[Proliferation and invasion of ovarian cancer SK-OV-3 cells inhibited by Tectoridin].

Objective: To study the effects of Tectoridin on the proliferation, migration and invasion of ovarian cancer SK-OV-3 cells and its mechanism. Methods: SK-OV-3 cells were treated with 0, 5, 10, 50 and 100 µg/L Tectoridin for 24 hours. The proliferation of SK-OV-3 cells treated with different concentrations of Tectoridin was detected by cell counting kit-8 (CCK-8). The migration was analyzed by wound healing test and the invasion was observed by Transwell. The effects of different concentrations of Tectoridin on the protein levels of phosphoinositide 3-kinase (PI3K)/serine-threonine kinase (AKT) signaling pathway were detected by western blot assay. Results: The A values of 0, 5, 10, 50, 100 µg/L Tectoridin groups were 0.857±0.043, 0.725±0.036, 0.611±0.032, 0.410±0.027, and 0.321±0.023, respectively. The relative migration distances of the cells were 1.000±0.083, 0.896±0.092, 0.644±0.075, 0.432±0.056, and 0.215±0.043, respectively. The numbers of cell invasion were (106.258±11.785), (93.241±10.251), (74.258±7.963), (40.236±5.210), and (32.147±3.458), respectively. The phosphorylated PI3K (p-PI3K) protein levels were 1.000±0.079, 0.875±.089, 0.727±0.075, 0.452±0.053, 0.365±0.052, respectively. The phosphorylated AKT (p-AKT) protein levels were 1.000±0.070, 0.816±0.072, 0.635±0.079, 0.463±0.065, 0.305±0.047, respectively. Compared with the 0 µg/L Tectoridin group, the differences of 5, 10, 50, 100 µg/L Tectoridin group were statistically significant (all P<0.05). The A values of the control group and the LY294002 group were 0.873±0.081 and 0.494±0.038, respectively. The relative cell migration distances were 1.000±0.081 and 0.523±0.051, respectively. The numbers of cell invasion were (106.228±11.783) and (61.243±9.251), respectively. The protein levels of p-PI3K were 1.000±0.075, 0.521±0.046, respectively. The protein levels of p-AKT were 1.000±0.070, 0.465±0.051, respectively. Compared with the control group, the difference in the LY294002 group was statistically significant (P<0.05). Conclusion: Tectoridin may inhibit proliferation, migration and invasion of SK-OV-3 cells by regulating PI3K/AKT signaling pathway.

COVID-19 with bronchogram - a potential indication of prolonged treatment.

Novel coronavirus disease-19 (COVID-19) has widely spread all over the world and seriously threatened people's health. This disease is currently diagnosed by clinical features, chest computed tomography (CT) scan, and nucleic acid test of severe acute respiratory syndrome coronavirus (SARS-CoV-2). Recently, some studies have suggested parenchymal consolidation and air bronchogram in severe cases. However, the effective treatment for COVID-19 patients with bronchogram has not been discussed. Herein, we report a case of 47-year-old woman who suffered from COVID-19 with bronchogram. These findings revealed that the body temperature and clinical laboratory test all returned to normal after this patient received a prolonged treatment. Furthermore, chest CT showed the bronchogram and consolidation resolved and nucleic acid retest of SARS-CoV-2 was also negative. These results provide an important reference for treatment option of COVID-19 with bronchogram.

MiRNA-338-3p regulates cervical cancer cells proliferation by targeting MACC1 through MAPK signaling pathway.

