RESUMO
Objective:To investigate the diagnostic value of long noncoding RNA (lncRNA) extracted from serum exosomes in epithelial ovarian cancer (EOC).Methods:(1) Patients with ovarian tumors who were hospitalized in the Affiliated Tumor Hospital of Guangxi Medical University from August 2018 to December 2019, including 35 cases of EOC patients (malignant group) and 20 cases of benign ovarian tumor patients (benign group) were collected; during the same period, 15 healthy women (normal group) who underwent physical examination in the Affiliated Tumor Hospital of Guangxi Medical University were used as controls. Fasting venous blood serum was collected from the above three groups of women, and serum exosomes were isolated and purified using commercial kits. The morphology of exosomal particles was observed with transmission electron microscope, and the particle size distribution of the exosomes was detected by NanoSight technology. The expression of specific proteins cluster of differentiation (CD) 63, CD 81, and tumor susceptibility gene 101 (TSG101) of exosomes were analyzed by western blot. (2) Four cases of EOC patients and three cases of healthy women were randomly selected. High-throughput sequencing technology was used to analyze the differentially expressed lncRNA in serum exosomes of these four EOC patients and three healthy women, and screen out the significantly differentially expressed lncRNA. The screened lncRNA with different expression levels was verified by quantitative reverse transcription-polymerase chain reaction (QRT-PCR) in these seven original clinical samples, furtherly confirmed and tested with QRT-PCR in larger clinical samples (a total of 70 serum samples). (3) The receiver operating characteristic (ROC) curve of the target lncRNA was drawn and its diagnostic indicators such as sensitivity and specificity were evaluated. By using logistic binary regression model, multi-factor joint diagnostic models were constructed and evaluated. Results:(1) Under transmission electron microscope, clear lipid bilayer structure was observed in serum exosomes, and one side presented a concave hemispheric or cup like structure; the peak diameter of the exosomal particles detected with NanoSight technology was 127.6 nm, and the particles between 30 and 150 nm accounted for 58.9%; western blot confirmed that the obtained (exosomal) particles could detect the expression of the marker proteins CD 63, CD 81, and TSG101. (2) Analysis of high-throughput sequencing technology showed that compared with the women in the normal sequencing group (3 cases), 425 differentially expressed lncRNAs (including 23 up-regulated and 402 down-regulated) were screened in the serum exosomes of the malignant sequencing group (4 cases). Six types of lncRNA with significantly abnormal expression levels (including FER1L6-AS2, LINC00470, LINC01811, CXXC4-AS1, LINC02343, and LINC02428) were randomly selected for original sample verification, and the results were consistent with the sequencing results. Subsequently, these six lncRNAs were used for larger samples QRT-PCR verification. Compared with the benign and normal groups, the expression of FER1L6-AS2, LINC00470 and LINC01811 in malignant group increased by 1.66 and 1.84-fold, 2.05 and 2.46-fold, 2.94 and 2.35-fold, respectively; the expressions of CXXC4-AS1, LINC02343 and LINC02428 were down-regulated to 29% and 34%, 40% and 46%, 42% and 42%, respectively. For the same lncRNA, there were statistical differences between the malignant group and the benign group, between the malignant group and the normal group (all P<0.05), and there were no statistical differences between the benign group and the normal group (all P>0.05). (3) The results showed that the area under curve (AUC) of these six lncRNAs ranged from 0.722 to 0.805, which had moderate diagnostic efficiency. To use logistic binary regression model to establish multi-indicator joint diagnostic models and establish different joint factor ROC curves. The results showed that the AUC of the joint factor prediction model 1 (composed of FER1L6-AS2 and LINC01811), the joint factor prediction model 2 (composed of CXXC4-AS1, LINC02343, and LINC02428), and the joint factor prediction model 3 (composed of FER1L6-AS2, CXXC4-AS1, LINC02343, and LINC02428) were 0.865, 0.934, and 0.962, respectively. The diagnostic efficacy of the combined factor prediction models was higher than that of the single lncRNA (all P<0.05). Conclusions:High-throughput sequencing technology is an effective method for screening out the different expression levels of lncRNA extracted from serum exosomes. The combined detection of multiple serum exosomal lncRNA indicators has a certain diagnostic efficacy for patients with EOC. Detection of serum exosomal lncRNA indicators will provide new ideas for the diagnosis of EOC.
RESUMO
Objective:To determine the content of albiflorin, paeoniflorin, benzoylpaeoniflorin, sophoricoside, prim-O-glucosylcimifugin, cimifugin, 5-O-methylvisammioside and sec-O-glucosylhamaudol in Huaiqin Salve with high performance liquid chromatography-quantitative analysis of multi-components by single-marker (HPLC-QAMS) method. Methods:The samples were separated with ZORBAX Eclipse XDB-C18 column by a gradient elution using methanol-acetonitrile (1:2) (A)-0.1% phosphoric acid solution (B) as mobile phase and the flow rate was 1.0 ml/min. The detection wavelength was 230 nm for albiflorin, paeoniflorin and benzoylpaeoniflorin, and 254 nm for sophoricoside, prim-O-glucosylcimifugin, cimifugin, 5-O-methylvisammioside and sec-O-glucosylhamaudol. The column temperature was 30 ℃. Using sophoricoside as an internal standard, the relative correction factors (RCFs) of albiflorin, paeoniflorin, benzoylpaeoniflorin, prim-O-glucosylcimifugin, cimifugin, 5-O-methylvisammioside and sec-O-glucosylhamaudol were calculated. The method was validated by comparison of the quantitative results between external standard method (ESM) and HPLC-QAMS method.Results:Albiflorin, paeoniflorin, benzoylpaeoniflorin, sophoricoside, prim-O-glucosylcimifugin, cimifugin, 5-O-methylvisammioside and sec-O-glucosylhamaudol showed good linear relationship within the range of 0.034 7-0.867 5, 0.063 6-1.590 0, 0.006 9-0.172 5, 0.198 6-4.965 0, 0.092 8-2.320 0, 0.026 6-0.665 0, 0.042 7-1.067 5, 0.020 9-0.522 5 μg ( r ≥ 0.999 1); whose average recoveries ( RSDs) were 98.85% (1.02%), 100.04% (0.67%), 96.92% (1.14%), 100.06% (0.85%), 99.31% (1.39%), 99.16% (1.22%), 98.59% (1.33%) and 97.58% (1.41%), respectively. No significant differences were found in the quantitative analysis of the components with ESM and HPLC-QAMS method. Conclusion:The HPLC-QAMS method could provide the reference for themulti-index component evaluation for Huaiqin Salve.