Background
Breast cancer is one of the most common cancers in women worldwide and is the leading cause of female disease-related mortality [
1,
2]. The most popular forms of breast cancer therapy were surgery, chemotherapeutic, and radiation therapy, considerably increasing therapeutic advantages [
3]. Based on numerous research, breast cancer regeneration and survival have been connected to stem cell characteristics [
4]. A subtype of malignant cells involved in the malignancy may be called cancer stem cells (CSCs) cells. CSCs might self-renew, differentiate in many directions, continue to divide indefinitely and heal malignancies. They were connected to radio-chemotherapy resistance, recurrence, cancer growth, multiplication, invasion, and metastasis [
5]. Breast cancer stem cells (BCSCs) have been shown to have a significant role in the ability of cancer cells to reproduce and self-renew [
6].
Reactive oxygen species (ROS) are involved in various biological systems as cytoplasmic signalling agents [
7]. NADPH-oxidases regulate reactive oxygen species (ROS) production [
8]. NADPH oxidases play various physiological roles, including cellular division, renal function control, and microbial immunological response; however, tumors are more likely to express them excessively than normal [
9]. According to current research, NOX4 is a produced NOX variant crucial for synthesizing H2O2 and is a member of the NOX class [
10]. A few studies demonstrate that NOX4 promotes cell growth through cell cycle regulation and apoptosis suppression, even if the methods through which NOX4 influences the proliferation, invasion, and survival of cancer cells are still unclear [
11,
12].
Genome editing methods are now possible because of developments in molecular biology, allowing us to alter genomes and investigate how genetic modifications affect organisms’ operations [
13]. Treatment for cancers impacting the body’s healthy cells has been carried out in various ways. Molecular biology research must continue to create effective cancer and oncology treatment strategies [
14]. The Clustered Regularly Interspaced (CRI) protein system has recently been linked to Short Palindromic Repeats (CRISPR), a powerful new therapeutic method with great precision and efficacy for treating cancer, have been adopted by the national cancer institute to lower the number of fatalities brought on by cancer [
15]. Numerous studies were planned and conducted to investigate the function of NOX4. Three different study kinds existed. Two antisense oligonucleotides were used to degrade produced RNA: RNA interference (RNAi) and antisense genomic regions. The second category includes CRISPR-mediated inhibition (CRISPRi) and activation (CRISPRa), which interact with the regulatory regions of the gene to control its degree of expression [
14]. Genes in the third group were deleted using adaptable nucleases like CRISPR/Cas9 (CRISPR-associated protein-9 nuclease), transcription activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs). Numerous parameters were recommended [
14,
15] for the ideal combination of a laboratory experiment for a particular transcription.
The main topics of these criteria were the targeted gene’s location and relationship to other genes, the positioning of its regulatory regions [
15,
16], and biological materials’ sub-cellular location. To investigate a NOX4’s function, partial transcript reduction caused by the method’s structure, the gene’s nuclear location, and possible off-target effects were at least two limitations [
17,
18]. The CRISPRi and CRISPRa techniques may not be able to inhibit or promote gene transcription when the expression of the target gene is regulated in a complex manner [
19,
20]. The CRISPR/Cas9 dual genotype homology-independent targeted integration method used a targeting segment to replace the genomic region between two Cas9 double-strand breaks (DSBs). The biological roles of NOX4 in breast cancer development and the underlying biochemical mechanisms remain a mystery. The oncogene NOX4 impacted the tumor’s poor prognosis. The histone modification profile on p57 was dictated by an interaction between the histone methyl-transferase EZH2 and NOX4 [
21]. It also functioned as an oncogene in cancer and pancreatic ductal adenocarcinoma [
21‐
23]. After screening and assessing the situation, we decided on the NOX4 as the target. In the current investigation, NOX4 was discovered to be a carcinogenic agent in the growth of breast cancer tumors. In the current investigation, a CRISPR/Cas9 version was employed. This method replaced the genomic region between two Cas9-induced double-strand breaks (DSBs) in each genotype with the targeted segment.
