MicroRNA-124-3p Functions as a Marker in Diagnosing Breast Cancer

doi:10.21203/rs.3.rs-61637/v1

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Research article

MicroRNA-124-3p Functions as a Marker in Diagnosing Breast Cancer

https://doi.org/10.21203/rs.3.rs-61637/v1

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posted 21 Sep, 2020

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Background

Early diagnosis of breast cancer can reduce the high fatality rate. Thus novel diagnostic methods are urgently required for breast cancer patients. The research aimed at checking potential correlation for serum microRNA-124-3p (miR-124-3p) expression with breast cancer, and exploring its competence as a promising potential biomarkers for breast cancer.

Methods

Quantitative real-time PCR (qRT-PCR) assay detected miR-124-3p levels. Possible relationship between miR-124-3p degree and clinical features of breast cancer patients was measured by Chi-square analysis. Sensitivity, specificity and area under the curve (AUC) for serum miR-124-3p degree were settled adopting receiver operating characteristics (ROC) analyses.

Results

Relative degree for serum miR-124-3p notably reduced among breast cancer sufferers in comparison with healthy persons (P=0.000). Moreover, miR-124-3p degree held tight connection to clinical stage (P=0.034), histological grade (P=0.002), and lymph node metastasis (P=0.001). ROC curve analyses unveiled at the optimal cut-off of 0.935, serum miR-124-3p expression reached a sensitivity of 78.4% and a specificity of 84.8% in discriminating between breast cancers sufferers and healthy persons, achieving an area under the curve (AUC) of 0.872 (95% CI: 0.752-0.871).

Conclusion

Serum miR-124-3p degree might represent one non-invasive indicator in diagnosing breast cancer.

Cancer Biology

Oncology

MicroRNA-124-3p

Breast cancer

Diagnosis

Biomarker

Breast cancer represents one most widespread malignancy among females around the world [1]. The incidence of breast cancer varies across the world, usually lower in less-developed countries than that in the more-developed countries. It is of great importance for early detection together with curative resection to improve breast cancer prognoses [2, 3]. Currently, the pathological observation are the main approaches to diagnose this disease. With considerable advancements in identifying serum indicators, it is essentially required to find indicators holding satisfactory specificity and sensitivity through serum detection, which is beneficial for precise screening, target treatment and outcome prediction [4]. Until now, no satisfactory indicators have been established in screening the malignancy. Hence, finding promising novel indicators would held considerable clinical significance.

MicroRNAs (miRNAs) impose negative or positive influences on gene expressions via antisense complimentarily, thus affecting RNA stability and protein translations [5-10]. Changes in miRNA degrees might alter biological courses such as cellular multiplication, differentiation, and apoptosis, which impose crucial influences on oncogenesis and tumour furtherance [11]. Reportedly, miRNAs take part in oncogenesis, assuming boosting or repressing functions [12-14]. According to existing evidence, miRNAs in serum or plasma were sufficient indicators in diseases [15-18]. As one member in miRNAs, microRNA-124-3p （miR-124-3p） could wield restraining effects against breast cancer, which negatively regulated the proliferation and invasion for malignant cells [19]. Nonetheless, diagnostic value for serum miR-124-3p in breast cancer is far from totally comprehended.

Through the present research, we detected relative degree for serum miR-124-3p in breast cancer, and evaluated possible connection for serum miR-124-3p to clinicopathological characteristics among cases with breast cancers. We also assessed the diagnostic performance of serum miR-124-3p in breast cancer.

Patients and serum samples

Our research acquired permission from the Ethic Committee of the 903rd Hospital of PLA. Enrolled subjects signed the informed consents.

Serum specimens came from 105 breast cancer sufferers receiving treatments in the 903rd Hospital of PLA. Histopathology for cases was examined. Cases would be removed from our study once they had other cancers at any sites or experienced chemotherapy or radiotherapy ahead of sampling. Detailed clinicopathological indexes for cases were manifested by Table 1. 97 normal serum samples from healthy volunteers was recruited as controls.

