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Objective:To determine the expression of circular RNA-SEC31A(circSEC31A) in pancreatic cancer and investigate the effects on the invasion and migration of pancreatic cancer cells and the underlying molecular mechanism.Methods:Differentially expressed circRNAs between pancreatic cancer cells (BXPC-3, PANC1, CaPan-2, SW1990) and human normal pancreatic cells (HPDE) were identified by qRT-PCR. Then, two cell lines with high circSEC31A expression were selected to conduct next experiments. According to the sequence of the back-splicing site in circSEC31A, siRNAs for downregulation of circSEC31A were designed and transfected by liposome to silence circSEC31A in pancreatic cancer cells, and grouped as followed siR-circSEC31A#1 and siR-circSEC31A#2. Meanwhile, siR-NC group transfected with non-specific siRNA served as control. Transwell assays and wound healing assays were operated to evaluate the functional role of circSEC31A on the invasion and migration of pancreatic cancer cells. RNA Pull-down assay with circSEC31A probe and oligo control probe was used to screen the miRNA combining with circSEC31A and the effects of miRNA on cell invasion and migration of pancreatic cancer cells were validated. The effect of miR-200c-3p and circSEC31A silencing on the expression of PDK1 mRNA was identified by qRT-PCR. The protein expression of PDK1, downstream Akt and p-Akt after circSEC31A silencing was verified by Western blotting assays.Results:The relative expression level of circSEC31A in HPDE (1.000±0.120) was obviously lower than that in BXPC-3 (1.920±0.130), SW1990 (2.93±0.528), PANC1 (4.557±0.692) and CaPan-2 (5.247±0.194), and all the differences were statistically significant ( P<0.001). Compared with the PANC1 siR-NC group (1301.3±94.6) and CaPan-2 siR-NC group (1835.0±70.1) per 100 high power field, transwell assays showed that the numbers of invasive pancreatic cancer cells was highly decreased in PANC1 siR-circSEC31A#1 group (727.3±92.9), siR-circSEC31A#2 group (792.0±18.1), CaPan-2 siR-circSEC31A#1 group (718.0±90.6), siR-circSEC31A#2 group (692.7±84.8). Wound healing assays showed that silencing circSEC31A decreased the wound healing rate of pancreatic cancer cells in PANC1 siR-circSEC31A#1 group (20.667±3.215)%, siR-circSEC31A#2 group (20.000±4.583)%, CaPan-2 siR-circSEC31A#1 group (28.000±8.185)%, siR-circSEC31A#2 group (29.667±5.686)%, compared with the PANC1 siR-NC group (55.000±4.359)% and CaPan-2 siR-NC group (69.000±3.606)%. RNA Pull-down assays showed that compared with PANC1 oligo probe group (1.000±0.091) and CaPan-2 oligo probe group (1.000±0.153), miR-200c-3p was significantly enriched in the PANC1 circSEC31A probe group (2.237±0.175) and CaPan-2 circSEC31A probe group (2.166±0.156). Compared with PANC1 siR-NC group (939.3±57.0) and CaPan-2 siR-NC group (786.7±51.5) per 100 high power field, the numbers of invasive pancreatic cancer cells were up-regulated in PANC1 siR-miR-200c-3p group (1206.0±99.1) and CaPan-2 siR-miR-200c-3p group (1838.0±105.7), while the low numbers of invasive pancreatic cancer cells were observed in PANC1 siR-miR-200c-3p+ siR-circSEC31A group (932.7±116.4) and CaPan-2 siR-miR-200c-3p+ siR-circSEC31A group (785.3±58.8). Compared with PANC1 siR-NC group (1.000±0.103) and CaPan-2 siR-NC group (1.000±0.107), the relative expression of PDK1 mRNA in PANC1 siR-miR-200c-3p group (1.898±0.159) and CaPan-2 siR-miR-200c-3p group (2.102±0.337) was upregulated. Furthermore, the expression of PDK1 mRNA was decreased in the siR-miR-200c-3p+ siR-circSEC31A group (0.980±0.070, 1.015±0.079). Western blot assays showed that the expression of PDK1 protein in PANC1 siR-NC group, siR-circSEC31A#1 group, siR-circSEC31A#2 group was 0.767±0.086, 0.281±0.191, 0.333±0.062 and in CaPan-2 siR-NC group, siR-circSEC31A#1 group, siR-circSEC31A#2 group was 0.712±0.038, 0.353±0.061, 0.308±0.018. The expression of p-Akt protein in PANC1 siR-NC group and siR-circSEC31A group was 0.741±0.050, 0.114±0.027, 0.139±0.041. In addition, p-Akt protein expression in CaPan-2 siR-NC group and siR-circSEC31A group was 0.823±0.052, 0.141±0.045, 0.280±0.089. PDK1 and p Akt expression in siR circSEC31A group was obviously lower than those in sir NC group. All the differences between either groups above were statistically significant ( P<0.05). Conclusions:circSEC31A is upregulated in pancreatic cancer cells, which facilitates the invasion and metastasis of pancreatic cancer cells via miR-200c-3p/PDK1/Akt signaling pathway, supporting that circSEC31A may function as a new diagnostic and therapeutic target for pancreatic cancer patients.
