AUTHOR TITLE PAGE

1. Aniqa Maryam
Department of Biochemistry and Biotechnology, University of Gujrat
19024160-001@uog.edu.pk

2. Meerab Bajwa
Department of Biochemistry and Biotechnology, University of Gujrat
19024160-011@uog.edu.pk

3. Misl e Noor
Department of Biochemistry and Biotechnology, University of Gujrat
19024160-014@uog.edu.pk

4. Zeesha Abbas
Department of Biochemistry and Biotechnology, University of Gujrat
19024160-017@uog.edu.pk

5. Israr Tariq
Department of Biochemistry and Biotechnology, University of Gujrat
19024160-034@uog.edu.pk

6. Muhammad Sufyan
Department of Biochemistry and Biotechnology, University of Gujrat
19024160-041@uog.edu.pk

CORRESPONDING AUTHOR
Muhammad Sufyan
Department of Biochemistry and Biotechnology, University of Gujrat
19024160-041@uog.edu.pk
Role of RNAi, CRISPR-Cas9, and miRNA in the diagnosis
and treatment of cancer
Aniqa Maryam1, Meerab Bajwa2, Misl e Noor3, Zeesha Abbas4, Israr Tariq5, Muhammad Sufyan6*

ABSTRACT
Mouse models are being used in the clinical study of cancer and another disease. Mouse models
are very beneficial for the comprehension of pathogenicity and molecular study of gastric cancer
but at a record level, only a few mice can develop gastric cancer. Enhancement of gene transfer
technology, scientists generated various transgenic knockout techniques and knocking mouse
model technique users. CRISPR is a widely used genome editing technique in the treatment of
cancer. Combining the helicobacter infection and carcinogens treatment, those transgenic mice
(knockout) evolve cancerous abrasion. CRISPR is marked out as RNA-mediate adaptive marked
system defense technique, which is spontaneously present in bacteria and archaea. CRISPR-Cas9
has set out a path great for the treatment of cancer in therapy, the machinations of the cancer
genome about its epigenome, and eradicating intoxicating carcinogenic viral infections. One of the
techniques used is microRNA (miRNA) which might be used as a biomarker in high GC is used
on. MicroRNA are endogenous non-coding RNA that have a significant role in post-transcriptional
gene regulations-transcriptional mRNA, results in degradation into a functional resulting rot
protein miRNAs silence the related target genes by inhibiting mRNA translation or degrade the
mRNA molecules by binding to 3′-untranslated (UTR) regions. The miRNA has a biologically
significant role in the cell cycle of life, differentiation, inflammation, cell growth, and
carcinogenesis. The thing which makes microRNA a biomarker is that they are highly conserved.
There are numerous types of small synthetic RNA being used in cancer therapy, those are, siRNA,
and shRNAs. There are no adverse effects to this technique as compared to other primary methods.
RNAi is a method in which the cells get breakdown the messenger RNA (mRNA) molecules, with
the help of the cell’s internal machinery, this stops the production of protein produced from
mRNAs that result in reduced cancer proliferation.

Keywords: gastric cancer; histology; mouse models; CRISPR/Cas9; application; cancer

treatment; technology.
INTRODUCTION
The CRISPR-Cas9 system is a vigorous gene-editing tool being used in various cell types and
organisms. CRISPR-Cas9 is an endonuclease that gets directed by a sgRNA (single guide RNA)
which creates highly particular double-strands breaks in between regions of target DNA this will
make knockout reagent production appreciably easier with other gene manipulation techniques
(Wang, Wei, Sabatini, & Lander, 2014). Due to its anonymous size and high negative charge which
delivers the CRISPR-Cas9 system into cells and tissues is difficult (usually exceeds 10,000 bp).
When plasmid DNA encoding the Cas9 and sgRNA has been sent without viruses, such as
membrane deformation, or hydrodynamic invasion the effectiveness of genome editing has been
detartrating the prospective for harm to targeted cells (Li et al., 2015). Although it seems to provide
an event or altered way to deliver DNA, there are a lot of concerns
about immunogenicity and RNA stability. Cas9 and sgRNA complex is being used for targeted
gene editing. The problem is with RNA instability (Esvelt, Smidler, Catteruccia, & Church, 2014).
It's also difficult to separate proteins because they are unstable, and plasmids can not get pass
through cell membranes without encapsulation. The best mechanism, which would be ideal is to
deliver proteins and plasmids. Genomic sequencing visualized that the protein-coding of genes of
mice and humans show a lot of similarities while Genome editing could be used to edit the genome
of any organism (Pennacchio & Rubin, 2003). It is the machination of the specific gene loci to
gain genome modifications of that gene, such as insertions, deletions, point mutations, etc.
CRISPR-Cas9 is the hallmark of a bacterial defense system that generates based on genome editing
technology. In the field of genome engineering, systems can be programmed to target particular
stretches of genetic code for editing DNA at particular locations, ad behave as new diagnostic tools
(Hryhorowicz, Lipiński, Zeyland, & Słomski, 2017). CRISPR-Cas9 is the most usual, economical,
faster, more precise, and effort efficient than other existing genome editing methods. The Cas9
endonuclease system consists of four parts which include two small RNA molecules named
CRISPR RNA (crRNA) and trans-activating CRISPR RNA (trans rRNA) (Feng et al., 2021).
Genetically- engineered mouse models for cancer were first developed in the 1980s. Gene-targeted
mutant animals such as knockout or transgenic mice have greatly improved our understanding of
gene function in vivo and the genetic diversity that characterizes health and disease. Therefore,
mice have attracted attention as a useful model to identify mechanisms underlying GPL and GC,
to identify protein and molecular biomarkers for prevention, diagnosis, prognosis, and treatment
of GC, and to contribute to drug discovery and development (Rotkrua et al., 2013).

