| BackgroundBreast cancer(BC)has the highest morbidity and mortality of various types of cancers among females across the world,seriously threatens women’s life safety and quality.A variety of factors,such as estrogen levels,susceptibility gene variation,living environment and styleand nutrition,are involved in the pathogenesis of breast cancer.Treatment strategies of Breast cancer are mainly based on surgical resection,supplemented by radiation therapy(RT),chemotherapy(CT),endocrine therapy(ET)and targeted therapy(TT),antineoplastic drugs can be used alone or in combination to reduce the risk of breast cancer recurrence.The precise therapy is a development trend in recent years,precise treatment of breast cancer is closely related to its molecular typing,according to the four key proteins estrogen receptor(ER),progesterone receptor(PR),human epidermal growth factor receptor 2(HER2)and proliferation marker Ki67,breast cancer is divided into 4 subtypes:Luminal A-like,Luminal B-like(HER2-/HER2+),HER2+and triple negative breast cancer(TNBC).About 70%of breast cancer patients have ER-positive cancer cells and rely on estrogen receptor amplification,so estrogen receptor is an ideal target for breast cancer hormone therapy.Tamoxifen(TAM)is the most commonly used first-line neoadjuvant chemotherapy for ER-positive breast cancer.In early-stage ER-positive breast cancer,5 years of adjuvant chemotherapy with tamoxifen halved the tumor recurrence rate and reduced annual breast cancer-related mortality by one-third.However,despite the use of tamoxifen adjuvant chemotherapy,one-third of patients will still relapse within 15 years,which shows that "resistance" is still a problem in breast cancer treatment.Therefore,further elucidating the molecular mechanism of BC resistance remains the urgent issue to be solved to improve the efficacy of TAM.The resistance mechanisms of TAM are complex,including abnormalities in ER structure,activation,and function,as well as dysregulation between estrogen signaling networks and other cellular pathways.Long non-coding RNAs(LncRNAs)are widely involved in the occurrence and development of various tumors,and are related to the drug resistance of tumor cells,which is a hot spot in the field of tumor research in recent years.In this study,the breast cancer tissues and adjacent normal breast tissues of breast cancer patients were collected and analyzed by RNA gene chip to find differentially expressed LncRNAs.After verification by reverse transcription quantitative polymerase reaction(RT-qPCR),it was found that LncRNA Hepatocyte nuclear factor 1 homeobox A-antisense RNA 1(HNF1A-AS1)had the greatest difference in expression between the two types of tissues.After cell experiments,it was found that the expression of HNF1A-AS1 was significantly increased in TAM-resistant breast cancer cells.At present,there is no report on the mechanism of HNF1A-AS1 involved in TAM resistance.In this study,we investigated the relationship between HNF1A-AS1,HNF1A-AS1 target miR-363 and miR-363 target gene SERTAD3 and TAM resistance through in vitro cell experiments.The effects of silencing HNF1A-AS1 on breast cancer tumor growth and tumor sensitivity to TAM therapy were investigated in vivo animal experiments.In this study,the mechanism of HNF1A-AS1,miR-363 and SERTAD3 in TAM resistance was studied through cell and animal experiments by silencing or overexpressing HNF1A-AS1,miR-363 and SERTAD3,in order to improve its therapeutic efficacy and help develop new therapeutic strategies to overcome the breast cancer TAM resistance.This subject is mainly divided into the following three parts:Part Ⅰ The expression of HNF1A-AS1 in breast cancer tissues and the relationship with prognosis of breast cancer patientsPurposes1.To analyzethe differentially expressed LncRNAs in breast cancer tissues.2.To determine the expression of 5 up-regulated LncRNAs in breast cancer tissues.3.To explore the relationship between LncRNA HNF1A-AS 1 and prognosis of breast cancer patients.Methods1.Collection