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Objective:To explore the feasibility and clinical value of sentinel lymph node (SLN) biopsy through cervix-uterine combined two-step injection with two tracers in patients with early stage endometrial cancer.Methods:From July 2019 to April 2021, a total of 73 patients, aged (54.2±3.3) year, who were preoperatively diagnosed as stage Ⅰ-Ⅱ endometrial cancer (including 56 low-risk patients and 17 medium-high risk patients) in Affiliated Hospital of Qingdao University were selected. According to the different sites of tracer injection, the patients were randomly divided into three groups: cervical injection group (25 cases): 1 ml of nano-carbon was used to inject at 3 and 9 o'clock in the cervix; uterine injection group (21 cases): the magnetic resonance imaging examination was performed to determine the location of the lesion, and 4 ml of methylene blue was injected into the uterine body at 2 sites where the lesion was located; combined injection group (27 cases): cervical injection of nano-carbon (1 ml) combined with uterine injection of methylene blue (4 ml). The SLN in all patients were identified under laparoscopy, removed, and followed by frozen pathological examination. Pathological ultra-staging was performed if the postoperative pathological outcome of SLN was negative. The total detection rate of SLN, bilateral pelvic SLN detection rate, sensitivity, negative predictive value, and location of SLN in each group were calculated and compared.Results:(1) In 73 patients with endometrial cancer, the overall detection rate of SLN was 88% (64/73), the detection rate of bilateral pelvic SLN was 67% (49/73), and the detection rate of para-aortic SLN was 49% (36/73). The overall detection rate of SLN (71%, 15/21) and bilateral pelvic SLN (43%, 9/21) in the intrauterine injection group were significantly lower than those in the cervical injection group [92% (23/25), 76% (19/25), respectively] and the combined injection group [96% (26/27), 78% (21/27), respectively; all P<0.05]; the detection rate of para-aortic SLN in the cervical injection group (28%, 7/25) was significantly lower than those in the intrauterine injection group and combined injection group [52% (11/21) and 67% (18/27), respectively; both P<0.05]. Among 73 cases with endometrial cancer, 9 had lymph node metastasis confirmed by postoperative pathological examination, 8 of them had lymph node metastasis detected by SLN and 1 had no lymph node metastasis detected by SLN, with a total sensitivity of 89% and a negative predictive value of 98%. The sensitivity and negative predictive value of cervical injection group and combined injection group were 100%, while the sensitivity and negative predictive value of intrauterine injection group were 67% and 95%. Among 56 low-risk patients, only one patient with lymph node metastasis was confirmed by postoperative pathology by SLN detection, and the metastasis rate was 2% (1/56), and the sensitivity and negative predictive value were 100%. Lymph node metastasis was confirmed in 8 of 17 patients (8/17) with a sensitivity of 88% and a negative predictive value of 90%. (2) A total of 459 SLN were detected in 73 endometrial cancer patients, with the highest proportion of external iliac (33.3%, 153/459).The obturator foramen was 25.3% (116/459), para-aortic 19.6% (90/459), iliac 12.0% (55/459), and presacral 9.8% (45/459). The proportion of para-aortic SLN in the cervical injection group was 12.4% (21/169), which were significantly lower than that in the intrauterine injection group and the combined injection group [27.4% (26/95) and 22.1% (43/195), respectively; both P<0.05]. (3) Pathological super-staging results: among 64 patients with negative SLN routine paraffin pathology, 4 cases of lymph node micro-metastases and 1 case of isolated tumor cell metastasis were detected, and the SLN micro-metastases rate was 8% (5/64), including 2 cases of low-risk patients and 3 cases of medium-high risk patients. Conclusions:SLN biopsy has high sensitivity and negative predictive value in patients with early endometrial cancer and could be used as an alternative to systematic lymph node dissection in low-risk patients. The SLN mapping through cervical-uterine combined injection could further improve the detection rate effectively and avoid the missed detection of positive para-aortic lymph node, especially for high-risk patients or patients with fundal tumor involvement.