Table 1 The correlation of serum miR-124-3p expression with clinicopathological parameters of breast cancer patients

Parameters	NO. of cases (n=105)	MiR-124-3p expression		c²	P values
Parameters	NO. of cases (n=105)	Low (n=54)	High (n=51)	c²	P values
Age (years)				0.351	0.554
≤60	67	33	34
>60	38	21	17
Tumor size				0.587	0.444
≤2	64	31	33
>2	41	23	18
Clinical stage				4.473	0.034
Ⅰ-Ⅱ	72	32	40
Ⅲ	33	22	11
Histological grade				9.583	0.002
Ⅰ-Ⅱ	81	35	46
Ⅲ	24	19	5
ER				0.561	0.454
Negative	62	30	32
Positive	43	24	19
PR				0.061	0.805
Negative	30	16	14
Positive	75	38	37
HER2				0.008	0.929
Negative	51	26	25
Positive	54	28	26
Lymph node metastasis				11.147	0.001
Absent	85	37	48
Present	20	17	3

ER: estrogen receptor; PR: progesterone receptor; HER-2: C-erbB-2.

RNA extraction and quantitative real-time PCR

Total RNA was separated for serum samples adopting Trizol reagent (Takara, Dalian, China) abiding by the producer’s guidance. RNA was reverse transcribed into cDNA employing One Step PrimeScript miRNA cDNA Synthesis Kit (Takara, Dalian, China). Then, we implemented quantitative RT-PCR assays (qPCR). miRNA degree was evaluated applying Applied Biosytems 7500 Fast Real-time PCR System (Applied Biosystems, Carlsbad, USA) with SYBR Premix Ex Taq (Takara, Dalian, China) abiding by corresponding indications. The relative expressions for miR-124-3p were quantified using 2^-ΔΔCt approach [20], namely ΔCt=Ct_miRNA-Ct_{endogenous control} in which Ct represents threshold cycle, or the amount of cycles where fluorescent signal surpass the threshold. RNU6B degree represented internal reference.

Statistical analysis

All the data syntheses were completed in SPSS 18.0 (SPSS. Chicago, IL, USA), and graphs were plotted by Origin 9.0. The statistical differences between two groups was checked through independent sample t test. analysis evaluated potential link for serum miR-124-3p degree to clinicopatholgical characteristics among included sufferers. Receive operating characteristic (ROC) curve calculated sensitivity and specificity for serum miR-124-3p expression and estimate the diagnostic capability in breast cancer. P under 0.05 meant the presence of statistical significance.

Serum miR-124-3p expression among breast cancer and healthy controls

As shown in Figure 1, qRT-PCR revealed serum miR-124-3p level strongly lowered among breast cancer sufferers in comparison to healthy persons (0.669±0.296 vs 1.130±0.434). The median miR-124-3p degree among all 105 patients with breast cancer was 0.64. We classified enrolled cases into low (n=54) and high (n=51) miR-124-3p level classes using the median degree as a cutoff.

Correlation for serum miR-124-3p degree with clinicopathological traits among sufferers with breast cancer

According to the results of Chi-square test, serum miR-124-3p degree exhibited strong connection to clinical stage (P=0.034), histological grade (P=0.002), and lymph node metastasis (P=0.001), but not to age, cancer dimension, ER, PR, or HER-2 (all P>0.05).

Diagnostic efficacy for serum miR-124-3p in breast cancers

ROC curves was plotted to identify whether miR-124-3p could discriminate between breast cancer sufferers and healthy persons. The results unveiled at the optimal cut-off (0.935), serum level of miR-124-3p achieved a sensitivity of 78.4% and a specificity of 84.8% to discriminate the cases and controls, reaching an area under the curve AUC of 0.872 (95% CI: 0.752-0.871, Figure 2).

Over the decades, survivals for breast cancer sufferers at early stages are lengthened [21]. Apart from timely diagnosis, adjuvant chemotherapy and endocrine therapy also considerably better cases’ overall survivals. There are two traditional indicators in diagnosing this malignancy, CA15.3 and CEA, and the previous studies have demonstrated sensitivity and specificity for them lie at 15-45% and 10-30%, separately. They are unsatisfactory for screening of breast cancer. Recently, miR-124-3p was reported to be generally reduced in multiple tumors while its transfection suppresses the malignant cellular growth and migration [22-25]. For example, Gov E et al. reported that miR-124-3p was a plausible indicator in ovarian cancer [22]. Yuan and colleagues demonstrated miR-124-3p expression down-regulated amid bladder cancer tissues and cell lines, which played important role on malignant cellular multiplication, emigration and apoptosis [23]. Moreover, in the study of Margolin-Miller Y and colleagues, high miR-124-3p degree held strong connection to shortened progression-free survivals among ependymoma patients, which might represent a self-sufficient indicator for prognoses in this disease [24]. Zhang and colleagues claimed miR-124-3p declined among breast cancer tissues and breast cell lines. Its declines promoted malignant cellular advancement primarily through elevating degree for autophapy relevant protein, Beclin-1 [25].

