**3. HPV nucleic acid detection**

Detection of HPV E6/E7 messenger RNA (mRNA) is an indicator of HPV oncogenic activity and may be used as a clinically predictive marker to identify women at risk of developing high-grade cervical dysplastic lesions and cervical carcinoma [63]. This method has been proposed as a more specific marker for cervical dysplasia and cancer than HPV DNA [64]. Several assays have been designed to detect mRNA of the E6 and E7 transforming genes of HPV. The level of E6 and E7 gene expression is increased in high-grade lesions compared with low-grade lesions. These tests have high specificity for detecting disease and could potentially serve as functional discriminators between high-risk and low-risk infections [19].

to reduce colposcopy referral in both ASCUS and low grade squamous intraepithelial lesion (LSIL). It is also more efficient than cytology for the triage of HPV DNA-positive women. Nevertheless, its low sensitivity demands a strict follow-up of HPV DNA positive-mRNA

Molecular Tools for Detection Human Papillomavirus

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37

APTIMA (Gen-Probe, San Diego, CA) is a commercially available mRNA test for HPV, which detectsmRNAofHR-HPVtypes (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and68),based on transcription-mediated amplification [66,74]. There is indication that the APTIMA test has clinicalsensitivitysimilartothatofHC2[74],butwithhigherspecificityfordetectionofdysplasia and improved clinical specificity over that of HC2, which is designed to detect DNA of the same HPV types, except for type 66 [66]. The germany study concluded that APTIMA had a sensitiv‐ ity similar to but a specificity significantly higher (P < 0.0001) than the HC2 test for the detec‐ tion of CIN2+. that he AHPV assay was significantly more sensitive and significantly more specific than cytology for the detection of disease [75]. The assay is sensitive and very specific for detection of high-risk HPV, which may improve patient management and reduce the cost of

In study carried out as part of a multicenter study in Canada which was assessed the clinical usefulness of testing for E6/E7 mRNA and other molecular biomarkers in cervical cancer screening in comparison with HPV DNA testing and cytology. A point worth noting is that 61 (15.9%) of 384 CIN 2+ cases were found in women with normal cytology at the time of enrollment, and 70.5% and 86.9% of these were positive by NASBA and HC2, respectively. This reinforces the importance of incorporating HPV testing or repeat cytology in cervical cancer screening. Also, a separate analysis of unsatisfactory cytology indicated an enriched

This reflects the inherent limitation of cytology-based evaluation. Regardless, the positivity with NASBA or HC2 was dependent on the number of genotypes covered by the respective tests and their prevalence in different grades of cytologic and histologic lesions. Furthermore, the lesion progression is more strongly associated with types 16 and 18, and there are indica‐ tions that an HPV test that distinguishes types 16 and 18 from other oncogenic types may be

Microfluidic approaches in diagnostics achieve significant reagent volume reduction and thus cost-drive innovation, potentially achieving widespread penetration in non-hospital, nonspecialized environments. However, microfluidic approaches are not without their challenges. The field of "lab-on-a-chip" (LOC) diagnostics has grown rapidly from this basic need, and it is fast accelerating towards a "sample-in answer-out" platform for molecular diagnostics. NASBA technology with real-time fluorescence measurement was adapted to detect HPV mRNA in cervical specimens and cervical cell lines. The isothermal nature of NASBA greatly simplifies amplification strategies for nucleic acid detection on chip. This platform has huge potential within "point-of-care"(POC) diagnostics as up to 16 different targets can be detected

In recent study from Norway, the prototype NASBA platform presented amplification efficien‐ cy of has been compared to an industry gold standard for HPV detection with encouraging

more useful as it could identify women at greater risk of cervical cancer [71].

simultaneously for each clinical sample analyzed [77].

care. However, APTIMA does not specify the individual HPV types detected [76].

negative cases [73].

population of CIN 2+ [71].

Initial studies comparing HPV DNA and RNA detection (using consensus PCR and PreTect HPV-Proofer) in women with ASCUS and mild dysplasia on cervical cytology showed that twice as many women were positive for HPV DNA compared with those with HPV RNA positivity. They also demonstrated that while both tests were highly sensitive for detection of CIN2+ (85.7%), HPV RNA detection was far more specific for detection of high-grade lesions (84.9% vs 50%) [65].

The PreTect HPV-Proofer (NorChip AS, Klokkarstua, Norway) is a commercially available real-time multiplex nucleic acid sequence-based amplification (NASBA) assay, also called NucliSENS EasyQ (bioMérieux, Marcy l'Etoile, France). This assay is based HPV detection using HPV DNA plasmids and detects E6/E7 mRNA of the five most common HPV types, HPV16, 18, 31, 33, and 45 [64,66]. These five types have been shown to account for about 82% of cervical cancer worldwide, but their prevalence varies between different geographical regions [67].

