MiR-708-5p/Pit-1 axis mediates high phosphate-induced calcification in vascular smooth muscle cells via Wnt8b/ß-catenin pathway.

Recently, the underlying mechanism of vascular calcification (VC) has been partially elucidated. However, it is still high incidence, and no effective treatment has been found. This study aims at figuring out the underlying mechanisms of microRNA-708-5p (miR-708-5p)/sodium-phosphate transporter 1 (Pit-1) axis in high phosphate (HP)-induced VC of T/G HA-VSMCs. Alizarin Red S staining was used to evaluate calcium salt deposition, and the activity of alkaline phosphatase (ALP) was determined by measuring the absorbance at 405 nm. RT-qPCR and Western blot were performed to assess the levels of miR-708-5p and Pit-1, the levels of ALP, Pit-1, ß-catenin, glycogen synthesis kinase 3 ß (GSK3ß), and p-GSK3ß proteins, respectively. The interaction between miR-708-5p and Pit-1 was validated by luciferase reporter assay. Our findings illustrated that miR-708-5p was downregulated and Pit-1was upregulated in HP-induced VC. MiR-708-5p mimics inhibited HP-induced VC. Further experiments demonstrated that miR-708-5p targets Pit-1. In addition, miR-708-5p inactivates the Wnt8b/ß-catenin pathway via targeting Pit-1 to reduce HP-induced VC. MiR-708-5p has a crucial effect on VC via targeting Pit-1 and inhibiting Wnt8b/ß-catenin pathway, it may serve as a new target for VC treatment.

Human telomerase RNA gene amplification detection increases the specificity of cervical intraepithelial neoplasia screening.

OBJECTIVES: The purpose of this study was to evaluate the clinical significance of genomic amplification of the human telomerase RNA gene (TERC) for cervical cancer screening and explore whether it can serve as a biomarker to improve the specificity of human papillomavirus (HPV) DNA testing for cervical cancer screening. METHODS: One hundred twenty women, including 20 cases of normal (control), 14 cases of cervical intraepithelial neoplasia grade I (CIN I), 35 cases of CIN II, 36 cases of CIN III, and 15 cases of squamous cervical cancer diagnosed by histopathologic evaluation, were subjected to cytopathologic examination, TERC detection by fluorescence in situ hybridization (FISH), and HPV DNA testing by Hybrid Capture II. RESULTS: TERC amplification was significantly associated with cytopathologic diagnosis (P < 0.001) and histopathologic evaluation (P < 0.001). The positive rate of TERC gain was significantly higher in patients with CIN III or squamous cervical cancer than in patients with CIN I or those in the control group (P < 0.001). The specificity and positive predictive value of FISH for detecting CIN II or more severe cervical lesions (> or = CIN II) were obviously higher than those of HPV DNA testing (97.1% vs 52.9%, 98.7% vs 83.8%). CONCLUSIONS: TERC amplification analyzed by FISH may serve as an adjunctive test to HPV DNA testing for improving the specificity and positive predictive value of cervical cancer screening.

[Randomized comparative study on the lipid lowering effects of Zhikang Granule and simvastatin].

OBJECTIVE: To compare the lipid lowering effects of Zhikang Granule (ZKG) and simvastatin. METHODS: Forty-five out-patients with hyperlipemia who met the entry criteria were enrolled and randomized into two groups in the ratio of 2: 1, 30 patients in the ZKG group and 15 patients in the simvastatin group. The lipid lowering effects and safety of treatment during the 24-week therapeutic period, as well as the influence of treatment on plasma high sensitivity C reactive protein (hs-CRP) level in patients were observed. RESULTS: No significant difference between the two groups was observed in serum levels of total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), high density lipoprotein-cholesterol (HDL-C) and triglyceride (TG) at the 4th, 8th, 12th and 24th week (P > 0.05). However, as compared with baseline, significant reduction of TC and LDL-C in both groups was shown at all the observing time points (P < 0.01), while the changes in TG and HDL-C were insignificant (P > 0.05). The control rates of LDL-C and TC in the ZKG group and the simvastatin group were 86.7% (26/30) versus 100% (15/15) at the 4th week, 80.0% (24/30) versus 100% (15/15) at the 8th week, 53.3% (16/30) versus 60.0% (9/15) at the 12th week, and 90.0% (27/30) versus 93.3% (14/15) at the 24th week, respectively, all showed insignificant difference between groups. No statistical differences were found between groups in levels of plasma transaminase, creatinine, uric acid and hs-CRP (P > 0.05). CONCLUSION: ZKG has a definite effect in lowering LDL-C and TC, and it is safe in long-term administration.