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Objective To investigate the effect of sulforaphane (SFN) on the proliferation and self-renewal of lung cancer stem cells and its regulatory mechanism. Methods MTT method was used to detect the effect of SFN on the proliferation of lung adenocarcinoma cell lines H460 and A549; tumor sphere formation experiment was used to detect the ability of tumor sphere formation; Western blot was applied to explore the expression of stemness-related proteins (such as β-catenin, Klf4, c-myc) in lung adenocarcinoma cells before and after SFN treatment; NGS sequencing was used to analyze the effect of SFN on the expression profile of tumor cell miRNAs. qRT-PCR verified the changes in the transcription level of key miRNAs by SFN. Western blot was used to detect the effect of SFN on the expression of DNMTs in tumor cells. We constructed miR-200c promoter-GFP plasmid, and applied IF, methylation PCR and DNA sequencing methods to detect the effect of SFN on the methylation level of tumor spheres and miRNA promoter. Results The miRNAs expression profile of lung adenocarcinoma tumor spheres changed significantly after SFN (5.0μmol/L) treatment, and miRNA-200c increased the most. Compared with the control group, the expression of β-catenin, Klf4, c-myc and Vimentin genes in H460 and A549 cells of SFN-S group decreased, and the protein expression levels of DNMT1 and DNMT3a were also significantly decreased. Compared with the control group, H460 and A549 cells stably expressing pEGFP-R200c plasmid in SFN-S group significantly reduced tumor sphere diameter, while tumor sphere fluorescence intensity increased, and GFP protein expression was up-regulated. There were 9 CpG-rich regions in the miR-200c promoter region in the above-mentioned pEGFP-R200c plasmid cell line, and the methylation levels were 88.9%, 44.4% and 38.8% in the control group, SFN-S group and 5-Aza-dC group, respectively. Conclusion SFN may downregulate the expression of stem-related genes in lung cancer stem cells by epigenetically decreasing the methylation level of miR-200c promoter and promoting the transcription of miR-200c.
