Long noncoding RNA LOC646029 functions as a ceRNA to suppress ovarian cancer progression through the miR-627-3p/SPRED1 axis / 医学前沿

Long noncoding RNAs (lncRNAs) play a crucial regulatory role in the development and progression of multiple cancers. However, the potential mechanism by which lncRNAs affect the recurrence and metastasis of ovarian cancer remains unclear. In the current study, the lncRNA LOC646029 was markedly downregulated in metastatic ovarian tumors compared with primary tumors. Gain- and loss-of-function assays demonstrated that LOC646029 inhibits the proliferation, invasiveness, and metastasis of ovarian cancer cells in vivo and in vitro. Moreover, the downregulation of LOC646029 in metastatic ovarian tumors was strongly correlated with poor prognosis. Mechanistically, LOC646029 served as a miR-627-3p sponge to promote the expression of Sprouty-related EVH1 domain-containing protein 1, which is necessary for suppressing tumor metastasis and inhibiting KRAS signaling. Collectively, our results demonstrated that LOC646029 is involved in the progression and metastasis of ovarian cancer, which may be a potential prognostic biomarker.

S100A7 promotes the metastasis and epithelial-mesenchymal transition on HeLa and CaSki cells / 中华妇产科杂志

Objective To elucidate the impact of over-expression of S100A7 on migration,invasion,proliferation, cell cycle, and epithelial-mesenchymal transition (EMT) in human cervical cancer HeLa and CaSki cells.Methods(1)Immunohistochemistry of SP was used to examine the expression of S100A7 in 40 cases of squamous cervical cancer tissues and 20 cases of normal cervical tissues.(2)The vectors of pLVX-IRES-Neo-S100A7 and pLVX-IRES-Neo were used to transfect human cervical cancer HeLa and CaSki cells, and the positive clones were screened and identified. Next, transwell migration assay, cell counting kit-8(CCK-8)assay and fluorescence activating cell sorter(FACS)were used to detect the effect of S100A7-overexpression on the migration, invasion, proliferation and cell cycle of cervical cancer cells. Furthermore, western blot was performed to observe the expression of epithelial marker(E-cadherin)and mesenchymal markers(N-cadherin,vimentin,and fibronectin)of EMT. Results(1)S100A7 expression was significantly higher in cervical squamous cancer tissues(median 91.6)than that in normal cervical tissues(median 52.1;Z=-2.948,P=0.003).(2)Stable S100A7-overexpressed cells were established using lentiviral-mediated gene delivery in HeLa and CaSki cells. S100A7 was detected by real-time quantitative reverse transcription PCR,S100A7 mRNA of S100A7-overexpressed cells were 119 ± 3 and 177 ± 16, increased significantly compared with control groups of median(P<0.01).Compared with the control cells, the number of S100A7-overexpressed HeLa and CaSki cells that passed the transwell membrane assay were increased significanatly(572 ± 51 vs 337 ± 25, P<0.01;100 ± 8 vs 41 ± 4, P<0.01).Matrigel invasion assay showed that the number of S100A7-overexpressed HeLa and CaSki cells that passed the transwell membrane were respectively 441±15 and 110±14,elevated significantly compared with control cells(156±21 and 59±7;P<0.05). However, S100A7 overexpression didn′t influence the proliferation and cell cycle progression of HeLa and CaSki cells(P>0.05). Expression of E-cadherin was dramatically decreased, while N-cadherin, vimentin, and fibronectin increased in S100A7-overexpressed cells. Conclusion S100A7 enhances the migration, invasion and EMT of HeLa cells and CaSki cells, and may be plays an important role in the development of cervical cancer.

