Published online Dec 21, 2018. doi: 10.3748/wjg.v24.i47.5338
Peer-review started: August 14, 2018
First decision: August 31, 2018
Revised: September 7, 2018
Accepted: October 5, 2018
Article in press: October 5, 2018
Published online: December 21, 2018
Gastric cancer (GC) remains a major public health problem, having the third highest incidence of death worldwide. Piwi like RNA-mediated gene silencing 1 (PIWIL1) is involved in regulation of widespread biological processes, including stem cell proliferation, embryogenesis, growth, and development, and has been found to be frequently over-expressed in various tumor types, including GC. Previous studies have demonstrated that PIWIL1 is implicated in improving tumor malignant behavior. PIWIL1 expression has been shown to be absent in normal somatic tissues, making it a very intriguing target for therapy. We attempted to investigate the role of PIWIL1 on the migration and invasion capacity of metastatic GC cells, using the AGP01 cell line, as well as checking the expression status of genes and proteins involved in these cellular processes, in order to elucidate the mechanisms by which PIWIL1 provokes tumorigenic effects and to shed light on potential new strategies to target PIWIL1
Many aspects of gastric carcinogenesis remain elusive, and much effort has been made to improve patient prognosis. The PIWIL1 has been identified as a novel extremely highly expressed gene in many types of cancer and its expression in GC tissue is related to poorer overall survival, suggesting that high expression of PIWIL1 is associated with poor prognosis and that it could be used as a predictive marker or even a target for therapy. Although PIWIL1 has been correlated with worse outcome, the involved mechanisms remain unclear, and many hypotheses are being tested. Once the upstream and downstream signaling pathways of PIWIL1 are elucidated, it will be possible to create new therapeutic strategies for gastric carcinogenesis, in order to improve the overall health of patients affected by this disease.
We performed permanent knockout of the PIWL1 gene to verify phenotypic modifications in the AGP01 metastatic GC cell line, as well as alterations in expression level of mRNA and protein, in an attempt to better understand the mechanisms by which PIWIL1 promotes tumor malignant behavior. This research demonstrates the importance of studying PIWIL1 in GC, since data obtained through the achievement of our objectives showed that this protein has a crucial role in gastric carcinogenesis, promoting molecular and phenotypic alterations compatible with enhanced tumor aggressiveness. The elucidation of the role of PIWIL1 protein in cancer cell invasion and migration will pave the way for developing potential clinical interventions, aiming to control GC dissemination.
We applied CRISPR/Cas9 technology to knockout the PIWIL1 gene in a metastatic GC cell line, and analyzed its phenotypic modifications, as well as alterations in gene and protein expression. CRISPR-Cas9 technology was considered in 2015 as one of the most important technological advances of science. Mainly, it allows permanent silencing of the target gene and also creates a stable and permanent cell line with the desired modification. By this way, multiple experiments can be carried on, including long term evaluation of the downstream events caused by the molecular alteration, as well as discovering potential pathways influenced by the studied gene. Therefore, after permanent knockout of PIWIL1 in the AGP01 cell line, we analyzed phenotypic modifications by performing wound-healing and Boyden chamber invasion assays, to assess migration and invasion, respectively. Moreover, aiming to shed light on the molecular mechanisms used by PIWIL1 to make changes in the migration and invasion capability of cells, we carried out proteomic and microarray assays, using multidimensional protein identification technology (commonly known as MudPIT) and a one-color microarray-based gene expression analysis kit, respectively.
PIWIL1 gene knockout was successfully performed and confirmed by Sanger sequencing, which revealed an insertion of seven adenines in the PIWIL1 gene sequence. In silico prediction of the encoded protein pointed to the appearance of a premature termination codon, suggesting that this insertion generates a truncated protein with a loss-of-function phenotype. PIWIL1 knockout promoted a significant decrease in cell migration and invasion capacity (P < 0.01 and P < 0.001, respectively), which is consistent with data present in the literature demonstrating that this protein is implicated in several signaling pathways that regulate cell motility. By comparing expression profiles after PIWIL1 knockout, a total of 251 mRNA were found to be differentially expressed, with 43 up-regulated and 208 down-regulated mRNA. A functional analysis grouping all differentially expressed mRNAs demonstrated that 35 genes encoded proteins were involved in invasion and migration cellular processes. After extensive review of data presented in the literature, we selected 9 of these 35 genes (DOCK2, ZNF503, PDE4D, ABL1, ABL2, LPAR1, SMAD2, WASF3 and DACH1) as possibly related to the mechanisms used by PIWIL1 to promote carcinogenic effects related to migration and invasion, since their functions are consistent with the changes observed (being up- or down-regulated after knockout). Additionally, the analysis of proteomic data revealed that PIWIL1 knockout caused modification in the expression of 27 proteins involved in epithelial-mesenchymal transition (EMT). Twenty-two oncoproteins related to EMT promotion, including FGFR1, PCNA, ACTN4, GSN and TUBB3, were expressed in the AGP01 cell line before knockout and reduced to a level that were not detectable by the technique after knockout. On the other hand, PIWIL1 knockout caused an increase in the expression of six proteins implicated in EMT suppression, such as ACSM3, ADGRG1 and ANPEP, that were absent in AGP01 before knockout. To the best of our knowledge, this is the first report describing molecular alteration compatible with phenotypic alterations after permanent knockout of PIWL1 in GC. Detailed mechanisms leading to PIWIL1 over-expression in cancer as well as the pathways by which this protein improves the malignant phenotype should be further investigated.
In the current study, we pioneered the performance of an in vitro knockout of the PIWIL1 gene by using the CRISPR-Cas9 system, and found that absence of this gene significantly impaired the migration and invasion capacity of the AGP01 cell line, besides modifying mRNA and protein expression of potential molecular targets involved in the EMT process. The results of such experiments contributed to understanding of the mechanisms used by PIWIL1 to promote alteration in migration and invasion capacity of gastric cells during tumorigenesis, and also revealed the participation of new players related to PIWIL1 expression, such as FGFR1, PCNA, ACTN4, PDE4D and SMAD2. Our results demonstrated that knockout of PIWIL1 promotes several changes in cell phenotype, suggesting the critical role of the PIWIL1 oncogene in GC, and confirmed the hypothesis that PIWIL1 expression provokes migration, invasiveness and EMT as potential mechanisms of improved tumor aggressiveness. The presented findings open new perspectives for molecular interventions in GC
Definite silencing of PIWIL1 by the CRISPR-Cas9 system resulted in robust findings favoring the discovery of new mechanisms involved in gastric carcinogenesis. The presented results must be validated by other researchers, and if confirmed, might lead to innovative interventions aiming to treat GC.