The Effect of Gene Editing by CRISPR-Cas9 of miR-21 and the Indirect Target MMP9 in Metastatic Prostate Cancer

Prostate cancer (PCa) has a high prevalence and represents an important health problem, with an increased risk of metastasis. With the advance of CRISPR-Cas9 genome editing, new possibilities have been created for investigating PCa. The technique is effective in knockout oncogenes, reducing tumor resistance. MMP9 and miR-21 target genes are associated with PCa progression; therefore, we evaluated the MMP-9 and miR-21 targets in PCa using the CRISPR-Cas9 system. Single guide RNAs (sgRNAs) of MMP9 and miR-21 sequences were inserted into a PX-330 plasmid, and transfected in DU145 and PC-3 PCa cell lines. MMP9 and RECK expression was assessed by qPCR, WB, and IF. The miR-21 targets, integrins, BAX and mTOR, were evaluated by qPCR. Flow cytometry was performed with Annexin5, 7-AAD and Ki67 markers. Invasion assays were performed with Matrigel. The miR-21 CRISPR-Cas9-edited cells upregulated RECK, MARCKS, BTG2, and PDCD4. CDH1, ITGB3 and ITGB1 were increased in MMP9 and miR-21 CRISPR-Cas9-edited cells. Increased BAX and decreased mTOR were observed in MMP9 and miR-21 CRISPR-Cas9-edited cells. Reduced cell proliferation, increased apoptosis and low invasion in MMP9 and miR-21 edited cells was observed, compared to Scramble. CRISPR-Cas9-edited cells of miR-21 and MMP9 attenuate cell proliferation, invasion and stimulate apoptosis, impeding PCa evolution.


Introduction
Prostate cancer (PCa) is one of the most common cancers among men, accounting for 26% of all cases diagnosed and 11% of estimated deaths worldwide. Metastatic PCa is challenging to treat because it presents considerable heterogeneity and limited therapeutic Int. J. Mol. Sci. 2023, 24, 14847 2 of 15 response [1]. Presently, standard PCa treatment is based on androgen receptor (AR) deprivation. However, this is ineffective when the disease advances to the metastatic stage due to androgen resistance [2].
The ECM-degrading enzyme metalloproteinase 9 (MMP9) plays an important role in promoting the invasion of tumor cells into other tissues; thus, it directly contributes to carcinogenesis. It has been proposed that altered MMP9 expression may contribute to PCa evolution and, consequently, high metastatic potential [3]. In addition to the extracellular matrix, MMP9 also regulates integrins, transmembrane proteins involved in adhesion to the extracellular matrix, such as CDH1, ITGB1 and ITGB3 [4]. It has also been shown that RECK negatively regulates MMP9. Notably, studies have demonstrated that MMP9 gene expression could be regulated by a microRNA (miRNA) that is upregulated in PCa, resulting in RECK expression downregulation and MMP9 upregulation [5][6][7]. miRNAs are 18-22 nucleotide stretches of noncoding RNA that regulate posttranscriptional gene expression. One miRNA can silence multiple genes and is largely tissue-specific. Concerning cancer, miRNAs can act as a tumor suppressor when they function to silence oncogenes [8].
Furthermore, miR-21 is highly expressed in PCa, which could attenuate tumor suppressor gene expression [9,10]. Studies that definitively block this miRNA and evaluate tumor evolution have not been conducted. This miR-21 regulates the expression of multiple tumor suppression-related genes, such as RECK, which regulates MMP9. miR-21 also regulates PDCD4 and BTG2, which are apoptosis-related, and MARCKS, which controls cellular invasion [11][12][13].
A new gene-editing tool named clustered regularly interspaced short palindromic repeat (CRISPR)-associated Cas9 (CRISPR/Cas9) is a revolutionary genome-editing technology that can modify the genome of cells with high specificity, correcting mutations or deleting oncogenes [14]. In this sense, employing the CRISPR-Cas9 system to inactivate metastasis-related genes to promote cell survival could represent a novel strategy for improving treatment efficiency. However, the effect of blocking MMP9 and miR-21 in PCa for therapeutic purposes is still controversial. Bodey B et al. and Babichenko II et al. did not observe any difference in MMP9 expression in prostate cancer patients compared to the control group [15,16]. Similarly, Folini M. et al., also found no difference in the expression of miR-21 in prostate cancer [17]. Therefore, in the present study, we sought to investigate the effect of downregulating the expression of these markers using the CRISPR-Cas9 system in a metastatic PCa model.