of breast cancer tissue specimens:eighty-two pairs of resected BC tissues(case group)and precancerous tissues(control group)specimens were collected from BC patients who diagnosed and never received radiotherapy,chemotherapy or endocrine therapy in the Third Affiliated Hospital of Zhengzhou University from May 2017 to May 2018.2.Collection of patients’clinical information:patients’age,tumor diameter,clinical stage,LNM(Lymph node metastasis),ER,PR and Ki-67.3.RNA microarrayanalysis was performed to find differentially expressedLncRNAs in 4 case and control groups.RT-qPCR was performed to measure the expression of the top 5 up-regulated LncRNAs in case and control groups.4.Data analysis was introduced using the SPSS 21.0(IBM Corp.,Armonk,NY,USA).Kolmogorov-Smimov method checked the data were in normal distribution.Data are expressed as x±s.The data between the two groups were compared using independent samples t test and paired samples t test,respectively.Survival analysis results were measured by kaplan-meier analysis.α=0.05 was the test level.Results1.Differential expression of LncRNAs in breast cancer by RNA microarrayRNA microarray analysis found that 143 LncRNAs were differentiallyexpressed,54 LncRNAs were up-regulated and 89 were down-regulated in breast cancer groups.2.Detection of relative expression levels of five LncRNAs by RT-qPCRThe top 5 up-regulated LncRNAs-HNF1A-AS1,CCAT2,CCHE1,LINC01540 and HOXA-AS3 were selected and detected by RT-qPCR in the case and the control groups.The relative expression of HNF1A-AS1 in the case group was 5.65±1.23,which was higher than that in the control group 1.12±0.78,the difference was statistically significant(P<0.01);the relative expression of CCAT2 in the case group was 6.03±1.34,which was higher than that in the control group 4.68±0.87,the difference was statistically significant(P<0.05);the relative expression of CCHE1 in the case group was 5.27±1.30,which was higher than that in the control group 4.98±0.91,the difference was statistically significant(P<0.05);the relative expression of LINC01540 in the case group was 4.85±0.92,which was higher than that in the control group 3.98±0.75,the difference was statistically significant(P<0.05);the relative expression of HOXA-AS3 in the case group was 4.76±1.02,which was higher than that in the control group 2.60±0.61,the difference was statistically significant(P<0.05).The expression difference of HNF1A-AS1 in case and control groups was the largest,and the difference was statistically significant(P<0.01).According to the Cancer Genome Atlas(TCGA)Database,BC patients with higher HNF1A-AS1 had lower survival rates.Therefore,we focused on HNF1A-AS1.3.Correlation of HNF1A-AS1 with survival and prognosis of breast cancer patlientsThe median value of HNF1A-AS1 expression in BC tissues was set as a critical value,with which the BC patients were divided into high-expression group(≥6.11,n=40)and low-expression group(<6.11,n=40).Then the relation between HNF1A-AS1 expression and BC clinic pathological features was determined.Patients with higher HNF1A-AS1 expression showedmore seriousclinical stages,higher LNM rate,PR,ER and Ki-67-positive rates(all P<0.05),while no correlation was found between the HNF1A-AS1 expression and the age and tumor size(P>0.05)ConclusionHNF1A-AS1 is highly expressed in breast cancer tissues and is negatively correlated with the survival and prognosis of breast cancer patients.Part Ⅱ HNF1A-AS1 regulates breast cancer cell proliferation and tamoxifen resistance via miR-363/SERTAD3in vitroPurposes1.To evaluate the effect of silencing HNF1A-AS1 on the proliferation and apoptosis of breast cancer cells.2.To clarify the effect of HNF1A-AS1,miR-363 and SERTAD3 on tamoxifen-resistant BC cells,3.To investigate the involvement of silencing or overexpression of HNF1AAS1,miR-363 and SERTAD3 in TAM resistance through the TGF-β/Smad3 pathway.Methods1.RT-qPCR was used to detect the expression of HNF1A-AS1 in human normal breast epithelial cellMCF-10A and breast cancer