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@#Objective: To investigate the effects of miR-200c on the proliferation, apoptosis and migration of triple negative breast cancer cell (TNBC) MDA-MB-231 and its metabolism-related molecular mechanism. Methods: miR-200c-231 (MDA-MB-231 overexpressing miR-200c) cells, miR-NC-231 (MDA-MB-231 transfected with miRNA-negative control) and the corresponding transplanted tumor models in nude mice were used as the study subjects. qPCR was used to detect the content of miR-200c and other related genes in cells and transplanted tumor tissues. The number of Ki67 positive cells in tumor tissue was analyzed by immunohistochemistry. The migration and apoptosis of cells were examined by Transwell chamber method and Flow cytometry, respectively. The expressions of proteins associated with proliferation, migration, and metabolism related signaling pathways in cells and tissues were confirmed by Western blotting. The changes of oxygen consumption rate (OCR), extracellular acidification rate (ECAR) and metabolic phenotype were detected by Seahorse energy metabolism detector. UPLC/LTQ-Orbitrap-MStechniquewasusedtoprofilethedifference of metabolites in cells. Results: The content of miR-200c in miR-200c-231cellswassignificantlyhigherthanthatinmiR-NC-231cells.Themass ofmiR-200c-231transplantedtumornotablydecreased,andthenumberofKi67positivecellsintumortissuesalsodecreasedsignificantly. The migration ability of miR-200c-231 cells decreased and the apoptosis rate increased (all P<0.01), accompanied with declined expressionsofZEB1/2,Vimentin,cyclinD1andincreasedexpressionofcleavedPARP(P<0.05orP<0.01),aswellasdecreasedphosphorylation leverofSTAT1/3andNF-κBbutincresedphosphorylationleverofCAMP(allP<0.05).OverexpressionofmiR-200cinMDA-MB-231cells increasedOCRandthecontentof10antitumor metabolites, but decreased ECAR and tryptophan 2,3-plus dioxidase (TDO2) expression (P<0.05 or P<0.01). Conclusion: miR-200c targeting TDO2 elevates the level of intracellular anticancer metabolites in TNBC MDAMB-231 cells, promotes the transformation from glycolysis to aerobic respiration phenotype, and inactivates STAT3 and NF-κB pathyway but activates cAMPpathway TNBC MDA-MB-231 cells, thus affects the malignant biological behaviors of MDA-MB-231 cells.
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Objective To explore the influence of miR-200 c on the biological behavior of laryngeal carcinoma Hep-2 cells and determine whether miR-200 c exerts its biological function through peptidyl-prolyl cis/trans isomerase (PIN1) in laryngeal carcinoma. Methods A qRT-PCR assay for the expression of miR-200 c was performed in laryngeal carcinoma tissues. Hep-2 cells were transfected with miR-200 c related small RNAs. Transwell assay detected the migration ability of the cells. Immunofluorescence assay was used to detect the abnormal amplification of the centrosome. A dual luciferase reporter gene system was used to detect the binding ability between miR-200 c and PIN1. Western blotting detected the protein expression level of PIN1. Results The expression of miR-200 c in laryngeal carcinoma was significantly increased. miR-200 c inhibited the migration of Hep-2 cells and could weaken the abnormal amplification of centrosome.PIN1 was confirmed as one of the target genes of miR-200 c. miR-200 c inhibited the expression of PIN1 at the translation level and could inhibit Hep-2 cell migration and abnormal centrosome amplification by regulating PIN1. Conclusion miR-200 c can inhibit the migration ability of laryngeal carcinoma cells and abnormal centrosome amplification by regulating PIN1.
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Objective: To detect the expression levels of microRNA200 (miR200) family in the plasma of the breast cancer patients and the normal controls, and to evaluate their potential values in the screening, progression and prognosis evaluation of breast cancer. Methods: A total of 82 cases of plasma samples of the patients with breast cancer (breast cancer group) and 30 cases of healthy plasma samples (control group) were collected. The microRNAs (miRNAs) were extracted from the plasma samples, the expression levels of miR200 family (miR200a, miR200b, miR200c, miR141 and miR429) were quantified by using real-time PCR technique. The receiver-operating characteristic (ROC) curve was used to assess the diagnostic value of miRNAs, and the associations between the levels of miRNAs and the clinicopathological characteristics were analyzed. Cox proportional hazard mode was used for survival analysis. Results: Compared with control group, the expression level of miR141 in the plasma of the patients in breast cancer group was decreased (P 0 . 05). Concuson: MiR200b and miR141 are likely to become the molecular biological parameters for the diagnosis of breast cancer.