Materials and methods
Search strategy and study selection
From April to June 2021, we read relevant and related publications in Medline, EMBASE, Web of Science, and Wanfang. The search term “NOX4” have been used. The articles that fit specified criteria were all added at once: (1) Participants were split into two groups according to their NOX4 transcription quantities (2) Risk ratios with 95% confidence intervals (CIs) for the association among NOX4 transcription and mortality percentages (3) The papers were published in English. The following conditions were not recommended:
-
Research studies that were letters, abstracts from conferences, meta-analyses, or review publications.
-
Research papers that used The Cancer Genome Atlas (TCGA) databases to analyze the prognostic importance of NOX4 level of transcription.
-
Research papers that used the same subjects.
Data collection and quality assessment
Duplicate studies were excluded according to the method of Kwon et al. (2015) [
24]. Two writers independently obtained data from eligible research. All of the authors discussed any disagreements and agreed. The following information was retrieved: first author, time of publication, geography, cancer type, therapeutic classification, statistical significance, detection method, quality controls, and cut-off parameters; HRs for OS with 95% confidence intervals; logistic regression type; and follow-up duration. The multivariate HRs were picked first when HRs were obtained using both univariate and multivariate assessments because they had fewer confounding factors. If not explicitly stated in the paper, HRs with 95% CIs would be produced from Kaplan-Meier survival graphs using the Engauge Digitizer tool. The high and low NOX4 expression cutoffs used for the survival analysis were 1.92. The qualitative aspects of the research were evaluated using the Newcastle-Ottawa scale (NOS) criteria, which employed a star grading scale from 0 to 9 [
25].
Validation using data that is readily available to the public
The Cancer Genome Atlas (TCGA) publication standards were used in this study. Gene Expression Monitoring Interactive Modeling evaluated the relationships between NOX4 transcriptional level and OS and DFS (GEPIA). The logistic regression was computed using the K-M approach and log-rank analysis, and the HRs and p values were shown in the K-M curve diagrams [
26].
Detection of expression levels and co-expression of apoptotic genes
To identify the genes expressed differently to examine the significance of gene abnormal expression in breast cancer, reads were mapped to the reference genome (human: GRCh38) using CLC Genomics Workbench Version 21 allowing two mismatches to be utilized to identify the genes expressed differently to examine the significance of gene abnormal expression in breast cancer. The information for invasive breast carcinoma, comprised 16,137 samples and downloaded the RNA expression counts, RNA expression counts were collected from The Cancer Genome Atlas (TCGA) from the cBioPortal for Cancer Genomics (cbioportal.org) can compare white our data. The HUGO Gene Nomenclature Committee’s database provided the gene names and symbols (genenames.org). All the genes involved in the apoptosis pathway were initially obtained from the QIAGEN website (
www.qiagen.com). It was done in the manner that gene expression was assessed in breast cancer to examine the co-expression of apoptotic genes highly expressed in breast cancer. Plots of the apoptotic genes with high expression and the gene expression network were made.
Identification of miRNAs and genes with variable expression
The differentially expressed genes were predicted using a bioinformatics technique to examine aberrant gene expression’s function in breast cancer. The HUGO Gene Nomenclature Committee’s database was used to get the gene names and symbols (genenames.org). The database for invasive breast carcinoma, comprising 1,105 samples, was collected from The Cancer Genome Atlas (TCGA) from the cBioPortal for Cancer Genomics (cbioportal.org). The retrieved genes can be input, and the data can be submitted by choosing the cancer study and genomic profiles. A similar method might be used to choose miRNAs. In the supplementary material, the exact method of selecting and identifying miRNAs was described.
Analysis of gene-miRNA interactions
The binding of genes to miRNAs was predicted utilizing bioinformatics software RegRNA 2.0 (regrna2.mbc.nctu.edu.tw/detection.html) to determine the miRNAs that could target genes. The National Center for Biotechnology Information’s GenBank database (ncbi.nlm.nih.gov/genbank) was used to find the protein sequence. The system value was also set to > 160, and the minimum folding free energy was adjusted under 20 to predict the miRNA target locations. A higher score showed a better capacity to bond. The selection of genes dropped above 3% modification frequency due to the vast number of genes differentially expressed in breast cancer. Additionally, the NCBI database was used in advance to look for the gene sequences connected to Homo sapiens.
