MiR-135b promotes proliferation of endometrial carcinoma cells by targeting FOXO1 / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the expression of miR-135b in endometrial carcinoma and the mechanism by which miR-135b promotes the proliferation of endometrial cancer cells.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSIONS</b>MiR-135b plays an important role in the occurrence and development of endometrial carcinoma partially by regulating its target gene FOXO1.</p>

Endometrial adenocarcinoma and clear cell carcinoma in a young woman with polycystic ovarian syndrome: a case report / 南方医科大学学报

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.

Study of integrated state of HPV-16 infection in cervical cancer and precancerous tissues / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the prevalence of physical state of HPV-16 DNA in cervical cancer and cervical precancerous carcinoma.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Construction of has-miR-335 lentiviral vector and verification of the target gene of miR-335 / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a lentiviral vector of miR-335 gene and verify the target gene of miR-335.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Long-term culture and identification of CD133⁺ hematopoietic progenitor cells from human umbilical cord blood / 南方医科大学学报

<p><b>OBJECTIVE</b>To isolate CD133(+) hematopoietic progenitor cells from human umbilical cord blood and optimize the culture condition for maintaining their stem cell characteristics.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>The optimized serum-free medium allows effective expansion of CD133(+) hematopoietic progenitor cells that maintain stem cell characteristics after a long-term culture.</p>

Selection and identification of specific-binding peptides for cancer stem cell surface marker CD133 / 南方医科大学学报

<p><b>OBJECTIVE</b>To select the peptides that specifically bind human cancer stem cell surface marker CD133 from the Ph.D.-7>(TM) phage peptide library.</p><p><b>METHODS</b>With a biotinylated extracellular fragment of human cancer stem cell surface marker CD133 as the target protein, the CD133 high-affinity peptides were screened from the phage peptide library by liquid phase panning. The clones with high-binding force with human CD133 were then identified by sandwich ELISA and their single-stranded DNA was extracted to test the specificity by competitive ELISA. The amino acid sequences of the selected peptides derived from the phage DNA sequences were synthesized after sequence alignment analysis, and their capacity of binding with colorectal carcinoma cells was assessed by immunofluorescence technique.</p><p><b>RESULTS</b>After 4 rounds of liquid phase selection, the phages capable of specific binding with human CD133 were effectively enriched, with an enrichment ratio of 388 times compared to that at the fourth and first rounds. Thirteen out of the 20 clones from the fourth round of panning were identified as positive clones, among which 11 had identical amino acid sequence of TISWPPR, and 2 had the sequence of STTKLAL, and the former sequence showed a stronger binding specificity to CD133.</p><p><b>CONCLUSION</b>We have successfully obtained a peptide that specifically binds human CD133 from the Ph.D.-7(TM) phage peptide library, demonstrating the feasibility of screening small molecule high-affinity polypeptides from phage peptide library by liquid-phase panning.</p>

Touchdown PCR and overlap extension PCR for generating CD133(+) cancer stem cell-selective adenovirus vector / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a replication-incompetent adenovirus vector targeting cancer stem cells by modified touchdown PCR and overlap extension PCR and investigate its infection efficiency in CD133(+) SW480 cells in vitro.</p><p><b>METHODS</b>The two portions of the fiber gene encoding the Ad5 fiber knob domain with the HI loop deleted were amplified using two pairs of designed primers and then linked by overlap extension PCR. The product obtained was identified by sequencing and inserted into prokaryotic expression vector pEGFP-N1. The product, pEGFP-N1 KNOBδHI, contained a unique EcoRV restriction site in the deleted portion of the sequence encoding the HI loop. The gene sequences of the adenovirus fiber were amplified using both common PCR and overlap extension PCR, then identified by sequencing and inserted into pNEB193, resulting in pNEB-F5. CD133(+) SW480 cells were infected with the generated adenovirus vectors Ad5-GFP and Ad5FHI-GFP to investigate the infection efficiency using fluorescent microscope.</p><p><b>RESULTS</b>The target fragments of expected sizes were amplified by touchdown PCR and overlap extension PCR, but not by common PCR. Ad5FHI-GFP showed a higher infection efficiency than Ad5-GFP in CD133(+) SW480 cells.</p><p><b>CONCLUSION</b>Compared with common PCR, touchdown PCR and overlap extension PCR can significantly improve the specificity and efficiency of the PCR products for constructing CD133(+) cancer stem cell-selective adenovirus type 5 vector, which provides carriers for tumor-targeted gene therapy.</p>

