[Expression of METTL14 in epithelial ovarian cancer and the effect on cell proliferation, invasion and migration of A2780 and SKOV3 cells].

Objective: To study the expression of methyltransferase-like protein 14 (METTL14) in epithelial ovarian cancer and its clinical significance, and to explore the effect of METTL14 expression on the proliferation, invasion and migration of ovarian cancer cells. Methods: Immunohistochemistry (IHC) was used to detect METTL14 expression in tumor tissue samples, and analyze the relationships among METTL14 expression, clinicopathological factors, and prognosis in ovarian cancer. Lentiviral vectors and small interfering RNA (siRNA) were used to up-regulate and down-regulate the METTL14 expression in ovarian cancer cell lines A2780 and SKOV3, respectively. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was used to detect the N6-methyladenosine (m6A) content in ovarian cancer cells. Cell counting kit-8 (CCK-8), wound healing assay, and transwell assay were used to examine the function of METTL14 expression in the cells. Results: (1) The IHC score of METTL14 protein was 6.2±3.7 in 20 samples of ovarian cancer tissues and 3.3±2.5 in 15 samples of normal ovarian tissues, and the difference was statistically significant (t=-2.64, P=0.012). Among the patients who suffered from ovarian cancer, there were 69 cases with high expression of METTL14 protein (IHC score≥6), accounting for 57.0% (69/121), and the cases with low expression of METTL14 protein (IHC score<6) accounting for 43.0% (52/121). Compared with the patients with low expression of METTL14, the patients with high expression of METTL14 had later stages, higher rates of lymph node metastasis, abdominal metastasis, and more ascite amount. The differences were statistically significant (all P<0.05). The overall survival rate was significantly lower in patients with high METTL14 expression than the low expression (P=0.009). (2) LC-MS/MS data showed that the relative expression of m6A in A2780 and SKOV3 cells in the lentivirus (LV)-METTL14 group were 0.213±0.024 and 0.181±0.018, which were significantly higher than those in the LV-normal control (NC) group (0.109±0.022 and 0.128±0.020; all P<0.05). While the relative expression of m6A in A2780 and SKOV3 cells in the si-METTL14 group were 0.063±0.012 and 0.069±0.015, which were significantly lower than the expression in si-NC group of 0.108±0.014 and 0.121±0.014 (all P<0.05). CCK-8 assay showed that the absorbance values were significantly lower in the si-METTL14 group compared with the si-NC group at 36, 48, 60 hours (all P<0.05); while were significantly increased in the LV-METTL14 group compared with the LV-NC group at 48, 60 hours (all P<0.01). Scratch wound assays showed that the migration rate of the si-METTL14 group was lower than those of the si-NC group, while the LV-METTL14 group were higher than the LV-NC group by 24 hours, the differences were statistically significant (all P<0.01). Cell migration and invasion were detected by transwell migration and invasion assays. After cultivated for 24 hours, the invasion cell number and the migration cell number in the si-METTL14 group were less than those in the si-NC group. While the invasion cell number and the migration cell number in the LV-METTL14 group were more than those in the LV-NC group, respectively. The differences were statistically significant (all P<0.01). Conclusion: Patients with high METTL14 expression have a worse prognosis in ovarian cancer, which may increase the m6A modification of ovarian cancer cells and promote cells proliferation, invasion and migration.

[Lentivirus media miR-1246 knockdown inhibits tumor growth and promotes apoptosis of SiHa cells].