[Endometrial adenocarcinoma and clear cell carcinoma in a young woman with polycystic ovarian syndrome: a case report].

A 26-year-old unmarried woman with irregular menstruation for 4 years was admitted for an intrauterine space-occupying mass. Pathological examination before surgery showed moderately to poorly differentiated endometrial adenocarcinoma. The patient underwent laparoscopically assisted epifascial panhysterectomy with bilateral salpingo-oophorectomy. Pathological examination of the surgical specimens reported moderately to poorly differentiated endometrial adenocarcinoma and stage II clear cell carcinoma. The patient then received chemotherapy and remained alive without evidence of recurrence. Young women with polycystic ovarian syndrome are at high risk of developing endometrial carcinoma, but concurrent clear cell carcinoma is rare. Careful evaluation before and after treatment are essential to improve the patients prognosis.

[MiR-135b promotes proliferation of endometrial carcinoma cells by targeting FOXO1].

OBJECTIVE: To explore the expression of miR-135b in endometrial carcinoma and the mechanism by which miR-135b promotes the proliferation of endometrial cancer cells. METHODS: The expressions of miR-135b and FOXO1 were using RT-PCR detected in 22 fresh endometrial cancer tissues and paired adjacent tissues and also in endometrial cancer cell lines JEC, Ishikawa, HEC-1-B, and RL-952. The RL-952 and Ishikawa cell lines were transfected with miR-135b mimics or inhibitors, and the changes in their proliferative activity were detected with MTT assay; the expressions of FOXO1 mRNA and protein were detected by RT-PCR and Western blotting, respectively. RESULTS: The expression of miRNA135b was significantly up-regulated and FOXO1 expression was down-regulated in endometrial carcinoma tissues as compared with the adjacent tissues (P<0.05). The mRNA expression of miR-135b was negatively correlated with the expression of FOXO1 in endometrial carcinoma. In RL-952 and Ishikawa cell lines, transfection with miR-135b mimics obviously promoted the cell proliferation (P<0.05). Up-regulation of miR-135b significantly decreased the expressions of FOXO1 protein and mRNA (P<0.05), and down- regulation of miR-135b increased FOXO1 expressions (P<0.05). CONCLUSIONS: MiR-135b plays an important role in the occurrence and development of endometrial carcinoma partially by regulating its target gene FOXO1.

[Significance of pelvic lymph node human papillomavirus DNA detection in cervical cancer].

OBJECTIVE: To assess the significance of human papillomavirus (HPV) DNA detection in the pelvic lymph nodes in cervical cancer. METHODS: HPV L1 gene fragment was amplified by HPV-specific PCR with general consensus primers from cervical cancer tissues. The types of HPV were identified by sequencing of the PCR product. The relationship between HPV positivity and pathological findings in metastatic pelvic lymph nodes of cervical cancer was analyzed. RESULTS: The positivity rate of HPV DNA was 40% in the pelvic lymph nodes in 40 cases of cervical cancer. HPV DNA was detected in the pelvic lymphatic nodes of all the 10 cases with pathologically confirmed lymph node metastasis of cervical cancer. HPV18 was detected in both of cervical and pelvic lymph nodes in some cases. CONCLUSIONS: HPV DNA can be detected in the pelvic lymph nodes before pathological identification of lymph node metastasis and indicates early pelvic lymph node metastasis of cervical cancer. The presence of HPV18 often indicates high likeliness of lymph node metastasis and suggests poor prognosis of the patients.

[Sequence polymorphism of human papillomavirus type 16 E7 in cervical cancer].

