| Endometrial carcinoma(EC)is one of the most common female malignancies that threaten health and life.In general,EC had a favorable outcome,but once metastasis or recurrence occurs,EC has a poor prognosis,regardless of the type or stage.Furthermore,the incidence of EC is increasing,highlighting the importance of investigating the underlying mechanisms of EC initiation.The majority of the human genome comprises noncoding DNA,and their products noncoding RNAs,such as long nc RNAs(lnc RNAs)and micro RNAs(miRNAs)have been shown to be important regulators in biological processes.The lnc RNA SNHG5 is a transcript of small nucleolar RNA host gene 5 In particular,increasing numbers of studies have demonstrated that abnormal expression of lnc RNA plays pivotal roles in tumorigenesis and representing promising targets for tumor diagnosis and treatment.Nevertheless,the clinical significance and molecular roles of SNHG5 in EC remains unclear.This study is aim to observe the expression of non-coding RNA SNHG5 on EC and to demonstrated the mechanism of SNHG5 modulates endometrial cancer progression via the miR-25-3p/BTG2 axis.The experiment contains three parts,shown as follows: Part 1 The expression of SNHG5 in ECObjective: Analysis of SNHG5 expression and assess the effect of SNHG5 in ECMethods: Real-time PCR was adopted to detect the expression of SNHG5 in endometrial cells and EC tissues Total RNA was isolated from cells or tissues using Trizol reagent(Thermo Fisher Scientific),and then was reverse transcribed to c DNA using Moloney murine leukemia virus reverse transcriptase(Promega,Madison,WI,USA)following the manufacturer’s instructions.Results: The results of Real-time PCR showed that SNHG5 exhibited significantly lower expression in EC tumor cells and tissues compared to normal cells and tissues,P<0.01.Conclusions: SNHG5 exhibited significantly lower expression in EC tumor cells and tissues indicated that SNHG5 is a tumor suppressor gene in EC cells.Part 2 The effect of SNHG5 proliferation,migration and invasion on EC cellsObjective: To evaluate the effect of SNHG5 proliferation,migration and invasion on EC cellsMethods:1.Cell culture transfection and transcription analysesAll cell lines were cultured in DMEM.To assess the effect of SNHG5 on EC development and progression,SNHG5 was knocked down by RNAi and overexpressed by transfection using a SNHG5 overexpression vector in both KLE and HEC-1-B cells.RT-qPCR to demonstrate whether the success of genetic manipulation of SNHG5 expression or knocked down in these two EC cell lines.2.Cell proliferation assayCell proliferation assays(MTS)were performed according to the manufacturer’s instructions.Subsequently,the absorbance of each well was measured using a microplate reader at 492 nm.3.Transwell assaysTranswell chambers(Corning Costar,Cambridge,MA,USA)were placed in 24-well plates(8-mm pore size).Cells that had migrated or invaded were fixed and stained with crystal violet.Subsequently,5 random selected fields of the stained cells were counted using a microscope.Results:1.Transcription analyses showed that SNHG5 m RNA levels were reduced in SNHG5-si RNA transfected cells,whereas SNHG5 levels were increased in EC cells transfected with the SNHG5-overexpression vector,demonstrating the success of genetic manipulation of SNHG5 expression in these two EC cell lines.P<0.01.2.The effect of SNHG5 for cells proliferationSNHG5 inhibits the proliferation of KLE and HEC-1-B cells.The proliferation of KLE and HEC-1-B cells was notably promoted by the inhibition of SNHG5 expression,whereas SNHG5 overexpression had the opposite effect.P<0.013.The effect of SNHG5 for cells migrationSNHG5 inhibits the migration of KLE and HEC-1-B cells.The migration of KLE and HEC-1-B cells was notably promoted by the inhibition of SNHG5 expression,whereas SNHG5 overexpression had the opposite effect.P<0.01.4.The effect of SNHG5 for cells invasionSNHG5 inhibits the invasion of KLE and HEC-1-B cells.The invasion of KLE and HEC-1-B cells was notably promoted by the inhibition of SNHG5 expression,whereas SNHG5 overexpression had