Currently, explorations on indicators wield essential functions in comprehending mechanisms for varied illnesses. Despite their employment in diagnosing breast cancer, CEA and CA15.3 merely possess insufficient sensitivity and specificity, especially for cases at early stages. MicroRNAs could generate pivotal influences on tumorigenesis. It has been revealed half of them seat at tumour-relevant genomic positions/fragile loci, which indicated their involvement in tumour origination and development [26]. Currently, miRNAs have great potential as novel biomarkers in diagnosing, therapeutic monitor, and predicting outcomes among breast cancer sufferers.

In this research, serum miR-124-3p degree dramatically reduced among breast cancer sufferers in comparison to controls. Moreover, we found that serum miR-124-3p degree possessed tight connection to clinical stage, histological grade, and lymph node metastasis, suggesting its involvement in breast cancer advancement and metastasis. Our findings were consistent with the previous studies [25]. ROC curve analyses confirmed serum miR-124-3p degree had a high sensitivity and specificity in discriminating between breast cancer sufferers and healthy persons, reaching high AUC values.

Multiple shortcomings of this research should be noted. Firstly, miR-124-3p might assume repressing effects against breast cancer, but potential mechanisms for such activities remained unknown. Future experiments are still necessary to ascertain unspecified influences for miR-124-3p and its aims in the malignancy onset and development. Another limitation is the relatively small size. Larger-scale researches would be indispensable to confirm efficacy for serum miR-124-3p expression to diagnose breast cancer for making more definitive conclusions.

Taken together, serum miR-124-3p expression dramatically decreased among breast cancer sufferers in comparison to healthy persons. Low miR-124-3p degree exhibited strong connection to the cancer development and progression. Our findings offered primary evidence of miR-124-3p affecting breast cancer and its potential to be adopted in early diagnosing this malignancy.

microRNA-124-3p (miR-124-3p)

Quantitative real-time PCR (qRT-PCR)

area under the curve (AUC)

receiver operating characteristics (ROC)

MicroRNAs (miRNAs)

Ethics approval and consent to participate

This study was supported by the Ethics Committee of the 903rd Hospital of PLA and also has been carried out in accordance with the World Medical Association Declaration of Helsinki.

The subjects had been informed the objective. Certainly, written consents were signed by every subject in this study.

Consent for publication

We obtaining permission from participants to publish their data.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by grant from National Natural Science Foundation of China (81902996), Fudan University Shanghai Cancer Center Fund (YJ201701), National Science and Technology Major Project (2020ZX09201-013).

Authors’ contributions

Y.M. design of the work; Q.X. the acquisition, analysis, L.C. interpretation of data; L.W. the creation of new software used in the work; X.H. have drafted the work or substantively revised it. All authors read and approved the final manuscript.

Acknowledgements

Not applicable.

Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D: Global cancer statistics. CA: a cancer journal for clinicians 2011, 61(2):69-90.
Cianfrocca M, Goldstein LJ: Prognostic and predictive factors in early-stage breast cancer. The oncologist 2004, 9(6):606-616.
Arriagada R, Rutqvist LE, Johansson H, Kramar A, Rotstein S: Predicting distant dissemination in patients with early breast cancer. Acta Oncol 2008, 47(6):1113-1121.
Li J, Zhang Z, Rosenzweig J, Wang YY, Chan DW: Proteomics and bioinformatics approaches for identification of serum biomarkers to detect breast cancer. Clinical chemistry 2002, 48(8):1296-1304.
Bartel DP: MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004, 116(2):281-297.
Bushati N, Cohen SM: microRNA functions. Annual review of cell and developmental biology 2007, 23:175-205.
Yekta S, Shih IH, Bartel DP: MicroRNA-directed cleavage of HOXB8 mRNA. Science 2004, 304(5670):594-596.
He L, Hannon GJ: MicroRNAs: small RNAs with a big role in gene regulation. Nature reviews Genetics 2004, 5(7):522-531.
Du T, Zamore PD: microPrimer: the biogenesis and function of microRNA. Development 2005, 132(21):4645-4652.
Ambros V: microRNAs: tiny regulators with great potential. Cell 2001, 107(7):823-826.
Esquela-Kerscher A, Slack FJ: Oncomirs - microRNAs with a role in cancer. Nature reviews Cancer 2006, 6(4):259-269.
Bartels CL, Tsongalis GJ: MicroRNAs: novel biomarkers for human cancer. Clinical chemistry 2009, 55(4):623-631.
Cortes-Sempere M, Ibanez de Caceres I: microRNAs as novel epigenetic biomarkers for human cancer. Clinical & translational oncology : official publication of the Federation of Spanish Oncology Societies and of the National Cancer Institute of Mexico 2011, 13(6):357-362.
Shenouda SK, Alahari SK: MicroRNA function in cancer: oncogene or a tumor suppressor?Cancer metastasis reviews 2009, 28(3-4):369-378.
Gilad S, Meiri E, Yogev Y, Benjamin S, Lebanony D, Yerushalmi N, Benjamin H, Kushnir M, Cholakh H, Melamed N et al: Serum microRNAs are promising novel biomarkers. PloS one 2008, 3(9):e3148.
Ng EK, Chong WW, Jin H, Lam EK, Shin VY, Yu J, Poon TC, Ng SS, Sung JJ: Differential expression of microRNAs in plasma of patients with colorectal cancer: a potential marker for colorectal cancer screening. Gut 2009, 58(10):1375-1381.
Wang K, Zhang S, Marzolf B, Troisch P, Brightman A, Hu Z, Hood LE, Galas DJ: Circulating microRNAs, potential biomarkers for drug-induced liver injury. Proceedings of the National Academy of Sciences of the United States of America 2009, 106(11):4402-4407.
Chim SS, Shing TK, Hung EC, Leung TY, Lau TK, Chiu RW, Lo YM: Detection and characterization of placental microRNAs in maternal plasma. Clinical chemistry 2008, 54(3):482-490.
Wang Y, Chen L, Wu Z, Wang M, Jin F, Wang N, Hu X, Liu Z, Zhang CY, Zen K et al: miR-124-3p functions as a tumor suppressor in breast cancer by targeting CBL. BMC cancer 2016, 16(1):826.
Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001, 25(4):402-408.
Nishimura R, Osako T, Nishiyama Y, Tashima R, Nakano M, Fujisue M, Toyozumi Y, Arima N: Evaluation of factors related to late recurrence--later than 10 years after the initial treatment--in primary breast cancer. Oncology 2013, 85(2):100-110.
Gov E, Kori M, Arga KY: Multiomics Analysis of Tumor Microenvironment Reveals Gata2 and miRNA-124-3p as Potential Novel Biomarkers in Ovarian Cancer. Omics : a journal of integrative biology 2017.
Yuan Q, Sun T, Ye F, Kong W, Jin H: MicroRNA-124-3p affects proliferation, migration and apoptosis of bladder cancer cells through targeting AURKA. Cancer biomarkers : section A of Disease markers 2017, 19(1):93-101.
Margolin-Miller Y, Yanichkin N, Shichrur K, Toledano H, Ohali A, Tzaridis T, Michowitz S, Fichman-Horn S, Feinmesser M, Pfister SM et al: Prognostic relevance of miR-124-3p and its target TP53INP1 in pediatric ependymoma. Genes, chromosomes & cancer 2017, 56(8):639-650.
Zhang F, Wang B, Long H, Yu J, Li F, Hou H, Yang Q: Decreased miR-124-3p Expression Prompted Breast Cancer Cell Progression Mainly by Targeting Beclin-1. Clinical laboratory 2016, 62(6):1139-1145.
Calin GA, Sevignani C, Dumitru CD, Hyslop T, Noch E, Yendamuri S, Shimizu M, Rattan S, Bullrich F, Negrini M et al: Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proceedings of the National Academy of Sciences of the United States of America 2004, 101(9):2999-3004.

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Version 1

posted 21 Sep, 2020

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MicroRNA-124-3p Functions as a Marker in Diagnosing Breast Cancer

Status:

Version 1

Abstract

Figures

Background

Methods And Materials

Results

Discussion

Conclusion

Abbreviations

Declarations

References

Status:

Version 1