1.The Function And Underlying Mechanism Of Long Non-coding RNA LINC00578 In Ovarian Serous Adenocarcinoma Metastasis 2.Detection Of Sentinel Lymph Node In Cervical Cancer And Endometrial Cancer Using Carbon Nanoparticles

Objective:This study aims to search for long non-coding RNA(LncRNA)with differential expressed between primary tumor and metastatic tumor in ovarian serous adenocarcinoma and to explore its expression in ovarian serous adenocarcinoma,its influence on the biological characteristics of ovarian cancer,and its molecular mechanism to regulate ovarian cancer metastasis.Methods:In this study,LncRNA significantly associated with ovarian serous adenocarcinoma metastasis were screened out by the high throughput LncRNA expression profiles of primary ovarian cancer and paired metastatic tumor.Quantitative real-time polymerase chain reaction(qRT-PCR)was then conducted to verify the LncRNA expression difference in fresh ovarian cancer tissues and paired metastatic cancer tissues.The expression of LncRNA in ovarian cancer tissue microarray was detected using in situ hybridization to analyze the correlation between the level of long non-coding RNA expression and clinic-pathological features and prognosis of ovarian serous adenocarcinoma patients.Subsequently,the effects of LncRNA on the invasion,migration and proliferation of ovarian cancer cells were explored in vitro by Transwell,wound healing assay and cell count kit-8(CCK-8)assay.In vivo,the constructed ovarian cancer cell lines with stable knockdown and overexpression of LncRNA were injected into nude mice by intraperitoneal injection.The effects of LncRNA on the growth,invasion and metastasis of ovarian cancer in vivo were observed by in vivo imaging methods and intraperitoneal anatomy.RNA pull-down was conducted to find the proteins directly bound to LncRNA,and RNA-sequencing(RNA-seq)combined gene set enrichment analysis(GSEA)analysis was conducted to find the cellular pathways involved in the LncRNA regulation to explore the molecular mechanism of LncRNA in ovarian cancer metastasis.Results:1)In this study,the transcriptome sequencing analysis of 5 pairs of primary ovarian serous adenocarcinoma and paired metastatic tumor revealed 13 IncRNA with significantly differential expression between primary and metastatic tumors,with 6 higher in the metastatic tumors compared with the primary tumors(M/P)and 7 higher in the primary tumors compared with the metastatic tumors(P/M).LINC00578 was the most significant gene between M/P expression.The expression of LINC00578 was validated in 26 ovarian serous adenocarcinoma patients with paired fresh tumors,and 16 patients(61.5%)showed LINC00578 was much higher in metastatic tumors than ovarian tumor.2)In ovarian serous adenocarcinoma tissue microarray,it was found that the level of LINC00578 was significantly related to the prognosis of the patients.The overall recurrence rate and mortality of high LINC00578 expression group was significantly higher than that of low LINC00578 expression(92.5%vs 75.4%,P<0.01;74.6%vs 53.6%,P<0.01).In terms of survival time,the median progression-free survival(PFS)and median overall survival(OS)of the high LINC00578 expression group was significantly lower than that of low LINC00578 expression(6 months vs 8 months,P<0.05;35 months vs 55 months,P<0.01).Log-rank test showed that LINC00578 expression was significant correlated with PFS and OS.The 5-year PFS of the high LINC00578 expression group was significantly lower than that of low LINC00578 expression(7.5%vs 22.8%,P<0.05;25.3%vs 47.3%,P<0.01).Multivariate Cox regression analysis showed that the expression level of LINC00578 was an independent influencing factor of PFS and OS in patients with ovarian serous adenocarcinoma(P<0.05).3)Then we did research about the effect of LINC00578 on the phenotype of ovarian cancer cells at the cellular level in vitro.Overexpressed Lentivirus LINC00578 and knockdown sh-LINC00578 were constructed by lentivirus packaging system to infect ovarian cancer cells.Transwell and wound healing assay showed that the overexpression of LINC00578 could promote the invasion and migration of ovarian cancer cells,and the invasion and migration decreased after knockdown of LINC00578.CCK-8 assay showed that the proliferation ability of ovarian cancer cells decreased after knockdown of LINC00578.4)In vivo,the constructed ovarian cancer cell with stable overexpression and knockdown of LINC00578 were injected into nude mice by intraperitoneal injection.The results showed that the metastatic ability of ovarian cancer cells increased after overexpression of LINC00578,and decreased after knockdown of LINC00578.5)RNA-seq analysis showed that LINC00578 probably involved in biological processes related to extracellular matrix and extracellular matrix receptor interaction pathway,and the process of DNA replication.Conclusion:The expression of LINC00578 is correlated with the prognosis of ovarian serous adenocarcinoma patients.Patients with high LINC00578 expression had worse progression-free survival and overall survival compared with patients with low LINC00578 expression.LINC00578 can act as a prognostic indicator of ovarian serous adenocarcinoma.In vitro,LINC00578 can significantly enhance the invasion,migration and proliferation of ovarian cancer cells.In vivo,LINC00578 can promote the metastasis of ovarian cancer in the abdominal cavity.The pro-proliferation and metastatic effects of LINC00578 may be related to abnormal DNA replication and extracellular matrix