Up-Regulation of Mir-105-5p Promotes The Development of Breast Cancer By Targeting AKT1 / GRB2 Genes In Patients With Breast Cancer

Objective: miR-105-5p either acts as an oncomiR or tumor suppressor that has been shown to have various expression levels in a wide range of diseases and targets RAC-alpha serine/threonine-protein kinase (AKT1) and Growth Factor Receptor-bound protein 2 (GRB2) genes. GRB2 is a signaling protein that takes part in various signaling pathways. Due to the fact that miRNAs by acting on target genes, and regulates the expression of them, so the aim of this study is to evaluate the expressions of miR-105-5P, AKT1, and GRB2 genes and the correlation between them in breast cancer (BC) tissue compared to normal-adjacent tissues (NATs) in women with breast cancer. Materials and methods: We utilized bioinformatics analysis to searching for target genes of miR-105-5p. Then, 35 BC and NATs tissues were taken. In this experimental study, quantitative Real-Time PCR was performed to evaluate miR-105-5P, AKT1 and GRB2 genes expression, and the correlation between them was evaluated. The relation between expression and features of clinicopathological was explored. Results: The miR-105-5p expression was increased signi�cantly in BC tissues in comparison to NATs (P 0.05). The expression levels of AKT1 and GRB2 genes were decreased signi�cantly in BC tissue in comparison to NATs (P 0.05). An inverse correlation was observed between miR-105-5p, AKT1 and GRB2 expression which was not signi�cant (P>0.05). Also, a direct correlation was observed between GRB2 and AKT1 gene expression (P 0.05). Conclusion: Our �ndings revealed that increased expression levels of miR-105-5p in patients with breast cancer caused a decrease in the expression level of its target genes, AKT1 and GRB2, and subsequently lead to tumor progression and invasion of the tumor. miR-105-5p may serve as a promising target for BC therapy.


Introduction
Breast cancer (BC) is widely assumed to be the main cause of cancer death in women around the world.
Every year, 2,088,849 new cases are diagnosed with BC with over 626,679 deaths estimated worldwide [1].It is a multifactorial disorder associated with the effects of multiple genes in combination with environmental factors [2,3,4].BC is classi ed into two forms of sporadic BC, which approximately attributes to 95 percent of BCs, and familiar BC which accounts for the rest of the BCs and it is caused by mutations in genes like Breast cancer susceptibility gene 1 (BRCA1) and Breast cancer susceptibility gene 2 (BRCA2) [5].There are ve major molecular subtypes of BC.They are grouped based on the genes expressed: Luminal A BC is hormone-receptor positive (progesterone-receptor positive and/or estrogenreceptor), HER2 negative, and the protein Ki-67 is in low levels.Luminal B BC is hormone-receptor positive (progesterone-receptor positive and/or estrogen-receptor), and either HER2 positive or HER2 negative with high levels of Ki-67.Triple-negative/basal-like BC is HER2 negative and hormone-receptor negative (progesterone-receptor and estrogen-receptor negative.HER2-enriched BC is HER2 positive and hormonereceptor negative (progesterone-receptor and estrogen-receptor negative).Normal-like BC is similar to luminal A disease: HER2 negative and hormone-receptor positive (progesterone-receptor and/or estrogenreceptor positive), and the protein Ki-67 is in low levels [6].
In the past decade, with the usage of genome-wide studies on DNA sequence, gene expression, and so forth, many aberrant genes and miRNAs have been identi ed which might contribute to breast tumor [7,8].miR-105-5P targets many genes in various cancers and can act either as tumor suppressor or oncomiR [9].Some studies showed that miR-1 to be associated with several diseases such as cancer (BC, human gliomas, colorectal cancer, gastric cancer, prostate cancer and hepatocellular carcinoma) and heart diseases [9].miR-105 functions as an oncogene in several of these cancers such as colorectal cancer and BC, and high expression of this miR causes metastasis and invasion.But in cancers such as prostate cancer, hepatocellular carcinoma, human gliomas, and lung cancer, miR-105 functions as a tumor suppressor.miR-105 can alter the expression levels of its target gene, so it has an impact on cancer progression [9].miR-105-5P is secreted by cancer cells and in BC, it works as oncomiR via targeting ZO1 protein, which acts as a tight junction adaptor protein, and therefore, it contributes to metastasis .miR-105-5Ptargets when this micro RNA is suppressed in BCs it can lead to a decrease in the metastasis rate [10,11].
Other genes such as AKT1 which are involved in different cell signaling pathways and any dysregulation of their expression may lead to creation of cancer cells [12].
AKT1 is serine-threonine protein kinase which regulates numerous processes such as proliferation, cell survival, growth, and so on [13,14].AKT1 is a mediator of various cellular pathways including PI3K / PTEN / AKT / mTORC1, therefore, it has distinct roles in the development of BC [15,16].It has been assumed that overexpression and activation of AKT1 might lead to BC progression by contributing to being resistant to anti-proliferative signals [17,18].
Growth Factor Receptor-bound protein 2 (GRB2) gene is an adapter protein that plays an important link between cell surface growth factor receptors and the Ras signaling pathway and it may have a vital role in cell survival, proliferation, differentiation and angiogenesis [19].It has been reported that GRB2 is involved in development and progression of various malignancies such as BC, lung cancer, gastric cancer, colorectal cancer and so forth [20,21,22].The aim of the current study is to evaluate the expression of miR-105-5p, AKT1, and GRB2 genes and the correlation between them in BC tissues from Iranian patients with BC and adjacent normal tissue (NATs) from same patients.