Randomized comparative study on the lipid lowering effects of Zhikang Granule and simvastatin / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To compare the lipid lowering effects of Zhikang Granule (ZKG) and simvastatin.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>ZKG has a definite effect in lowering LDL-C and TC, and it is safe in long-term administration.</p>

Proteinchip-flight mass spectrometry for screening differentially expressed proteins in the serum of cervical cancer patients / 南方医科大学学报

<p><b>OBJECTIVE</b>To identify the differentially expressed proteins in the serum of patients with cervical cancer for use as the biomarkers for early diagnosis of cervical cancer.</p><p><b>METHODS</b>Surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) with weak cationic chips (CM10) was used to examine the serum samples of 24 patients with cervical squamous cell carcinoma and 25 age-matched healthy women. The protein fingerprints were obtained, and bioinformatic analysis was performed to identify the differentially expressed proteins in the serum of the patients.</p><p><b>RESULTS</b>Fifty-two differentially expressed proteins were detected in the serum of cervical cancer patients (P<10(-5)), among which 6 proteins with mass/charge ratio of 4173.77, 5903.09, 6087.12, 10716.9, 6109.61 and 3397.41, respectively, showed lowered expression in the serum of cervical cancer patients. Two diagnostic models for cervical cancer were generated using software, including one consisting of the 4173.77(M/Z) protein with the diagnostic specificity of 96% and sensitivity of 75% for cervical cancer and the other consisting of 3 proteins at 5335.81(M/Z), 7562.99(M/Z), and 9287.89(M/Z) with specificity of 91.67% and sensitivity of 96.0%.</p><p><b>CONCLUSION</b>Cervical cancer patients show different serum protein expression profile from healthy women. The 6 differential proteins identified may serve as specific serum biomarkers in close relation to the origin and progression of cervical cancer.</p>

Correlation between human papillomavirus DNA in the lymph nodes and metastasis of early-stage cervical carcinoma / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the correlation between the presence of human papillomavirus (HPV) DNA in the lymph nodes and histopathologically confirmed metastasis of early-stage cervical carcinoma.</p><p><b>METHODS</b>HPV L1 gene fragment in paraffin-embedded tissue specimens of the primary tumor and pelvic lymph nodes from 31 patients with cervical cancer was amplified using HPV-specific PCR with general consensus primers GP5+/GP6+. The type of HPV was identified by sequence analysis of the PCR products, and the correlation between the presence of HPV DNA in the lymph node and the clinicopathological indices of cervical carcinoma was analyzed.</p><p><b>RESULTS</b>The positivity rate of HPV DNA in the pelvic lymph nodes was 58.1% in the 31 patients, and in 13 of the patients with confirmed metastasis, the detection rate was 84.6% as compared with the rate of 27.8% in the other 18 patients without metastases. The presence of HPV DNA in the lymph node was associated with histologically confirmed metastases. The results of both HPV DNA detection and pathological examination indicated that the obturator, internal iliac and external iliac lymph nodes were more liable to be positive for HPV DNA, accounting for over 90% of the positivity.</p><p><b>CONCLUSION</b>HPV DNA detection in the pelvic lymph nodes is a helpful predictive factor of metastases, and the obturator, internal iliac and external iliac lymph nodes are the among the most vulnerable lymph nodes of metastatic involvement by early-stage cervical carcinoma.</p>

Preliminary study of gene expression profiling in human type I and II endometrial carcinoma / 南方医科大学学报

<p><b>OBJECTIVE</b>To study gene expression profiling in human type I and II endometrial carcinoma.</p><p><b>METHODS</b>Six Affymetrix human genome genechips were utilized to investigate the differences in gene expression profiles between type I and II endometrial carcinoma with bioinformatic analysis.</p><p><b>RESULTS</b>Many genes were highly expressed in estrogen-dependent endometrial carcinoma, and some of them were involved in the metabolism and conversion of estrogen, while some others in estrogen regulation. CYP2C9, for instance, was involved in the conversion of estrogen sulfate to 16-hydroxy sulfate metabolite, DDC in estrogen-dependent pathogenesis of endometrial carcinoma possibly by DDC interaction with AR to enhance steroid receptor transcription.</p><p><b>CONCLUSION</b>High expression of these genes in estrogen-dependent endometrial carcinoma may provide insights into their roles in the pathogenesis and prognosis of this malignancy.</p>