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[Objective] To investigate the expression of microRNA-200c (miR-200c) in colorectal carcinomas (CRC),and analyze its role on tumor cell migration and invasion.[Methods] The expression levels of miR-200c in CRC tissues and adjacent normal mucosa were assessed by real-time quantitative RT-PCR (qRT-PCR).miR-200c mimics were transiently transfected into human colorectal cancer cells,and their roles on cell migration and invasion were analyzed by Transwell assay.Cell proliferation was measured using the Cell Counting kit-8.The expression levels of epithelial and mesenchymal markers as well as related transcription factor ZEB1 were detected by Western blotting.[Results] Lower miR-200c expression was found in primary CRC tissues with lymph node metastasis compared to those without lymph node metastasis and adjacent normal mucosa.Transfection of miR-200c mimics suppressed proliferation,and reduced invasion and migration in SW620 cells.Furthermore,up-regulation of miR-200c inhibited ZEB1,and resulted in increased E-cadherin and reduced Vimentin gene expression.[Conclusion] miR-200c was associated with invasive and metastatic behavior of CRC.These effects may be mediated through regulation of epithelial-mesenchymal transition.
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Objective:To study the effect of miR-200c on the migration and proliferation abilities of breast cancer MDA-MB-231 and BT-549 cells,and to clarify the mechanism of miR-200c in inhibiting the epithelial-mesenchymal transiton (EMT) of triple negative breast cancer.Methods:The human triple negative breast cancer cell lines (MDA-MB-231 and BT-549) were chosen in this study.The cells were transiently transfected with miR-200cmimics and Lipo2000 (experimental group),miR-200c negative control and Lipo2000 (negative control group),and Lipo2000 alone (reagent control group);at the same time,blank control group was set up.The expression levels of vimentin and β-catenin mRNA and protein were detected by RT-PCR and Western blotting method.The proliferation rates and migration abilities of MDA-MB-231 cells and BT-549 cells after transfection of miR-200c were analyzed by CCK8 assay and wound healing assay.Results:The RT-PCR and Western blotting showed that the expression levels of vimentin and β-catenin mRNA and proteins in experimental group were decreased,and the differences were statistically significant compared with blank control group,negative control group and reagentcontrol group (P＜0.05).The CCK8 results showed that the proliferation rates of the cells in experimental group were lower than those in negative control group and reagent control group (P＜0.05).The wound healing assay results showed that the recovery rate of scratch width in experimental group was lower than those in negative control group and reagent control group (P＜0.05).Conclusion:miR-200c might inhibit EMT in triple negative breast cancer by regulating the expressions of β-catenin and vimentin mRNA and proteins in MDA-MB-231 and BT-549cells and decreasing the abilities of migration and proliferation of triple negative breast cancer cells.
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Objective: To study the effect of miR-200c on the migration and proliferation abilities of breast cancer MDA-MB-231 and BT-549 cells, and to clarify the mechanism of miR-200c in inhibiting the epithelial-mesenchymal transiton (EMT) of triple negative breast cancer. Methods: The human triple negative breast cancer cell lines (MDA-MB-231 and BT-549) were chosen in this study. The cells were transiently transfected with miR-200c mimics and Lipo2000 (experimental group), miR-200c negative control and Lipo2000 (negative control group), and Lipo2000 alone (reagent control group); at the same time, blank control group was set up. The expression levels of vimentin and (3-catenin mRNA and protein were detected by RT-PCR and Western blotting method. The proliferation rates and migration abilities of MDA-MB-231 cells and BT-549 cells after transfection of miR-200c were analyzed by CCK8 assay and wound healing assay. Results: The RT-PCR and Western blotting showed that the expression levels of vimentin and (3-catenin mRNA and proteins in experimental group were decreased, and the differences were statistically significant compared with blank control group, negative control group and reagent control group (P<0. 05). The CCK8 results showed that the proliferation rates of the cells in experimental group were lower than those in negative control group and reagent control group (P<0. 05). The wound healing assay results showed that the recovery rate of scratch width in experimental group was lower than those in negative control group and reagent control group (P<0. 05). Conclusion: miR-200c might inhibit EMT in triple negative breast cancer by regulating the expressions of (3-catenin and vimentin mRNA and proteins in MDA-MB-231 and BT-549 cells and decreasing the abilities of migration and proliferation of triple negative breast cancer cells.