Identification of miRNA targets and creation of the gene-miRNA mRNA network
Mirtarbase databases’ miRNA testing may be used to determine which genes to target (
http://mirtarbase.mbc.nctu.edu.tw/php/index.php). Genes discovered by utilizing this database were utilized. To enhance the network diagram’s clarity, it was also validated that the NOX4 gene showed High expression. Utilizing the Cytoscape program (version 3.7.1; available for free at
http://www.cytoscape.org/download.php), the NOX4-miRNA mRNA association network was created. In addition to the expression network, genes associated with apoptosis were examined.
Examination of the Gene Ontology (GO)
The target gene underwent GO enrichment using the GOrilla tool to examine better the biological impact of abnormal expression levels and miRNAs in breast cancer (cbl gorilla. cs. Technion.ac.il). A list of related genes is returned for each GO word, with the best-performing genes listed first. Each gene name is followed by a brief description of the gene and the gene symbol [
27].
B. In-Vitro analyzes
Cell Culture
The cells were cultured per ATCC recommendations after receiving them from Iran’s National Cell Collection (Pasteur Institute, Iran). There were used two distinct breast cancer cells. MCF-7 and MDA-MB-231 cells were grown in Dulbecco’s modified Medium containing (DMEM; Gibco) enhanced with 10% FBS (Gibco, USA), 50 U/ml penicillin, and 50 µg/ml streptomycin at 37 °C in 5% CO2 (Sigma-Aldrich, USA). The Control group consisted of regular human breast epithelial cells (HMEC). The human breast epithelial cells (HMEC) were purchased from Iran’s National Cell Collection (Pasteur Institute, Iran).
DNA Development and Gene Targeting
The NOX4 gene was silenced in human breast carcinoma cell lines using the CRISPR/Cas9 technique. The GenBank sequence database of the National Center for Biotechnology Information contains the NOX4 gene sequence (National Biosciences, Inc., Plymouth, MN). To create single guide RNA (sgRNA) sequences that targeted various regions of the NOX4 gene, the specifically designed CRISPR CHOPCHOP websites (
https://chopchop.cbu.uib.no) and (
http://crispr.mit.edu/) were utilized. Three vectors were constructed: pX459 (which contained the U6 promoter-sgRNA intubation site-sgRNA scaffold and the CAG promoter-Cas9-T2Apuromycin N-acetyltransferase gene-bovine hormonal polyadenylation signal); pX460-1 (which contained the U6 promoter-sgRNA intubation site-sgRNA scaffold and the CAG promoter-enhanced GFP); sgRNA-encoding oligonucleotides with steaky ends were generated (Macrogen Inc., South Korea), synthesized, phosphorylated, and inserted into BbsI-digested and gel isolated carriers to accomplish this purpose (using Gel Extraction Kit; DENAzist Asia Co., Iran).
In “CRISPR du-HITI” approaches plasmids, the PAM domain was added following the sgRNA coding portion. DsRed2, the herpes simplex virus thymidine kinase polyadenylation signal, the CMV promoter, PuroR, IRES2, EGFP, SV40 polyadenylation transmitter, and the right homologous arm (550 bp) were all present in the plasmid used for “CRISPR HDR” targeting (808 bp). Table
1 demonstrates the plasmids employed to transfect breast malignant cell lines utilizing Lipofectamine 2000 chromophore (Thermo Fisher Scientific, USA). Before being selected by PCR test two weeks after transfection for “CRISPR excision” and eradicating the NOX4 exon, each culture (50 colonies) was monitored to grow.
Table 1
The DNA constructs that were employed in this work
pX459-1 | hU6 promoter- sgRNA (downstream of NOX4 Exon1)-sgRNA scaffold-CAG promoter-Cas9-T2A- PuroR-bGH polyA | CRISPR Excision CRISPR du-HITI |
pX459_2 | hU6 promoter- sgRNA (upstream of NOX4 Exon 1)-sgRNA scaffold-CAG promoter-Cas9-T2A- PuroR-bGH polyA | CRISPR Excision CRISPR du-HITI |
pX460_11 | hU6 promoter-sgRNA (downstream of NOX4 Exon) plus PAM-sgRNA scaffold-CAG promoter-EGFP-bGH polyA | CRISPR du-HITI |
pX461_11 | hU6 promoter-sgRNA (downstream of NOX4 Exon) plus PAM-sgRNA scaffold-CAG promoter-PuroR-bGH polyA | CRISPR du-HITI |
DNA extraction and sequencing
Both wild-type and knockout cell lines had their DNA molecules extracted using the DNA-extraction Kit (Cinnacolon, Tehran, Iran), which was then subjected to PCR assay. PCR-amplified results were presented to Sanger Sequencing technology following a smooth reaction recovery (Macrogen Inc., South Korea).