celllines MCF-7,BT474,ZR-75-30,MDA-MB-231,HCC1937 and MDA-MB-436.2.MCF-7 and BT474 cells were transfected with HNF1A-AS1 siRNA vectors si-HAS1-1 and si-HAS1-2,siRNA blank vector(si-NC),and divided into 3 groups:si-NC group,si-HAS1-1 group and the si-HAS1-2 group.3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay,5-ethynyL-2’-deoxyuridine(EdU)labeling assay,colony formation assay and Flow cytometrywas performed to detect the proliferation and apoptosis of breast cancer cells.3.The TAM-resistant cell line MCF-7/TAM was constructed,MTT assays were used to identify the drug resistance of breast cancer cells to TAM;RT-qPCR was used to detect HNF1A-AS1 expressionin the tamoxifen-resistant cell line MCF-7/TAM;transfect HNF1A-AS1siRNA vectors si-HAS1-1 and si-HAS1-2,siRNA blank vector(si-NC)into MCF-7/TAM,divided into 3 groups:si-NC group,si-HAS1-1 group and si-HAS1-2 group;the proliferation activity andthe sensitivity to TAM of drug-resistant cells were detected by MTT and Hoechst assays.4.Transfect HNF1A-AS1 overexpression vector and NC negative control plasmid into MCF-7 and BT474,the cells were divided into two groups:control group and HAS1 group,MTT was used to detect the sensitivity to TAM.The online database StarBase and miRcode Pearson were used to analyze the correlation between HNF1A-AS1 and miR-363,and the correlation between HNF1A-AS1 and miR-363 was further confirmed by dual-luciferase reporter gene experiments.RT-qPCR method was used to detect the expression of miR-363 in breast cancer tissue,breast cancer celllines MCF-7 and BT474,and drug-resistant cellline MCF-7/TAM.The correlation of miR-363 and SERTAD3 was analyzed by TargetScan,StarBase and miRTarBase software,and the correlation of miR-363 and SERTAD3 was further confirmed by dual-luciferase reporter gene experiments.RT-qPCR method was used to detect the expression of SERTAD3 in breast cancer tissue.5.MCF-7HAS1 group cells were transfected with miR-363 mimics,and the experiment was divided into two groups:co-transfection HASl+mimics NC group(HAS1+NCgroup)and co-transfection HAS1+miR-3 63 mimics group(HAS1+miR-363 group),the sensitivity of the two groups of cells to TAM was compared using MTT assay.The SERTAD3 overexpression vector and empty vector(Empty Vectors)were introduced into MCF7 cells and MCF/TAM+si HAS 1-1 group cells,then they were divided into 4 groups:MCF7 cells Empty Vectors group and SERTAD3 group,MCF/TAM cells si HAS 1-1+Empty Vectors group and si HAS 1-1+SERTAD3 group,compare the effect of the four groups of cells on TAM resistance.6.Western blot was performedto detect the expression of TGF-β,total Smad3 and phosphorylated Smad3 in the TGF-β/Smad3 signaling pathway in breast cancer cell lines withsilencing or overexpression HNF1A-AS1,miR-363 and SERTAD3.7.Graph Pad Prism SPSS 21.0 and5.0 were used for statistical analyses.Data were expressed as x±s,and data were tested for normality test.T-Test(Independent Samples)was used for comparison between two groups.The significance level wasα=0.05.Results1.Expression of HNF1A-AS1 in breast cancer cell linesThe relative expression levels of HNF1A-AS1 in breast cancer celllines were:MCF-7(3.61±0.32),BT474(4.12±0.41),ZR-75-30(2.14±0.23),MDA-MB-231(3.25±0.30),HCC1937(2.21±0.19),and MDA-MB-436(2.67±0.21),which were higher than those of normal breast epithelial cell MCF-10A(P<0.05).Since the expression of HNF1A-AS1 is particularly high in MCF-7 and BT474 cells,we selected MCF-7 and BT474 for HNF1A-AS1-related intervention experiments.2.Silencing HNF1A-AS1 inhibits the growth of breast cancer cells2.1 The results of MTT,EdU and colony formation assay.suggested that silencing HNF1A-AS1 reduced the viability of BC cells(all P<0.05)2.2 The results of flow cytometry showed that the apoptosis rate(%)of MCF-7 cells transfected with si-HAS1-1 and si-HAS1-2 were 21.12±0.42 and 21.98±0.56respectively,which were significantly higher than si-NC group 9.04±0.42.The apoptotic rates(%)of BT474 cells transfected with si-HAS1-1 and si-HAS1-2 were 22.11±0.46 and 