Let-7d and miR-185 Impede Epithelial-Mesenchymal Transition by Downregulating Rab25 in Breast Cancer

Objective: MicroRNAs (miRNAs) expression has deregulated in several cancer types including breast cancer (BC). The present study aims at investigating the role, mechanism, clinical value of let-7d and miR-185 in BC, and the possible correlation these miRNAs with Rab25. Materials and Methods: Tumor samples as well adjacent normal tissues (ANT) were acquired from fresh surgical specimens from 110 patients and the expression levels of let-7d, miR-185, Rab25, and snail were evaluated using real-time PCR. The immunohistochemical (IHC) process and western blot were done to detect the level of Rab25 and Snail protein expression in BC samples. Results: By comparing miRNAs expression profiles in clinical tissues of 110 patients using real-time PCR, let-7d, and miR-185 expression were dramatically downregulated in BC tissues (P < 0.05). Tumor size, stage, and lymph node metastasis were significantly related to miRNAs expression. Based on qRT-PCR and bioinformatics database analyses, we also recognized Rab25 as a possible target of miR-185 and let-7d. Rab25 expression was enhanced in BC cells and associated inversely with the expression level of mentioned miRNAs. qRT-PCR, immunohistochemistry, and western blot studies verified that Rab25 upregulation increased the levels of the snail, that key transcription factor of epithelial-mesenchymal transition (EMT). Conclusion: These findings demonstrated that let-7d and miR-185 inhibited EMT by targeting Rab25 expression in BC. Therefore, targeting the let-7d and miR-185/Rab25 interaction may offer new therapeutic opportunities for treating BC patients.


Introduction
Breast cancer (BC) is the most prevalent malignancy among all women, and it is identified to be one of the deadliest cancers (Chatran et al., 2018;Jafari-Gharabaghlou et al., 2018;Rasouli et al., 2020). Despite the noticeable progress in the diagnosis of the early BC stage, most of the patients diagnosed with BC (~90%) die as a result of metastasis (Hosseini et al., 2016). Metastasis occurs through multi-step processes such as local invasion, migration, transport, and colonization (Montazeri et al., 2017;Mellatyar et al., 2018). One of the crucial factors for metastatic dissemination of cancer is a developmental process termed epithelial-mesenchymal transition (EMT) (Brabletz et al., 2018;Bahmanpour et al., 2019). EMT is a phenomenon in which cells lose their epithelial features and exhibit the migratory phenotype of mesenchymal cells (Aiello et al., 2018;Mittal, 2018). Snail, a master EMT-transcription factor, plays essential roles in multiple stages of embryonic development and is positively associated with tumor metastasis. Recent researches have highlighted that microRNAs (miRNAs) are important factors in EMT and cancer cell metastasis (Mohammadian et al., 2017a;Mohammadian et al., 2017b;Sheervalilou et al., 2017a). MiRNAs are short non-coding RNAs (containing ~22 nucleotides) that control target genes by sequence-specific binding with their 3′-untranslated regions (UTRs) (Mohammadian et al., 2016a;Mohammadian et al., 2016c;Sheervalilou et al., 2017b). Several reports have indicated that miRNAs interfere in several physiological and pathological processes such as cell growth, apoptosis, angiogenesis, and metastasis (Mohammadian et al., 2016b;Sheervalilou et al., 2016;Sheervalilou et al., 2019). Additionally, investigations have demonstrated the role of miRNAs in the metastatic cascade of BC. For example, miR-132 prevents proliferation and invasion of BC cells by targeting FOXA1 (Wei et al., 2018). MiR-200c suppresses metastasis of BC by decreasing Foxf2 (Zhang et al., 2017). MiR-19b-1 inhibits angiogenesis and migration by targeting VEZF1 in BC (Yin et al., 2018b). Thus, miRNAs and their targets can be considered a complex network wherein they can affect metastatic BC. According to studies, reduced expression of let-7d and miR-185 has been detected in different types of cancers, such as colon, lung, gastric and ovarian cancer (García-Vázquez et al., 2018;Jiang et al., 2018). Let-7d belonging to the let-7 miRNA family has been recognized as a tumor suppressor miRNA, most likely by suppressing the expression of the high mobility group A2 (HMGA2) or k-RAS (Wang et al., 2016). Furthermore, miR-185 regulates several types of cellular signaling that are necessary for the biological properties of tumor cells (Chai et al., 2017). However, the molecular mechanisms of these miRNAs in regulating EMT in BC remain largely unknown. Many studies showed that these miRNAs regulated various genes, which one of the most well-known ones is the Ras family. Rab25 (Ras-related protein Rab-25) is known as a molecular switch that is implicated in various biological processes and signaling pathways under normal and cancerous conditions (Mitra et al., 2016). In the current study, Rab25 was found to be a target gene of both let-7d and miR-185. Over-expression of Rab25 can counteract the effect of let-7d and miR-185, restore BC malignant phenotypes, and mediate its efficacy in BC progression that can be associated with Snail expression. Our findings suggest that let-7d, miR-185, and Rab25 might be appropriate tools for intervention development to improve BC diagnosis and treatment.