T7 endonuclease test for mismatched duplex identification
A mismatch-sensitive T7 endonuclease 1 assay (New England Biolabs) was utilized to verify that DNA breakage and specific nucleotide disruption occurred at the designated location. Following the directions provided by the manufacturer, DNA extraction from the colonies using the FavorPrepTM GEL Purification and DNA extraction kit. Each Purified DNA was divided into 10 µl (200 ng), 2 µl (10X NE-Buffer 2) buffer, and 19 µl (nuclease-free water) and placed in separate microtubes. The samples were warmed at 95 °C for 10 min. It was then given time to cool gradually to room temperature. T7 endonuclease I (5 units/ml) was added to 19 µl of each test, which was then incubated at 37 °C for 15 min before being analyzed on an agarose gel. Tanon-electrophoretic software detected band strengths, and the desired disruption was observed.
Quantitative reverse transcription PCR
Total RNA was extracted from wild-type and knockout cells using the Y-Tizol RNA extraction Kit (Yekta-Tajhiz, Iran). The quality and variety of the collected RNA were assessed using a 2000 Nanodrop spectrometer (Thermo Scientific, USA). 1 µg of total RNA was reverse transcribed, and cDNA was produced using random hexamer primers and MMLV reverse transcriptase (Thermo Fisher Scientific, USA). Premix Ex-Taq (Probe qPCR) master mix, 2 µl of cDNA, 500 nM primers, and 100 nM probe (dual-labelled hybridized probes, 5’FAM-3’BHQ1-labeled for NOX4 and 5’CY5-3’BHQ2 for GAPDH) in a 20 µl reaction medium were used in quantitative RT-PCR experiments (Qiagen, USA). The amplification stages employed were 95 °C for five minutes, followed by 40 cycles of 94 °C for thirty seconds, 62 °C for thirty seconds, and 72 °C for thirty seconds. Sanger sequencing was employed to confirm the PCR results’ identity. By subcloning amplified portions and serial dilution, standard curves were produced. Three PCR experiments were conducted on each quantity, and two real-time observations were made. The cycle threshold (Ct) values were then evaluated to the log of the copy numbers. The following formulas were used to determine efficiency (E) for each qPCR strategy based on the computed gradient of standard curves created using 5-fold serial dilution vector compounds: E = (10–1/slope-1) 100%.
All calibration curves in the investigated range were standard and had high correlation coefficients (R2). The matching calibration graph determined the ratio of NOX4 and GAPDH transcription copies. The outcomes of dividing the amounts of the target gene (NOX4) and the standard gene (GAPDH) for two sets of cDNAs were displayed.
Breast cancer cells MCF-7 and MDA-MB-231 transfected with plasmids was seeded in six-well plates and cultured (Corning, NY, USA). As previously stated [
19], cells (2 × 10
5) were grown in serum-free DMEM medium with added EGF, hFGF, and penicillin/streptomycin (Gibco). Prototypes of spheroids were fixed, stained with crystal violet dye solution, and then recognized using a light stereomicroscope (Olympus, Tokyo, Japan).
Analyze the colony growth and proliferation of CCK-8
With a CCK-8 detection kit, the CCK-8 assessment was completed (Dojindo Japan). Before the cell lines received the CCK-8 chemical treatment, the transfected cells were seeded into culture plates and grown for 10 h. 450 nm was used to measure absorbance.
Transwell invasion screenings
Cancer cells were placed on a pre-coated plate with adherent cells in a 24-well transwell tube (Corning) (BD Biosciences, San Jose, CA, USA). After 24 h of treatment, the top surfaces were brushed, and the invaded regions were fixed with 4% paraformaldehyde and stained with Giemsa. Afterwards, the optical microscope was used to observe the cells.