In comparison to HPV DNA tests, NASBA-based HPV detection showed better results in terms of specificity for high-grade cervical lesions, while its sensitivity is lower. Currently, it is difficult to know whether the differences in diagnostic accuracy are a result of the larger type detection range and high analytical sensitivity of the HPV DNA tests. To elucidate this issue, experimental confirmation that RNA is the sole target of HPV NASBA is required [64].

NASBA-positive women are 69.8 times more likely to be diagnosed with CIN2+ within 2 years of testing than NASBA negative women. Thus, the addition of HPV mRNA triage to cervical screening programmers may decrease the incidence of false-negative results while also allowing the time interval between screening events to be increased for those women who are HPV DNA and RNA negative [63]. Several studies on the relative performance of NASBA have been conducted in Europe with an indication that this test is more specific than other tests, including the APTIMA and HC2 assays to identify CIN 2+ [65,68-70]. This indicates that the NASBA assay can be used reliably to obtain simultaneous type-specific information for the five genotypes targeted by the test. This is an appealing feature given the indication for the identification of types 16 and 18 in risk stratification and better clinical management of women with a positive HPV test [71].

On the other hand, there have been reports that the NASBA technique can detect DNA, causing false-positive results [64,72]. However, NASBA can serve as a better triage test than HPV DNA to reduce colposcopy referral in both ASCUS and low grade squamous intraepithelial lesion (LSIL). It is also more efficient than cytology for the triage of HPV DNA-positive women. Nevertheless, its low sensitivity demands a strict follow-up of HPV DNA positive-mRNA negative cases [73].

**3. HPV nucleic acid detection**

(84.9% vs 50%) [65].

regions [67].

with a positive HPV test [71].

Detection of HPV E6/E7 messenger RNA (mRNA) is an indicator of HPV oncogenic activity and may be used as a clinically predictive marker to identify women at risk of developing high-grade cervical dysplastic lesions and cervical carcinoma [63]. This method has been proposed as a more specific marker for cervical dysplasia and cancer than HPV DNA [64]. Several assays have been designed to detect mRNA of the E6 and E7 transforming genes of HPV. The level of E6 and E7 gene expression is increased in high-grade lesions compared with low-grade lesions. These tests have high specificity for detecting disease and could potentially

Initial studies comparing HPV DNA and RNA detection (using consensus PCR and PreTect HPV-Proofer) in women with ASCUS and mild dysplasia on cervical cytology showed that twice as many women were positive for HPV DNA compared with those with HPV RNA positivity. They also demonstrated that while both tests were highly sensitive for detection of CIN2+ (85.7%), HPV RNA detection was far more specific for detection of high-grade lesions

The PreTect HPV-Proofer (NorChip AS, Klokkarstua, Norway) is a commercially available real-time multiplex nucleic acid sequence-based amplification (NASBA) assay, also called NucliSENS EasyQ (bioMérieux, Marcy l'Etoile, France). This assay is based HPV detection using HPV DNA plasmids and detects E6/E7 mRNA of the five most common HPV types, HPV16, 18, 31, 33, and 45 [64,66]. These five types have been shown to account for about 82% of cervical cancer worldwide, but their prevalence varies between different geographical

In comparison to HPV DNA tests, NASBA-based HPV detection showed better results in terms of specificity for high-grade cervical lesions, while its sensitivity is lower. Currently, it is difficult to know whether the differences in diagnostic accuracy are a result of the larger type detection range and high analytical sensitivity of the HPV DNA tests. To elucidate this issue, experimental confirmation that RNA is the sole target of HPV NASBA is required [64].

NASBA-positive women are 69.8 times more likely to be diagnosed with CIN2+ within 2 years of testing than NASBA negative women. Thus, the addition of HPV mRNA triage to cervical screening programmers may decrease the incidence of false-negative results while also allowing the time interval between screening events to be increased for those women who are HPV DNA and RNA negative [63]. Several studies on the relative performance of NASBA have been conducted in Europe with an indication that this test is more specific than other tests, including the APTIMA and HC2 assays to identify CIN 2+ [65,68-70]. This indicates that the NASBA assay can be used reliably to obtain simultaneous type-specific information for the five genotypes targeted by the test. This is an appealing feature given the indication for the identification of types 16 and 18 in risk stratification and better clinical management of women

On the other hand, there have been reports that the NASBA technique can detect DNA, causing false-positive results [64,72]. However, NASBA can serve as a better triage test than HPV DNA

serve as functional discriminators between high-risk and low-risk infections [19].

