Candidate Oligo Therapeutic Target, miR-330-3p, Induces Tamoxifen Resistance in Estrogen Receptor-Positive Breast Cancer Cells via HDAC4

Tamoxifen is a drug used for treating breast cancer (BC), especially for individuals diagnosed with estrogen receptor-positive (ER+) BC. Its prolonged use could reduce the risk of recurrence and significantly lengthen the survival rate of BC patients. However, an increasing number of patients developed resistance to tamoxifen treatment, which reduced therapeutic efficiency and caused substandard prognosis. Therefore, the exploration of the molecular processes involved in tamoxifen resistance (TR) is urgently required. This investigation aimed to elucidate the relationship of microRNA-330 (miR-330-3p) with the TR of BC. There is little information on miR-330-3p′s link with drug-resistant BC, although it is well known to regulate cell proliferation and apoptosis. Primarily, miR-330-3p expression in parental BC (MCF7/T47D), TR (MCF7-TR), and T47D/TR cell lines was detected by qRT-PCR. Then, the impact of miR-330-3p on the TR of BC cells was assessed by a cell proliferation assay. Lastly, dual-luciferase reporter, qRT-PCR, and western blot assessments were carried out to identify histone deacetylase 4 (HDAC4) as the potential miR-330-3p target gene. The data indicated that miRNA-330 was overexpressed in TR ER+ BC cells and its overexpression could induce TR. Furthermore, miRNA-330 could also reduce the expression of HDAC4, which is closely linked to TR, and overexpression of HDAC4 could reverse miRNA-330-induced drug resistance. In summary, miR-330-3p could induce TR of ER+ BC cells by downregulating HDAC4 expression, which might be a novel marker of TR and a possible treatment target against BC patients who are tamoxifen-resistant.


Introduction
In women, breast cancer (BC) is the most frequent and threatening cancer type, accounting for 7-10% of all malignant tumors.BC incidence has increased since the late 1970s [1,2].Estrogen receptor positive (ER+) has been reported to be expressed in 70-80% of BC patients, with increasing incidence every year [3,4].BC has substantially endangered women's mental and physical health; therefore, the investigation of BC has attracted the attention of scientists worldwide.
Tamoxifen is a nonsteroidal antihormonal antineoplastic agent [5][6][7], is used for the clinical treatment of BC, and is particularly efective in ER+ patients.Tamoxifen could signifcantly alleviate the recurrence risk of BC and is very efective among metastatic ER+ BC patients [8,9].Longterm use of tamoxifen could prolong survival and reduce recurrence in BC patients.However, more and more patients gradually developed tamoxifen resistance (TR) in recent years [10].TR has been reported in 30 to 40% of BC patients treated for 5 years.Cancer cells are generally more aggressive when they get resistant and more difcult to treat with conventional therapies, making it even worse to continue treatment [11,12].Terefore, TR is very important for treating BC.Understanding the bio-molecular mechanism of tamoxifen can solve the drug-resistance problem.
Tis investigation focuses on elucidating the link between miR-330-3p and TR.It was revealed that miR-330-3p upregulation was substantially related to TR and that miR-330-3p could cause drug resistance by downregulating HDAC4.Tis investigation might provide useful information to fnd new therapeutic targets and strategies for TR BC patients.
Antibodies used were β-actin (Sigma-Aldrich), HDAC4 (#15164, 140kd, Cell Signaling Technology, USA), and Lipo2000 (Invitrogen, USA).Assay.Cell viability was elucidated by CCK-8 (Dojindo Laboratories, Kumamoto, Japan).Cells were propagated for 24h, the media were refreshed by only RPMI-1640, and then 150 nM miR-330-3p mimics and miR-330-3p sponge (negative control; NC) were transfected via the Lipo 2000.After 48 h, 3000 cells/wells were propagated in 96-well plates in 100 μL of media +10% FBS at 37 °C for 24 h.Ten, the supernatant was refreshed by 200 μL full growth medium with tamoxifen; each treatment had three replicates.Ten, 10 μL of CCK-8 solution was incorporated in the medium for 2 h at 37 °C, and their absorbance (OD) was measured via a microplate spectrophotometer (Biotek, Germany) at 490 nm.Each sample's OD was subtracted from the blank value, and cell growth reduction was measured as a percentage of control OD (no drug).Te data are depicted as the mean ± SD for repeated experiments thrice.

Dual-Luciferase Reporter
Assays.Cells at the concentration of 10000/well were propagated in 48-well plates overnight in 100 μL of media +10% FBS.Ten, cells were cotransfected by HDAC4 3′-UTR luciferase vector and pre-miR-330-3p or miRNA negative control via DharmaFECT Duo Transfection Reagent (Dharmacon) for 48h at 37 °C and 5% CO 2 .Ten, with the help of phosphate-bufered saline (PBS), cells were rinsed before lysis with 100 ul PLB lysis solution and collection cracking fuid.Te luciferase function was tested via the Dual-Luciferase Reporter Assay System (Promega, USA) and a luminometer.Te experiments were repeated thrice.

