OBJECTIVE:Existence of miRNAs in plasma exosomes of lung cancer patients were screened to develop new methods for the screening and auxiliary diagnosis of lung cancers. METHODS:Plasma samples were collected from 6 lung cancer patients (3 adenocarcinomas,2 squamous carcinomas and 1 small cell lung cancer) and 4 healthy controls. Agilent Bioanalyzer was used for qualitative inspection of the extracted RNA. Plasma exosomal miRNA was analyzed by high-throughput sequencing. Target gene prediction was performed by TargetScan,PITA and miRNAorg database,and R software was applied for KEGG and GO analysis of target genes. RESULTS:The exosomal differential miRNA expression analyses showed that 142 miRNAs were expressed abnormally in the lung cancer group compared with to the normal group,with 62 miRNAs up-regulated and 80 miRNAs down-regulated. GO analysis showed that these abnormally-expressed (target) genes were mainly involved in DNA-based template transcription,transcriptional regulation and signal transduction,etc. The target KEGG analysis indicated that these target genes were mainly involved in axon guidance signaling pathway,calcium signaling pathway,and actin cytoskeleton regulation. CONCLUSION:Our data suggest that differentially expressed miRNAs may have good predictive potential for lung cancer metastasis. In addition,they may be candidate biomarkers for the screening and diagnosis of lung cancers.

OBJECTIVE:To investigate synergistic antitumor effects between lentivirus-mediated short hairpin RNAs (shRNA) targeting HPA and nadroparin in A549 cells,and to determine their underlying molecular mechanisms. METHODS:The lung cancer A549 cell line with down-regulated HPA expression by shRNA was used. These cells were also treated with six different conditions and then evaluated. CCK-8 assay was used to detect cell viability,enzyme-linked immunosorbent assay (ELISA) to measure concentrations of transforming growth factor (TGF-β1) in the culture medium,and the transwell assay to determine cell migration and invasion ability. Western Blot was used to detect expression of TGF-β1,phosphorylated- Smad2/3,ZEB-1,SNAIL,TWIST,E-cadherin,N-cadherin and MMP-2 in the TGF-β signaling pathway. RESULTS:HPA-silenced A549 cell line was successfully established by transfection of shRNA with lentivirus. Results from the CCK-8 assay demonstrated that shRNA inhibiting HPA or nadroparin alone slightly inhibited cell viability(all P<0.05),while the combined treatments significantly inhibited cell viability (P<0.05). ELISA results demonstrated that secretion of transforming growth factor (TGF)-β1 was decreased with inhibition of HPA and/or treatment with nadroparin (all P<0.05),particularly in the combined treatment group (P<0.05). Results from the transwell assay demonstrated that both HPA-silencing and nadroparin suppressed cell migration and invasive abilities (all P<0.05),these effects from the combined treatments were significant (all P<0.05). Western blot analyses demonstrated the similar results. Down-regulation of TGF-β1,phosphorylated-Smad2/3,Zinc-finger E-box-binding 1 (ZEB-1),twist-related protein (TWIST),snail family transcriptional repressor (SNAIL),N-cadherin and MMP-2,and upregulation of E-cadherin were induced to varying degrees by HPA-silencing or nadroparin alone or in combinations (all P<0.05). CONCLUSION:HPA silencing,especially when combined with nadroparin significantly inhibited cell invasion and migration,and up-regulated E-cadherin expression which was associated with inhibition of ZEB-1,TWIST and SNAIL,and the TGF-β1-mediated EMT process.