36 Human Papillomavirus and Related Diseases – From Bench to Bedside A Diagnostic and Preventive Perspective

APTIMA (Gen-Probe, San Diego, CA) is a commercially available mRNA test for HPV, which detectsmRNAofHR-HPVtypes (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and68),based on transcription-mediated amplification [66,74]. There is indication that the APTIMA test has clinicalsensitivitysimilartothatofHC2[74],butwithhigherspecificityfordetectionofdysplasia and improved clinical specificity over that of HC2, which is designed to detect DNA of the same HPV types, except for type 66 [66]. The germany study concluded that APTIMA had a sensitiv‐ ity similar to but a specificity significantly higher (P < 0.0001) than the HC2 test for the detec‐ tion of CIN2+. that he AHPV assay was significantly more sensitive and significantly more specific than cytology for the detection of disease [75]. The assay is sensitive and very specific for detection of high-risk HPV, which may improve patient management and reduce the cost of care. However, APTIMA does not specify the individual HPV types detected [76].

In study carried out as part of a multicenter study in Canada which was assessed the clinical usefulness of testing for E6/E7 mRNA and other molecular biomarkers in cervical cancer screening in comparison with HPV DNA testing and cytology. A point worth noting is that 61 (15.9%) of 384 CIN 2+ cases were found in women with normal cytology at the time of enrollment, and 70.5% and 86.9% of these were positive by NASBA and HC2, respectively. This reinforces the importance of incorporating HPV testing or repeat cytology in cervical cancer screening. Also, a separate analysis of unsatisfactory cytology indicated an enriched population of CIN 2+ [71].

This reflects the inherent limitation of cytology-based evaluation. Regardless, the positivity with NASBA or HC2 was dependent on the number of genotypes covered by the respective tests and their prevalence in different grades of cytologic and histologic lesions. Furthermore, the lesion progression is more strongly associated with types 16 and 18, and there are indica‐ tions that an HPV test that distinguishes types 16 and 18 from other oncogenic types may be more useful as it could identify women at greater risk of cervical cancer [71].

Microfluidic approaches in diagnostics achieve significant reagent volume reduction and thus cost-drive innovation, potentially achieving widespread penetration in non-hospital, nonspecialized environments. However, microfluidic approaches are not without their challenges. The field of "lab-on-a-chip" (LOC) diagnostics has grown rapidly from this basic need, and it is fast accelerating towards a "sample-in answer-out" platform for molecular diagnostics. NASBA technology with real-time fluorescence measurement was adapted to detect HPV mRNA in cervical specimens and cervical cell lines. The isothermal nature of NASBA greatly simplifies amplification strategies for nucleic acid detection on chip. This platform has huge potential within "point-of-care"(POC) diagnostics as up to 16 different targets can be detected simultaneously for each clinical sample analyzed [77].

In recent study from Norway, the prototype NASBA platform presented amplification efficien‐ cy of has been compared to an industry gold standard for HPV detection with encouraging

results. By this, we have demonstrated the subcomponents of a complete integrated in vitro diagnosticsystem:fromclinicalsampleinputtosamplepreparationtoamplificationtodetection, thus advancingtowards a "sample-in, answer-out"diagnosticplatform.TheprototypeNASBA platform combined extraction and amplification in a microfluidic device consisting of extrac‐ tion and NASBA chips. In addition, the adopted NASBA method offers the unique characteris‐ tics of isothermal amplification, greatly simplifying the thermocycling requirements for the system: effectively allowing a "blackbox" technology to bedevelopedencompassing amplifica‐ tion and detection simultaneously in a real-time format. The Authors described this technolo‐ gy platform is not limited to diagnostics of cervical precancer and cancer, but it has enormous potential in the monitoring and diagnosis of gene activity in areas such as infectious disease, oncology, immune response to allergens, immunotherapies, and chemotherapies [77].

miRNA export), Dicer (an RNase-III enzyme that produces mature miRNA from pre-miRNA),

Molecular Tools for Detection Human Papillomavirus

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39

In the study [81] was profiled 455 miRNAs via miRNA microarray in 4 cervical cancer tissues and 4 normal cervical cancer tissues and they found significant overexpression of 18 miRNAs and underexpression of 15 miRNAs cervical cancer tissues compared to normal cervical cancer tissues [85]. In the analysis from157 cellular miRNAs via the TaqMan MicroRNA Human Early Panel Kit (Applied Biosystems) in 10 early stage squamous cell carcinomas and 10 normal cervical samples. The authors found overpression of 68 miRNAs and underexpression of two