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Objective:To investigate the effects of ovarian cancer ascites-derived exosomes on the stem cell properties and invasion ability of ovarian cancer stem-like cell (OCS-LC).Methods:(1) A2780 cells were induced into OCS-LC in serum-free medium, and authenticating their stem-like properties by sphere-forming test, differentiation test and CD 133 marker detection. (2) Exosomes from ascites and A2780 cell were extracted by ultracentrifugation, then authenticating them. The morphology of exosomes was observed by the transmission electron microscope, exosome particle size was measured by nanoparticle tracking analysis (NTA). The expressions of heat shock protein 70 (HSP-70), CD 63 and CD 9 were detected by western blot. (3) The exosomes from ovarian cancer ascites and tumor cell supernate were co-cultured with OCS-LC. The groups were divided into control group, ascites-derived exosomes (ADE) group (ADE+OCS-LC group), and cells-derived exosomes (CDE) group (CDE+OCS-LC group). The sphere-forming ability was evaluated by sphere-forming cycle, maximum sphere diameter and sphere-forming rate of each group; the expression of CD 133 was detected by immunofluorescence staining under microscope; quantitative real-time (qRT)-PCR was used to detected the expression levels of octamer-4 (Oct-4), Nanog mRNA of the signature genes in the stem cells of each group; the metastasis ability of each group was measured by transwell assay. Results:(1) Identification of OCS-LC: sphere-forming experiment showed that the suspension of OCSC single cells was grown in serum-free medium in secondary sphere-forming. Differentiation function experiment showed that OCS-LC were differentiated into adherent A2780 cells by changing the growth mode in serum containing medium. Flow cytometry showed that the proportion of CD 133 positive ( CD133+) cells in OCS-LC group was (18.9±0.9)%, significantly higher than that of control group (0.6±0.5)% ( t=38.570, P<0.01). (2) Under transmission electron microscope, clear lipid bilayer structure was observed in ADE and CDE, and one side presented a concave hemispheric or cup like structure. NTA showed that the diameter of exosomes mainly ranged from 30 to 100 nm, with an average diameter of 67.2 nm. Western blot analysis showed that the specific protein molecules HSP-70, CD 63 and CD 9 were positive. (3) Three groups′ OCS-LC could continue to grow into spheres, and the group of ADE+OCS-LC showed two growth modes, most of the cells continued to grow into spheres, while a small part of cells grew in adherent differentiation. The sphere-forming rate of OCS-LC in the control group, ADE+OCS-LC group, and CDE+OCS-LC group were (1.05±0.20)%, (4.15±0.10)%, and (10.45±0.25)%, the sphere-forming cycle of OCS-LC in the three groups were (15.3±1.5), (10.3±0.6), and (6.7±0.6) days, and the maximum diameters of OCS-LC were (100.3±3.2), (145.2±5.1) and (170.0±2.1) μm, respectively. And the differences were statistically significant (all P<0.05). After co-culture of exosomes with OCS-LC, the sphere-forming ability of cells in the group of CDE+OCS-LC was prior to ADE+OCS-LC group (all P<0.05). Immunofluorescence staining showed that the number of CD 133 green fluorescent chromophore cells in OCS-LC groups [(46.2±2.1)%, (58.4±2.2)%] was significantly higher than that in the control group [(26.6±1.5)%] after the addition of exosomes in co-culture, the positive rate of CD 133 was higher than that in the control group( F=187.588, P<0.05). The qRT-PCR results showed that the expression levels of Oct-4 mRNA in ADE+OCS-LC and CDE+OCS-LC groups were 3.46±0.24, 4.03±0.31, compared with that in control group (1.04±0.12), the differences were statistically significant ( F=134.932, P<0.05). The mRNA expression levels of Nanog were 1.57±0.32, 2.66±0.15, which were significantly higher than that in the control group (1.00±0.07), and the differences were statistically significant ( F=49.329, P<0.05). And the expression of both in CDE+OCS-LC group increased more significantly than ADE+OCS-LC group (all P<0.05). The number of invasive cells in the three groups of OCS-LC were: control group 30±5, ADE+OCS-LC group 102±4, CDE+OCS-LC group 210±7, and there were statistically significant differences among three groups ( F=820.800, P<0.05). Compared with the control group, the number of invaded cells in the co-culture group were significantly increased ( P<0.05), and the CDE+OCS-LC group had the higher cell invasion ability then the ADE+OCS-LC group ( t=23.202, P<0.05). Conclusions:Exosomes derived from ovarian cancer ascites could enhance and maintain the stemness of OSC-LC, and promote the invasion of tumor cells. Moreover, CDE is superior to ADE.
