LINC02544 modulates the RAB/RAS signaling pathway by potential regulation of DENND2A expression level in the breast cancer patients: integrated bioinformatics and experimental analyses

doi:10.21203/rs.3.rs-2125671/v1

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LINC02544 modulates the RAB/RAS signaling pathway by potential regulation of DENND2A expression level in the breast cancer patients: integrated bioinformatics and experimental analyses

https://doi.org/10.21203/rs.3.rs-2125671/v1

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Background: One of the most common female malignancies is breast cancer (BC) and is recognized as a second death factor for women population among other cancer-related diseases. Dysregulation of RNA expression levels can lead human status to some different pathological statuses, including breast cancer. In this study, we have investigated the expression level of DENND2A and lncRNA LINC02544 in the BC tissue samples among Iranian population. Also, the clinicopathological and biomarker analysis of these RNAs were investigated.

Method: Microarray analysis was performed on GSE61304 in order to find the differentially expressed genes in the BC samples compared to controls. Multiple online and offline software, including R programming language and several relative statistical and visualization packages, demonstrated the differential expression level of genes,relative lncRNAs, and single nucleotide polymorphisms. The Real-time PCR results have demonstrated that the expression level of DENND2A has been reduced in the breast cancer samples compared with controls. In order to investigate the genotype frequency of rs6852 region in the DENND2A gene, high-resolution melt (HRM) method has been used.

Results of microarray and Real-time PCR analysis have been shown that the expression of DENND2A and lncRNA LINC02544 have significantly down-regulated in the breast cancer samples. These data suggested that these two RNAs are likely to respond to alter the expression level of each other.

Therefore, DENND2A could be an important prognostic biomarker in breast cancer samples among Iranian population. lncRNA LINC02544 could have an activator effect on the DENND2A expression. There is strong evidence that both of these RNAs could perform as tumor suppressors in breast cancer.

Molecular Biology

Bioinformatics

SNP analysis

DENND2A

lncRNA

RNA Interactions

Over the last 50 years, most cancer investigations have been dedicated to examining the differences in the expression of several genes in tumor cells compared to normal cells[1]. These differences could have crucial effects on different human diseases, like hepatitis[2], alzheimer [3], diabetes[4], MS[5], and different types of human cancers, including hepatocellular carcinoma[6], retinoblastoma[7], lung cancer[8], head and neck squamous cell carcinoma[9], and breast cancer (BC)[10]–[13]. The extensive research into gene expression profiles in various pathogenic conditions - including BC - can accelerate the treatment of these diseases and help predict the occurrence of these situations.

Among the various human cancer types, BC is the most common female malignancy and the leading cause of cancer mortality in women. Worldwide more than 2.1 million females are diagnosed with breast cancer annually, with about 62000 death, making it the most frequent female malignancy and the leading cause of cancer mortality in women. Nearly 60% of BC death occurs in developing countries[14]. While the success in early screening and recent advancements in anti-cancer therapies could bring changes in the key outcomes of breast cancer patients[15], [16], the recurrence rate of BC remains significant[17]. Accordingly, measuring the expression of genes associated with BC, identifying the diagnosis and prognosis biomarkers, and understanding the patterns of expression of different genes in various clinical and pathological conditions related to BC, can contain valuable information about BC and help prevent the disease as well.

A variety of tools have been developed to measure gene expression. Among them, real-time PCR and microarray are two essential methods for measuring gene expression. Analysis of DNA microarray results is really helpful in profiling gene expression and genome-wide gene expression. In addition, these mentioned tools have given valuable information about the expression level and expression of relative genes and RNAs in multiple groups, like "tumor" and "control" or "treated" and "untreated"[18].