Gene Editing with CRISPR/Cas9
We inserted sgRNAs into the PX-330 plasmid and sequenced them to validate the construct ( Figure 1A). Before transfecting the plasmids into PC-3 and DU145 cell lines, we performed a puromycin dose-response curve and observed that 150 µg/mL for 10 days was the ideal concentration and time for selecting plasmid-transfected cells (Supplementary Figure S2). Cells were co-transfected with the plasmids containing MMP9 sgRNAs 1 and 2, targeting MMP9 Exon 1, or miR21 sgRNA 1 or 2. The transfections were validated with GFP images (Supplementary Figure S3).
After confirming the downregulation of miR-21 expression in the CRISPR/Cas9-edited PCa cell line, the gene expression of other miR-21 targets was assessed in the PC-3 and DU145 cells. The cells edited with miR-21 sgRNA1 displayed greater MARCKS expression (p = 0.0302) than Scramble ( Figure 3A). On average, the mean BTG2 gene expression values in PC-3 cells with miR-21 sgRNA1 gene editing were greater than Scramble cells, but the results were not significantly different ( Figure 3B). Additionally, the expression of apoptosis programmer PDCD4 was upregulated in miR-21 sgRNA1-edited samples compared to Scramble (p = 0.0453) ( Figure 3C). Samples edited with MMP9 sgRNAs 1/2 did not present any significant differences compared to Scramble cells ( Figure 3A-C).

Discussion
CRISPR-Cas9 is a novel gene-editing technique that has brought several possibilities for gene therapies and corrections in multiple mutation-related diseases [18]. It has been proposed that this technique could potentially correct molecular mutations described in the literature or even downregulate or edit genes that may be possible candidates in molecular target therapy [19][20][21].
In this study, we sought to evaluate the role of MMP9 and the indirect regulator, miRNA-21, in metastatic PCa cell lines (PC-3 and DU145) using the CRISPR-Cas9 technique. We first standardized all the initial experiments by inserting the sgRNAs into the px-330 plasmid and expanding these samples in bacteria to generate a sufficient quantity of sgRNA-containing plasmids.
The sgRNA-containing plasmids were transfected into the PC-3 and DU-145 cell lines following the protocol of Zhang [22]. We opted to co-transfect two MMP9 sgRNAs to edit two different regions of MMP9 Exon 1, because a previous study showed that gene editing in more than one region could be more efficient [23]. Our approach resulted in a significant

