, while the cell migration was detected by transwell assay. Immunofluorescence was used to identify intracellular location of Rac1 in SiHa cell. Results: (1) The expression of Rab11, intergrin α5, intergrin ß3, p-FAK, p-PI3K and Rac1 in the normoxic blank group were 0.56±0.04, 0.33±0.03, 0.32±0.03, 0.36±0.03, 0.35±0.03 and 0.47±0.03, respectively. In the hypoxic blank group, they were 0.73±0.03, 0.74±0.03, 0.61±0.03, 0.62±0.03, 0.60±0.03 and 0.73±0.03, respectively. In the negative control group, their expressions were 0.72±0.03, 0.73±0.03, 0.59±0.03, 0.61±0.03, 0.59±0.03 and 0.72±0.03, respectively. While in the Rab11-siRNA group, they were 0.44±0.03, 0.30±0.03, 0.29±0.03, 0.30±0.03, 0.30±0.03 and 0.34±0.04, respectively. The expressions of Rab11, α5, ß3, p-FAK, p-PI3K and Rac1 were significantly higher in the hypoxic blank group than in the normoxic blank group (P<0.05), and were significantly lower in the Rab11-siRNA group than in the hypoxic blank group and the negative control group (P<0.05). (2) The expressions of Rab11-mRNA were 1.000±0.000, 1.454±0.114, 1.442±0.101, 0.570± 0.046 in the normoxic blank group, the hypoxic blank group, the negative control group and the Rab11-siRNA group, respectively. It was significantly higher in the hypoxic blank group than in the normoxic blank group (P<0.05), and was significantly lower in the Rab11-siRNA group than in the hypoxic blank group and the negative control group (P<0.05). (3) By Matrigel, the invasion cell number in the normoxic blank group, the hypoxic blank group,the negative control group and the Rab11-siRNA group were 65±12, 106±16, 104± 17 and 50±11, respectively. The invasion capacity was significantly higher in the hypoxic blank group than in the normoxic blank group (P<0.05), and was significantly lower in the Rab11-siRNA group than in the hypoxic blank group and the negative control group (P<0.05). (4) By transwell assay, the migration cells in the normoxic blank group, the hypoxic blank group, the negative control group and the Rab11-siRNA group were 127±12, 169±15, 161±13 and 77±13, respectively. The capacity of invasion was significantly higher in the hypoxic blank group than in the normoxic blank group (P<0.05), and was significantly lower in the Rab11-siRNA group than in the hypoxic blank group and the negative control group (P<0.05). (5) The immunofluorescence showed that the red fluorescence intensity around nucleus was significantly increased in the normoxic blank group, the hypoxic blank group and the negative control group than in the Rab11-siRNA group. Conclusions: Hypoxia could promote the invasion and migration of SiHa cells. In hypoxia, the down regulation of Rab11 expression could inhibit the invasion and migration of SiHa cells. This might be due to the decreased expression of the intergrin α5, intergrin ß3, p-FAK, p-PI3K and Rac1 protein.

Suppression of Frizzled-2-mediated Wnt/Ca²âº signaling significantly attenuates intracellular calcium accumulation in vitro and in a rat model of traumatic brain injury.

Traumatic brain injury (TBI) can dramatically increase levels of intracellular calcium (Ca²âº). The association between Wnt5a/Frizzled-2 (wingless-type mouse mammary tumor virus integration site family member 5a/Fzd2) signaling and Ca²âº cellular homeostasis in lower vertebrates has been well documented. However, little is known about Wnt5a/Fzd2 signaling in mammalian nerve cells, or whether Ca²âº accumulation after TBI is mediated through this pathway. We hypothesized that an activated Wnt5a/Fzd2 pathway following TBI may play a role in Ca²âº overloading. To elucidate the influence of Fzd2 and the Wnt5a signal transduction pathway on an increase in intracellular Ca²âº, we assessed the expression of Wnt5a/Fzd2 in rat hippocampal cells both in vitro and in vivo. We found that transfection of the rat Fzd2 gene in rat neonatal hippocampal astrocytes significantly increased gene expressions of both Wnt5a and Fzd2 by fourfold when compared to non-transfected cells (P<0.01 in both cases). Expressions of the proteins Wnt5a and Fzd2 were significantly increased approximately two- and threefold, respectively, when compared to non-transfected control cells (P<0.01 in both cases). Moreover, intracellular Ca²âº, as manifested by the fluorescent intensity of the intracellular Ca²âº indicator Fluo-3/AM, was significantly increased by 1.75-fold (P<0.01). The blocking of Fzd2 signaling using Stealth RNAi markedly inhibited the elevated gene and protein expression of Wnt5a in the transfected cells by two- and fourfold, respectively (P<0.01), and suppressed intracellular Ca²âº by 1.5-fold (P<0.01). Furthermore, in vivo, we demonstrated that TBI-induced dramatic upregulation of gene and protein expression of Wnt5a/Fzd2 by two- and fivefold (P<0.01) in injured hippocampi, and intracellular Ca²âº increased in isolated injured hippocampal cells. Whereas, the in vivo blocking of Fzd2 signaling by hippocampal delivery of Stealth RNAi and Invivofectamine significantly suppressed the increased gene and protein expression of Wnt5a and Fzd2 induced by TBI by 1- to 3.5-fold (P<0.01) and also inhibited Ca²âº accumulation by 1.5-fold (P<0.01). These findings demonstrated that the Wnt5a/Fzd2 signaling pathway contributed to increasing intracellular Ca²âº in nerve cells under physiological and pathological conditions. Furthermore, our findings provide evidence that specifically expressed components of this signal pathway, such as Wnt5a and Fzd2, are potential therapeutic targets following brain trauma.