Among several genes involved in BC, DENND2A is considered for investigation in this study based on microarray data analysis. DENND2A is a member of DENN domain family that binds to actin filaments. DENN domain-containing proteins which interact directly with Rab family small GTPases. And also, DENN domains have enzymatically function as Rab-specific guanine nucleotide exchange factors[19]. This gene located on chromosome 7, is involved in the Vesicle-mediated transport signaling pathway. The role of this protein-coding gene in breast cancer is still unknown. According to the microarray analysis, DENND2A possibly has a tumor suppressor effect in BC by significantly reducing expression. Therefore, in this research the relative gene expression of DENND2A was evaluated by a real-time PCR experiment for a reliable and accurate understanding of the role of DENND2A in BC in Iran. Also, the genotype frequency of rs6852 single nucleotide polymorphism was examined in order to understand the role of this SNP in the different expression levels of DENND2A in BC samples.

LncRNAs are a type of RNA molecule that is longer than 200 nucleotides and found in the nucleus or cytoplasm. The rest of them do not code any proteins, and only a handful of them can encode a small number of polypeptides. LncRNAs regulate a wide range of biological processes, including tumor invasion, metastasis, and apoptosis, at pre-transcriptional and also transcriptional stages. As a result, lncRNA irregularities in BC patients' peripheral blood can promote breast cancer development, which leads to early diagnosis and treatment. The regulatory effects of lncRNAs on malignancy activities in tumorigenesis have been confirmed in numerous studies[20]–[22]. Another important aim of this study is to understand the expression pattern of LINC02544 as a correlated lncRNA with DENND2A in breast cancer samples among Iranian populationan. In addition, they were finding the experimental correlation result of these two RNAs based on their expression data.

3.1 Microarray analysis

58 BC tumors and four control normal microarray samples in the GSE61304 dataset have been analyzed by R Studio (4.0.2). By means of the GEOquery package, the microarray data has been provided by GEO online database. Statistical analysis and differential gene expression evaluations have been performed by Limma package. Pheatmap and ggplot2 packages were used for drawing microarray plots. Log FC bigger than one and smaller than -1 was considered as a relative expression threshold. Adjusted P. Value < 0.05 was considered as a significant level.

3.2 Bioinformatics analyses

Using the Co_LncRNA database, which shows the coefficient between gene and LncRNA based on TCGA database in BC compared with normal control. The following criteria were applied, the connection between lncRNA and selected genes based on the Spearman Rank Correlation statistical method, with a coefficient below two and a p-value of less than 0.01 as significant. The literature mining was conducted to collect the most effective lncRNAs as pathogenic factors in BC. Also, all of the SNPs that were located in 3UTR in genes that such variation could result in gain or loss of function of miRNA hybridization were studied with the miRNASNP database. Survival analysis was performed by GEPIA2 online software based on TCGA RNA-seq data. Protein-protein interaction analysis was performed by STRING online database.

3.3 Clinical features of tissue samples

Tissue samples were collected from 36 patients who were diagnosed with BC from the Alzahra hospital in Isfahan and the Dey hospital in Tehran. The collection was enrolled for the study after signing an informed consent approved by the Ethics Committee (IR.MUBAM.REC.1398.032). BC and control samples are paired. Samples were transferred to the lab with Liquid nitrogen in sterile conditions.

3.4 Real-time PCR experiment

Total RNA from tissues was extracted by adding 1.0 mL Trizol (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's protocol. The RNA concentration was estimated using Nanodrop Spectrophotometer (ND-1000, ThermoFisher, MA, USA). Purified RNA was stored at -70C for further steps.

RNA was reverse transcribed into cDNA using a reverse transcription kit (TAKARA cDNA Synthesis Kit, Tokyo, Japan) based on the manufacturer's protocol. The cDNA synthesized was stored at -20 C.

All sequences were downloaded from NCBI, and primers were designed using Gene Runner software (table 1). Primer-BLAST web tool was applied for further analysis of primer specificity. Also, GAPDH mRNA is used as a housekeeping gene. Primers were ordered from TAQ Copenhagen Company (Denmark). The Magnetic Induction Cycler (MIC) was used to perform the qRT-PCR process (Biomolecular Systems, Australia).

3.5 HRM

Total DNA was extracted with a Genetbio kit (Korean) from tissue samples. Then High-resolution melting technique was done with mic Real-time PCR, and EVA green Master Mix (Solis BioDyne Co., Estonia) was used for genotyping.