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Objective To investigate the expression of long intergenic non-protein coding RNA-regulator of reprogramming (Linc-ROR) in high-grade ovarian serous cancer, and explore the relationship between Linc-ROR expression and biological function of high-grade ovarian serous cancer. Methods A total of 34 high-grade ovarian serous cancer tissue samples and 19 normal fallopian tube tissue samples were collected between June 2014 and February 2016. Real-time reverse transcription (RT)-PCR was used to detect the Linc-ROR mRNA expression in different samples. The relationship between Linc-ROR expression level and ovarian cancer International Federation of Gynecology and Obstetrics (FIGO) stage, lymph node metastasis was analyzed. Constructed Linc-ROR small interference RNA (siRNA) and pIRES2-EGFP-Linc-ROR plasmid, then Linc-ROR siRNA and pIRES2-EGFP-Linc-ROR plasmid were respectively transfected into SKOV3 cells. Cell proliferation, migration and invasion ability were assessed by cell counting kit-8 (CCK-8), wound healing assay and transwell invasion assay. Results (1) The expression level of Linc-ROR mRNA was significantly higher in high-grade ovarian serous cancer than normal fallopian tube tissues (4.31± 0.38 vs 1.03 ± 0.21; t=25.842, P<0.01). With the progression of FIGO stages, the expression of Linc-ROR was increased (F=95.702, P<0.01), and it was associated with lymph node metastasis (t=7.397, P<0.01). (2) The results of RT-PCR showed that the expression level of linc-ROR in Linc-ROR-i group was significantly lower than that in Linc-ROR-NC-i group (0.30 ± 0.11 vs 1.02 ± 0.10; t=15.269, P<0.01). The expression level in Linc-ROR-p group was significantly higher than that in Linc-ROR-NC-p group (8.90 ± 0.45 vs 1.03 ± 0.17;t=21.934, P<0.01). The CCK-8 assay showed that when the cells were cultured for 3, 4, 5 and 6 days, the A value in Linc-ROR-i group was significantly lower than that in Linc-ROR-NC-i group (P<0.05). And the A value in Linc-ROR-p group was significantly higher than that in Linc-ROR-NC-p group (P<0.05). Wound healing assay showed that, after 48 hours incubation, migration rate of cells in Linc-ROR-i group was significantly less than that in the Linc-ROR-NC-i group [(52±4)%vs(67±5)%;t=5.720,P<0.01]. The migration of cells in Linc-ROR-p group was significantly greater than that in the Linc-ROR-NC-p group [(84±4)%vs(66±4)%;t=7.330,P<0.01]. Cell transwell invasion assay showed that, after 48 hours of incubation, the number of invasive cells in Linc-ROR-i group was lower than that in Linc-ROR-NC-i group (74 ± 3 vs 104 ± 3; t=15.810,P<0.01). And the number of invasive cells in Linc-ROR-p group was higher than that in Linc-ROR-NC-p group (217 ± 4 vs 108 ± 5; t=38.060, P<0.01). Conclusion Highly expressed Linc-ROR could enhance the proliferation, migration and invasion ability of high-grade ovarian serous cancer cells, which may be one of the important molecules in the occurrence and development, invasion and metastasis of high-grade ovarian serous cancer.