the opposite effect.P<0.01.Conclusions: The proliferation,migration and invasion abilities of EC cells increased when SNHG5 was knocked down,but decreased when SNHG5 was overexpressed,these results indicated that SNHG5 is a tumor suppressor gene in EC cells.Part 3 lnc RNA SNHG5 modulates endometrial cancer progression via the miR-25-3p/BTG2 axisObjective: A mechanistic study of SNHG5 can directly bind to miR-25-3p and abolish its suppressive function against its target BTG2,and the SNHG5/miR-25-3p/BTG2 axis plays an important role in EC cells and clinical tissues,indicating its potential for applications in EC diagnosis and therapy.Methods: 1 Assessment the binding targets micro RNAs of SNHG5 was miR-25-3p To elucidate the underlying mechanisms by which SNHG5 influences the development progression of EC,two micro RNAs(miR-25-3p and miR-92a-3p)were predicted to be the binding targets of SNHG5 by the star Base,v2.0 program database(http://starbase.sysu.edu.cn).RT-qPCR to determine whether miR-25-3p was the binding targets of SNHG5.2 To assess the effect of miR-25-3p on EC development and progression 2.1 Real-time PCR was adopted to detect the expression of miR-25-3p in EC tissues 2.2 The effect of miR-25-3p for cells proliferation,migration and invasionCell proliferation assays(MTS)were performed according to the manufacturer’s instructions to assess to effect of SNHG5 for cells proliferation;Transwell chambers were placed in 24-well plates,Cells that had migrated or invaded were fixed and stained with crystal violet.Subsequently,5 random selected fields of the stained cells were counted using a microscope.3 SNHG5 primarily functions by inhibiting mir-25-3p.SNHG5 and mir-25-3p have been shown to have opposite roles in KLE and HEC-1-B cells.To confirm whether SNHG5 suppresses the development of EC through its repression of miR-25-3p,mir-25-3p mimics were transfected into SNHG5-overexpressing KLE and HEC-1-B cells.And then assessment proliferation,migration and invasion function of KLE and HEC-1-B cells.4 BTG2 is a direct mir-25-3p target.4.1 we searched for potential targets of miR-25-3p using Target Scan(http://www.targetscan.org),the most commonly used software for miRNA target prediction.4.2 Luciferase assays for assessment the binding targets m RNAs of mir-25-3p.For the luciferase reporter assay,complementary sequences for miR-25-3p in the 3′-UTR of BTG2 m RNA were predicted using Target Scan,chemically synthesized and inserted upstream of the Renilla luciferase(Rluc)gene.Subsequently,the luciferase reporter plasmidor the empty vector was co-transfected into EC cells with miR25-3p mimics in a 24-well plate.Luciferase activity was determined according to the manufacturer’s protocol after transfection using the Dual-Luciferase Reporter Assay System(Promega).The Rluc activity was normalized to that of firefly luciferase activity and presented as the percentage of inhibition.4.3 The level of BTG2 protein was assessed by western blot assays Cells were harvested and lysed in lysis buffer on ice,after centrifugation,protein concentrations were measured using a BCA protein assay kit(Beyotime,Nanjing,China).Protein extracts were resolved by SDS-PAGE and transferred to PVDF membranes.The antibodies used for western blotting included Anti-BTG2(Proteintech)and peroxidase-conjugated anti-mouse or rabbit Ig G(Cell Signaling Technology).The antigen–antibody reaction was visualized by an ECL assay(EMD–Millipore,Billerica,MA,USA).5 To determine whether SNHG5 can affect BTG2 expression through miR-25-3p 5.1 RT-qPCR were performed to analyze BTG2 expression in KLE and HEC-1-B cells after overexpressing SHNG5 or both SHNG5 and mir-25-3p.5.2 western blot assays were performed to analyze BTG2 expression in KLE and HEC-1-B cells after overexpressing SHNG5 or both SHNG5 and mir-25-3p.6 Clinical Significance of the SNHG5/miR-25-3p/BTG2 Axis.6.1 Real-time PCR was adopted to detect the expression of BTG2 in EC tissues,then correlation analysis to revealed the relationship between BTG2、mir-25-3p and SNHG5 expression in EC tissues.6.2 Ninety eight patients with