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Objective:This work aims to detect the levels of miR-200c/141 methylation and miR-200c/141 in gastric cancer tissue and investigate the relationship between miR-200c/141 expression and clinical parameters. Methods:The methylation status of miR-200c/141 CpG island and miR-200c/141 in gastric cancer tissue specimens was evaluated by qRT-PCR or BS-MSP method. We analyzed the relationship among the methylation status of miR-200c/141 CpG island, expression level of miR-200c or miR-141, and clinical parame-ters. Results:The status of miR-200c/141 CpG island methylation in gastric cancer tissue was significantly higher compared with that in paracarcinoma tissue. MiR-200c and miR-141 were markedly decreased in gastric cancer tissue compared with those in adjacent tis-sue. MiR-200c/141 CpG island methylation was negatively related with the expression of miR-200c and miR-141 in gastric cancer speci-mens. Conclusion:The upregulation of miR-200c/141 CpG methylation inhibits miR-200c/141 expression in gastric cancer tissue.
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Objective:To clarify the effect of miR-200c on the proliferation of retroperitoneal fibroblast, and analyze its molecular mechanism, which provide theoretical basis for the inhibition of retroperitoneal fibrosis. Methods: 36 cases of retroperitoneal tissues were collected, and the primary fibroblasts were cultured. The cells stimulated by 5 ng/ml transforming growth factor-β1 (TGF-β1) were used as TGF-β1 group;the cells were transfected with pcDNA3.1-miR-200c first, and then stimulated by 5 ng/ml TGF-β1 were used as miR-200c group;the cells transfected only by pcDNA 3.1 were used as pcDNA 3.1 group;the normal cultured cells were used as control group. The MTT assay, cell scratch and Transwell chamber were used to detect the proliferation and migration of fibroblast, respectively. The ELISA experiment was used to detect the content of Akt protein in the lysis liquid of each group. Results: The OD value of fibroblast proliferation in miR-200c group was obviously lower than that of TGF-β1 group (P<0.01);compared with TGF-β1 group, the cell migration rate was decreased significantly in miR-200c group (P<0.01);the content of Akt protein in miR-200c was obviously lower than that of TGF-β1 group (P<0.01). Conclusion: miR-200c inhibits the effect of TGF-β1 on the proliferation and migration of fibroblast by down-regulating Akt signaling pathway, which has important significance in inhibiting peritoneal fibrosis.
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Objective To study the mechanism of miR?200c in regulating RMP7?induced increases of blood?tumor barrier(BTB)permeability by targeting Ras homolog gene family member A(RhoA). Methods Endogenous expression of miR?200c was detected by real?time PCR in hu?man cerebral microvascular endothelial cell line hCMEC/D3(ECs)after RMP7 treatment. miR?200c mimic and miR?200c inhibitor were transfect?ed into GECs(ECs with U87 glioma cells co?culturing),respectively. Transfection efficiency of miR?200c mimic and miR?200c inhibitor were de?termined by real?time PCR. HRP flux and TEER assays revealed BTB permeability. The protein expression level of RhoA was assessed by West?ern blotting. The distribution of RhoA was assessed by immunofluorescence microscopy. RhoA luciferase assays were performed using the Dual?Lucif?erase reporter assay system. Results RMP7 significantly induced a decrease in miR?200c expression in GECs of BTB. miR?200c mimic and miR?200c inhibitor were successfully transfected into GECs. Overexpression of miR?200c inhibited endothelial leakage and restored normal transendo?thelial electric resistance values. Simultaneously ,overexpression of miR?200c significantly reduced the protein expression level of RhoA. In addi?tion,immunofluorescence analysis revealed that the distribution of RhoA in the cytoplasm and nuclei of GECs were decreased in miR?200c mimic group. RhoA was one of the direct targets of miR?200c with the specific binding site being located at the seed sequence. The results of miR?200c si?lencing were opposite to that of the miR?200c overexpression group. Conclusion miRNA?200c regulated RMP7?induced increases in BTB perme?ability by targeting RhoA.