23.94±0.45 respectively,which were significantly higher than the si-NC group11.35±0.21(P<0.01).3.Effects of silencing HNF1A-AS1 on proliferation and drug sensitivity ofTAM-resistant cells3.1 MCF-7/TAM cells were induced by low-dose of TAM.The result of MTT showed the IC50(20μg/mL)ofMCF-7 to TAM was significantly lower than that of MCF-7/TAM,and the difference was statistically significant(P<0.05),which confirmed that the TAM-resistant cellline MCF-7/TAM was successfully constructed.3.2 Compared to MCF-7 cell,the expression of HNF1A-AS1 in drug-resistant cellline MCF-7/TAM(5.98±0.64)was higher,the difference was statistically significant(P<0.01).3.3 The results of Hoechst showed the proliferation ability of MCF-7/TAM cells in si-HAS1-1 group and si-HAS1-2 group were significantly lower than that in si-NC group.The sensitivity of MCF-7/TAM cells to TAM was significantly increased(P<0.05).4.HNF1A-AS1 regulates the effect of miR-363/SERTAD3 on TAM resistance in breast cancer cells4.1 The results of MTT showed that compared with the control group,MCF-7 and BT474 cells in HAS1 group had increased resistance to TAM(P<0.05).The results of Hoechstshowed that compared with the control group,the proliferation ability of MCF-7 and BT474 cells in HAS1 group was significantly increased(P<0.05).4.2 The potential target miR-363of HNF1A-AS1 was predicted using the online database StarBase and miRcode Pearson.The dual-luciferase reporter gene experiment further confirmed that HNF1A-AS1 binded to miR-363.4.3 The expression of miR-363 in breast cancer tissues was lower than that in the normal control group(1.05±0.23 vs 3.16±1.28)(P<0.01).The expression of miR-363 in breast cancer drug-resistant cells MCF-7/TAM(0.42±0.07)was significantly lower than that in non-drug-resistant cellline MCF-7(P<0.01).4.4 The expression of miR-363 in MCF-7/TAM transfected with siRNA HAS1-1 and siRNA HAS1-1was significantly increased(respectively 2.64±0.41 and 2.49±0.26),the difference was statistically significant(P<0.01).However,the relative expression of miR-363 in MCF-7 and BT474 cells HAS1 groups were significantly lower than that in control group(0.43±0.07 and 0.48±0.06,respectively)(P<0.01).5.Silencing or overexpression of HNF1A-AS1,miR-363 and SERTAD3 was involved in breast cancer TAM resistance through the TGF-β/Smad3 pathway5.1 MCF7 cells HAS1 group cells were co-transfected with miR-363 mimics(miR-363 mimics)and empty control(NC mimics).The results of MTT experiment showed that the sensitivity of the cells to TAM was significantly increased in the co-transfected MCF7 cells in the HAS1+miR-363 group compared with the HAS1+NC group(P<0.05).The IC50 values of TAM in the two groups were 78μg/mL vs 20 μg/mL,the difference was statistically significant(P<0.05).The results of Hoechst experiment showed that the proliferation ability of MCF7 cells inHAS1+miR-363 group was significantly lower than that in HAS1+NC group(P<0.05).5.2 Co-transfection of SERTAD3 expression vector in MCF-7 and MCF-7/TAM si-HAS1-1 group cells.The results of the MTT experiment showed that resistant to TAM was increased in the SERTAD3 group compared to the Empty Vectors group in MCF7,the IC50 values of TAM in the two groups were 79 μg/mL vs 20 μg/mL(P<0.05).The resistant to TAM was increased in the si HAS 1-1+SERTAD3 group compared to the si HAS1-1+Empty Vectors group.The IC50 values of TAM in the two groups were 92 μg/mL vs 40 μg/mL,the differences was statistically significant(P<0.05).5.3 The results of Western blot showed that in MCF-7/TAM cells si-HAS1-1 group,si-HAS 1-2 group and si-NC control group,the expression of TGF-βwas significantly lower in si-HAS1-1 group(0.17±0.02)and si-HAS1-2 group(0.18±0.02)than in the si-NC group(0.48±0.08),and the expression of total Smad3(t-Smad3)in the si-HAS1-1 group(0.49±0.07)and si-HASl-2 group(0.50±0.08)were not significantly different from that in the si-NC control group(0.52±0.08).The expression of phosphorylated Smad3(p-Smad3)in si-HAS1-1 group(0.21±0.06)and si-HAS 1-2 group(0.23±0.05)was lower