endometrial carcinoma confirmed pathologically and eighty patients with endometrial benign disorder undergoing surgical treatment in our hospital were enrolled in this study,protein expression of BTG2 were evaluated by immunohistochemical assay According to the protein expression of BTG2 evaluated by immunohistochemical assay,the subjects were categorized to 2 groups,that is,low(+/ ++)and high(+++)groups.6.3 The correlation of BTG2 expression level with clinical pathological factors were analyzed.Results: 1 The results of Real-time PCR showed miR-25-3p expression was repressed after SNHG5 overexpression,but was elevated in the SNHG5-knockdown KLE and HEC-1-B cells,and miR-25-3p may be the binding targets of SNHG5.2 To assess the effect of miR-25-3p on EC development and progression 2.1 The results of Real-time PCR showed that SNHG5 exhibited significantly higher expression in EC tissues compared to para-tissues,P<0.01.2.2 miR-25-3p promotes the proliferation,migration and invasion of KLE and HEC-1-B cells.The results of MTS assays,transwell migration assays,and matrigel invasion assays suggested that overexpression of miR-25-3p accelerated the proliferation,migration and invasion of KLE and HEC-1-B cells.P<0.01.3 The inhibition of KLE and HEC-1-B cell proliferation,migration and invasion caused by SNHG5 overexpression were almost completely rescued by the transfection of mir-25-3p mimics.These results indicated that mir-25-3p is the central downstream target of SNHG5 in the suppression of EC cell progression.P<0.01.4 BTG2 is a direct mir-25-3p target.4.1 According to Target Scan the predication results,B-cell translocation gene 2(BTG2)was selected for further analysis due to the identification of an miR-25-3p binding site on the 3′-UTR of BTG2 m RNA.4.2 Luciferase assays results showed that the putative miR-25-3p binding sites notably decreased the luciferase activity in miR-25-3p-overexpressing KLE and HEC-1-B cells compared to the negative control.In contrast,the mutation of putative miR-25-3p binding site had no effect on the luciferase activity,indicating that miR-25-3p suppressed BTG2 expressing by directly binding to the 3’UTR of BTG2 m RNA P<0.01.4.3 The level of BTG2 protein was assessed by western blot assays.In agreement with the reporter assay results,BTG2 protein levels were decreased in KLE and HEC-1-B cells transfected with miR-25-3p mimics compared with that observed in the negative control.These results firmly demonstrated that BTG2 is a direct target of miR-25-3p and that its expression is suppressed by miR-25-3p P<0.01.5 SNHG5 can affect BTG2 expression through miR-25-3p 5.1 According the result of RT-qPCR the m BTG2 expression was elevated by SNHG5 overexpression,whereas the elevated BTG2 expression decreased when we overexpressed SNHG5 while overexpressing miR-25-3p,P<0.01.5.2 According the result of western blot assays the BTG2 expression was elevated by SNHG5 overexpression,whereas the elevated BTG2 expression decreased when we overexpressed SNHG5 while overexpressing miR-25-3p,P<0.01.6 The expression profile of SNHG5,miR-25-3p,BTG2 in EC tissues 6.1 The results of RT-qPCR which showed that the level of BTG2 m RNA was down regulated in EC tissues compared to that observed in para-tissues,P<0.01.In addition,the result of correlation analysis revealed a positive relationship between BTG2 and SNHG5 expression in EC tissues(r =0.376 P=0.017);a negative correlation between SNHG5 and miR-25-3p expression was observed by correlation analysis(r =-0.369 P=0.019).6.2 The results of immunohistochemistry which showed that BTG2 protein levels were notably decreased in EC tissues compared to the noncancer tissues,P<0.01.6.3 Low expression of BTG2 is related to some of clinicopathologic feature.Conclusions: These results demonstrated that the SNHG5/miR-25-3p/BTG2 axis is of great significance in EC cancer tissues.And the SNHG5/miR-25-3p/BTG2 axis plays an important role in EC cells and clinical tissues,indicating its potential for applications in EC diagnosis and therapy. |
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