Objective: To study the effect of lentivirus-mediated microRNA (miR) -1246 RNA interference (RNAi) on biological characteristics and behaviors in cervical cancer cells as well as to identify the downstream signaling pathways affected. Methods: MiR-1246 specific cDNA was synthesized and cloned into the recombinant lentiviral vector (LV-miR-1246-inhibitor) . The SiHa cells were devided into three groups: no viral infection (negative control, NC) , infection with control virus (LV-NC) , and infection with miR-1246-inhibitor virus (LV-miR-1246-inhibitor) . The expression of the miR-1246 was detected by reverse transcription (RT) -PCR. Cell growth was analyzed by cell counting kit 8 (CCK-8) assay. The invasion was dectected by transwell matrige gel. Cell apoptosis was detected by flow cytometer. The growth of xenograft tumors was also investigated. Expression of thrombospondin-2 (THBS2) , matrix metalloproteinase (MMP) 2, 9 were also evaluated in the cells. Results: (1) The expression level of miR-1246 in SiHa cells (0.11±0.13) was significantly lower in group LV-miR-1246-inhibitor than those in the group LV-NC and the group NC (1.14±0.86 and 1.30±0.73, respectively; P<0.01) . (2) The proliferation of SiHa was also markedly suppressed in CCK-8 at 96 hours (P<0.01) . (3) The number of group LV-miR-1246-inhibitor was significantly less than those in the LV-NC and NC groups in transwell invasion assay (71±4, 162±5 and 188±5, respectively; P<0.01) . (4) The apoptosis rate of SiHa cells in the group LV-miR-1246-inhibitor [ (16.10±3.37) %] was significantly lower than those of group LV-NC and group NC [ (5.67±0.89) % and (1.78±0.08) %,P<0.01]. (5) The tumor volume in the nude mice group LV-miR-1246-inhibitor [ (287±59) mm(3)] was significantly lower than those in the LV-NC and NC groups [ (571±137) and (657±144) mm(3), respectively; P<0.01]. (6) Compared with the LV-NC group and the NC group, THBS2 protein expression in the tumor tissue of the nude mice in the group LV-miR-1246-inhibitor was significantly increased (P<0.05) , while the expression levels of MMP-2 and MMP-9 protein were significantly decreased (P<0.01) . Conclusion: These results suggest that miR-1246 functions during cervical cancer pathogenesis and tumor formation via the THBS2, MMP signaling pathway.

[Comparison of the short-term and long-term outcomes of laparoscopic surgery and open surgery for early-stage cervical cancer].

Objective: To evaluate the short-term and long-term outcomes after laparoscopic surgery compared with traditional laparotomy in cases of stage ⅠA2-ⅡA2 cervical cancer. Methods: We conducted a retrospective study on the clinical data of 1 863 patients diagnosed as FIGO stages ⅠA2-ⅡA2 cervical cancer in 6 third-grade class-A hospitals in Guangxi province between January 2007 and May 2014. One thousand and seventy-one received laparoscopy, and 792 received laparotomy. T-test, U-test and χ(2) test were used to compare the short-term and long-term outcomes. The short-term outcomes included surgical related outcomes and operative complications, and the long-term outcomes included quality of life (pelvic floor functions and sexual functions), survival and recurrence. Pelvic floor function and sexual function were assessed with the International Consultation on Incontinence Quesonnaire Female Lower Urinary tract(ICIQ-FLUTS) and the Female Sexual Function Inventory (FSFI), respectively. Survival rates were estimated by Kaplan-Meier analysis. The survival curves were compared with Log-rank test. Cox regression analysis was used to evaluaterisk factors for prognosis. Results: (1)The short-term outcomes : There were significant difference in operative time([(257±69) vs(238±56)min], estimated blood loss[(358±314) vs(707±431)ml], anus exhausting time[(2.5±0.9) vs (2.9±0.8)d], preserved days of catheter[(15±7) vs(18±9)d], and post-operative length of stay[(19±16) vs (30±21)d] between the laparoscopic surgery group and the opensurgery group(P<0.05). There was no significant difference in lymph nodes yielded[(21±9) vs (21±11)], left parametrial width[(2.5±0.8) vs (2.7±0.7)cm], right parametrial width [(2.6±0.3) vs (2.7±0.2)cm], vaginal cuff length[(2.4±0.7) vs (2.2±0.7)cm] between the laparoscopic surgery group and the opensurgery group(P>0.05). The intra-operative complications occurred in 8.1%(87/1 071)in the laparoscopic surgery group and in 10.7%(85/792)in the open surgery group(P>0.05). However, the complications of vascular injury in the laparoscopic surgery group[2.6%(28/1 071)]was lower than that in the open surgery group[7.7%(61/792), P<0.001]. The laparoscopic surgery exhibited lower post- operative complication rate [33.8%(362/1 071)vs 40.2%(318/792), P<0.05] and poorer wound healing rate [0.7%(7/1 071)vs 4.0%(32/792), P<0.05]. (2)The long-term outcomes(Hierarchical analysis): The overall incontinence in ICIQ-FLUTS questionnaire in nerve-sparing laparoscopic group [28.4%(67/236)] was lower than that in the open surgery group [35.9%(71/198), P=0.004] . However, There was no significant difference in degree of incontinence between the two groups(P>0.05). The overall sexual dysfunction in FSFI questionnaire after 12 months of postoperative in the nerve-sparing laparoscopic group [47.0%(111/236)]was lower than that in the open surgery group [58.6%(116/198), P=0.001], and the six different dimension scores in the laparoscopic surgery group were higher than that in the open surgery group (P<0.05). The recurrence rate was 3.5%(35/1 007)in the laparoscopicsurgery group and 4.7%(35/740)in the open surgery group(P>0.05). The 5-year OS was 94.0% for the laparoscopic surgery group and 90.2% for the open surgery group(P>0.05), and the 5-year DFS was 93.9% for the laparoscopic surgery group and 89.1% for the open surgery group(P>0.05). (3) Prognostic fators: In univariate analysis, tumor dimension, clinical stage, deep stromal invasion, LVSI, and retroperitoneal lymph node metastasis signficantly affected 5-year OS and 5-year DFS(P<0.05); In multivariate analyses, LVSI, deep stromal invasion and LN metastasis were independent prognostic factors(P<0.05). Conclusions: Laparoscopy can reduceestimated blood loss, accelerate postoperative recovery and improve the quality of life after surgery compared to laparotomy, and it ensures the same oncological results as open surgery. Laparoscopic approach is a safe and effective treatment for early-stage cervical cancer.