OBJECTIVE:To screen for key genes affecting prognosis of cervical cancer (CC),and to explore expression and prognostic values of AURKA in cervical squamous cell carcinomas (CSCC). METHODS:mRNA expression profiles and clinical data from GEO database of CC patients were downloaded,and the differentially expressed genes (DEGs) between cervical and normal cervical tissues were screened by bioinformatics technology. PPI network and survival analyses showed that AURKA was a potential biomarker associated with CC staging and prognosis. GEPIA2 and HPA databases were used to verify expressions of AURKA mRNA and its encoded protein in both cervical tissues,and in different stages of cancer. Kaplan-meier method was used to analyze whether the high and low expression of AURKA (median truncated value) was significantly correlated with survival in patients with cervical cancer. Subsequently,quantitative real-time PCR (qPCR) and Enzyme-linked immunosorbent assay (ELISA) were used to detect the mRNA and protein levels of AURKA in serum of CSCC patients,cervical intraepithelial neoplasia patients and healthy female subjects. RESULTS:GEPIA2 database analyses showed high expression of AURKA mRNA in cervical cancer tissues (P<0.05). Further analysis of the relationship with cervical cancer stages showed that the expression levels in different cancer stages were significantly different (P<0.05),and the levels increased with the progression of tumor staging. Compared with normal cervical tissues,expression of the AURKA protein was also increased using the HPA database analyses,and the difference was statistically significant (P<0.05). Kaplan-Meier survival analyses showed that patients with higher AURKA gene expressions had shorter survival times. qPCR and ELISA showed that the relative expressions of AURKA mRNA in CSCC patients and cervical intraepithelial neoplasia were significantly higher than that in healthy subjects,the difference was statistically significant (P<0.05). Expressions of serum AURKA protein in CSCC patients was significantly higher than that in patients with cervical intraepithelial neoplasia and health subjects (P<0.05). Results from ROC curve and χ² test showed that the diagnostic value of AURKA mRNA was high. The FIGO staging,lymph node metastasis,tumor size and muscle layer infiltration depth of the serum AURKA mRNA high expression group were higher than those in the AURKA mRNA low expression group (P<0.05). CONCLUSION:Expressions of AURKA mRNA and protein in the serum of CSCC patients were elevated,and the combination of the two tests had potential application value for the diagnosis of CSCC and the monitoring of prognosis.

OBJECTIVE:To study expression of the cystatin 6 (CST6) protein in esophageal squamous cell carcinomas among Kazakhs,and to explore its relationship with clinicopathological parameters and prognosis. METHODS:From December 2010 to December 2020,tissues from 55 Kazakh patients who had esophagectomy at the First Affiliated Hospital of Xinjiang Medical University were collected. Expression of CST6 in cancer and corresponding paracancerous tissues was detected by immunohistochemical method. Relationships between its expression and clinicopathological features was analyzed by χ² test,Kaplan-Meier univariate analysis and COX multivariate analysis to evaluate their relationships with prognosis of esophageal cancer. RESULTS:Expression of the CST6 protein was mainly located in the cytoplasm,cell membrane and keratinized area. The levels of expression of CST6 were 36.4% in the cancer and 63.6% in the corresponding paracancerous tissues. The difference was statistically significant (χ²=9.032,P=0.003). CST6 expression was related to family history (P=0.045) and tumor differentiation (P=0.036):higher in patients with no family history of tumor and with well-differentiated tumors. Kaplan-Meier survival analysis showed that the expression was related to the survival rates of patients (P<0.05):the average survival time of patients with high CST6 expression was longer. Cox multivariant survival analysis showed that negative expression was an independent risk factor for poor prognosis. CONCLUSION:CST6 protein was significantly under-expressed in esophageal squamous cell carcinoma among Kazakh patients. The protein therefore may play a tumor suppression role and its negative expression was a risk factor for poor prognosis.

OBJECTIVE:To elucidate functions of the PTEN C-tail in nasopharyngeal carcinoma (NPC). METHODS:Fluorescence quantitative PCR was used to detect expressions of PTEN in NPC cell lines and nasopharyngeal epithelial cell lines,NP69.5-8F and HONE1. Cells were cultured with 5 ng/mL TGF-β1. Negative control (NC),PTEN WT(wild-type) and PTEN 1-353 (lacking C-tail structure of PTEN) plasmids were over-expressed in the cells. Western blot were used to verify their transient efficiency. After 48 h,effects of PTEN WT and PTEN 1-353 on metastasis abilities were verified by Transwell experiments. Effect of PTEN WT and PTEN 1-353 on proliferation capacities were verified by CCK-8 assays. RESULTS:Compared with NP69,expressions of PTEN in NPC cells were reduced. Compared with the NC group,the migration abilities of TGF-β1-induced NPC cells were inhibited by PTEN WT and PTEN 1-353 (P<0.01),but there was no statistical difference between PTEN WT and PTEN 1-353 (P>0.05). Compared with the NC group,the proliferation abilities of TGF-β1-induced NPC cells were inhibited by PTEN WT and PTEN 1-353 (P<0.05). Interestingly,the inhibition of PTEN 1-353 was weaker-than PTEN WT(P<0.05). CONCLUSION:The PTEN C-tail was not involved in the regulation of metastasis of NPC cells. However,the PTEN C-tail could be involved in regulating the proliferation ability of NPC cells.

