2. Cervical cancer is a female-specific disease with a
high incidence and mortality behind breast & lung
cancer
A disease in which malignant cells form in the
tissues of the cervix
It rises in 30–34 years of age and peaks at 55–65
years
The worldwide incidence of cervical cancer is
~510,000 new cases with ~288,000 deaths
annually
Cervical cancer is a complex disease caused by the
interaction of viral, host, and environmental factors
3. Prevention, diagnosis, and treatment of female-specific diseases are
increasingly important issues due to lifestyle changes of women
Human pappilomavirus (HPV) has been detected in more than 90% of
cervical cancers and therefore implicated as the main cause of cervical
cancer
HPV infection alone is not sufficient to induce the malignant
transformation of HPV-infected cells
Other unidentified genetic alterations, such as microRNAs the master
switches, are required.
MiRNAs are found to be associated in many cases of cervical cancer with
changed expressions.
4. Signs and Symptoms
The most common symptoms are:
Bleeding between periods
Bleeding after sexual intercourse
Bleeding in post-menopausal women
Discomfort during sexual intercourse
Smelly vaginal discharge
Vaginal discharge tinged with blood
Pelvic pain
5. Causes
Risk factors which are known to increase the risk of
developing cervical cancer-
Human pappilomavirus (HPV)
Many sexual partners, becoming sexually active early
Smoking
Weakened immune system
Giving birth at a very young age
Several pregnancies
Contraceptive pill
Other sexually transmitted diseases (STD)
Chlamydia, gonorrhea
Genetic Alterations
6. Stages of Cervical Cancer
Mild Dysplasia
Moderate Dysplasia
Severe Dysplasia
Invasive Cervical Cancer
Stages of cervical cancer according to WHO classification-
7. Types of Cervical Cancer
Ectocervix-
Squamous Cell
Carcinoma
Endocervix-
Adenocarcinoma
8. Tests To diagnose Cervical Cancer
Phisical Exam & History
Pelvic Exam
Pap Test
HPV Test
Endocervical Curettage
Colposcopy
Biopsy
9. Cervical Cancer Vaccines
Two Vaccines are Licensed globally
which are also available in India:
A Quardivalent vaccine- Gardasil
(Merk)
HPV Serotypes 16, 18, 6 & 11
A bivalent vaccine- Cervarix
(Glaxo Smith Kline)
HPV serotypes HPV 16 & 18 Gardasil
10. Human Pappilomavirus (HPV)
One of the most common STIs
Papillomaviruses are small viruses approximately 52-55nm in size
HPVs are circular double stranded DNA viruses & belongs to family-
Papillomaviridae
In 1981, Zur Hausen et al. reported the detection of HPV in cervical
neoplasia
In 1995 the WHO declared HPV as a known carcinogen for causing
cervical cancer, because HPV DNA types could be detected in almost all
cervical cancers
12. Classification Of HPV
More than 100 types of human papilloma viruses (HPVs)
are known today, and they are generally classified according
to their potential to induce malignant transformation-
13. HPV Life Cycle and Infection
The HPV life cycle consists of initial infection, uncoating, genome
maintenance, genome amplification, and packaging to form new viral
particles
14. HPV Induced Dysregulation
Once viral DNA is incorporated into host DNA, oncogenic transformation
is induced
Oncoproteins E6 & E7 inactivate tumor suppressor genes p53 & pRB.
E6 protein binds to E6-associated protein (E6-AP), a ubiquitin protein
ligase , & forms E6/E6-AP complex & targets p53
E6 degrades p53, inhibits p53 binding to DNA & binds to p53 enhancers &
suppressed p53 function
E6/E6-AP complex also activates telomerase & contributes to cell
immortalization
E7 protein contributes to carcinogenesis by degrading Rb family proteins
necessary for cell cycle progression
15. HPV Determition
PCR-based methods are commonly used for HPV Detection
after DNA isolation
Most PCR assays utilize consensus primers, directed to a
conserved L1 gene, and hence are able to amplify most of the
mucosal HPV types
The other PCR type used to detect specific HPV is HPV type
specific PCR such as HPV 16 or 18
16. MicroRNA
MicroRNAs are small (~18-24 nt), non-coding RNAs that regulate
gene expression & associated with cancer
First miRNA, lin-4, was discovered in C. elegans in 1993, is found
in most eukaryotes, including humans
It is predicted that miRNA account for 1-5% of the human genome
and regulate at least 30% of protein-coding genes
To date, more than 17,000 miRNAs have been annotated in 142
species, including over 1900 human miRNAs
18. Classification of MiRNAs
MicroRNAs are classified in two group depending on their
origin-
Intergenic or Exonic miRNAs: located between the introns of
genes & transcribed by RNA pol II or pol III as a stem loop structure called
pri-miRNA
Interagenic or Intronic miRNAs: miRNAs located within an
intron of a protein coding gene & transcribed by RNA pol II as part of pre-
mRNA
20. MiRNA Expression Profiling
Initially it was conducted on samples extracted from tissues, now stable
miRNAs are found in readily available body fluids including, serum ,
plasma, urine and saliva
MiRNA expressions are generally analyzed by microarray & qRT-PCR
using microRNA specific primers
U6 RNA, RNU44, and RNU48 is usually used as reference control & A.