Epidermal growth factor promotesproliferation of endometrial adenocarcinoma cell line Ishikawa / 基础医学与临床

Objective To investigate the effect of epidermal growth factor (EGF) on the proliferation of endometrial adenocarcinoma cells,phosphorylation of estrogen receptor α (ERα)and Ack1 in the absence of estrogen.MethodsIshikawa cell line was stimulated by EGF without estrogen settings, Cell Counting Kit-8 (CCK-8) was used to evaluate cell proliferation, Western blot was used to detect ER α phosphorylation and Ack1 phosphorylation.Giving tyrosine inhibitor dasatinib to assess the effect of EGF on cell proliferation,phosphorylation of ERα and Ack1 in Ishikawa cells.Results EGF enhanced the proliferation of endometrial adenocarcinoma cells (P<0.05).EGF induced ERα phosphorylation at Tyr-537 and phosphorylation of Ack1.Compared with untreated control, Dasatinib inhibited the proliferation of endometrial adenocarcinoma cells (P<0.05), phosphorylation of ERα Tyr-537 and Ack1.Conclusions EGF promotes Ishikawa cells proliferation in the possible way of activating ER α site-specific phosphorylation at Tyr-537 and phosphorylation Ack1, which could be blocked by dasatinib.

Expression and function of long intergenic non-protein coding RNA-regulator of reprogramming in high-grade ovarian serous cancer / 中华妇产科杂志

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.

MicroRNA-16 regulates the proliferation,invasion and apoptosis of ovarian epithelial carcinoma cells in vitro / 中华妇产科杂志

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.

Role of microRNA-21 in the proliferation and apoptosis of ovarian epithelial carcinoma cells / 中华妇产科杂志

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.

The synthesis and shedding of transmembrane CXCL16 in first-trimester human trophoblast / 中华微生物学和免疫学杂志

Objective To detect the regulation of CXCL16 synthesis and shedding in first-trimester human trophoblasts. Methods Firstly, we analyzed the expression and secretion of chemokine CXCL16 in primary cultured trophoblasts by immunochemical staining and ELISA. Then we determined the soluble and cell-associated CXCLI6 respectively with and without treatments of cytokine IFN-γ, TNF-α, IL-4 and ADAM10 by ELISA. Results Trophoblast expressed and secreted CXCL16 in a stable level. Cytokine IFN-γ induced both synthesis and secretion of CXCL16 significantly ( P <0. 01 ) in trophoblasts. ADAM10 increased the shedding of chemokine domain of CXCL16 from trophoblasts but didn't influence the synthesis of CXCL16 protein in trophoblast. Conclusion IFN-γ and ADAM10 play important roles in production and shedding of transmembrane CXCL16 in first-trimester trophublasts.

Establishment of the drug resistant cell line of choriocarcinoma and the reversal of drug resistance by transfection of human interleukin 2 gene / 中华妇产科杂志

Objective To establish the drug resistant cell line of choriocarcinoma and to study the transfection of the human interleukin 2 (hIL 2) gene into the established drug resistant cell line and investigate the reversal of the multidrug resistance Methods The resistant cell line was established by pulse exposed choriocarcinoma cell line JEG 3 to etopside (VP 16) for ten months The recombinant plasmid containing pcDNA3 1(+) hIL 2 gene was constructed The drug resistant cell line was transfected with the constructed plasmid by lipofectin, and the tumor cell colonies containing the IL 2 sequence were selected by genetin The expression of hIL 2 and drug resistant related genes was detected by reverse transcript polymerase chain reaction The chemosensitivity of the gene transfected tumor cells and the non transfected cell lines to methetraxate, VP 16, kengshengmycine, paclitaxol and 5 fluorouracil was determined by the methyl thiazolyl tetrazolium cytotoxicity assay Results The transfected cells expressed human hIL 2 gene, and showed the reversal of multidrug resistance by methyl thiazolyl tetrazolium assay The transfected cells expressed no multidrug resistance gene 1 (MDR1) on mRNA level Drug resistance index to VP 16 decreased from 38 7 to 6 0 and 6 1, the index to methetraxate decreased from 14 5 to 2 6 and 2 5, to methetraxate from 13 0 to 2 0 Conclusion The transfection of hIL 2 gene into the drug resistance cell line of choriocarcinoma can modulate the MDR1 expression on the mRNA level, and reverse the drug resistance