RNA Extraction and Real-Time Quantitative PCR.
For obtaining whole RNA, Trizol (Takara, Japan) reagent was utilized by following the instructions provided by the manufacturer (Invitrogen, CA, USA) [30].Quantity and quality were assessed via Nano-drop and Agilent 2100 Bioanalyzer (Agilent Technologies).Ten, purifed RNA (1 μg) was reversely transcribed with the help of PrimeScript RT Reagent Kit (Takara, Japan).cDNA and total RNA were quantifed via a biophotometer (Eppendorf, Germany).Te acquired cDNA was stacked at −20 °C.Te 20 μL reactions were prepared in 96-well plates comprising 10 μL of SYBR Green PCR Master Mix (Takara, Japan), 1 μL of each primer (2 μM) (supporting information (SI) Table S1), and 8 μL of template DNA.β-actin was utilized as an endogenous gene.Each reaction was repeated thrice.Te relative expression levels were elucidated by the 2 − ^^Ct method.
2.5.Western Blot Assessment.Proteins were acquired by lysing cells with RIPA bufer.Ten, standard Western blot protocol was conducted to elucidate protein expression.Briefy, with the help of KEYGEN Protein Extraction Kit (KEYGEN, China), protein samples were obtained at the indicated time point, boiled in sample bufer comprising sodium dodecyl sulfate (5 * SDS) for 5 minutes, exposed to 8% SDS-PAGE electrophoresis, and then translocated to PVDF (polyvinylidene fuoride) membranes (Bio-Rad), which were then blocked for 2 hours in 5% milk-Tris-Bufered Saline Tween-20 (TBST) at ambient temperature, kept with HDAC4 and β-actin (Cell Signaling Technology, USA) monoclonal antibodies at 4 °C overnight, washed thrice in TBST, and incubated again for 2 hours in appropriate horseradish peroxidase-linked secondary antibodies (Cell Signaling Technology, USA) at ambient temperature.

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Te Breast Journal Enhanced chemiluminescence (Termo, USA) was utilized for visualizing blots and assessed via the scanning densitometer using molecular analysis software FluorChem M system (Protein Simple, USA).
2.6.Statistics.Te variabilities in protein levels and invasion rate between the treated and control cells were elucidated by one-way ANOVA and standard student t-test.SPSS (20 version) (SPSS Inc.Chicago, Illinois, USA) was utilized for statistical measurements.P value <0.05 was termed statistically important.Each experiment was repeated thrice.

miR-330-3p Is Highly Expressed in Tamoxifen-Resistant
Cell Lines.Research suggests that miR-330-3p is closely linked with cell proliferation and apoptosis [31][32][33].To investigate whether its expression is associated with the sensitivity of ER+ BC cells to tamoxifen, real-time RT-PCR identifed miR-330-3p in constructed tamoxifen-resistant and parental cell lines.Te data revealed that its levels in T47D-TR and MCF7-TR cells were notably increased than in T47D and MCF7 cells.Its expression in MCF7-TR cells was 5.12 times more than that in MCF7 cells, and in T47D-TR cells, it was about 4.21 times increased than that in T47D cells (Figure 2).Furthermore, miR-330-3p was upregulated in tamoxifen-resistant cells, expressing a positive correlation with the ER+ BC cells' TR.

Gene Ontology and Pathway Analysis of miR-330-3p
Target Genes.Te target prediction of miR-330-3p was performed, and bioinformatics tools assessed its function.
Analyzed GO data suggested that targeted genes were enriched in the biological processes of DNA-templated transcription, nervous system development, cell adhesion, ubiquitin-dependent protein catabolic process, Notch signaling pathway, protein dephosphorylation, protein polyubiquitination, homophilic cell adhesion via plasma membrane adhesion molecules, BMP signaling pathway, and bicellular tight junction assembly (Figure 4(a)).Te target genes' molecular functions included zinc, calcium, and metal ion binding, ubiquitin-protein ligase activity, transcription factor activity, nucleotide binding, ubiquitin-protein   4 Te Breast Journal transferase activity, ligase activity, sequence-specifc DNA binding, thiol-dependent ubiquitin-specifc protease activity, and SH3 domain binding, (Figure 4(b)).Te product of these genes is primarily composed of cell junction, bicellular tight junction, intracellular ribonucleoprotein complex, nuclear pore, and PcG protein complex (Figure 4(c)).Tese genes were classifed into diferent biological pathways, including signaling pathways of insulin, neurotrophin, estrogen, ErbB, and GnRH and pathways of chronic myeloid leukemia, ubiquitin mediated proteolysis, spliceosome, melanogenesis, and renal cell carcinoma (Figure 4(d)).