Tissue samples
Tumor samples as well adjacent normal tissues (ANT) were acquired from fresh surgical specimens from 110 patients who were treated at Noor-e-Nejat hospital, Tabriz, Iran from April 2016 to July 2018. The present research was ethically confirmed through the Ethics Committee of the Tabriz University of Medical Science (NO. IR.TBZMED.REC.1396.55). The clinicopathological data of these patients were collected with their signed written informed consent before inclusion in the current study. All fresh tissue specimens were gathered and instantly kept in liquid nitrogen at −80 °C. The histological studies were judged by two independent pathologists who were uninformed of the medical information of the corresponding patient.

RT-PCR analysis
Total cellular RNA was extracted from tissue samples according to the TRIzol reagent instructions (Invitrogen, USA). RNA was reverse-transcribed to complementary DNA (cDNA) by using QuantiTect cDNA synthesis kit (Qiagen, Germany) based on the manufacturers' recommendations. RT-PCR with real-time quantitation was carried out by SYBR Green Master Mix and assessed on a Roche light cycler 96 (Germany) in triplicate. The sequence and details of the specific primers used to RT-PCR amplification of let-7d, miR-185, Rab25, snail, and GAPDH, as a reference gene, are listed in Table 1. PCR cycling conditions was as follow: 1 cycle at 95 ºC for 5 min, followed by 40 cycles of 95 ºC for 20 seconds and 72 °C for 30 seconds to 1 min. All samples were performed in triplicate and findings were computed by using the 2 −∆∆Ct method .

Immunohistochemical staining
The immunohistochemical (IHC) process was done to detect the level of Rab25 and Snail protein expression. Tumor tissues were first fixed in 10% formalin-PBS buffer, embedded in paraffin, sectioned (4 μm thickness), and was mounted on glass slides. Then, diluted hydrogen peroxide (3%) was utilized to hinder the endogenous peroxidase activity. All the retrievals were performed in citrate buffer (pH 6·0) using microwave after that 1 h incubation with a mouse anti-Rab25 monoclonal antibody (1: 250, Abcam Inc, MA, USA) and rabbit anti-snail monoclonal antibody (1:100, Santa Cruz, Santa Cruz, CA, USA) overnight at 4°C. Negative controls were also obtained by the omission of specific primary antibodies. Then, tissue specimens were incubated with anti-mouse and the anti-rabbit secondary antibody, conjugated to HRP, at ambient temperature for 30 min. 3,3′−diaminobenzidine (DAB) tetrahydrochloride substrate was utilized for about 5 min to color development. Samples were counterstained with 0.1% hematoxylin and, finally, IHC stained slide investigated by a light microscope .

Western blotting
We performed western blot to identify Rab25 and Snail protein expression in BC samples. The protein content of tissue lysates was separated using 10% SDS-polyacrylamide gels and moved to polyvinyl difluoride membranes (Roche Diagnostics GmbH). Once being blocked with 5% defatted dry milk, the membranes were incubated with the primary antibodies against Rab25, Snail, and GAPDH at 4˚C overnight. Afterward, the immunoreactive proteins were incubated with HRP-conjugated secondary antibodies (1:3,000, Razi Biotech Co, Tehran, Iran) at ambient temperature for 2 h and then visualized by using ECL detection Kit (Bio-Rad). The intensity of each western blot band was counted via ImageJ, version 1.44 software (National Institutes of Health, Bethesda, USA) and normalized to the respective GAPDH loading control (Sadeghi-Soureh et al., 2020).