receptor interaction pathways,which deserves further investigation.Objective:This study aims to explore the feasibility and effectiveness of sentinel lymph node biopsy(SLNB)using carbon nanoparticles(CNPs)during laparoscopic surgery for cervical cancer patients.Methods:CNPs are lymphatic system tracers and are widely used in lymphography.This study analyzed the cervical cancer patients who received SLN biopsy using CNPs tracer in the Gynecological Department of Cancer Hospital,Chinese Academy of Medical Sciences between December 2015 and September 2018.SLN mapping was conducted by means of an intracervical injection at the 3 o'clock and 9 o'clock locations using a 25-mg CNPs tracer after the induction of general anesthesia.Then,the black-stained lymphatic channels were observed and carefully dissected,and the first lymph nodes in channels were identified as SLNs under laparoscopy.Afterwards,the SLNs was excised separately,followed by system lymphadenectomy and radical hysterectomy.The SLNs and other resected specimens separately underwent routine pathological examinations.The effectiveness of CNPs was evaluated by the detection rate and accuracy parameters.Factors associated with SLN laterality and SLNs localizations were also analyzed.Results:Forty-five women with cervical cancer staged ?B1-?A1 according to 2009 International Federation of Gynecology and Obstetrics staging scheme were retrospectively reviewed.The median age of all patients was 43 years(30-63 years)and the median body mass index(BMI)was 23.59 kg/m2(17.5-30.0 kg/m2).All procedures were successfully completed without conversion to open laparotomy,and no intraoperative or postoperative complications occurred.Among these 45 patients,at least one SLN were identified in 42 patients(93.3%),and bilateral SLNs were identified in 27 patients(64.3%).A total of 225 SLNs were harvested,with a median count of 5.0(0-18).In our study,81.3%of SLNs located in the usual localizations,which included the external iliac(39.1%)region,internal iliac(25.8%)region and obturator(16.4%)region,while the rest 1 8.7%of SLNs were in unusual localizations,which included the common iliac(10.7%)region,parametrial(7.6%)region and pre-sarcal(0.4%)region.According to the pathology examination,four positive lymph nodes were found in four patients(8.9%)without a false negative case.The sensitivity and negative predictive values of SLN detection were 100%.In further analysis,univariate and multivariate analyses showed that increased BMI was associated with the decrease in bilateral SLN detection rate(P=0.015).However,age,histological type,conical history,learning curve,degree of tumor differentiation and LVSI had no effect on bilateral SLN detection rate(P?0.05).Conclusion:Laparoscopic SLN mapping with CNPs appears to be simple and efficient for patients with early-stage cervical cancer.Objective:This study aims to explore the feasibility and effectiveness of carbon nanoparticles in sentinel lymph node biopsy for endometrial cancer.Methods:This study analyzed patients with endometrial cancer who underwent SLN biopsy using carbon nanoparticles in the gynecological department of Cancer Hospital,Chinese Academy of Medical Sciences from January 2016 to September 2018.SLN biopsy was conducted by means of superficial injection(1-3mm)combined with interstitial intracervical injection(1-2cm)at the 3 o'clock and 9 o'clock locations using a 50-mg CNPs suspension tracer after the induction of general anesthesia.Then,the black-stained lymphatic channels were observed and carefully dissected,and the first lymph nodes in channels were identified as SLNs under laparoscopic mapping.Afterwards,the excision of SLNs was conducted,followed by standard endometrial staging surgery.The SLNs and other resected specimens separately underwent pathological examinations.The overall detection rate,bilateral detection rate,sensitivity,negative predictive value and false negative rate were calculated to evaluate the efficacy of carbon nanoparticles.SLNs localizations were also analyzed.Results:Fifty-two patients underwent SLNB using CNPs were analyzed.The median age of all patients was 53 years(28-66 years)and the median body mass index was 25.2kg/m2(20.3-35.3kg/m2).All these patients were preoperatively classified as clinical stage ?(44/52,84.6%)or stage ?(8/52,15.4%).The main histology type was endometrioid adenocarcinoma(48/52,92.3%)?According to the 2009 FIGO scheme:30 patients in stage IA(57.8%),8 patients in stage IB(15.4%),6 patients in stage ?(11.5%),1 patient in stage IIIA(1.9%),5 patients in stage IIIC 1(9.6%),2 patients in stage IIIC2(3.8%).All procedures were successfully completed without conversion to open laparotomy,and no intraoperative or postoperative complications occurred.Among these At least one SLN were identified in 47(90.4%)cases,of which bilateral SLNs were identified in 37(71.2%)cases,only left hemi-pelvic SLNs in 4(7.7%)cases and only right hemi-pelvic SLNs in 6(11.5%)cases.A total of 220 SLNs were harvested,with a median number of 4.0.In this study,the majority of SLNs were in the external iliac(40%)region,followed by obturator(24.5%),internal iliac(21.8%),common iliac(11.4%)and para-aortic(2.3%)regions.Metastatic lymph nodes were confirmed in 7(13.5%)cases with one false negative case,yielding false negative rate of 14.3%(1/7),sensitivity of 85.7%(6/7)and negative predictive value of 97.9%(47/48).Conclusion:SLN mapping with carbon nanoparticles for endometrial cancer appears to be safe,efficient,and deserves further investigation.