OBJECTIVE:To investigate expression of hypoxia-inducible factor-1α (HIF-1α),vascular endothelial growth factor (VEGF),Ki67,serum protein albumin,IgG1 and IgM,and their relationship with numbers of functional blood vessels in large-cell lung cancer (LCLC) Lu65 and Lu99 cell line xenografts in nude mice. METHODS:The LCLC cells were injected subcutaneously into the back of nude mice to establish a xenograft model,and the in vivo specimens were prepared by cryotechnique (IVCT). Protein (albumin,IgG1,IgM) protein expressions were determined. According to the expression of IgM protein in the extracellular matrix around blood vessels,the number of functional blood vessels and non-functional blood vessels were distinguished and counted. t test,[χ²] test and Spearman rank correlation analyses were used for statistical evaluations. RESULTS:Expression of HIF-1α protein was found in a few tumor cell nuclei in the Lu65 but not in the Lu99 xenograft tumors and the difference was statistically significant (P<0.05). Both VEGF and Ki67 proteins were highly expressed in the tumors. The expression levels of HIF-1α protein were not significantly correlated with the VEGF and Ki67 proteins. The numbers of functional blood vessels in Lu65 and Lu99 tumors were higher than that of non-functional blood vessels (P<0.05). CONCLUSION:The expression levels of HIF-1α protein in the Lu65 and Lu99 tumors had no significant correlation with the VEGF and Ki67 proteins but were related to cellular heterogeneity. On the other hand,the IVCT technology was useful in distinguishing functional from non-functional blood vessels. The numbers of functional blood vessels in both Lu65 and Lu99 tumors were higher than that of non-functional blood vessels. Whether serum proteins were capable of penetrating blood vessel walls may be related to their molecular weights and structures of blood vessels.

OBJECTIVE:To investigate pharmacokinetics siRNA drugs in vivo and to analyze advantages of liposomes as drug carriers. METHODS:A previously constructed siRNA expression plasmid was loaded into nano-invisible cationic liposomes and used for investigations:bare siRNA plasmid group and siRNA plasmid liposomes group. DNase I digestion and serum stability were evaluated. Then,20 μg bare plasmid siRNA and liposome siRNA plasmid were injected into mice through their tail veins,and blood samples were collected at 0 min,5 min,15 min,30 min,1 h,2 h,4 h,12 h,and 24 h,respectively. Quantitative real-time PCR (qPCR) was used to quantitatively detect liposomes in mice,and the concentrations of siRNA plasmid at each time point was calculated. RESULTS:The bare plasmids were completely degraded in DNaseⅠwithin 20 min,and the degradation of plasmid liposomes was slow,with (20.16±2.47)% DNA remaining at 24 h. The bare plasmid only remained (11.03±0.92)% at 20 min in 80% serum,and was basically degraded at 1 h. The plasmid liposomes remained (10.26±1.04)% at 24 h in 80% serum. The stability of siRNA expression plasmid in DNaseⅠand serum was significantly improved by using liposomes as a carrier (P<0.05). The half-life of liposomal encapsulated plasmids in mice was about 2 h,while that of naked plasmids was only 6 min,and the difference was statistically significant (P<0.05). CONCLUSION:In vivo detection of siRNA liposomes can be accomplished by qPCR. Cationic liposomes can protect nucleic acid drugs inside the body,which is an efficient delivery carrier. At the same time,liposomes can improve stability of drugs in vivo and prolong the action time of drugs.

OBJECTIVE:To investigate effects of dehydrated icaritin (DICT) on the stemness of human urine-derived stem cells (hUSCs). METHODS:hUSCs were isolated by centrifugation at room temperature and cultured in vitro. Morphological observations of hUSCs was performed under inverted phase contrast microscope. The growth curve of hUSCs from 1-7 days was plotted according to MTT experiment. The surface markers of hUSCs were detected by flow cytometry. The control group of hUSCs culture medium and DICT treatment groups with concentrations of 0.5,1.0,1.5,2.0,2.5,3.0 μmol/L were set up. Cell viability of each group was measured by the tetramethylthiazole blue (MTT) assay from 1-7 days. Effects of 2.0 μmol/L DICT treatment of hUSCs on the expression of OCT-4 and C-MYC mRNA were measured by quantitative real-time PCR (qPCR) after 3 days. RESULTS:hUSCs with good morphology and high growth activity were successfully obtained. hUSCs expressed CD44 and CD90,surface markers of MSCs,but not CD34,surface marker of hematopoietic stem cells,and CD31,surface marker of endothelial cells. 2.0 μmol/L DICT treatment for 3-6 days significantly promoted proliferations of hUSCs,enhanced their growth viability (P<0.01),and up-regulated the mRNA expression of hUSCs stemness genes OCT-4 and C-MYC (P<0.01). CONCLUSION:DICT enhanced the stemness of hUSCs in vitro.