OBJECTIVE: Aberrant expression of miR-338-3p has recently involved in the progression and development of various types of malignant tumors, but its role in the progression of cervical cancer remains unknown. This study aims to investigate the role of miR-338-3p/MACC1 axis in the progression of cervical cancer. PATIENTS AND METHODS: MiR-338-3p and metastasis-associated in colon cancer 1 (MACC1) expression was determined in cervical cancer by quantitative real-time PCR (qRT-PCR). We explored the association of miR-338-3p expression with pathology and prognosis in cervical cancer patients. We explored the function of miR-338-3p and MACC1 on cell proliferation. A luciferase reporter assay was conducted to confirm the target gene of miR-338-3p in cervical cancer cells. RESULTS: In the present work, our data showed that the expression of miR-338-3p was substantially decreased in cervical cancer tissues and associated with advanced FIGO stage, lymph node metastasis, depth of cervical invasion and poor overall survival. However, the MACC1 had an opposite expression. Mechanistically, we identified that MACC1 which acted as a functional downstream target for miR-338-3p. Furthermore, overexpression of miR-338-3p decreased expression of MACC1 in cervical cancer cells could significantly inhibit cervical cancer cell proliferation and induce cells apoptosis. Interestingly, miR-338-3p and MACC1 had proven to be involved in the progression of cervical cancer cells by regulating mitogen-activated protein kinase (MAPK) signaling pathway. CONCLUSIONS: Our results suggested miR-338-3p/MACC1/MAPK regulatory pathway play an important role in the progression of cervical cancer.

[Effect and regulation of metformin on endometrial carcinoma subline of progestin-resistance].

OBJECTIVE: To investigate the effect and mechanism of metformin on endometrial carcinoma subline of progestin-resistance and find whether metformin could regulate progestin-resistance in endometrial carcinoma. METHODS: Ishikawa endometrial carcinoma cells were cultured for a long period in the presence of the synthetic medroxyprogesterone 17-acetate (MPA) to generate a subline refractory to the growth-suppressive effects of MPA, named Ishikawa/MPA cells. The effect of MPA (1, 5, 10, 20, 40 and 60 µmol/L) or metformin (1, 10, 20, 40, 60, 70 and 80 mmol/L) on proliferation of the Ishikawa/MPA and parental Ishikawa cells was detected by cell counting kit-8 (CCK-8) method. Western blot was used to detect the effect of metformin and (or) MPA on the expression of PR-B in the Ishikawa/MPA and Ishikawa cells. RESULTS: The Ishikawa/MPA showed that growth stimulation rather than inhibition in the Ishikawa cells after low MPA concentration treatment. The doubling time of Ishikawa/MPA cell lines were (43±4) hours and that of Ishikawa cell line were (47 ± 3) hours. No changes in doubling time were observed (t=0.349, P= 0.572). Low concentration (1 and 5 µmol/L) of MPA could promote the growth of Ishikawa/MPA cells (by 3% and 13%). High concentration (10, 20, 40 and 60 µmol/L) of MPA could inhibited the growth of Ishikawa/MPA cells (by 4%, 3%, 9% and 40%) and the growth of Ishikawa cells (by 41%, 55%, 65% and 66% ). At the same concentration, the difference of inhibition rates between the two cell lines were statistically significant (P<0.01). Metformin (1, 10, 20, 40, 60, 70 and 80 mmol/L) could inhibite the growth of Ishikawa/MPA (by -10%, 20%, 56%, 89%, 97%, 98% and 99%) greater than those in parental Ishikawa cells (by -6%, 19%, 37%, 54%, 70%, 72% and 83%), and the inhibitory effect was dose-dependent manner. Western blot assay showed that for Ishikawa cells, the protein expression levels of PR-B in metformin group and MPA+metformin group were respectively (53.5±4.0)% and (37.7±5.2)%, which were higher than that in the control group [(23.4 ± 3.0)%], and there were significant the differences (P<0.01). For Ishikawa/MPA cells, the protein expression levels of PR-B in metformin group and MPA+metformin group were respectively (38.6±1.7)%, (36.3±2.5)%, which were higher than those in the control group [(6.4±1.6)%], and there were also significant differences (P<0.01). CONCLUSION: Metformin may regulate the progestin-resistance in endometrial carcinoma by increasing the expression of PR-B.

Downregulation of MACC1 expression enhances cisplatin sensitivity in SKOV-3/DDP cells.