Table 1: sequence of primers

Gene/Lnc RNA/SNP name	Primers	Sequense
DENND2A	Forward Reverse	5'-GTC CAC CCT TCC TGA GAA C-3' 5'-CGA GCC CCT ATA CAC GTT C-3'
LINC02544	Forward Reverse	5'-CAG AAA ATC ACG GTG GGC AC-3' 5'-TGT CTA CAT CAA CGC CTG TCC-3'
GAPDH	Forward Reverse	5'-GCT CTC TGC TCC TCC TGT TC-3' 5'-ACG ACC AAA TCC GTT GAC TC-3'
rs6852	Forward Reverse	5'-GTCTGCCTTGTGAAATGTTCTC-3' 5'-AGTTCCGCACCGTGACAAC-3'

3.6 Statistical analysis

Statistical analysis of real-time PCR data was performed by GraphPad Prism (version 8) software using the -ddCt method. The expression plots were drawn by GraphPad software. The Kolmogorov-Smirnov test was performed to check the normality of the expression data. Wilcoxon test and paired t-test was done with the expression data to examine our results and compare the control with tumor samples, respectively. Genotype frequency analysis was done by SPSS software. P. Value < 0.05 was considered the significant level. To examine the correlation between the expression level of two mentioned RNAs, Pearson correlation analysis was used.

4.1. Results

4.1.1. Bioinformatics analyses

Microarray analysis on GSE61304 dataset was performed (Fig. 1, 2). DENND2A has a significant down-regulation in BC samples compared to normal was shown in differential expression analysis. (logFC = -1.9, adj.P.Value < 0.05, Fig. 3). This analysis revealed that DENND2A could be a significant tumor suppressor for BC. Survival analysis displayed that the low expression of DENND2A could reduce the survival rate of BC patients (Fig. 4).

Protein-protein analysis revealed that DENND2A has a remarkable interaction with RAB9B, GDI1, GDI2, CHM, and FAM19A1 (Fig. 5).

According to the results of the Co-lncRNA online database, on the one hand, LINC02544 could have a significant regulatory effect on DENND2A mRNA. On the other hand, miRNA SNP analysis revealed that rs6852 SNP in the DENND2A plays a noticeable role in proportion of tumor cells and pathogenicity in breast cancer. According to mentioned online software analysis, binding of hsa-miR-186-5p to the rs6852 region of DENND2A can cause a reduction of DENND2A expression level in tumor cells. In this case, higher expression of miRNA has directly led to lower DENND2A expression in tumor cancer compared to control groups.

4.1.2. The relative expression level of DENND2A and LINC02544

According to real-time PCR relative expression analysis, DENND2A and lncRNA LINC02544 have a slower rate of expression in BC tumor cells compared to normal cells (Fig. 6). ROC analysis exposed that DENDD2A mRNA may be an effectual prognostic biomarker (Fig. 7).

Pearson correlation analysis revealed that DENDD2A and LINC02544 have a strong positive correlation in tumor samples. (Fig. 8).

4.1.3. Clinicopathological analysis

The expression level of DENND2A and LINC002544 in the different clinicopathological statuses such as stage, lymph node metastasis, age, Menopausal status, PR receptor, ER receptor, and HER2/neu receptor were examined, which suggested that the expression level of LINC02544 has down-regulated in the lymph node-negative samples and in the patients who reached the menopausal age compared to normal samples (Fig. 9).

4.1.4. Genotype-frequency analysis

Analysis of genotype frequency for rs6852 exhibited that there was no difference in rs6852 genotype frequency in BC samples compared to normal (Table 2, Fig. 10).

Table 2

Genotype-frequency table.
			Genotype
			CC	AC	AA	Total
Group	Tumor	Count	66	15	36	117
		% within Group	56.4%	12.8%	30.8%	100.0%
	Control	Count	78	26	56	160
		% within Group	48.8%	16.3%	35.0%	100.0%
Total		Count	144	41	92	277
		% within Group	52.0%	14.8%	33.2%	100.0%
P. Value	0.435
not consistent with Hardy-Weinberg equilibrium

4.2. Discussion

To obtain more accurate and reliable results about the gene and lncRNA expression patterns, we have used a high-throughput bioinformatical analysis to find a differentially expressed hub gene. Based on our microarray analysis, DENND2A has showed a significantly down-regulated expression. Microarray analysis has also demonstrated that the expression of DENND2A has co-expression with relevant lncRNAs (LINC02544 ). We designed the real-time PCR based on the result of bioinformatics analyses, to find out the relative expression level of these two RNAs.