Statistical analysis
The statistical data analysis was conducted applying the relative level of let-7d and miR-185 expression in 110 specimens of BC and ANT tissues. Let-7d and miR-185 were suppressed 4 and 1.8-fold, respectively, in BC compared to ANT (both P < 0.05) ( Figures 1A and B).
SPSS 16.0 (Chicago, IL, USA) or Graph Pad Prism 6 (San Diego, CA, USA). Comparisons between the difference in means values among mRNA levels of BC and ANT samples were determined using the Mann-Whitney U test. One-way analysis of variance (ANOVA) was applied to assess the statistical differences in mRNA expression levels between the different histologic tumor grades. The statistical relationship among mRNA or protein levels of each group and clinicopathological features were analyzed using a Chi-Square, Student's T-test or Mann-Whitney test. p-value <0.05 was considered to show a statistically significant difference.

Let-7d and miR-185 expression was decreased in BC samples and was associated with clinicopathological criteria
We investigated the potential effect of let-7d and miR-185 in BC development. We first examined the Gene Primer sequences

Rab25 and Snail expression was increased in BC samples and was associated with clinicopathological criteria
The protein levels of Rab25 and Snail were detected by the IHC staining intensity in both BC and paired ANT tissues. In this case, Rab25 was expressed primarily in the cytoplasm, along with minimal expression in the nuclei of BC cells (Figure 2A). On the contrary, Snail staining was localized predominantly in the nucleus, and no positive staining was detected in the cytoplasm of BC cells. ( Figure  3A). The positive expression rates of Rab25 and Snail expression in BC tissues were 82.7% (91/110) and 85.4% (94/110), respectively, is markedly higher than those of the relevant ANT (19%,21/110) and (35.4%, 39/110). To confirm IHC results, we also conducted quantitative-PCR to examine the Rab25 s and Snail mRNA expression level in all BC and ANT cases. The level of Rab25 and Snail mRNA was higher in the BC group than in the ANT group (both P =0.001) (Figures 2Band 3B). Table 3 summarizes the associations of Rab25 and Snail protein expressions with various clinicopathological criteria. Our results indicated that Rab25 overexpression was associated with the tumor size (P = 0.021), lymph-node metastasis (P = 0.032), histological grade (P = 0.047) and pathological stage (P = 0.038) of BC. Besides, we detected a positive association between the Snail expression level and the tumor size (p=0.036), pathological stage (P =0.049), and lymph node metastasis (P =0.018). It is worth noting that similar results were found when the relationship was investigated between mRNA expression levels and clinicopathological parameters. Table 2 shows the data. The results showed that the Rab25 and Snail expression in BC tissues was significantly higher than that in ANT tissues. To confirm the results of IHC and qPCR analyses and to further investigate the effect of Rab25

Rab25 expression is positively correlated with the Snail expression in BC tissues
The Spearman's rank correlation coefficient was used to examine the relationship between Rab25 and snail protein expression levels. As Table 4 shows, Rab25 protein expression is positively correlated with the expression level of Snail. Out of sixty-seven Rab25 positive (++/+++) cases, fifty-two (77.6%) BC patients positively expressed  Snail (++/+++), while the co-expression of Rab25 and Snail was detected in two (25%) of the eight Rab25 positive cases in ANT tissues. Moreover, from sixteen BC samples with negative staining (−/+) for Snail, fourteen (87.5%) patients indicated IHC negative (−/+) for Rab25. Our finding revealed that the Rab25 and Snail expression levels were significantly associated with each other in all BC (r = 0.513, p =0.011) and ANT samples (r= 0.263, p = 0.046).

Correlation between let-7d and miR-185 and Rab25 in BC tissues
Recent studies have reported that the reduction of some miRNAs was found to be related to higher levels of    (Shahabi et al., 2019;Yan et al., 2019). First, to ascertain the miRNAs targeting Rab25, we excavated target genes through two bioinformatics databases (miRDB and Target Scan) and provided a list of miRNAs targeting Rab25. Afterward, data mining techniques were performed, and miRNAs that significantly implicated the development in BC progression and metastasis phenotype were selected.
According to the findings, we found that Rab25 might be one of the target genes of let-7d and miRNA-185. Moreover, the qPCR analysis showed that diminished expression of these miRNAs significantly downregulated the Rab25 expression in BC tissues rather than in ANT (Figures 4Aand 4B). Correlation analysis of let-7d, miR-185, and Rab25 expression in BC tissues demonstrated an inverse relationship between declared miRNAs and Rab25 ( Figure 4C). Overall, these results suggest that Rab25 is downregulated by let-7d and miRNA-185.