Discussion
CRISPR-Cas9 is a novel gene-editing technique that has brought several possibilities for gene therapies and corrections in multiple mutation-related diseases [18]. It has been proposed that this technique could potentially correct molecular mutations described in the literature or even downregulate or edit genes that may be possible candidates in molecular target therapy [19][20][21].
In this study, we sought to evaluate the role of MMP9 and the indirect regulator, miRNA-21, in metastatic PCa cell lines (PC-3 and DU145) using the CRISPR-Cas9 technique. We first standardized all the initial experiments by inserting the sgRNAs into the px-330 plasmid and expanding these samples in bacteria to generate a sufficient quantity of sgRNA-containing plasmids.
The sgRNA-containing plasmids were transfected into the PC-3 and DU-145 cell lines following the protocol of Zhang [22]. We opted to co-transfect two MMP9 sgRNAs to edit two different regions of MMP9 Exon 1, because a previous study showed that gene editing in more than one region could be more efficient [23]. Our approach resulted in a significant reduction in MMP9 gene and protein expression in PC-3 and DU-145 cells when using sgRNAs 1 and 2 for MMP9 in the same cell rather than with the individual sgRNAs.
Furthermore, we also showed that miR-21 gene editing significantly decreased the expression of this gene in both PC-3 and DU145 cell lines. In both cell lines, miR-21targeted downregulation was accompanied by the upregulation of target genes such as RECK, MARCKS, and PDCD4. It should be pointed out that the expression of the target gene BTG2 was increased in the DU145 cell line. These results are particularly interesting given that miR-21 is reportedly upregulated in patients with high-grade PCa [24].
The miR-21 target genes play important roles in key molecular mechanisms like cell migration and apoptosis. Indeed, Kim et al. (2020) demonstrated that miR-21 inhibition improved the stability and therapeutic efficacy and reduced metastasis in PCa xenografts in mice [25], a result consistent with our data. It is plausible that the increased expression of these key factors in miR-21 CRISPR-Cas9-edited cells could lead to reduced cell migration rates and higher cell apoptosis. We also observed upregulated BAX and PDCD4 gene and protein expression, two important apoptosis-related factors. In the PC-3 cell line, proliferation was decreased, and there were higher percentages of cells in early, late, and total apoptosis. In contrast, cell proliferation was unaffected in the DU145 cell line, but there were significant increases in apoptosis rates.
The results with MMP9 gene editing using CRISPR-Cas9 were similar to miR-21 editing. For example, MMP9 edited cells displayed altered gene and protein expression, reduced proliferation and increased apoptosis compared to the Scramble group. Until now, downregulated MMP9 gene and protein expression have not been linked to apoptosis. Thus, studies to investigate this cellular response were developed.
These experiments showed that MMP9-edited PC-3 and DU145 cells exhibit upregulated integrin gene expressions, including ITGB3 in PC-3 and CDH1 and ITGB1 in DU145 cells. The same response was observed in miR-21-edited cells, where CDH1, ITGB3, and ITGB1 were upregulated in PC-3 cells, and ITGB3 and ITGB1 were upregulated in DU145 cells. These results are particularly interesting because the relevance and importance of integrins in cancer progression are unclear.
A previous study found that the loss of CDH1 expression induces oncogenic cell transformation and facilitates tumor development [26]. Additionally, Werb et al. (2007) showed that metalloproteinases are responsible for cleaving the extracellular matrix and proteins such as CDH1 and other integrins [27]. Furthermore, Kurozumi et al. (2016) demonstrated that miRNA-223, which targets ITGA3 and ITGB1, acts as a tumor suppressor inhibiting these integrins [28]. Our data indicate that MMP9 downregulation may lead to upregulated integrin expression in PC-3 and DU145 cell lines, consequently reducing cell proliferation and stimulating apoptosis.
We  [3,29]. In this sense, our study results implicate MMP9 and miR-21 in metastatic PCa. Furthermore, we demonstrated that knocking these molecules out of metastatic cell lines using the CRISPR-Cas9 system slows cell proliferation and inhibits cell invasion, thus, highlighting the utility of this emerging technique for hard-to-treat diseases and conditions. Future studies in animal models are necessary to corroborate these in vitro findings.
The present study successfully downregulated the MMP9 and miR-21 gene and protein expression PCa cell lines using the CRISPR-Cas9 system. CRISPR-Cas9-edited PC-3 and DU145 cells displayed reduced cell proliferation, increased apoptosis and inhibited cell invasion, responses that could potentially trigger a regression in the metastatic PCa evolution.