Luciferase gene reporter experiment
Luciferase reporter complexes were created using the wild-type and mutant genotypes for the E2F1 connection of the Nanog promoter region. MCF-7 and MDA-MB-231 cells were co-transfected with the vectors and E2F1 using the Lipofectamine 2000 reagent (Thermo Fisher, USA). Promega’s Dual-Luciferase Reporter Assay Kit evaluated the Renilla vector’s performance (Promega).
MTT analysis
The MTT cytotoxicity Kit I (Roche, Switzerland) was used to verify the cell viability. 5 × 103 cells/well on a 96-well flat-bottomed plate were seeded and cultivated at 37 °C in a 5% CO2 incubator. The samples in each well were washed thoroughly with PBS over three days (24 h, 48 h, and 72 h), during which the cell viability was assessed. 100 µl of serum-free medium and 5 µg/ml Sigma MTT were added to each well, and they were then cultured for 4 h at 37 °C in a CO2 incubator. The media was gradually removed and replaced with DMSO. Using a State Fax-2100 Microplate reader, the optical density at 570 nm was compared to the background at 690 nm.
Cell cycle evaluation
The cells were fixed in absolute ethanol for 24 h. Before being labelled for 15 min using PI/RNase staining solution from BD Bioscience Pharmingen, the cells were washed twice in PBS. FACS flow cytometry was performed to ascertain the cell population’s DNA composition. FlowJo V10 software (Tree Star, Ashland, OR) assessed the cell cycle statistics.
Quantitative real-time PCR analysis
A quantitative real-time PCR method with SYBR green amplification was employed to measure the levels of the proapoptotic genes P57, P21, Prkca, MDM4, and FADD, as well as the antiapoptotic genes BCL2 and SURVIVIN. The manufacturer performed quantitative real-time PCR as directed using an SYBR® Premix Ex TaqTM II kit (TaKaRa, Japan) and particular primers (Table
2). By adjusting the corresponding GAPDH amount, relative genomic levels were measured. The experiments were carried out twice.
Table 2
List of specific primers used in this research
NOX4 | NOX4-F NOX4R | TTGGCTTTGGATTTCTGGAC TGGGTCCACAACAGAAAACA | 59 |
P57 | P57-F P57-R | CTTCTTTGACCCTGACACC CTGAACATGGAGAGATAGTGC | 59 |
Survivin | Sur-F Sur-R | GAGAACGAGCCAGACTTGG GCTTTCCTTTCTGTCAAGAAGC | 62 |
BCL2 | BCL2-F BCL2-R | TGTGGCCTTCTTTGAGTTCG TACAGTTCCACAAAGGCATCC | 58 |
Prkca | Prkca-F Prkca-R | ′CGACTGTCTGTAGAAATCTGG CACCATGGTGCACTCCACGTC | 59 |
MDM4 | MDM-F MDM4-R | AATGATGACCTGGAGGACTCTA ACTGCCACTCATCCTCAGAGGTA | 59 |
FADD | FADD-F FADD-R | C GGAAATGGGACAAAACATCCT TGCGGGAGTAGTTGGAAAGT | 59 |
GAPDH | GDH-F GDH-R | GCCAAAAGGGTCATCATCTCTGC GCCAAAAGGGTCATCATCTCTGC | 62 |
E2F1 | E2F1-F E2F1-R | TGATTGTGGCAAAGGAGGA CTCTTCCTTGCTCGTTGTTGGTAT | 62 |
Statistic assessment
Every study was conducted twice, and the data were presented as mean ± SD. The group variations were examined using one-way ANOVA and independent variables t-tests. The analysis was done with SPSS software, and the graphics were made with Graph-Pad Prism. A P value of less than 0.05 was used to indicate significance.
Discussion
Numerous tumors showed downregulation of NOX4, associated with metastasis and survival in human malignancies [
25,
26,
33]. However, the clinical relevance of NOX4 upregulation in human malignancies was still up for discussion. As a result, we conducted a comprehensive investigation of the diagnostic value of NOX4 overexpression using data from The Cancer Genome Atlas (TCGA) and computational studies. We thoroughly investigated the biological functions of NOX4 in cancerous tumors and the associated processes. This study found that increased NOX4 expression was associated with worse OS and DFS and an increased incidence of lymph node metastases. The intricate mechanisms underlying NOX4’s carcinogenic actions may help to explain why NOX4 increased expression in malignancies was clinically significant [
34]. By sponging miRNAs and altering the synthesis of their targets, including miR-185-3p/E2 F1/Nanog [
35], miR-625/CCND1 [
36], miR-524-5p/YB1/ZEB1 [
37], miR-124-3p/EZH2 [
38], and miR-765/APE1 [
39], the majority of researchers discovered that NOX4 could increase malignant cells’ aggressiveness. This study is a meta-analysis, and the main limitations of a meta-analysis are that it combines different types of studies and that the summary effect may ignore important differences between studies.