[An experimental study of the effect of extraction and retention of canine primary intruded teeth to the permanent successors].

Objective: To compare the influence of extraction and retention of the intruded dogs' teeth on permanent successors. Methods: Nine healthy 45-days-old Chinese rural puppies were selected, and six were submitted to the intrusion of the bilateral canine. Intruded teeth on the left side were extracted 30 minutes later and the teeth on the right side were kept in their sockets. After 8 months, all dogs were sacrificed. General observation, periapical radiograph and cone beam CT were used to observe the preoperative and postoperative deciduous teeth, permanent germs and permanent teeth development. The structure and content of successors' enamel were observed by scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS). Results: In the extraction groups, the enamel hypoplasia was found in 19 permanent successors, ectopic eruption in 2 cases and abnormal teeth were in 19 cases in total (79%). In the retention groups, enamel hypoplasia of the permanent successors occurred in 2 cases, root dilaceration in 6 cases, and ectopic eruption in 5 cases, retained deciduous teeth in 3 cases, and there were 22 abnormal teeth in total (92%). In blank control group, there was no abnormal teeth. The major effect of intruded deciduous teeth on the permanent successors exhibited enamel hypoplasia [85% (41/48)], presented as enamel coloration and enamel defect (P=0.416). Conclusions: The intruded deciduous teeth should be removed early in order to reduce the effect on the permanent teeth.

Differential expression of telomerase activity in human cervical cancer and cervical intraepithelial neoplasia lesions.

PURPOSE: Telomeres are tandem arrays of repeated DNA sequences located at the ends of eukaryotic chromosomes, and are synthesized by the enzyme telomerase. Loss of telomeric DNA may play an important role in the development of human cancers. However, very little is known about the status of telomerase during human cervical cancer development. PATIENTS AND METHODS: Telomerase activity was measured by telomere repeat amplification protocol (TRAP) assay in 24 cervical cancers, one carcinoma in situ (CIS), and 20 cervical intraepithelial neoplasia (CIN) lesions. Adjacent nontumor cervical tissue from the same 24 cervical cancer patients and normal cervical tissues from 11 control individuals also were examined for the presence of telomerase activity. RESULTS: Twenty two of the 24 (91.7%) cervical cancer specimens and the single CIS tissue were strongly positive for telomerase activity. Relatively weak but distinctive telomerase activity also was detectable in one of four CIN-I (25%), two of eight CIN-II (25%), and two of eight CIN-III (25%), respectively. However, telomerase activity was not found in the 24 corresponding nontumor cervical tissues from the same cervical cancer patients and the 11 normal cervical tissues from control individuals. CONCLUSION: The majority of cervical cancers contain strong telomerase activity. Significant proportions of noncancerous CIN tissues also contain telomerase activity, although weaker than that in cervical cancer. It seems that there is a progressive increase of telomerase activity in association with an increased degree of cervical malignancy. These results seem to suggest that the expression of telomerase may play a crucial role in cervical cancer carcinogenesis.