thaliana miRNA as negative control
Quantification is done using the 2 delta Ct method, where fold change in
expression of a gene in an experimental sample is quantified relative to the
same gene in a reference sample
21. MiRNAs In Cervical Cancer
MiRNAs play a vital role in cancer regulating pathways, like controlling
cell proliferation, differentiation and survival
MiRNAs involved in carcinogenesis are classified into oncogenic miRNAs
(oncomiRs) and tumor suppressor miRNAs(TSG)
Involved in cancer pathogenesis by posttranscriptional regulation of gene
expression
50% of miRNA genes are localized in cancer-associated genomic regions
or in fragile sites or integration sites of high-risk HPVs
Expression patterns of miRNAs suggested that beyond HPV, microRNAs
play a major role in cervical cancer
22. Altered Expressions of MiRNAs in
Cervical Cancer
MicroRNAs expressions were analyzed for normal cervix and cervical
cancer tissues, by microarray in combination with RT-PCR verification &
found to be deregulated
On comparision, many miRNAs with cancer-specific upregulation or
downregulation have been found-
MiR-21 is overexpressed in cervical cancer and is a negative regulator of
expression of the tumor suppressor gene programmed cell death 4
(PDCD4)
23. MiR Let-7a was found to be downregulated by HPV & this
downregulation of miR let-7a leads to the aberrant expression of STAT3
(validate target of let-7a) developing CC
MiR- 218 is found to be underexpressed in CC tissues compared to the
normal cervix & leads to the decreased expression of LAMB3, which is
involved in cell migration and tumorigenicity
MiR-34a was identified as a direct transcriptional target of cellular
transcription factor p53, since HPV E6 oncoprotein destabilizes p53 during
virus infection, it causes down-regulation of miR-34a expression in most
CC tissues
Cont..
24. Regulation of miRNAs by HPV
Oncoproteins
Deletions or mutations in miRNA genes, as well as
aberrant expression of oncogenic or tumor-
suppressive miRNAs, are common in human
cancers
Deregulation of oncogenic and tumor suppressive
miRNAs in human cervical cancer is associated
with HR-HPV integration
Cervical cancer represents a unique tumor model
for understanding how viral E6 and E7
oncoproteins deregulate the expression of the
microRNA
MiRNAs
HPV
28. HPV-Oncoproteins are able to regulate the
expression of miRNAs
HPV Proteins MiRNAs Up/Downregulated Target Gene
E5 mir-146a Up-regulated ZNF813
E5 mir-324-5p Down-regulated CDH2, CTNNB1
E5 mir-203 Down-regulated p63
E6 mir-34a Down-regulated p18Ink4c, CDK4,
CDK6, Cyclin E2
E6 mir-218 Down-regulated LAMB3
E6 mir-23b Down-regulated uPA
E6/E7 mir-29 Down-regulated YY1 and CDK6
E7 mir-15b Down-regulated CCNA2, CCNB1,
CCNB2 MSH6
E7
miR-15a/
miR-16-1 and
miR-203
Down-regulated c-Myc, c-Myb, PPAR
29. Diagnosis & Treatment
of Cervical Cancer
Using miRNAs
Many studies have examined the use of
miRNAs as cancer diagnostic marker and as
anticancer therapy.
30. Diagnosis of Cervical Cancer Using
miRNAs in Serum
MiRNAs with expression changes in cancer have the potential to be
diagnostic biomarkers based on plasma & serum tests
miR-21 and miR-126 are found to be overexpressed in serum and are
associated with cervical cancer.