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Objective To investigate the expression of miR-200c in endometrial carcinoma and its correlation with ZEB2 protein. Methods The expression levels of miR-200c and ZEB2 gene and protein in tissues were detected. The expression of miR-200c in endometrial carcinoma RL95-2 cell line was inhibited by using antisense oligonucleotides and its effect on cell invasiveness was tested. The expressions of ZEB2 gene and protein in cells were detected. Results The expression levels of miR-200c and ZEB2 mRNA and protein in endometrial tissues were significantly higher than those in adjacent tissues and control group (P < 0.05). The average penetrating number in antisense miR-200c transfected group was significantly less than negative control group and liposome group (P < 0.05). The expression levels of ZEB2 gene and protein in antisense miR-200c transfected group were lower than the negative control group and liposome group (P < 0.05). Conclusion The expression of mRNA-200c in endometrial carcinoma was high and it might be promoting tumor cell invasion and metastasis by regulating ZEB2.
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Objective To investigate the effects of miR-200c on proliferation and apoptosis of tongue squamous cell carcino-ma (TCCS)Tca8113 cells.Methods The mimics of miR-200c were transfected into Tca8113 cells using liposome.The Tca8113 cell proliferation was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)assay.The flow cytometry as-say was used to determine the cell cycle and the apoptosis rate of Tca8113 cell.The protein expression levels of Bcl-2 and Caspase-3 in Tca8113 cell was detected by Western-blot.Results The 20,40,80 nmol/L miR-200c mimics groups inhibited the growth of Tca8113 cells,the difference compared with the control group showing statistical significance(P <0.05).The greater the miR-200c mimics concentration and the longer duration of action,the more significant the inhibition effect(P <0.05).After 48h transfecting by miR-200c mimics,the Tca8113 cells were arrested in the G0/G1 phases of cell cycle,and the apoptosis rate of the miR-200c mim-ics groups was significantly increased,the difference compared with the control group showing statistical significance(P <0.05);Western blot verified that the expression amount of Bcl-2 protein in the 20,40,80 nmol/L miR-200c groups was significantly lower than that in the control group,while the expression amount of Caspase-3 protein was significantly higher than that in the control group(P <0.05).Conclusion The overexpression of miR-200c might inhibit the proliferation of Tca8113 cell and induces their ap-optosis.
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Objective:To investigate the effect on the expression of Slug for the trasfection of miR-200c combined with the research on the ability of migration of breast cancer cell BT549.Methods:Chemically synthesized miR-200c mimic was trasfected into BT549 cells,which have high metastatic potential.The effect on the ability of migration of breast cancer cell BT549 for the transfection of miR-200c was analyzed by Transwell migration assay and Wound healing assay.The expression of Slug and E-cadherin mRNA was detected by Real-time PCR.The expression of Slug protein was detected by Western blot.Results:Transfection with miR-200c mimic significantly down-regulated the expression of Slug as compared with the control group (P<0.05).BT549 cell tranfected with miR-200c mimic had lower levels of migration capacity than cells in the control group (P<0.05).Conclusion:miR-200c inhibits Epithelial-mes-enchymal transition by suppressing Slug leading to down-regulation of migration capacity of breast cancer cell BT549.
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BACKGROUND: MicroRNA (miRNA) is a class of noncoding protein RNA as a promising biomarker for various diseases. In this study, the expression of let-7b, miR-30c, and miR-200c was studied in breast cancer tissues to evaluate the potential relationship with known clinicopathological parameters. METHODS: Quantitative real-time polymerase chain reaction was performed to determine the expression level of three miRNAs in 37 pairs of noncancerous normal and cancer tissues and an additional 38 cancer tissues from patients with invasive ductal carcinoma. RESULTS: miR-200c expression was higher in cancer tissues compared to noncancerous normal tissues, and its ratio was correlated with patient age at surgery, type of surgery, and Ki-67 expression. The expression level of let-7b in cancer tissues was inversely correlated with lymph node metastasis, histological grade, and Ki-67 expression but positively correlated with estrogen and progesterone receptor expression. miR-200c expression level was positively correlated with Her-2 expression. The miR-30c expression level in breast cancer was not correlated with any parameters. CONCLUSIONS: miR-200c and let-7b could be used as biomarkers in patients with breast cancer, but its pathological mechanism should be determined.