Subjects
In this experimental study, fresh breast cancer (BC) tissues and paired normal adjacent tissues (NATs) (>5 cm from tumor) from 35 women with BC were collected from Imam Khomeini Hospital, Tehran, Iran.Tissues were carried to the laboratory via liquid nitrogen tank.The BC patients had the average age of 50 years when diagnosed (range 30-55 years).Patients did not undergo any radiation therapy or chemotherapy.The diagnosis, histological grade and the clinical stage of each case were independently con rmed by a pathologist.Patients had no other diseases and the ones that did, were excluded from the study.The study was approved by the Ethics Committee and all of the contributors provided written informed consent.

Statistical analysis
All experimental information was expressed as mean ± SEM.Statistical comparisons between the BC tissues and NATs were carried out using paired t-test and one-way analysis of variance was carried out to compare the differences among more than two groups followed by Tukey's test.Differences were considered statistically signi cant at P ≤ 0.05.The correlation between AKT1, and GRB2 genes and miR-105-5p in BC patients was assessed by Pearson's correlation.In this study, GraphPad Prism was used to carry out all statistical analyses (GraphPad Software, La Jolla California USA).

Results
The clinicopathological characteristics of patients are presented in Table 1

AKT1 is downregulated in Breast cancer
We rst investigated the expression of AKT1 in BC tissues and NATs by quantitative RT-PCR.We observed the expression levels of AKT1 was decreased in BC tissues compared to that in NATs signi cantly (Fig. 2) (p= 0.0141).Next, we compared the expression levels of AKT1 with the clinicopathological features including histological grade, clinical stages and patients' age.The lowest expression of AKT1 was observed in Stage III and grade I. But, the AKT1 gene expression was not signi cantly related to clinicopathological features including histological grade, and clinical stages.
To better understand the relationship between AKT1 expression and patients' age, we considered the expression of this gene in younger and older groups (≤ 50 year and > 50 year).No signi cant difference was observed between aged ≤ 50 year and aged > 50-year groups (P=0.680).However, there was a trend toward decreasing AKT1 expression in aged > 50-year groups (Table 3).

AKT1 is a target of miR-105-5p
Bioinformatics prediction revealed potential binding sites of miR-105-5p on the 3′-untranslated region (3′-UTR) of AKT1 (Fig. 3a).Spearman's rank order correlation was used to determine the relation between miR-105-5p expression and its target gene.We observed a negative correlation between miR-105-5p and AKT1 in BC tissues (slope=-0.008862,P=0.9602) but not signi cantly (Fig. 3b).In the other hand, the expression of miR-105-5p was up-regulated, whereas AKT1 was downregulated in the tumor tissues.These ndings collectively indicate that AKT1 is a target of miR-105-5p in BC.