METHODS

CRISPR CAS:
The CRISPR/Cas-based strategies provide an innovative avenue toward clinical applications in
cancer gene therapy and immunotherapy. The common-investigated CRISPR/Cas systems for
cancer therapy are almost based on the nucleases including Cas9, Cas12a, Cas13a,d their
orthologues. It is critical for successful CRISPR-based antitumor therapy to select an appropriate
gene target to maximize efficacy as well as minimize toxicity. The consideration of therapeutic
targets for cancer therapy involves elaborate interactions between the tumor and the host
environment which will influence the treatment effect of CRISPR/Cas-based systems (Xia et al.,
2019).
The/Cas9 is carried out extremely precise cutting of all examined DNA strands. Following PAM
rearrangement, the sgRNA attaches to the target gene in this procedure, causing Cas9 endonuclease
to cleave the DNA strands upstream of the PAM site and create double-stranded breaks (DSBs)
(Xia et al., 2019). After DSBs are created, two pathways—Non-Homologous End Joining (NHEJ)
and Homology Directed Repair—can start the genome's repair process (HDR). NHEJ frequently
leads to the insertion/deletion (InDel) of altered DNA strands and it's the most common process to
happen. In the end, the production of InDel results in frameshifts and premature stop codons (Xia
et al., 2019). While NHEJ can directly join the broken sequences, HDR is capable of correctly
inserting the correct DNA into the target spot with the use of a volunteer DNA template. Giving a
neighboring foreign DNA template to create specially built and modified DNA can be used to
introduce the desired gene (Xia et al., 2019).

RNAi:
RNAi is a very successful technology for gene therapy application for the treatment of different
cancers (Xia et al., 2019). The activation of proto-oncogenes tips the scale b/w oncogenes and
tumor-suppressing genes that give rise to cancer (Abdelrahim, Safe, Baker, & Abudayyeh, 2006).
So to inhibit tumor growth, RNAi technology represses the activated oncogenes mRNA (Xu, A
Lee, & Chen, 2011). By transferring the m-bcr/abl (siRNA) to the myelogenous leukemia K562,
the chronic expression of mRNA of this type is inhibited, which will result in decreasing cell
malignancy and cell apoptosis will also induce (Valencia-Serna, Gul-Uludağ, Mahdipoor, Jiang,
& Uludağ, 2013).
The efficiency of chemotherapy can be improved by using RNAi molecule, this is how when there
is a specific combination b/w Bcl-2 and c-raf of siRNA gene it will induce the apoptosis in HL-
60, U937, and THP-1 leukemia and also it increases their sensitivity to etoposide (Baiquan,
Yuanhua, Ran, & Ling, 2010).

MDR1 is a multidrug- resistance gene have a very vital role in resisting chemotherapy tumors
(Takara, Sakaeda, & Okumura, 2006). When MDR1 (siRNA) transfects the gastric cancer cells
then the MDR1 mRNA and protein got inhibited and decrease the resistance to daunomycin (Nieth,
Priebsch, Stege, & Lage, 2003). This is how RNAi is used to reverse multidrug resistance (Stege,
Priebsch, Nieth, & Lage, 2004).