Hepatitis C virus in peripheral blood mononuclear cells.

RNA extracted from plasma and peripheral blood mononuclear cells of patients with chronic hepatitis C were used as the template for reverse transcription followed by double in vitro enzymatic amplification with nested primers. Hepatitis C virus was detected in 14 of 15 (93.3%) plasma specimens and in 8 of 15 (53.3%) peripheral blood mononuclear cell specimens obtained from patients with chronic hepatitis C and abnormal liver functions. The results suggest that hepatitis C virus could be found frequently in peripheral blood mononuclear cells of patients with chronic hepatitis C. Whether the presence of hepatitis C virus in peripheral blood mononuclear cells plays any role in the pathogenesis of diseases associated with hepatitis C virus infection remains to be determined.

Serum hepatitis B virus DNA in hepatitis B virus seropositive and seronegative patients with normal liver function.

The presence of hepatitis type B virus (HBV) DNA in serum specimens from 926 apparently healthy people with normal liver functions was determined by polymerase chain reaction; 41.2% of people with positive results for HBV surface antigen (HBsAg) (94 of 228) and 95.2% of people with positive results for HBV e antigen (HBeAg) (60 of 63) were found to have positive results for serum HBV DNA. On the other hand, serum HBV DNA was found in 11.0% (77 of 698) of HBsAg-negative people and in 13% (69 of 530) of those who had positive results for serum antibodies directed against HBsAg. The results seem to suggest that HBV DNA can be found in a significant portion of apparently healthy people with normal liver function who are either seronegative for HBsAg or seropositive for antibodies directed against HBsAg.

Population growth and predation interference between two species of predatory phytoseiid mites (Acarina: Phytoseiidae) in interactive systems.

Populations of two species of phytoseiid mite predators, Phytoseiulus persimilis Athias-Henriot and Amblyseius degenerans (Berlese), feeding on a tetranychid prey, Tetranychus pacificus McGregor, were allowed to grow separately as well as together on bush lima bean (Phaseolus lunatus Var.) arenas in the laboratory. The population plateau attained by P. persimilis was nearly 5 times higher than that for A. degenerans when each species was on separate leaf arenas. When they were on the same arena, P. persimilis was outcompeted by A. degenerans after about 70 days of population growth. When dispersal to other arenas was necessary for the predators to find prey in another experiment, P. persimilis survived well but not A. degenerans. The mechanisms underlying species displacement were explored further. The differential mortality of immature predators at different developmental stages due to interspecific predation was concluded to be responsible for the population decline of P. persimilis, and the decline of A. degenerans in another experiment was attributed to its sedentary tendency regardless of prey distribution and to the lack of alternative food sources in the system. The implications to biological control of mutual predation between predator species is discussed briefly.

Amplification of viral RNA for the detection of dengue types 1 and 2 virus.

In vitro DNA amplification by means of the polymerase chain reaction (PCR) was used to amplify dengue types 1 and 2 viral genomes in cultured cells and in the serum of persons infected with dengue virus. Results of the present investigation suggest that the PCR method is type-specific in detecting dengue virus and has a detection sensitivity of less than 100 plaque-forming units (pfu) for both serotypes of the virus. The PCR method may be useful for detecting and typing dengue virus in clinical and epidemiological specimens.

Detoxification of aflatoxin B1 by enzymes isolated from Armillariella tabescens.

Detoxification of aflatoxin B1 (AFB1) by Armillariella tabescens multienzyme, which was isolated from mycelium pellets of A. tabescens, was confirmed by thin-layer chromatography (TLC) and rat assay. The results of toxicology and pathology studies showed that toxicity of AFB1 was minimized after treatment with A. tabescens multienzyme. The result of the Ames test indicated that the mutagenic activity of multienzyme-treated AFB1 was greatly reduced (or inactivated) compared with that of untreated controls. TLC determinations showed that AFB1 at an initial concentration of 16 microM was completely detoxified (100%) by the fungal multienzyme. The infrared spectrum suggests that the multienzyme is responsible for opening the difuran ring of AFB1.