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Objective To study the role and mechanism of microRNA-16(miR-16)in the proliferation,invasion and apoptosis of ovarian epithelial carcinoma cells in vitro.Methods The SKOV-3 cells were transfected with miR-16 mimics or negative control RNA(NC)by lipofectamine 2000.The expression of miR-16 was detected by real-time reverse transcription(RT)-PCR in SKOV-3 cells,and western blot was used to detect the expression of vascular endothelial growth factor(VEGF),matrix metalloproteinase-2(MMP-2)and bcl-2 protein.Methyl thiazolyl tetrazolium(MTT),5-ethynyl-2'-deoxyuridine(EdU)and transwell assay were used to determine the proliferation and invasion abilities.And the rate of apoptotic cell was detected by flow cytometry method.Results(l)The expression level of miR16 in the transfection cells group was significantly higher than that in NC group(125.93 ± 15.30 versus 0.78 ± 0.16,P ＜ 0.01).(2)The rclative expression level of VEGF protein in transfection cells,NC and blank control group was 0.58 ± 0.05,1.22 ± 0.03,1.20 ± 0.03,MMP-2 protein was 0.63 ± 0.03,1.16 ±0.03,1.21 ± 0.03,and bel-2 protein 0.52 ± 0.03,1.19 ± 0.05,1.28 ± 0.06,respectively.The level of VEGF,MMP-2 and bcl-2 protein in the transfection group were lower than those in other control groups,and there were significantly differences among them(all P ＜0.01).(3)After transfected 4 days,the inhibition rate of cell proliferation in the transfection group was dramatically higher than that in NC group[(37.2 ±6.2)％ versus(3.6 ± 3.2)％,P =0.001].(4)The percentage rate of proliferative cells in the transfection,NC and blank control group was(12.3 ± 0.8)％,(23.4 ± 1.8)％,(31.1 ± 4.9)％.And it was lower in the transfection group(P ＜ 0.05).(5)Decreased cells via the transwell member in the transfection group(6 ± 3)were detected as compared with NC group(40 ± 9)and blank control group (48 ± 8,P ＜ 0.01).(6)Twenty-four hours after cultured in serum starvation and hypoxia,the rate of the viable and late apoptotic cells in the transfection group were significantly higher than those in NC group and blank control group[the rate of viable apoptotic cell was(16.9 ± 2.1)％,(10.3 ± 1.7)％ and(9.0 ±0.8)％ respectivcly,P＜0.01;the rate of late apoptotic cell was(13.4±3.3)％,(3.2 ±1.8)％ and (0.7 ±0.6)％ respectively,P ＜ 0.01].After cultured 48 hours,total apoptotic cells in the transfection group was significantly more than those in other groups(P ＜ 0.01).Conclusion miR-16 might inhibit the proliferation,invasion of ovarian epithelial carcinoma cells and enhance their sensitivity to apoptotic stimuli via downregulation of the expression of VEGF,MMP-2 and bcl-2 protein.
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ObjectiveTo investigate the role and mechanism of microRNA-21 (miR-21) in the proliferation and apoptosis of ovarian epithelial carcinoma cells. MethodsA short-hairpin RNA specifically targeting miR-21 plasmid was constructed, and the recombinant was identified by restriction endonuclease analysis and DNA sequencing. Three experimental groups were included, transfection group (transfected with pSIREN-miR-21 ), negative control group ( transfected with pSIREN-miR-21-neg) and blank control group (without transfection plasmid ). The expression of miR-21 was detected by stem-loop real-time reverse transcription (RT)-PCR in OVCAR3 cells ,and western blot was used to detect the expression of programmed cell death 4 ( PDCD4 ) protein. Tethyl thiazolyl tetrazolium (MTT) and flow cytometry method were used respectively. ResultsRecombinant plasmid (pSIREN-miR-21) was constructed successfully and identified by restriction endonuclease analysis and DNA sequencing. The relative expression level of miR-21 in cells transfection, negative control and blank control group was 0.26 ± 0.08, 1.26 ± 0.21and 1.00 respectively. The level of miR-21 in the cells in transfection group was significantly lower than those in the negtive control and blank control group(P ＜0. 01 ). The gray scale of PDCD4. protein was 1443 ±33,858 ± 19 and 846 ± 16 in the transfection group, negative control and blank control group respectively. The value of PDCD4 in transfection group was higher than other control groups, and there were significantly difference among them( P ＜0. 01 ). Moreover, the optical density of the cells in transfection group was 0. 661 ±0. 015,significantly lower than those in two control groups (0. 848 ± 0. 150 for negative control, 0. 935 ± 0. 133 for blank control,P ＜ 0. 01 ). Forty-eight hours after tranfection, the rate of viable apoptotic cell was significantly higher than negative control and blank control group [(25.821 ± 0. 763 )％ vs.(0. 010 ± 0. 003 ) ％ vs. (0. 238 ± 0. 023) ％ ; P ＜ 0. 01];72 hours after tranfection, the rates of viable apoptotic cell and necrotic cell were all higher than the two control groups [the rate of viable apoptotic cell was ( 30. 480 ±0. 821 ) ％, ( 7. 792 ± 0. 312 ) ％ and ( 7. 033 ± 0. 257 ) ％ respectively ( P ＜ 0. 01 ) ; the rate of necrotic cell was (3.558 ±0.211)％, (1.557 ±0.067)％ and (1.049 ±0.028)％, respectively (P＜0. 01)].ConclusionmiR-21 might play an important role in the proliferation and apoptosis of ovarian epithelial carcinoma cells through negatively control the expression of PDCD4.