Overexpression of other miRs including miR-27a, miR-34, miR-34a, miR-
146a, miR-155, miR-196a, miR-203, and miR-221 was detected in the
serum of CC samples
These results indicate that miRNA levels in serum can be used for
diagnosis of cervical cancer
31. Diagnosis of Lymph Node Metastasis
Using miRNAs in Serum
Several miRNAs in serum have been identified as candidate markers for
lymph node metastasis in CC
Zhao et al. analyzed expression of miR-20a and miR-203 in serum
collected before surgery and treatment in 80 patients:
The miR-20a level in serum of patients with CC was markedly higher than
that in healthy volunteers and was overexpressed in patients with lymph
node metastasis
The expression level of miR-203 in patients with CC was higher in
comparison with healthy volunteers, however, lymph node metastasis was
found only when miR-203 expression was suppressed
33. Treatment with miRNA
Supplementation
Anticancer treatment may be achieved by regulating the expression
level of miRNAs
The function of tumor suppressor miRs with reduced levels may be
recovered by supplementation of the miRNA itself
Atelocollagen, is being examined as a potential delivery system for
nucleic-acid-based drugs
This protein is extracted from calf dermis and then digested with
protease to reduce antigenicity & can be transferred in to tissues or
cells
34. Supplementary agents can be classified as:
o Hairpin single-stranded pre-miRNA
o Double-stranded RNA
Liu et al. introduced miR-143 into HeLa CC cells and showed that cell
growth was inhibited and apoptosis was enhanced with increased miR-143
expression & maintained Bcl2 (oncogene) expression
o Therefore, miR-143 has an association with Bcl2 and treatment targeting
this pathway may be possible
Similarly, supplementing anti-let7a miR in HPV16 positive CC cases can
increase the expression of down-regulated let-7a miR & maintain the
expression of STAT3
Cont..
35. Treatment by Inhibition of
miRNA Function
One strategy for overexpressed miRNA in cancer is to inhibit the miRNA
function using agents with complementary binding to the miRNA
Antisense miRNA oligonucleotides (AMOs) or ‘antagomirs’ are the most
common miRNA inhibitors based on Antisense technology
• For miR-21, an oncomiR in cervical cancer, anti-miR-21 was developed as
a modified 2’-O-methoxyethyl (2’-O-MOE) phosphorothioate antisense
agent
• Wang et al. found, miR-21 expression was downregulated and tumor
growth was markedly suppressed by the AMO in comparison with a control
group
36. • MiR-21 inhibition may be achieved with other approaches,
including miRNA sponges, miRNA erasers, and tough decoys
• Tough decoys may have particularly potent inhibitory activity
• Haraguchi et al. inhibited miR-21 using a tough decoy and
recovered expression of PDCD4, a target gene of miR-21
Cont..
37. Difference MiRNA Based Drug &
Traditional Drug
Miravirsen, first miR based drug, made for silencing
miR-122 in HCV infected patients.
39. miRNAs Associated with Therapeutic
Resistance for Cervical Cancer
Expression of various miRNAs is up- or downregulated in cervical cancer
and these expression levels can increase or decrease sensitivity to
chemotherapy and radiotherapy
Phuah et al. showed that the expression patterns of 25 miRNAs, including
miR-138, miR-210, and miR-744, altered the sensitivity to 1’S-1’-
acetoxychavicol (ACA) and cisplatin
Lei et al. found that miR-155 negatively regulates the EGF-induced
epithelial-mesenchymal transition(EMT), inhibits proliferation, metastasis,
invasion, and increases sensitivity to cisplatin
Thus, miRNAs may have an important role in the response to
chemotherapy
40. Summary & Conclusion
HPV
Cervical
Cancer
Progression
Cervical cancer remains as a leading cause of
morbidity and mortality for women worldwide
HPV Integration may alter miRNA expression via
deletion, amplification, or genomic rearrangement
which may have implications for their expression in
cervical cancer
Depending on the nature of their targets, miRNAs
can function as either tumor suppressor miRs or
oncogenic miRs.
These findings suggest many approaches to miRNA-
specific personalized treatment and molecular targeted
therapy
Therefore, miRNAs are likely to be important in
diagnosis and treatment of cervical cancer.
Target
MiRNA
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