OBJECTIVE: To explore the genetic polymorphism of E7 open-reading frame of human papillomavirus (HPV) type 16 in cervical cancer. METHODS: The types of HPV was identified by sequence analysis of the PCR product of HPV in cervical cancer tissues. HPV 16 gene fragment in the cervical cancer tissue was amplified by HPV-specific PCR with general consensus primers. RESULTS: The positivity rate of high-risk HPV types in 50 cases of cervical cancers was 78%. Mixed infection of HPV16 and HPV18 was found in 18 cases, and the infection with HPV16 alone occurred in 15 cases. HPV16 E7 was amplified from 25 out of the 34 cases positive for HPV16. A T-to- C change occurred in the 647th nucleotide in the viral nucleotide sequence, causing conversion of the Asn codon of E7 gene into a Ser codon. CONCLUSIONS: The most frequently observed substitution in HPV16 E7 open reading frame occurs in the discrete regions of 647-846, and some substitutions result in the same-sense mutation. The hot-spot mutation of HPV16 E7 in cervical cancers in Guangdong Province occurs at the nucleotide 647 and 846 in the DNA sequence.

Selection of choriocarcinoma-associated genes using bioinformatics.

OBJECTIVE: To analyze the data of gene expression profiles of choriocarcinoma and screen for choriocarcinoma- related genes. METHODS: Human cDNA expression microarray containing 4 096 genes was used to study the gene expression profiles in specimens of complete hydatidiform moles (n=3) and choriocarcinomas (n=3), with normal placental villi serving as the control group. The candidate genes with similar expression profiles were identified by hierarchical cluster analysis, and their expressions in normal and neoplastic tissues analyzed by electronic Northern analysis and other bioinformatics methods. Three selected genes were analyzed by beta-actin semiquantitative reverse transcriptase-PCR to confirm the data. RESULTS: A total of 52 coexpressed candidate genes were identified from the gene expression data derived from the choriocarcinoma specimens, of which 19 genes were selected as choriocarcinoma-related genes by further cluster analysis, such as dynamin (L07807), katanin p60 (AF056022), zinc finger protein ZNF184 (U6656), calmodulin (U12022), carboxypeptidase M (BC022276), calcineurin-binding protein cabin 1 (NM_012295) and transducin-like enhancer protein TLE1(M99435). CONCLUSION: The identification of choriocarcinoma-related genes by cluster analysis provides new clues for seeking the key genes associated with the progression and metastasis of choriocarcinoma.

[Detection of human papillomavirus DNA in cervical cancer tissue by PCR and direct sequencing].

OBJECTIVE: To explore an effective method for detecting human papillomavirus (HPV) DNA in cervical cancer tissue. METHODS: HPV L1 gene fragment in cervical cancer tissue was amplified by HPV-specific PCR with consensus primers, and typing of the HPV strains was performed on the basis of sequence analysis of the PCR product. RESULTS: The positivity rates of HPV DNA was 78% in the 50 cases of cervical cancer, and mixed infection with HPV16 and HPV18 strains was the most common, which accounted for 48% on the total infections. Infection with HPV58 was detected in one case. The sequencing results showed no difference in L1 sequence between the detected samples and the standard German HPV58 strain. CONCLUSION: PCR and direct sequencing approach is effective for detecting and typing of HPV DNA in cervical cancer tissue, through which rare HPV strain or mutants of known HPV strains may not escape detection.

[Study of integrated state of HPV-16 infection in cervical cancer and precancerous tissues].

OBJECTIVE: To investigate the prevalence of physical state of HPV-16 DNA in cervical cancer and cervical precancerous carcinoma. METHODS: Multiplex PCR was adopted to detect the physical state of HPV in samples from 252 patients with cervical carcinoma, including 48 samples of cervical cancer, 204 cervical intraepithelial neoplasia (CIN ) (125 CIN I, 46 CIN II and 33 CIN III) and 20 normal samples from the subjects with hysteromyoma undergoing hysterectomy, respectively. RESULTS: Among 48 patients with cervical cancer, 31 (65.6%) were infected with HPV-16. Eighteen among 31 (58.1%) HPV-16 infected patients with cervical cancer were found to have integrated infection of HPV-16. The positive rates of HPV-16 infection in the patients with CIN I, CIN II and CIN III were 19.2%, 34.8% and 42.4%, and the integrated infection rates of HPV-16 were 16.7%, 18.8% and 35.7%, respectively. Compared with patients with different grades of CIN, the integrated rate of HPV-16 infection in those with cervical cancer was significantly elevated. CONCLUSION: Among the patients with HPV-16 infection, the integrated state of HPV-16 is positively correlated with the severity of cervical lesions. Combined HPV typing test and detection of integrated viral state contribute to predicting the prognosis of patients with cervical precancerous lesions and increasing the accuracy of screening cervical cancer on the basis of HPV DNA detection.