The findings of this study showed that breast carcinoma cell lines produced more NOX4, which may be associated with a poorer prognosis. These results suggested that NOX4 was a regulator of breast cancer. It investigated how NOX4 affected breast carcinoma cells’ biological morphology and stemness. Gain and loss-of-functional studies showed that NOX4 accelerated sphere growth, produced more stem factors, and helped maintain the features of breast cancer CSCs, indicating that NOX4 was involved in malignant cell differentiation [
40]. Breast, lung, colorectal, malignant, and glioblastoma were previously linked to CSCs [
37]. Important biological properties of cancer that cause invasion and metastasis. Various investigations [
41,
42] linked CSCs to tumor metastasis and invasion. Researchers conducted experiments to learn more about how NOX4 regulated the aetiology of breast cancer. E2F1 connected with the promoter of the Nanog gene, demonstrating that it served as a transcriptional activator. A luciferase reporter assay, ChIP, was used to validate the proactive approach of E2F1 and Nanog while establishing the NOX4/E2F1/Nanog regulatory circuit. The transcription factor E2F1 has been identified as an oncogenic element in colorectal and bladder cancer [
43,
44]. Additionally, it was shown that E2F1 was up-regulated and active in the carcinogenesis of breast cancer, contributing to the creation of Nanog in this disease. The importance of functional studies in defining proteins’ biological roles increased as more molecules were found. Similar to how protein-coding genes were studied, approaches meant to limit the production of proteins were crucial for comprehending the biological functions of NOX4. However, two frequently utilised strategies, RNA interference and antisense methods proved ineffectual for molecules with nucleus localization [
45]. These strategies are also connected to insufficient target gene suppression, off-target consequences, and technical variations. A fantastic possibility was employing recently developed genome editing technology to move a gene’s chromosomal position so that the associated NOX4 became inactive [
46].
Furthermore, changes in the related genomic areas must be significant enough to negatively affect the growth and biological operation of the synthesized RNA. For instance, a direct excision followed by NHEJ could not sufficiently modify a particular gene’s structural makeup [
47‐
52]. Three different CRISPR-mediated KO methods, including “CRISPR excision,“ “CRISPR HDR,“ and “CRISPR du-HITI,“ were used to change the position of NOX4 in several breast cancer cell lines. As previously described, two sgRNAs were used to delete a genomic area of genes [
41‐
29]. The genome contained no reporter sequences since no donor plasmids were used to apply this approach. Selecting Knockout cells thus took much time. On the other hand, “CRISPR HDR” uses donor plasmids, enabling the user to select the molecular pathways depending on fluorescence and antibiotic resistance [
50‐
54].
However, homology components must be cloned into the donor vector for this method to function. Cancer cells might be chosen using the “CRISPR du-HITI” technique, which does not need the presence of homologous bands. Using this technique, we managed the generation of Knockouts so that each cell had antibiotic resistance and fluorescence traits that made it simple to recognize a double allele Knockout. The NOX4 Knockout cells generated in this study were verified using classical PCR, sequencing, and RT-qPCR. Because the qPCR forward and reverse primers were equal to an exon, we did not anticipate any stimulation results from “CRISPR excision” and “CRISPR du-HITI” Knockout cells. Each of these processes included the deletion of an Exon. The initial part of the exon was labelled with a reporter/transcription termination signal-containing component in “CRISPR HDR,“ nonetheless. An amplifying outcome was therefore predicted. On the NOX4 Knockout cells, RNA-seq, a soft agar colony formation test, and MTT studies were carried out. In these cells, many genes were expressed in various ways. MTT and soft agar colony growth confirmed the decreased proliferative capacity of the NOX4 Knockout cells. This outcome aligned with earlier findings that NOX4 inhibition affected the genes controlling cell proliferation.
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