miRNA/LncRNAs
Gene marker HOTAIR involves GC functioning and expression level (J. Zhang et al., 2020).
Knockdown of HOTAIR by Si-RNA leads to the inhibition of GC cell proliferation, migration,
and invasion (L. Zhang et al., 2020). HOTAIR proves as a carcinogenesis cutter as well as it targets
other miRNAs so acts like competitive endogenous RNA (Cai & Wan, 2018). Other LncRNAs-
related genes such as lncRNA H19, MALAT1, and MALAT12 also involve in high expression in
GC cell lines (Zhou, Yin, Dang, Ye, & Zhang, 2015). If there is overexpression MALAT induces
epithelial-mesenchymal transition through the kinase pathway (Ying et al., 2012). If lncRNA
LEIGC is overexpressed in the cell line, it will suppress tumor growth and cell proliferation. So
there is an enhancement of sensitivity in GC cells. If LEIGC knockdown reverse process starts.
(Han et al., 2014) HOTAIR dysregulation shows its reverse relationship between expression and
downregulation (Fattahi et al., 2020). It also increases in vivo metastasis in xenograft GC tumors.
In addition, it also mediates gene expression (Ma et al., 2020). One such example is hypoxia-
mediated cell metastasis. SNGG is a novel gene that upregulates hypoxia. Whereas HULC
regulates the migration of cells deletion impact reversely (Lin, Song, & Ding, 2018). It takes into
notice that lncRNA dysregulation results in cell metastasis and invasion. It not only competitively
behaves but also plays a certain role in posttranscriptional activities by binding with mRNA. Thus
all these lncRNA and related genes prove biomarkers in GC diagnosis (Qin et al., 2021).

RESULTS

CRISPR technology has been utilized to identify new therapeutic targets and medications for the
treatment of GC, from the growth of tumor cells to the development of resistance to
chemotherapeutic-induced cell death (Goodspeed, Jean, & Costello, 2019). To examine the impact
of 5-fluoracil on the production of pyroptosis in these cells, Wang and colleagues used
CRISPR/Cas9 to engineer the deletion of the gastrin E (GSDME) gene in a GC cell line. The
authors discovered that the GSDME deficit converted the 5-FU-induced pyroptosis into apoptosis,
which is characterized by cell death without lysis, shrinkage, and fragmentation in apoptotic bodies
(Xia et al., 2019). The CRISPR/Cas9-mediated deletion of the legumain (AEP) gene was employed
in the study by Cui and colleagues to evaluate the ability of these cells to proliferate in the presence
of various chemotherapeutic drugs (Almeida, Wisnieski, Takao Real Karia, & Smith, 2022). This
gene has previously been demonstrated to be an oncogene connected to GC metastasis and
invasiveness. The scientists showed that 5-FU, paclitaxel, docetaxel, and T-DM1 therapy greatly
reduced the proliferation of AEP knockout GC cells (Digklia & Wagner, 2016). These findings
suggest that AEP may be a useful treatment target for GC (Cui et al., 2017). Likewise for RNAi,
according to Cioca et al., the use of RNAi molecules might increase the effectiveness of
chemotherapy. For example, when a certain combination of Bcl-2 and c-raf siRNA genes are used,
it will cause apoptosis in the HL-60, U937, and THP-1 leukemia and increase those cells'
sensitivity to etoposide (Cioca, Aoki, & Kiyosawa, 2003). The MDR1 mRNA and protein were
suppressed, decreasing the daunomycin resistance when MDR1 (siRNA) was transfected in gastric
cancer cells (Stege et al., 2004). The use of gene-knockout technology is crucial for the growth,
division, metastasis, and drug resistance of cancer cells. Researchers discovered numerous cancer-
related genes as possible targets for RNAi-based treatment last year. The given outcome
demonstrates that miRNA and GC growth and proliferation are closely related in the case of
miRNA used for diagnosis as biomarkers. miRNA cell proliferation and accumulation happen as
a result of variations and modifications in cell lines following a diagnosis (H. S. Liu & Xiao, 2014).
By hypermethylation, miRNA dysregulations cause the downregulation of tumor-suppressive
genes. The level of the proteins (JAK2, MeCP2, NOTCHA) involved in GC development is
regulated (Yang, Zhang, Sui, He, & Ding, 2015). There are two categories of pathogenic infection
that GC causes. The first factor, in this case, is the inhibition of TsmiR and onco-miR expression.
Although chemotherapy is a subtype that is implicated (B. Liu, Shyr, Cai, & Liu, 2019).

DISCUSSION
The development of many malignancies can be stopped, reduced, or inhibited thanks to RNAi
knockout technology. The MDR1 gene, which confers multidrug resistance, is crucial for tumor
resistance to chemotherapy. The MDR1 mRNA and protein were suppressed and the resistance to
daunomycin was decreased when MDR1 (siRNA) was transfected in gastric cancer cells. CRISPR
technology has been utilized to find new therapeutic targets and medications for the treatment of
GC, from the formation of tumor cells to the creation of resistance to chemotherapeutic-induced
cell death. One of the most prevalent biomarkers in GC is microRNA-21 it is one of the progressive
biomarkers that identify the gene involved in expression and regulation in GC. It behaves as a gene
suppressor biomarker in gastric cancer diagnostics.

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