Transcriptional gene expression profiles of HGF/SF-met signaling pathway in colorectal carcinoma.

AIM: To explore the transcriptional gene expression profiles of HGF/SF-met signaling pathway in colorectal carcinoma to understand mechanisms of the signaling pathway at so gene level. METHODS: Total RNA was isolated from human colorectal carcinoma cell line LoVo treated with HGF/SF (80 ng/L) for 48 h. Fluorescent probes were prepared from RNA labeled with cy3-dUTP for the control groups and with cy5-dUTP for the HGF/SF-treated groups through reverse-transcription. The probes were mixed and hybridized on the microarray at 60 degrees for 15-20 h, then the microarray was scanned by laser scanner (GenePix 4000B). The intensity of each spot and ratios of Cy5/Cy3 were analyzed and finally the differentially expressed genes were selected by GenePix Pro 3.0 software. 6 differential expression genes(3 up-regulated genes and 3 down-regulated genes) were selected randomly and analyzed by beta-actin semi-quantitative RT-PCR. RESULTS: The fluorescent intensities of built-in negative control spots were less than 200, and the fluorescent intensities of positive control spots were more than 5000. Of the 4004 human genes analyzed by microarray, 129 genes (holding 3.22 % of the investigated genes) revealed differential expression in HGF/SF-treated groups compared with the control groups, of which 61 genes were up-regulated (holding 1.52 % of the investigated genes) and 68 genes were down-regulated (holding 1.70 % of the investigated genes), which supplied abundant information about target genes of HGF/SF-met signaling. CONCLUSION: HGF/SF-met signaling may up-regulate oncogenes, signal transduction genes, apoptosis-related genes, metastasis related genes, and down-regulate a number of genes. The complexity of HGF/SF-met signaling to control the gene expression is revealed as a whole by the gene chip technology.

[Age and prognosis: analysis of their association in 93 cases of cervical cancer].

OBJECTIVE: To investigate the relationship between the patient's age and clinical prognosis of cervical cancer. METHODS: Ninety-three patients with cervical cancer selected from those hospitalized in Nanfang Hospital from Jan 1997 to Dec 2002 were divided into younger age (less than 35 years old) and elder age group ( more than 35 years old). Comparison was made in the clinical stages, pathological grade and differentiation between the 2 groups. RESULTS: Statistical difference was found in the clinical stages and pathological grade between the 2 groups, and in patients below the age of 35 years, 82.61% were below stage II a and 17.39% above stage II b, had squamous cell carcinoma and 13.39% had adenocarcinoma; In those above 35 years, 51.43% were classified below stage II a stage and 48.73% above stage II b, and squamous cell carcinoma were identified in 82.98% and adenocarcinoma in 17.02% of these patients. However, no statistical difference was found in the cell differentiation between the two groups. CONCLUSIONS: Patients above 35 years have poorer prognosis than their younger counterparts, which might be due to more advanced clinical stage and higher rate of adenocarcinoma.

[Clinical management of vaginal delivery with previous cesarean section].

OBJECTIVE: To explore the feasibility of vaginal delivery with previous cesarean section. METHODS: Eighty-seven randomly chosen pregnant women who had previously received cesarean section were enrolled in this study, and their consent was obtained to go through vaginal delivery. RESULTS: Eighty cases (91.95%) completed vaginal deliveries successfully. Examination of the uterine cavity after labor found partial rupture of the uterine cicatrix in 1 case and immediate surgical repair was performed. Death occurred in none of the cases. Of all the cases of vaginal delivery, the rate of spontaneous labor was 35% and the rate of instrumental vaginal delivery rate was 65%. The Apgar score of 80 neonates one minute after birth was above 8 in 76 and 4 to 7 in 4 cases. CONCLUSION: In pregnant women who have previous cesarean section, vaginal delivery can be possible when there are indications without contraindications.