The aim of this study was to investigate the correlation between MACC1 expression and resistance to cisplatin (DDP) in DDP-resistant human epithelial ovarian cancer SKOV-3 cells (SKOV-3/DDP). MACC1 mRNA and protein expression levels in SKOV-3 and SKOV-3/DDP cells were detected by reverse transcriptase polymerase chain reaction and western blot. The SKOV-3/DDP cells were divided into 5 groups: control, shVect (transfected with p-super-EGFP-1 plasmid), pshMACC1 (transfected with psuper-EGFP-shMACC1 plasmid), PD (pretreated with 20 µM PD98059), and combined (transfected with psuper-EGFP-shMACC1 plasmid and pretreated with 20 µM PD98059) groups. Cisplatin sensitivity and cell apoptosis in SKOV-3/DDP cells were assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and flow cytometry. ERK1/2 and p-ERK1/2 expression was determined by western blot. MACC1 mRNA and protein expression levels in SKOV-3/DDP cells were 2.66 ± 0.54 and 1.95 ± 0.45 times those seen in SKOV-3 cells (P < 0.05). Cisplatin sensitivity of pshMACC1 group was much higher than that in the control and shVect groups. Cisplatin-induced cell apoptosis rates increased significantly in the pshMACC1, PD, and combined groups, compared to the control and shVect groups. Moreover, the apoptosis rate was the highest in the combined group among the 5 groups (IC50 = 20.836 ± 0.629 µM). p-ERK1/2 expression decreased significantly in the pshMACC1, PD, and combined groups (this decrease was the most obvious in the combined group). In conclusion, downregulation of MACC1 expression could enhance cisplatin sensitivity and decrease drug resistance in SKOV- 3/DDP cells.

Prenatal diagnosis of Chinese families with phenylketonuria.

The aim of this study is to investigate the ability to prenatally diagnose phenylketonuria (PKU) by using phenylalanine hydroxylase (PAH) gene mutation analysis combined with short tandem repeat (STR) linkage analysis in 118 fetuses from 112 Chinese families. Genomic DNA was extracted from the peripheral blood from members of 112 families and the exons and exon-intron boundaries of the PAH gene were amplified by PCR. PCR products were analyzed by bi-directional Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). The three variable number of tandem repeat (VNTR) markers PAH-1, PAH-26, PAH-32 were used in the prenatal diagnosis for the PKU families. We identified a spectrum of 63 different mutations, including 61 point mutations and indels, two large exon deletion mutations, and five novel mutations. A substantial proportion of mutant alleles were accounted for by p.R243Q (15.62%), EX6-96AG (9.82%), p.V399V (7.59%), p.Y356X (6.70%), and p.R413P (5.36%). The same mutations were identified in 31 prenatally genotyped fetuses. We identified 58 fetuses that carried only one mutant allele and 29 fetuses that carried no mutations of PAH and were presumed normal. PAH gene mutation analysis combined with STR linkage analysis can provide rapid and accurate prenatal diagnosis for PKU families.

A novel 3-base pair deletion of the CRYAA gene identified in a large Chinese pedigree featuring autosomal dominant congenital perinuclear cataract.

Congenital cataract is caused by reduced transparency of the lens resulting from metabolic disorders during the fetal period. The disease shows great heterogeneity both clinically and genetically. We identified a 4-generation ethnic Han Chinese family affected by autosomal dominant congenital perinuclear cataract. The patients underwent full clinical and ophthalmologic examinations to rule out any concomitant disorders. Blood samples were collected and genomic DNA was extracted. Potential mutations in the candidate gene alpha A crystallin (CRYAA) were screened. Prenatal diagnosis was then provided for a fetus of the affected proband by chorionic villus sampling. In all patients, DNA sequencing of the CRYAA gene revealed a novel 3-bp deletion mutation in exon 3 (c.246_248delCGC), which led to deletion of codon 117 encoding arginine (p.117delR) in the peptide chain. The same mutation was not found among unaffected and healthy individuals. Bioinformatic analysis revealed that although the c.246_248delCGC is an 'in-frame' mutation, removal of arginine resulted in a significant change in the protein structure. The fetus did not possess this mutation and was confirmed to be healthy at 1-year follow-up. A novel disease-causing mutation, c.246_248delCGC (p.117delR), of the CRYAA gene has been identified in a Chinese family with autosomal-type perinuclear congenital cataracts. This is also the first report of prenatal diagnosis of this type of congenital cataract.