OBJECTIVE:To explore effects of intramuscular injection of glycerol on kidneys,the main blood storage organ,of rats. METHODS:Twenty-four healthy adult SPF SD rats (half male and half female) were randomly divided into four groups of six rats per group:control and test groups for each sex. After 24 hours of water deprivation,rats were given either normal saline or 50% glycerol normal saline at the dose of 10 mL/kg. After another 24 hours,anesthesia was performed and blood was collected through the abdominal aorta. The kidney,liver and spleen were removed surgically and examined for pathological damage. Urine samples were collected from their metabolic housing and examined for serum biochemistry,blood routine,urine routine and hemagglutination. RESULTS:The results showed that albumin (ALB) and red blood cell (RBC) in the glycerol male and female groups were reduced significantly (P<0.05),while creatinine (Crea),urea nitrogen (Urea),alanine aminotransferase (ALT) and platelet (PLT) were increased significantly (P<0.05). Total protein (TP) and specific gravity of urine (SG) of female animals in the glycerol group were reduced significantly,while aspartate aminotransferase (AST) was increased significantly (P<0.05). Pathological results showed that the renal tubules in the experimental groups were dilated,including clear tube type and protein tube type. The hepatic sinuses were dilated and the renal injury was the most obvious. CONCLUSION:Intramuscular injection of glycerol not only caused kidney damage,but also caused slight liver damage. In this glycerol-induced acute renal injury model,the degree of renal injury in male rats was lower than that in female rats.

OBJECTIVE:To evaluate teratogenicity of cultured ginseng fibrous roots in rats. METHODS:80 female and 40 male SD rats were caged in a ratio of 2:1 for mating. Pregnant rats were randomly divided into high-,medium-,and low-dose groups (2,4,8 g/kg,respectively,equivalent to the recommended amount of 60.5,121,242 times according to body mass) and control group,with 16 in each group. The rats were continuously given ginseng between 6-15 days of conception,and were executed on the 20th day. Caesarean sections were conducted to remove the uterus to inspect the quality,and to check the number of implantations,absorption of fetuses,early stillbirth,late stillbirth,and the numbers and abnormalities of live fetuses. Some fetal pups were examined for appearance,and bone and visceral abnormalities. RESULTS:Compared with the control group,there were no significant differences in the body mass,uterine quality,average number of live births,and length and body quality of pregnant rats. The appearance,organs and bones of the fetal rats were examined. Each dose group,there was no obvious abnormalities and deformities in the negative control group. CONCLUSION:The cultured fibrous roots of ginseng showed no teratogenic effects under the conditions of our investigation.

OBJECTIVE:To study teratogenic effects of chestnut shell extract in Sprague-Dawley (SD) rats. METHODS:Pregnant SD rats were randomly divided into five groups:low,middle and high dosages of chestnut shell extract groups,negative control group and positive control group. The negative control group was given distilled water and the positive control group was given 13 mg/kg vitamin A. The extracts were given intra-gastrically at the 6th to 15th day of gestation,and the pregnant rats were killed at the 20th day of gestation. RESULTS:Compared with the negative control group,the positive control group showed reduced body weight,weight gain and net weight gain from the 9th to the 20th day. In the positive control group,the body weight and body length were obviously decreased,and the rate of deformity,of viscera malformation,and of skeleton malformation were obviously increased. The positive control group showed obvious teratogenic effects. However,there was no significant difference between each dose group and the negative control group. CONCLUSION:Under our experimental conditions,the no observed adverse effect level (NOAEL) of chestnut shell extract on female rats and fetus was above 3.33 g/kg.

OBJECTIVE:Safety of glutathione with vitamin C tablets was preliminarily evaluated by feeding rats with the tablets for 30 days. METHODS:Glutathione with vitamin C tablets were given 417,834,1667 mg/kg (equivalent to 25,50,100 times of the recommended dose in human) to rats for 30 days. General activities of rats were observed every day,and their body weights and food intakes were recorded every week. After 30 days,serum biochemical and hematological indexes were detected. The main organs such as liver,kidney,spleen,stomach,testis were weighed and organ coefficients were calculated,and histopatho-logical examination was performed. RESULTS:During the test,the rats were generally in good condition. Compared with the control group,the body weights of rats in the test group were not significantly abnormal (P>0.05). Results of hematology and blood biochemical indexes were within the normal range (P>0.05). There was no significant difference in organ coefficients (P>0.05) and no obvious pathological changes were found in the organs of the high-dose group. CONCLUSION:Under the experimental conditions,glutathione with vitamin C tablets had no harmful effects in rats.