Cell Culture
The present study utilized two metastatic PCa cell lines from the American Type Culture Collection (ATCC): PC-3 and DU145. These cells were cultured in MEM medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic and antimycotic solution (Sigma Co., St. Louis, MO, USA). The cultures were incubated at 37 • C with 5% CO 2 , and the culture medium was changed every two days. The PC-3 and DU145 cell lines were certified (Supplementary Figure S1).
The phosphorylated sgRNAs were annealed and cloned into the px330-U6-GFP vector (Addgene, Watertown, MA, USA) using Addgene's website instructions. The products of the cloning reactions were transformed into DH5α competent E. coli (Invitrogen, Waltham, MA USA). Individual bacterial colonies were selected and expanded, and the PureLink™ HiPure Plasmid Filter miniprep Kit (Invitrogen) was used to extract and isolate the plasmid. The constructs were sequenced with the following primers: FWD 5 -GGGCCTATTTCCCATGATTCC-3' and REV 5'-CGCGCTAAA AACGGACTAGC-3'. Colonies harboring the px330-U6-GFP vector with sgRNA were again expanded, and the plasmids were extracted using the PureLink™ HiPure Plasmid Filter maxiprep Kit (Invitrogen).
Next, these plasmids were transfected into the PC-3 and DU145 cell lines with the Xfect™ Transfection Reagent (Takara Bio, San Jose, CA, USA) following the manufacturer's instructions. Both cells lines were exposed to 150 µg/mL puromycin dihydrochloride (Sigma Co.) for 10 days to select transfected cells. All the experiments were performed in three replicates of three independent experiments and standardized according to Zhang et al. [22].

Flow Cytometry for Cell Proliferation and Apoptosis
After CRISPR-Cas9 editing, the PC-3 and DU145 cells were evaluated with the Muse™ cell death kits (MCH100105) for Annexin V and a Proliferation kit (MCH100114) for Ki67, according to the manufacturer's recommendations. The apoptosis and proliferation analyses were performed on a Muse ® Cell Analyzer (Merck Millipore, Burlington, MA, USA).

Western Blotting
Protein analysis was conducted using the total PC-3 and DU145 cell extracts. The samples were macerated in RIPA lysis buffer (Millipore, Billerica, MA, USA) with a TissueLyser (Qiagen, Germantown, MD, USA) and incubated at 4 • C for 20 min. These samples were then centrifuged at 15,000 rpm at 4 • C for 30 min.
The resulting supernatant was collected and mixed with 2× Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA, USA). Total protein samples (100 ng/mL) from both cell lines were loaded onto 10% acrylamide gels and subjected to SDS-PAGE. The separated proteins were then transferred to 0.45 µm pore-size nitrocellulose membranes at 120 V for 1 h. The SNAP apparatus (Millipore) was used for antibody incubation. Membranes were blocked with 3% bovine serum albumin (BSA, Sigma) diluted in Tris-buffered saline-Tween 20 (TBS-T) for 15 min. As suggested by the manufacturer, the primary antibodies were diluted in TBS-T with 1% BSA. The monoclonal MMP9 antibody utilized herein was applied at 1:1000 (Cloud-Clone). β-actin (1:1000, Millipore) was used to normalize the protein loading.
The membranes were incubated for 20 min at room temperature, washed with TBS-T 3 times for 15 s, and incubated with an anti-mouse IgG (H + L) secondary antibody, Human Serum Adsorbed, and Peroxidase labeled (KPL). Blots were developed using an ECL Western blotting detection system (Millipore), and the immunoblot images were captured with an Alliance 4.7 device (Uvitec, Cambridge, UK). Quantitative analyses were performed using the Alliance 16.06 software.

Invasion Assays
PC-3 and DU145 cell lines edited with CRISPR-Cas9 for MMP9 and miR-21, as well as their respective controls, were plated [approximately 12,000 cells in 250 µL of MEM serum-free culture medium (Gibco TM )] in Transwell chambers (Becton-Dickinson, São Paulo, SP, Brazil) with an 8 µm pore size containing 50 µL of Matrigel diluted in MEM serum-free culture medium (1:5). Next, 750 µL of culture medium containing 10% FBS was added to the lower chamber of the plate. The cells were maintained in a CO 2 incubator for 48 h at 37 • C. Cells were fixed with 4% formaldehyde in PBS, stained with a 1% crystal violet solution in methanol, and counted on an optical microscope at 20× magnification.

Statistical Analysis
A Kolmogorov-Smirnov normality test was performed for the expression and correlation analyses. A t-test was used when comparing two groups, and ANOVA was used for comparing three groups. The GraphPad Prism 9.0 software was used for all statistical analyses. Statistical significance was set at p < 0.05.  Data Availability Statement: The datasets used and/or analyzed during the current study are available to the corresponding author on reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.