[Comparative study of the impacts of laparoscopic and open laparotomic surgeries on oxidative stress in patients with uterine myomas].

OBJECTIVE: To evaluate the impact of laparoscopic surgery on oxidative stress in patients with uterine myomas. METHODS: Plasma malondialdehyde (MDA), antioxidant activity (AOA), and glutathione peroxidase (GPx) activity in 20 patients with uterine myomas were measured before, at the end of (5 min after deflation), and 24 h after laparoscopic surgery, for comparison with the measurements in 20 patients receiving laparotomic surgery at the same time points. RESULTS: Immediately following laparoscopic surgery, plasma MDA level increased significantly as compared with the preoperative level (P<0.01) and recovered the normal level 24 h after the operation; but in laparotomic surgery, plasma MDA level underwent a slight increase in the immediately early postoperative stage and continued to increase 24 h after the operation. In laparoscopic surgery, GPx and AOA decreased significantly in the early postoperative stage (P<0.01) and recovered to some extent 24 h postoperatively, whereas in laparotomic surgery, GPx and AOA slightly decreased immediately following the operation and further decreased 24 h after the operation. Compared with patients receiving laparotomic surgery, laparoscopic patients had significantly lower plasma MDA level (P<0.01) and significantly higher GPx activity (P<0.01) with comparable AOA level (P=0.33) 24 h after the operation. CONCLUSION: Free radical produces at the end of a laparoscopic procedure, possibly as a result of ischemia-reperfusion induced by the inflation and deflation of the pneumoperitoneum.

Preoperative CT findings of thymoma are correlated with postoperative Masaoka clinical stage.

RATIONALE AND OBJECTIVES: Both preoperative computed tomography (CT) staging and postoperative surgical Masaoka clinical staging are of great clinical importance for diagnosing thymomas. Our study aimed to investigate the relationships between these two staging systems. MATERIALS AND METHODS: This was a retrospective review of 129 patients who had undergone thymoma surgery. Helical CT and 16-slice CT were performed preoperatively. Surgical findings were evaluated according to the Masaoka clinical staging system. RESULTS: A significant association was shown between Masaoka clinical staging and CT staging, especially of features including tumor size (P = .004), tumor shape (P < .001), tumor density (P < .001), capsule completeness (P < .001), and involvement of surrounding tissues (P < .001). Based on the CT findings, there were 35.09% of Masaoka stage I patients who had a tumor size <5 cm as compared to 14.81% of stage IV patients. Only 8.77% of Masaoka stage I patients had a tumor size ≥10 cm as compared to 40.74% of stage IV patients. In stages III and IV, most tumors were irregularly shaped with an uneven density and incomplete capsule. Invasive tumors were more frequently found in stages III (81.48%) and IV (88.89%) than in stages I (0%) and II (38.89%). The incidence of myasthenia gravis was comparable in different stages. Consistency between CT and Masaoka clinical stages was higher in stage I (37.98%) than other stages (approximately 10%). CONCLUSION: This study documented a close relationship between preoperative CT thymoma staging and postoperative Masaoka clinical staging. Thus, preoperative CT findings can be beneficial for determining the proper management and prognosis of thymoma patients.

[Long-term culture and identification of CD133⁺ hematopoietic progenitor cells from human umbilical cord blood].

OBJECTIVE: To isolate CD133(+) hematopoietic progenitor cells from human umbilical cord blood and optimize the culture condition for maintaining their stem cell characteristics. METHODS: CD133(+) hematopoietic progenitor cells were isolated from human umbilical cord blood using magnetic cell sorting system, and the cells were detected by flow cytometry. Four methods were used for culturing cells. After 8 weeks' culture, cytomorphology, flow cytometry, immunocytochemistry and immunofluorescence assay were used to identify the characteristics of the stem cells. RESULTS: Over 80% of CD133(+) hematopoietic progenitor cells were isolated from human umbilical cord blood using magnetic cell sorting system. The cells were effectively expanded using optimized serum-free medium after 8 weeks of cell culture, whereas the cells in other media differentiated into adherent cells in a poor state. CONCLUSION: The optimized serum-free medium allows effective expansion of CD133(+) hematopoietic progenitor cells that maintain stem cell characteristics after a long-term culture.