Discussion
Breast cancer ranks 1 st among the most malignant tumors threatening women's health.Recently, it has been identifed that the incidence of BC is gradually increasing, especially in ER+ patients.Tamoxifen is the frst-line agent against ER+ BC [36,37].However, the recurrence increases after 10 years of tamoxifen treatment without clear rationales [38,39].Te growing number of drug-resistant patients causes clinical therapeutic obstacles and greatly burdens society.Clinical trials have revealed that most patients could beneft from novel drugs which uncover drug resistance.FDA has approved 10 oligo agents for clinical use.Tis investigation aimed to elucidate the possible roles of highly expressed miR-330-3p in tamoxifen-resistant BC (Figure 2).Te Tam-resistant cells used here showed less sensitivity to tamoxifen, suggesting that it is better to use 4OH-Tam, a more active derivative [40,41].
Te literature suggests that based on the cell types, miRNAs play multi-roles in regulating cellular processes.Te literature reveals that miR-330-3p induces tumor progression in various cancers, including nonsmall-cell lung cancer, gastric cancer, colorectal cancer, and pancreatic cancer.It also promotes BC cell migration through CCBE1, Myc, and PDCD4 pathways, predicting substandard prognosis in BC patients.Tis investigation reveals that miR-330-3p overexpression could induce TR in ER+ BC cells (Figure 3); however, the underlying information on the mechanism is still limited.
Based on the rationales of miRNAs negatively regulating target genes, possible miR-330-3p targets were predicted via target scan e-tools, and then gene ontology and pathway analysis were carried out in the David database.Te data suggested that miR-330-3p might regulate diverse biological processes through neurotrophin signaling, spliceosome, insulin signaling, estrogen signaling, ubiquitin-mediated proteolysis, and ErbB signaling pathways (Figure 4).Furthermore, HDAC4 was determined as the miR-330-3p target gene and was involved in miR-330-3p-mediated TR of ER+ BC cells (Figures 5(a) and 5(b)).It is essential to elucidate how miR-330-3p regulates HDAC4 expression and induces TR to better understand and explore novel strategies to solve this clinical obstacle.
HDAC4, a key member of the classic HDAC family, regulates transcriptional activity by modulating histone in the nucleus [42].Aberrant HDAC4 expression has been correlated with multiple biological processes in cancers, including tumorigenesis, migration, and drug resistance.Recently, HDAC4 has been found to increase gastric cisplatin resistance via the p53-p73/BIK pathway [43].In addition, HDAC4 might cause 5-FU resistance in BC cells Te Breast Journal through deacetylation of the SMAD4 promoter [44].Also, HDAC4 interacts with ERα N-terminus in the nucleus and then suppresses the transcription activity of estrogen-responsive genes by estrogen and tamoxifen [34].Moreover, Ahmad et al. found that miR-10b could induce HDAC4-mRNA degradation, resulting in BC cell's TR [35].

Conclusion
In conclusion, it was revealed that aberrant miR-330-3p expression could increase ER+ BC resistance to tamoxifen, that miR-330-3p levels reversely correlate with HDAC4 in ER+ BC cells, and that miR-330-3p induces TR in an HDAC4-dependent manner.Tis research provides brief information that explores the underlying mechanism of BC cells' TR and indicates that miR-330-3p might be a prognostic index for ER+ BC patients and could be a candidate therapeutic target to overcome TR.

Figure 1 :Figure 2 :Figure 3 :
Figure1: Tamoxifen-resistant cells were constructed based on parental cells with tamoxifen treatment.CCK-8 test was performed to identify the vitality of acquired tamoxifen-resistant breast cancer cell lines (MCF7/TR and T47D/TR) and their parental cell lines (MCF7 and T47D).Cells were treated with the indicated tamoxifen dose for 72 h (data are represented as mean ± S.D of triplicate experiments; p < 0.05).

Figure 4 :
Figure 4: Gene ontology and pathway analysis of miR-330-3p.Te potential mechanism involved in miR-330-3p induced tamoxifen resistance as predicted after analyzing the roles of miR-330-3p targeted substrates.(a) miR-330-3p participates in DNA-template transcription, cell adhesion, and nervous system development biological processes.(b) miR-330-3p possesses the ability to regulate metal ion binding, zinc ion binding, and transcription factor activity.(c) Te downstream products are mainly located on cell junction, bicellular tight junction, intracellular ribonucleoprotein complex, nuclear pore, and PcG protein complex.(d) Te targeted genes were enriched in insulin/ neurotrophin/estrogen/ErbB/GnRH signaling pathways and ubiquitin-mediated proteolysis.