| Objective: In this study,long non-coding RNA(lnc RNA)associated with Estrogen receptor(ER)-positive breast cancer were screened by DNA microarray,and the expression levels of the lnc RNAs in tissues and cells were examined.The correlation between the lnc RNA expression level and the prognosis was analyzed through related functional experiments.This study preliminarily explored the possible biological functions of lnc RNA in ER-positive breast cancer and provided experimental evidence for finding new lnc RNAs as biomarkers of ER-positive breast cancer.Methods: 40 pairs of biopsy specimens,consisting of ER-positive breast cancer tissue and adjacent non-carcinoma tissue(confirmed by immunohistochemistry results),obtained from surgical resection from January to October 2020,and underwent Yantai Yuhuangding Hospital,were included in this research.Four were selected for initial screening of differential expression of lnc RNAs using DNA microarray.For lnc RNAs with significantly differential expression detected by the DNA microarray,the expression level of the lnc RNA in the cancer tissue and adjacent non-carcinoma tissue was assessed by the quantitative Real-Time Polymerase Chain Reaction(q RT-PCR).Then,the expression level of the specific lnc RNAs was evaluated in the 40 pairs of ER-positive breast cancer tissues and adjacent non-carcinoma tissues.The further cellular level evaluation was carried out by analyzing the expression level of the specific Inc RNAs detected by DNA microarray,verified by the q RT-PCR results,in two ER-positive breast cancer cell lines,MCF-7 and T47 D,and normal breast epithelial cell line MCF-10 A.The relationship between the lnc RNAs and the patient prognosis was analyzed by Kaplan-Meier survival analysis,using data from the TCGA database.lnc RNA(LINC01614),which was found related to the patient prognosis,was selected for further study.The expression level of LINC01614 in estrogen-stimulated MCF-7 cells was assessed using q RT-PCR,and the correlation between the Inc RNA expression and estrogen level was analyzed.Knockdown of LINC01614 expression was done by using MCF-7 cells with transfected si RNA.Later,by using q RT-PCR to detect the expression level of LINC01614,the si RNA with the highest interference efficiency was selected.A CCK8 assay,a scratch assay,and a transwell migration assay were performed to examine the ability of the breast cancer cell to proliferate,migrate and invade under the influence of knockdown separately.Results: Through the initial DNA microarray done on the four pairs of biopsy specimens,4050 lnc RNAs were identified(fold change ≥ 2,P < 0.05),of which 1589 lnc RNAs were significantly upregulated in cancer tissues,and 2461 lnc RNAs were significantly downregulated(P < 0.05).Next,the expression level of 7 differently expressed lnc RNAs(fold change > 7),including LINC01614,CTD-2116N17.1,KIAA0101,linc-BCL2A1-3,AC044784.1,AL365436.2,and CTD-2510F5.4 in the 40 groups of biopsy specimens were evaluated.The results suggested that the expression of LINC01614,AC044784.1,and CTD-2510F5.4 was significantly increased in ER-positive breast cancer tissues(P < 0.05),which was consistent with the results from the DNA microarray screening.The expression of LINC01614,AC044784.1,and CTD-2510F5.4 was further evaluated using ER-positive breast cancer cell lines MCF-7,T47 D,and normal breast epithelial cell line MCF-10 A.High expression of these Inc RNAs was also found(P < 0.05).The prognostic analysis of LINC01614,AC044784.1,and CTD-2510F5.4 using the TCGA database revealed that the expression level of LINC01614 in ER-positive breast cancer patients was significantly correlated to the patient prognosis.Patients with high expression of LINC01614 had a shorter survival time(P < 0.01),while the expression level of AC044784.1 and CTD-2510F5.4 has no significant correlation with the prognosis(P > 0.05).Significantly high expression of LINC01614 in cells was also found in estrogen-stimulated MCF-7 cells(P < 0.05).Compared with the control group,the expression of LINC01614 in MCF-7 cells was significantly decreased after the transfection of si RNA(P < 0.001).The CCK8 assay,the scratch assay,and the transwell migration assay showed that,by inhibiting the expression of LINC01614,the capability of the breast cancer cell to proliferate,migrate and invade decrease significantly(P < 0.05).Conclusion: 1.LINC01614,AC044784.1,and CTD-2510F5.4 were increasingly expressed in ER-positive breast cancer tissues and breast cancer cell lines.Thus,LINC01614 might be a new biomarker of the prognosis of ER-positive breast cancer.3.Expression of LINC01614 in breast cancer cells might be regulated by estrogen level.It promotes the proliferation,migration,and invasion of breast cancer cells,which provide new theoretical evidence of possible pathogenesis of ER-positive breast cancer. |
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