Relationship between computed tomography manifestations of thymic epithelial tumors and the WHO pathological classification.

OBJECTIVE: To explore the relationship between computed tomography (CT) manifestations of thymoma and its WHO pathological classification. METHODS: One hundred and five histopathologically confirmed cases were collected for their pathological and CT characteristics and results were statistically compared between different pathological types of thymoma. RESULTS: Tumor size, shape, necrosis or cystic change, capsule integrity, invasion to the adjacent tissue, lymphadenopathy, and the presence of pleural effusion were significantly different between different pathological types of thymomas (P<0.05). Type B2, B3 tumors and thymic carcinomas were greater in size than other types. More than 50% of type B3 tumors and thymic carcinomas had a tumor size greater than 10 cm. The shape of types A, AB, and B1 tumors were mostly round or oval, whereas 75% of type B3 tumors and 85% of thymic carcinomas were irregular in shape. Necrosis or cystic change occurred in 67% of type B3 thymomas and 57% of thymic carcinomas, respectively. The respective figures for capsule destruction were 83% and 100%. Increases in the degree of malignancy were associated with increases in the incidence of surrounding tissue invasion: 33%, 75%, and 81% in type B2, type B3, and thymic carcinomas, respectively. Pleural effusion occurred in 48% of thymic carcinomas, while calcification was observed mostly in type B thymomas. CONCLUSIONS: Different pathological types of thymic epithelial tumors have different CT manifestations. Distinctive CT features of thymomas may reflect their pathological types.

[Construction of has-miR-335 lentiviral vector and verification of the target gene of miR-335].

OBJECTIVE: To construct a lentiviral vector of miR-335 gene and verify the target gene of miR-335. METHODS: The precursor sequence of miR-335 gene was amplified from the genomic DNA by PCR and cloned into the lentiviral vector PLVTHM labeled with GFP. Real-time quantitative RT-PCR was used to detect miR-335 and RASA1 expression in different colorectal cancer cell lines. A recombinant vector psiCHECK-2-RASA1 containing RASA1 3'UTR was constructed followed by site-directed mutagenesis of RASA1 3'UTR to establish the vector psiCHECK-2-RASA1-Mut. Co-transfection of hsa-mir-335 or a NC with these recombined vectors in HEK293A and SW480 cells was performed, and dual-luciferase reporter assay was utilized to examine the changes in luciferase activities. The recombinant PLVTHM-miR335 plasmid was packaged into mature lentivirus by 293FT cells and used to infect SW620 cells. Flow cytometry was employed for sorting the GFP+ cells. The expression of miR-335 and RASA1 were determined by qRT-PCR, and Western blotting was used to detect the expression of RASA1 protein in SW620 cell lines. RESULTS: The recombinant lentiviral vector PLVTHM-miR335, psiCHECK-2-RASA1 and the mutation expression vector psiCHECK-2-RASA1-Mut were successfully constructed. Dual-luciferase reporter assay showed that miR-335 decreased luciferase activity in cells co-transfected with psiCHECK-2-RASA1. The expression of miR-335 in SW620 cells infected with the lentivirus PLVTHM-miR335 was significantly increased, but the expression of RASA1 showed only slight changes. Overexpression of miR-335 suppressed the expression of RASA1 protein in SW620 cells. CONCLUSION: We have successfully constructed the lentiviral vector containing mir-335 gene and a SW620 cell line with miR-335 overexpression. MiR-335 can suppress RASA1 gene expression by targeting the specific sequence of RASA1 3'UTR.

Primary retroperitoneal malignant melanoma: A case report.

Primary malignant melanoma occurring at an extra cutaneous site is rare. A case of primary malignant melanoma located in the retroperitoneum of an 18-year-old female is presented in this study. Histopathological examination of the tissue biopsies at laparotomy with immunohistochemical stains confirmed a diagnosis of malignant melanoma. Further extensive clinical and radiological investigations proved the retroperitoneum to be the primary site.