Prenatal diagnosis based on HPRT1 gene mutation in a Lesch-Nyhan family.

We explored the feasibility of applying gene diagnosis in prenatal diagnosis by analysis of hypoxanthine-guanine phosphoribosyltransferase-1 (HPRT1) gene mutation in a Chinese Lesch-Nyhan family. A homozygous mutation of p.R170X (c.508C＞T) in HPRT1 gene was detected in the proband, and a heterozygous mutation of p.R170X was detected in his mother. This mutation failed to be found in the 50 unrelated healthy individuals. Prenatal diagnosis indicated that the foetus was male and also carried p.R170X (c.508C＞T) mutation, same as the proband. Parents of the foetus decided termination of pregnancy, and the result of gene analysis for the aborted tissue was consistent with that of prenatal diagnosis. We can see that Lesch-Nyhan syndrome (LNS) is caused by non-sense mutation p.R170X（c.508C＞T）in HPRT1 gene in this family. Prenatal gene diagnosis is a valid strategy to prevent LNS because it can avoid the birth of LNS foetuses.

Mutation analysis and prenatal diagnosis for three families affected by isolated methylmalonic aciduria.

Isolated methylmalonic acidemia (MMA) is a genetically heterogeneous disorder caused mainly by deficiency of methylmalonyl-CoA mutase. In the present study, we analyzed MUT gene mutations in 3 Chinese couples with a birth history of isolated MMA. We also provided prenatal diagnoses for the detected mutation. Exons and exon-intron boundaries of the MUT gene were analyzed by polymerase chain reaction and direct sequencing. Prenatal genetic diagnoses were performed by chorionic villus sampling after the genotypes of parents were determined. Six heterozygous mutations in the MUT gene were identified in the 3 families, including c.1880A>G (p.H627R) and IVS9-1G>A for family 1, c.1741C>T (p.R581X) and c.729insTT (p.D244fX39) for family 2, and c.616C>T (p.Q206X) and c.1280G>A (p.G427D) for family 3. Among these, c.616C>T (p.Q206X), c.1280G>A (p.G427D), IVS9-1G>A, and c.1741C>T (p.R581X) were novel mutations. These mutations were not detected in 100 normal controls. The fetus in pedigree 3 was free of the mutations carried by the parents, while the fetuses in pedigrees 1 and 2 were heterozygous mutation carriers. All 3 families decided to continue with their pregnancies and the neonates did not show any symptoms of MMA after birth. Our results indicated that mutations in the MUT gene are the primary cause of isolated MMA, and that most mutations were novel. For families with early-onset isolated MMA, direct sequencing of the MUT gene is crucial for genetic counseling, prenatal diagnosis, and identification of carriers.

Clinical analysis of 65 cases of laparoscopic treatments on tubal infertility caused by tubal distortion.

OBJECTIVE: This study aims to investigate the clinical value of laparoscopic treatment on tubal infertility caused by tubal distortion. MATERIALS AND METHODS: A total of 65 cases of patients with tubal infertility were divided into three groups based on tubal distortion degree, i.e., 21 cases had a minimum angle of tubal distortion < 45 degrees (A group), 39 cases had a distortion angle between 45 degrees and 90 degrees (B group), and five cases had a distortion angle between 90 degrees and 145 degrees (C group). The pregnancy outcome and the impact of tubal distortion degree on pregnancy outcome were analyzed 6 to 24 months after operation. RESULTS: The total pregnancy rate of these 256 cases were 43.75% with an intrauterine pregnancy rate of 40.23% and an ectopic pregnancy rate of 3.52%. In the simple distortion tubal infertility cases, the total pregnancy rate was 44.62%. In Group A, five cases became pregnant after operation (33.33%); in Group B, 19 cases (48.72%); and in Group C, three cases (60%). The differences in pregnancy rate between Groups A and B and Groups A and C were statistically significant (p < 0.05), whereas that between Groups B and C was not (p > 0.05). CONCLUSION: Tubal plastic surgery via laparoscopy is an effective way to treat infertility caused by tubal distortion by restoring the normal shape of oviducts, especially in cases when the minimum angle of tubal distortion is greater than 45 degrees.