Production, purification, and characterization of an intracellular aflatoxin-detoxifizyme from Armillariella tabescens (E-20).

Some Armillariella tabescens (E-20) multienzymes have previously been reported to present detoxifying activities against aflatoxins. In this paper, we describe the isolation purification of an intracellular enzyme, named aflatoxin-detoxifizyme, which exhibited detoxification activity on aflatoxin B(1) (AFB(1)). This aflatoxin-detoxifizyme exhibited a specific activity of 7.09 nmol min/mg at pH 6.0 and 28 degrees C. The apparent molecular mass was 51.8 kDa as determined by SDS-PAGE. The isoelectric point was estimated to be 5.4 and optimum activity for the enzyme was found at pH 6.8 and 35 degrees C. The activity of the purified enzyme was confirmed by Ames test.

Hepatitis B virus DNA in cervicovaginal cells.

Hepatitis B virus DNA sequences were detected in seven (12.1%) of 58 cervicovaginal cell specimens that were obtained from pregnant women by polymerase DNA amplification assay. The presence of hepatitis B virus DNA in these cells raises the possibility that infected cervicovaginal cells may be a source through which hepatitis B virus can be transmitted from infected mothers to their newborns and between heterosexual partners.

Prenatal transmission of hepatitis B virus to neonates born to serum hepatitis B virus DNA-positive mothers.

Hepatitis B virus (HBV) DNA was detected by in vitro enzymatic DNA amplification techniques in 66.7% (six of nine) of hepatitis B virus surface antigen (HBsAg)-positive and in 21.1% (7 of 33) of HBsAg-negative pregnant women. Five of the HBV DNA and HBsAg-positive women and one HBV DNA-positive but HBsAg-negative woman gave birth to infants positive for serum HBV DNA at time of birth. These results suggest that HBsAg-negative pregnant women are potentially capable of transmitting HBV DNA to their infants.

Differential expression of cytokine genes in cervical cancer tissues.

The expression of genes coding for inflammatory cytokine interleukin-1-alpha (IL-1 alpha), IL-6, interferon-gamma and tumor necrosis factor-alpha from 15 normal cervix, 11 cervical intraepithelial neoplasia and 13 cervical cancer tissues was investigated. The cytokine messenger ribonucleic acids were reverse transcribed and amplified in the presence of biotinylated and dinitrophenylated primers. Amplified DNA was then captured onto streptavidin-coated microwell plate and quantitatively measured in a colorimetric reaction using ant-DNP antibodies conjugated to horse radish peroxidase. There is no change of IL-1 alpha, IL-6 and tumor necrosis factor-alpha gene expression in either cervical intraepithelial neoplasia or cervical cancer tissues. But the transcription of interferon-gamma gene is significantly reduced in both cervical intraepithelial neoplasia and cervical cancer tissue as compared to normal cervix. This study demonstrated that reverse transcription and quantitative polymerase chain reaction coupling to colorimetric microwell plate assay is a sensitive and useful method to quantitate multiple cytokine gene expression. Our results also suggest that cervical epithelial cells are capable to express cytokines and that interferon-gamma may play a role in the pathogenesis of cervical cancer since its reduced expression may influence inflammation and immunity of the cervical tissues.

Genomic aberrations of human papillomavirus recovered from cervical cancers.

Human papillomaviruses (HPV) contribute to the development of malignancies of the uterine cervix and the viral E6 and E7 oncogenes are invariably retained and expressed in cervical cancer tissues. Minor, but not major, structural aberrations have been found quite frequently in viral DNA recovered from cervical cancer tissues. We examined the presence of the DNA sequence of HPV type 18 in 33 cervical cancer tissues by polymerase chain reaction. HPV type 18 DNA sequences was found in 24 of these 33 cervical cancer tissue specimens, and at least 21 of these 24 specimens did not appear to retain all the region and open reading frames examined. Twelve of these 24 tissues seemed to harbor only the E6 and/or E7 genes. These results can be construed to suggest that the absence of viral genes other than E6 and E7 is quite frequent in HPV recovered from cervical cancer tissues and that the E6 and E7 genes are important in the carcinogenesis of cervical carcinoma. It is possible that the E6 and/or E7 alone may be sufficient to maintain the transformed phenotype of cervical cancer.