GRB2 is downregulated in Breast cancer
The expression level GRB2 was quanti ed by quantitative real-time PCR.We observed that there was a signi cant decrease in GRB2 expression in BC tissue samples in comparison to NATs (p= 0.0473) (Fig .4).
We next explored the relationship between GRB2 and clinicopathological characteristics of BC patients.The lowest expression of GRB2 was observed in Stage III and grade I.But the signi cant difference was not found when the gene expression was compared between different histological grades (I to III) and different clinical stages (I to III).Also, the expression of GRB2 in patients aged > 50-year groups was lower than aged< 50 year, but no correlation was observed between GRB2 expression and age (< 50 year and > 50-year groups) (P= 0.585) (Table 4).GRB2 is a target of miR-105-5p Bioinformatics prediction indicated that GRB2 is the potential target gene of miR-105-5p.The 3′-UTR of GRB2 mRNA has a speci c sequence which is complementary to the seed region of miR-105-5p (Fig. 5a) We analyzed 35 BC tissues and NATs to explore the relationship between miR-105-5p and the target GRB2 gene.The Spearman's correlation demonstrated that miR-105-5p expression had negative correlation with the GRB2 expression (slope = -0.02614,p=0.4563) but not signi cantly shown in (Fig. 5b).Showed miR-105-5p was up-regulated in BC tissues while GRB2 was down-regulated BC tissues.
Moreover, we explored the relationship between two target genes, AKT1 gene and GRB2 gene.We observed a signi cant positive correlation between AKT1 gene and GRB2 gene (slope = 3.582, p<0.0001) in BC tissues.This nding indicates that AKT1 gene and GRB2 gene can be a regulator of signaling pathway in BC progression.

Discussion
Our study indicated that miR-105-5p expression was up-regulated in BC tissues in comparison to NATs. that it was statistically signi cant.In some studies, it was observed that miR-105-5p acts as tumor suppressor.Honeywell et al. and Liu et al. found out that miR-105-5p inhibits prostate tumor growth and human glioma cell progression respectively and its reduced expression can lead to these types of cancers [10,30].The same result was found with non-small cell lung cancer [31].It was also revealed that in some studies, miR-105-5p acts as onco-miR.miR-105-5p expression level was increased in gastric cancer cells and colorectal cancer cells [9,32].Our results indicated miR-105-5p acts as onco-miR in BC tissue because the expression level of miR-105-5p is increased in BC tissue compared with NATs, and it is revealed that miR-105-5p may be relevant with cell proliferation.miRNAs have been known to have a role in regulating most biological processes and therefore, they can be used in order to gain an insight into the complex processes, including cell proliferation in cancer.Our bioinformatics studies showed that miR-105-5p targets AKT1 pathway and GRB2 gene.AKT signaling pathway has been a major signaling node within the cells [33].AKT1 pathway dysregulation could possibly lead to disruption of the balance between survival and death and it can also have a major impact on cancer development and therapy.Our results demonstrated that AKT1 expression level signi cantly decreased in BC tissue compared with NATs even the expression of this gene is more in the advanced stages of BC tissues.Our results agreement with previous studies.Heng et al. found out that AKT1 expression level was signi cantly lowered in breast tumor in comparison to matched adjacent normal tissues [34].We also observed miR-105-5p had negative correlation with expression of AKT1 in BC tissues.This result can be justi ed by Shen et al. 's nding in which they demonstrated that miR-105-5p can inhibit PI3K/AKT pathway in hepatocellular cancer [12].
GRB2 is an essential signaling mediator in various oncogenic signaling pathways and it has role-playing in several human malignancies [35].The GRB2 has a wide association with a number of irregularities.So, it is an important target for the design of remedial anticancer strategies [36].Lin-Yan et al. found that overexpression of GRB2 had correlation lymph node metastasis [37].Yu et al. demonstrated that GRB2 is over expressed in gastric cancer [22].Watanabe et al. found that GRB2 expression was signi cantly increased in human bladder cancer cell lines [38].Lim et al. found out that GRB2 downregulation led to AKT inactivation and consequently it caused BC [38].Our results revealed that GRB2 expression level signi cantly decreased in BC tissue compared with NATs.Its expression level lowered more in the advanced stages of BC.We demonstrated that miR-105-5p negatively modulated GRB2 by combining with the 3′-UTR of GRB2 gene.
Furthermore, another important aspect of this study showed that strong positively correlated between the expression levels of AKT1 and GRB2 genes.So, that downregulation of GRB2 genes suppressed the activation of AKT1 pathway.Lim et al. found that downregulation of GRB2 can lead to AKT1 inactivation and subsequently can cause lowered expression of AKT1 [39].These ndings support the pivotal role of AKT pathway in modulating the BC development.Based on our results, we speculate that miR-105-5p may contribute to the development of BC via downregulation of AKT1 and GRB2 genes.miR-105 affects the genes which are involved in intracellular signaling pathways and this leads to the downregulation of GRB2 and GRB2 downregulates AKT1 through p13k signaling pathway.Consequently, these alterations lead to more invasion and metastasis in BC.