Expression of budding uninhibited by benzimidazoles-1 and mitotic arrest deficient-2 in endometrial carcinoma and its significance.

OBJECTIVE: The aim of this study was to explore the expression of budding uninhibited by benzimidazoles-1 (Bub1) and mitotic arrest deficient-2 (Mad2) in endometrial carcinoma and its significance. MATERIALS AND METHODS: The expression of Bub 1 and Mad2 in 30 human normal endometrial tissues (group A), 30 complexly-hyperplastic endometrial tissues (group B), and 63 endometrial carcinoma tissues (group C) was observed using immunohistochemistry (the streptavidin-peroxidase method). RESULTS: The positive expression rates of Bub1 in groups A, B, and C were 86.67%, 56.67%, and 28.57%, respectively. The positive rate of Bub1 protein was correlated with the differentiation degree and clinical stage of endometrial carcinoma (p < 0.05) other than lymph node metastasis (p > 0.05): A higher differentiation degree and a more advanced stage of endometrial carcinoma indicated a higher positive rate of Bub1 protein. The positive rates of Mad2 protein in groups A, B, and C were 23.33%, 56.67%, and 85.71%, respectively. The positive rate of Mad2 protein was correlated with the differentiation degree of endometrial carcinoma (p < 0.05) other than its clinical stage and lymph node metastasis (p > 0.05): A lower differentiation degree indicated a higher positive rate of Mad2 protein. Bub1 and Mad2 proteins were negatively correlated in the endometrial carcinoma tissues (r = - 0.719, p < 0.001). CONCLUSION: Bub1 and Mad2 proteins interact with each other. They may play an important role in the initiation and development of endometrial carcinoma.

Non-nuclear activation of signal transducer and activator of transcription 3 by 17ß- estradiol in endometrial cancer cells.

Why estrogen hyperstimulation can lead to endometrial carcinogenesis has not been fully clear yet. Non-nuclear action of estrogen has arised much attention of many experts. Signal transducer and activator of transcription 3 is a very important signal molecule, which plays vital role in endometrial canver. The present study is oriented to the problem whether estrogen can activate STAT3 by non-nuclear action in endometrial cancer cells. So, the levels of phosphorylated STAT3 (P-STAT3) and total STAT3 were examined by western blot in endometrial cancer cells including Ishikawa with rich-expressed estrogen receptor (ER) and HEC-1A with poor-expressed ER after stimulation with 1µM estradiol (E2) at different time points and at varied doses of E2 for optimal time. Inhibitory role of AG490 on activation of STAT3 induced by E2 was also tested. P-STAT3/STAT3 was used as a measure of activation of STAT3. We found that maximum P-STAT3/STAT3 took place at 15 min in both Ishikawa cells and HEC-1A cells. The activation of STAT3 elicited gradually with increasing doses of E2. AG490 stopped the activating STAT3 in the same dose-dependent manner in both endometrial cancer cells. The results demonstrate that E2 is able to activate STAT3 in both Ishikawa with rich-expressed ER and HEC-1A with poor-expressed ER endometrial cancer cells by non-nuclear action, which provides the preliminary laboratory basis for the probability of endometrial adenocarcinoma treatment with blockage of STAT3 signaling, especially for ER-poor endometrial adenocarcinoma.

One-stage reconstruction of the penis with free skin flap: report of three cases.

Three cases of penile defect of varying extent were reconstructed with free skin flaps from the forearm based on the radial artery. In two cases, part of the forearm flap was used to reconstruct the urethra, and the rest of the flap to reconstruct the corpus penis. A piece of autogenous cartilage was then inserted between the two layers. All procedures were completed in a one-stage operation. In the third case, a free skin graft was used for urethral reconstruction. Surgery in all three cases was successful, providing a penis of acceptable appearance as well as excellent urinary function.