than that in si-NC group(0.42±0.06).But after exogenous SERTAD3 was introduced into MCF-7/TAM si HAS 1-1 cells,the expression of TGF-β in MCF-7/TAM cells si HAS1-1+SERTAD3 cells was significantly increased(0.34±0.07).There was no significant difference in the expression of t-Smad3(0.523±0.08),the expression of p-Smad3 was also significantly increased(0.38±0.06),and the differences were statistically significant(P<0.01).In MCF-7 cells control group,HAS1 group,HAS1+miR-363 group and SERTAD3 group,Western blot detection showed that the expression of TGF-β in HAS1 group(0.38±0.04)was higher than that in control group(0.22±0.08),the protein expression of TGF-β in SERTAD 3 group(0.41±0.07)was higher than that in HAS1+miR-363 group(0.31±0.03),and the differences were statistically significant(P<0.01).The expression of p-Smad3 in HAS1 group(0.40±0.06)was higher than that in control group(0.23±0.04),the difference was statistically significant(P<0.01).The protein expression of p-Smad3 in the SERTAD3 group(0.42±0.05)was higher than that in the HAS1+miR-363 group(0.31±0.04),the difference was statistically significant(P<0.01).ConclusionHNF1A-AS1 competed for binding to miR-363,regulated the expression of SERTAD3,and involved in the proliferation of breast cancer cells and tamoxifen resistance.Part Ⅲ Silencing of HNF1A-AS1 inhibits the growth of breast cancerxenografts and increases the efficacy of tamoxifen in vivoPurposesIn this part,we further explored whether silencing HNF1A-AS1 could inhibit tumor growth in vivo and increase the therapeutic sensitivity of TAM by constructing an animal model of breast cancer xenografts in nude mice.Methods1.Establishment of a nude mouse model of breast cancer.Twenty specificpathogen-free-gradefemale BALB/c nude mice were randomly assigned into four groups:si-NC group(implanted with MCF-7 cells from the si-NC group),si-HAS1-1 group(implanted with MCF-7 cells from the si-HAS1-1 group),si-NC+TAM group(implanted with MCF-7 cells from si-NC group and intraperitoneally injected with TAM)and si-HAS1-1+TAM group(implanted with MCF-7 cells from the si-HAS1-1 group and intraperitoneally injected with TAM).2.Each mouse was injected with 100 μL cell suspension through the axillarymammary gland.3.After injection,the growth of subcutaneous tumor was observed regularly.When the tumor reached 4-5 mm in diameter,each mouse was intraperitoneally injected with TAM,20mg/kg.The injection was conducted once every 3 days for 5 times in all.Meanwhile,the major diameter(a)and minor diameter(b)of the subcutaneous tumors were detected using a vernier caliper,and the tumor volume(V)was calculated as:(V)=a×b2/2.4.Mice were euthanized after5 injections,and the tumor tissues were separatedand weighed.5.The expression level of Ki-67 in breast cancer tumor was detected bylmmunohistochemical method.6.Data analysis was introduced using the SPSS 21.0.All measurement datawas expressed as x±s,and the data were tested for normality.The data between the two groups was compared using an independent sample t test.The significance level wasα=0.05.Results1.Effects of silencing HNF1A-AS on tumor growth and sensitivity to TAM therapy in nude mice.The volume and weight of implanted tumor in nude mice in si-HAS1-1 group all was smaller than that in si-NC group,the volume and weight of implanted tumors in nude mice in the si-HAS1-1+TAM group all was smaller than that in the si-NC+TAM group,and the difference were statistically significant(P<0.05).2.Expression of Ki67 in transplanted tumor cells.The immunohistochemical results of Ki-67 showed that the rate(0.41±0.05)of Ki-67 positive cells in the si-HNF1A-AS1 group was lower than that in the si-NC group,and the rate(0.26±0.08)of Ki-67 positive cells in the si-HNF1A-AS1+TAM group was lower than that in the si-NC+TAM group,the difference was statistically significant(P<0.05).ConclusionSilencing HNF1A-AS1 inhibits breast cancer tumor growth and increases tumor sensitivity to TAM therapy. |
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