This experiment revealed that DENND2A could be considered as a strong diagnostic biomarker (AUC = 0.8151). Based on the Pearson correlation test, we have understood that DENND2A and LINC02544 expression levels have a remarkably positive correlation with each other. These results can be interpreted as a meaning that these RNAs can promote breast cancer status by affecting each other's expression patterns and disturbing the transcription and translation of opposite RNA. In addition, we have observed that lncRNA LINC02544 induced Lymph node metastasis and affected menopausal status. These findings could prove our hypothesis about the expression level of DENND2A and LINC02544 and the correlation of these RNAs with breast cancer progression and survival rate of patients in our samples.

It is important to note that previous studies revealed that DENND2A could have a key role in Hepatitis B [23] and non-small cell lung cancer[24]. However, the main role of DENND2A in breast cancer remained unclear. Nevertheless, based on previous studies, LINC02544 was over-expressed in breast cancer[25]. Our findings about the expression pattern of LINC02544 in the Iranian population were the opposite of previous studies about this lncRNA.

We had considerable limitations in finding and analyzing breast cancer samples. To achieve more reliable results, we would like to suggest that the same experiments will be performed on this mRNA and lncRNA but on a bigger Scale. Also, it is possible that due to the small number of samples, the results of our polymorphism analysis were non-significant. So, it is suggested that the genotype frequency analyses and different genotype model analyses will be performed on BC samples with a bigger Scale. Heterozygous, dominant, co-dominant, and recessive genotype models analyses could give us valuable information about the role of rs6852 SNP and the effect of this polymorphism on the expression level of DENND2A and breast cancer progression as well. According to the available evidence, this gene and LncRNA may play a role in causing cancer through the RAB/RAS pathway [19].Therefore, it is suggested that the expression level of RAB9B, GDI1, GDI2, CHM, and FAM19A1 mRNAs and those correlations be analyzed. According to our protein-protein interaction analysis, these proteins have a high score interaction with DENND2A protein. In all, the transcription level of these mRNAs in tumor samples and differences in their expression level of them could give us crucial data about the impact of these genes on breast cancer progression. It also provides researchers with a new and effectual diagnostic and therapeutic strategy.

DENND2A gene and LINC02544 LncRNA have a significantly low expression in Breast cancer patients as compared to normal cases in the Iran population. Therefore, changes in gene expression and LncRNA in BC samples can act as biomarkers to identify this disease. But since DENND2A and LINC02544 may have an effect on other cancers, it is suggested to check their expression level in other cancers.

Authors contribution

Maria Nezamnia, Sayedeh Zahra Shirdeli, Fatemeh Sobhani, and Mina Moein equally contributed to this research as the first authors.

Data Availability

The data are provided by the responsible author upon justifiable request.

Conflicts of Interest

The authors declare that there is no conflict of interest regarding the publication of this paper.

Funding Statement

In this scientific research that was carried out, no financial assistance was received from financial, commercial, public, or non-profit organizations.

Acknowledgments

We wish to extend our special thanks to Mr. Mohammad Rezaei for helping with data analysis and bioinformatics.

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LINC02544 modulates the RAB/RAS signaling pathway by potential regulation of DENND2A expression level in the breast cancer patients: integrated bioinformatics and experimental analyses

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Abstract

Figures

Introduction

Materials And Methods

Results And Discussion

4.1. Results

4.1.1. Bioinformatics analyses

4.1.2. The relative expression level of DENND2A and LINC02544

4.1.3. Clinicopathological analysis

4.1.4. Genotype-frequency analysis

4.2. Discussion

Conclusions

Declarations

References

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