Discussion
miRNAs play vital roles in regulating the expression of cancer-related genes expression and are implicated in cancer development and progression (Kafshdooz et al., 2018;Tiwari et al., 2018;Norouzi et al., 2019;Sheervalilou et al., 2020). Previous studies have indicated revealed that the let-7d level is strongly down-regulated downregulated in various cancer cells. Let-7d directly targets c-Myc, HMGA2, CCL7, PBX3 and COL3A1 (Ramberg et al., 2011;Su et al., 2014;Ye et al., 2014), and so inhibits thereby inhibiting cancer cell invasion and metastasis. Another reports report found that miR-185 was able to regulate cellular proliferation, as well as DNA-damage response and repair (Wang et al., 2013;He et al., 2016). The relative expression of let-7d and miR-185 are lower in a variety of various cancer tissues. Besides, low expression of the mentioned miRNAs is related to large tumor size and advanced metastatic cancer (Wei et al., 2018). Consistent with previous reports (Fu et al., 2014;Wei et al., 2018), we discovered that let-7d and miR-185 expression was markedly reduced in BC tissues versus than in their paired ANTs, and proven both serve served as a tumor suppressor gene in BC. EMT is considered an important molecular event in tumor invasion and migration (Jolly et al., 2017). It is now well known that miRNAs can target the EMT regulatory factors to promote or inhibit EMT in BC cells . For instance, miR-497 suppresses EMT and metastasis of BC cells by targeting SMAD7 (Liu et al., 2016). MiR-125b negatively regulates EMT and influence influences BC invasion and migration by targeting MAP2K7 (Hong et al., 2016). Other miRNAs, such as miR-206, miR-381, miR-520c-3p, miR-143, and miR-153 inhibit the EMT-related BC and exhibit anticancer properties via several target genes and signaling pathways (Li et al., 2015;Zhai et al., 2016;Tang et al., 2017;Xue et al., 2017). Our results prove that Let-7d and miR-185 reverse EMT in BC cells. Rab25 has been implicated in diverse diseases, many of which are linked to the hijacking and use of intracellular trafficking pathways though through types of pathogens (Cheng et al., 2004). Additionally, overexpression of Rab25 increased the tumorigenicity and invasiveness in BC cancer (Wang et al., 2015). Our findings indicate that Rab25 stimulates the EMT-related cell invasion of BC. In accordance with our findings, Yin et al., (2018a) also exhibited unraveled that downregulation of the level of Rab25 in BC cells substantially suppressed suppresses the invasive motility and is related to the induction of vimentin and reduction of E-cadherin, which are the key markers of EMT). It is well known that Obviously, miRNAs could negatively regulate target genes via binding to 3′-UTR mRNAs, and let-7d and miR-185 downregulated downregulate the Rab25 expression. In conclusion, this study implied demonstrated that let-7d and miR-185 downregulation was associated with markedly significantly increased Rab25 expression, thereby reinforces reinforcing the BC invasion and metastasis via the snail-driven EMT. These results describe the mechanism underlying the tumor suppressor functions of let-7d and miR-185 and demonstrate that the signal pathway let-7d and miR-185/Rab25 might be a potential target for BC treatment.
In conclusion, this study demonstrated that let-7d and miR-185 downregulation was associated with significantly increased Rab25 expression, thereby reinforcing the BC invasion and metastasis via the snail-driven EMT. These results describe the mechanism underlying the tumor suppressor functions of let-7d and miR-185 and demonstrate that the signal pathway let-7d and miR-185/ Rab25 may be a potential target for BC treatment.

Funding
This study was financially supported by grant No: 960205 of the Infectious and Tropical Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

Ethical approval
All procedures performed in studies involving human participants were in accordance with the ethical standards of the Ethics Committee of Tabriz University of Medical Science (NO. IR.TBZMED.REC.1396.55) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Author's contribution
NZ and BN designed and directed the project; AS and VM processed the experimental data and performed the analysis, drafted the manuscript and designed the figures; NZ, BN and KA contributed to the final version of the manuscript. All authors provided critical feedback and helped to shape the research, analysis and manuscript.

Informed consent
Informed consent was obtained from all individual participants included in the study.