Conclusion
Our ndings demonstrated miR-105-5p was up-regulated in human BC.miR-105-5p promotes cell proliferation in BC by targeting AKT1 and GRB2 genes.Up-regulation of miR-105-5p could promote BC growth by decreasing the activity of AKT1 signaling pathways and GRB2.
Our Insilco analyses showed that AKT1 and GRB2 are the direct targets of miR-105-5p and basically micro RNAs effect on the cell through regulating their target genes.Increased expression of miR-105-5p downregulate the expression of GRB2 and AKT1 and therefore these changes cause tumor progression and tumor invasion.These two genes are important genes in intracellular signaling pathways and reduced expression of them can dysregulate the cell cycle and cause tumor formation.Therefore, identifying a speci c biomarker, and gaining an understanding into the underlying mechanism might provide feasible therapeutic approaches to overcome cancer.By targeting miR-105-5p, it might be considered as a possible therapeutic strategy for BC treatment.However, the detailed mechanism of miR-105-5p in BC progression needs further investigations.
F.F. Participated in study design.F.R. and F.A. collected the samples.F.F, F.R. and F.A. participated in data collection and evaluation, drafting.F.F and F.R. participated in RT-qPCR analysis and statistical analysis.F.R. prepared lab working, and doing molecular experiments.F.F. and F.R. Contributed extensively in the interpretation of the data and the conclusion.All authors performed editing and approving the nal version of this paper for submission, also participated in the nalization of the manuscript, and approved the nal draft.

Figures Figure 1 miR
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Table 1 .
. Clinicopathologic characteristics of patients with BC -5p expression and clinicopathological characteristics of BC patients including histological grade, clinical stages and patients' age were examined.An increase in the expression level of miR-105-5p was observed in stage I and grade I compared with other grade (II -III) and stage (II-III).BC patients were divided according to age into the younger and older groups (women aged ≤50 and >50 years, respectively).In older women (aged >50 years) miR-105-5p expression was increased compared with younger women (aged ≤50 years) but no difference was found in the rate of miR-105-5p mRNA expression in according to age.In our study, statistical analysis showed that the miR-105-5p expression was not signi cantly related to clinicopathological features including histological grade, clinical stages and patients' age (p >0.05) (Table2).
Quantitative real-time PCR was used to evaluate the expression levels of miR-105-5p in breast cancer (BC) and paired normal adjacent tissues (NATs).We observed that there was a signi cant up-regulated in BC samples relative to that in NATs as the control group (Fig.1) (p=0.0194).This nding indicates that miR-105-5p may be a regulator of molecular mechanisms in BC progression.The relationship between miR-105

Table 2 :
Correlation between miR-105-5p expression and Clinicopathological Characteristics in Breast Various target prediction programs (TargetScan, miRBase, miRanda, mirDIP, miRWALK, and miRDB) were used to explore the potential mechanism and analyze the AKT1 and GRB2 target genes of miR-105-5p (ACESSION: MIMAT0000102).Based on the predicted genes, score class of AKT1 gene is top up 5% as high-throughput miR-105-5p targeting and Score class of GRB2 gene is top up 1% as very highthroughput miR-105-5p targeting.

Table 3 :
Correlation between AKT1 expression and Clinicopathological Characteristics in Breast Cancer

Table 4 :
Correlation between GRB2 expression and Clinicopathological Characteristics in Breast Cancer