| AimTriple negative breast cancer(TNBC)is a highly aggressive disease characterized by rapid tumor progression,high incidence of recurrence and metastasis,and poor prognosis.In the process of cancer occurrence and development,the dysregulation of microRNA(miRNA)usually leads to abnormal gene expression.In previous studies,it was found that the expression of miR-126-3p in TNBC tumor cells was significantly lower than that in other molecular types of breast cancer,and studies have shown that tumor vascular integrity may be affected by miR-126-3p.Therefore,this project is intend to explore the function of miR-126-3p in TNBC cell lines and its effect on TNBC vascular mimicry,to determine whether miR-126-3p has dual anti-angiogenesis and anti-tumor effects,and to provide references for better TNBC targeted therapy.MethodsIn vitro culture normal breast epithelial cells MCF-10 A and TNBC cell lines MDA-MB-231 and HCC1937;real-time quantitative polymerase chain reaction(PCR)was used to determine the expression level of miR-126-3p;in vitro experiments to evaluate the effect of miR-126-3p on the proliferation,invasion,migration and vascular mimicry in TNBC,including cell counting kit-8(CCK-8)assay,colony formation assay,transwell assay and angiogenesis simulation assay;using multiple online bioinformatic databases to predict and overlap the target genes of miR-126-3p;construct expression vector of target gene and its 3’-UTR point mutation vector,verify the target gene by luciferase activity;use the online survival tool(Kaplan-Meier plotter,KM ploter)to analyze the prognosis of the m RNA expression of target gene in breast cancer;collect clinical samples and construct tissue microarrays of female breast invasive ductal carcinoma and its paired adjacent,and determine the expression of target gene of each sample by immunohistochemistry and immune response score(IRS)standards,combine the clinicopathological profile and follow-up data to analyze the correlation between target gene and clinical factors and the impact on the prognosis of patients.ResultsIn vitro cell experiment: compared with normal breast epithelial cell MCF-10 A,the expression of miR-126-3p was down-regulated in TNBC cell lines;the results of functional studies showed that,compared with the negative control group,the overexpression of miR-126-3p inhibited the proliferation(at 72 hours timepoint,overexpression vs.negative control: MDA-MB-231 1.28±0.05 vs.1.64±0.05;HCC19372.22±0.06 vs.2.63±0.03),migration(overexpression vs.negative control:MDA-MB-231 32.00 ±1.41 vs.70.00±4.08;HCC1937 14.33±1.25 vs.34.00±1.63),invasion(overexpression vs.negative control: MDA-MB-231 37.33±3.30 vs.65.67±3.30),colony forming ability(overexpression vs.negative control:MDA-MB-231 63.67±3.30 vs.125±3.56)and angiogenesis ability(overexpression vs.negative control: MDA-MB-231 0.38±0.22 vs.1.31±0.50)in TNBC cells,the differences were statistically significant(P<0.05,respectively).Bioinformatics analysis: three databases of TargetScan,miRDB and miRTarBase were used to predict and take the intersection.According to the cumulative weighted score ranking of the target genes of the intersection in TargetScan,the first G protein signaling 3(RGS3)was selected for further verification.The dual luciferase report experiment confirmed that miR-126-3p directly binds to the 3’-UTR region of RGS3,and q RT-PCR confirmed that RGS3 was up-regulated in the TNBC cell line.Silencing the RGS3 gene inhibited the proliferation(at 72 hours timepoint,si-RGS3 vs.negative control: MDA-MB-231 1.42±0.07 vs.1.74±0.05;HCC1937 1.66±0.04 vs.2.35±0.04),migration(si-RGS3 vs.negative control: MDA-MB-231 39.00±1.63 vs.71.00±4.32),invasion(si-RGS3 vs.negative control: MDA-MB-231 36.33±1.24 vs.76.33±4.64),colony forming ability(si-RGS3 vs.negative control: MDA-MB-231 74.00±6.16 vs.131.67±2.49)and angiogenesis ability(si-RGS3 vs.negative control: MDA-MB-2310.58±0.19 vs.1.50±0.41)in TNBC cells,the differences were statistically significant(P<0.05,respectively).The inhibitory effect of miR-126-3p overexpression on TNBC cell line can be reversed by introducing exogenous RGS3 expression plasmid.The analysis of TCGA database and KM plotter showed miR-126-3p was low expressed in breast cancer comparing to the healthy group and the expression difference was statistically significant(P<0.001);the 25-year overall survival(OS)rate of breast cancer patients with low-expressing of miR-126-3p was significantly higher than those with higher expression,presenting 173.69 vs.198.02(low expression group vs.high expression group,unit: month)in median survival time,and the difference in prognosis was statistically significant(P=0.00025).The online survival tool(KM-ploter)analysis suggested that the m RNA level of RGS3 gene was related to the recurrence-free survival(RFS)and overall survival(OS)in TNBC patients: TNBC patients with higher RGS3 expression got worse RFS and OS than those patients with low RGS3 expression,and the differences were statistically significant(RFS: P=0.032,OS: P=0.0076).The results of immunohistochemical analysis showed that RGS3 gene was highly expressed in breast cancer tissues(65.2%,88/135)and lowly expressed in normal breast tissues(22.2%,30/135),and the difference was statistically significant(P<0.0001);The high expression of RGS3 was significantly related to dangerous clinical factors such as lymph node positive metastasis(P=0.027),ER negative expression(P=0.028),and triple-negative breast cancer phenotype(P=0.016);patients with positive RGS3 expression had worse OS than those with negative RGS3 expression in 10-year follow-up(P=0.0455),and in both subtypes of HER2 overexpression and TNBC showed that RGS3-positive patients had a worse prognosis.Cox single factor regression analysis revealed that positively expressed RGS3(HR 2.257;95%CI 1.176-4.333;P=0.019),grade 3 in tumor histology(HR 1.891;95%CI 1.015-3.521;P=0.045),lymph nodes positive metastasis(HR 1.808;95%CI 1.033-3.194;P=0.038)and negative estrogen receptor(ER)expression(HR 1.967;95%CI 1.058-3.654;P=0.032)were statistically significant factors leading to the poor prognosis in breast cancer;In the results of cox multivariate regression analysis,the positive expression of RGS3(HR2.216;95% CI 1.048-4.688;P=0.037)and negative expression of ER(HR 1.914;95%CI 1.008-3.634;P=0.047)were indicated poor prognostic factors in breast cancer.ConclusionsMiR-126-3p is down-regulated in TNBC,characterized as tumor suppressor.MiR-126-3p inhibits the proliferation,invasion and migration of TNBC cells by binding to the 3’-UTR region of RGS3 gene in TNBC;miR-126-3p suppresses the vascular mimicry in TNBC by regulating RGS3,indicating that the signal transduction pathway regulated by RGS3 may be involved in the formation of TNBC vascular mimicry.High expression of RGS3 gene is significantly related to prognostic risk factors such as lymph node positive metastasis,ER negative expression and triple negative breast cancer phenotype,suggesting RGS3 is a cancer-related gene and RGS3 is related to the aggresive biological behavior of breast cancer.Those patients with highly expressed RGSs in m RNA or protein level in breast cancer have a worse RFS and OS.Comprehensive cox regression analysis indicate that RGS3 is independent factor of the poor prognosis of breast cancer. |
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