A recent review published [81] presented that cervical cancer represents a unique tumor model forunderstandinghow viralE6 andE7 oncoproteinsderegulate the expressionofthemiR-15/16 cluster, miR-17-92 family, miR-21, miR-23b, miR-34a, and miR-10b/93/25 cluster via the E6 p53 and E7-pRb pathways. miRNAs may influence the expression of papillomavirus gene in a differentiation-dependent manner by targeting viral RNA transcripts. Genome-wide profiling of miRNA signatures has indicated that aberrant, increased or decreased, miRNA expression is common in most human tumors [83,86]. The table 2 [81] presented a summary of miRNA expression profiling studies in cervical cancer, as illustrated below, in three distinct studies.

In the study conducted in 2011 was found significant overexpression of 18 miRNAs and underexpression of 2 miRNAs in cervical cancertissues comparedto normal cervicaltissues via TaqMan MicroRNA arrays. Those authors demonstrated, via individual qRT-PCR assays, significantoverexpressionofmiR-16,miR-21,miR-106b,miR135b,miR-141,miR-223,miR-301b and miR-449a, and underexpression of miR-218 and miR-433 in cervical cancer and dysplasia from normal cervical compared to normal cervical tissue. Their results showed that miR-21, miR-135b, miR-223 and miR-301b were overexpressed in the cervical cancer tissue compared to both normal and cervical dysplasia tissue. So, those authors concluded that such miRNAs are

Another recent study published in 2011 was analyzed six paired normal and cervical cancer tissues by using the same miRNA array plataform and showed an increased expression of 12 miRNAs and decreased expression of 11 miRNAs, as presented in table 2 [87]. The study presented by [88] found increased expression of miR-21 in cervical cancer and a decreased expression of both miR-143 and miR-145. Those data together indicated that cervical cancer expresses no or very little of the miR-143/145 cluster. A PCR-based miRNA assay was used to analyze 102 cervical cancer samples [89] and the authors identified miR-200a and miR-9 as two

In the analysis [90] was investigated 802 unknown and 122 predicted human miRNAs via CaptialBio mammalian miRNA arrays V 3.0 in 13 HPV-16 and HPV-18 positive cervical cancers and their adjacent normal tissues. The authors found that miR-141 is overexpressed and miR-218 is underexpressed in cervical cancer compared to normal cervical tissue. In the study [111] analyzed miRNA expression via microarray in 5 cervical squamous cell carcinomas that appeared to be HPV-negative and 5 normal cervical tissues and they found that miR-21 to be

good candidates for markers of progression from normal to dysplasia to cancer [85].

molecular markers that could be used to predict cervical cancer survival.

overexpressed in the cervical cancer samples.

TRBP (a Dicer partner), and Ago2 (a major component for RISC) [81,83-84].

in the cervical carcinomas versus normal cervical samples [86].

The mRNA assays with real-time PCR may be a useful tool in investigation of as well as in primary screening for cervical neoplasias, and it might be worthwhile to consider which genotypes to include in further investigations to optimize sensitivity and specificity, especially in a post vaccine era, when it may be necessary to reconsider HPV testing strategies [66].

#### **3.1. MicroRNA analysis in Human Papillomavirus (HPV)**

MicroRNAs (miRNAs) are noncoding regulatory RNAs 18-25 nucleotides in size that are derived from RNA polymerase II (pol II) transcripts of coding or noncoding genes. MiRNAs most often function by binding to the 3´ UTRs of target messenger RNAs, whereby they induce mRNA degradation or translational repression [78]. Many miRNAs are tissue- or differentia‐ tion-specific, and their temporal or short-lived expression modulates gene expression at the posttranscriptional level by base-pairing with complementary nucleotide sequences of target mRNAs [79-80]. The functions of miRNAs are still largely unknown, but it seems that they are important in the regulation of cellular gene expression and behavior [81,82].

As of August 2012, the miRBase database (http://www.mirbase.org/) [112] had collected 21,264 entries representing hairpin precursor pre-miRNAs. Human genome contains ~ 416 miRNA genes encoding 1048 distinct mature miRNAs from every chromosome except Y. Approxi‐ mately 113 miRNA genes encode a cluster of miRNAs and produce ~ 390 miRNA sequences. Bioinformatics prediction shows that each miRNA targets ~ 200 RNA transcripts directly or indirectly, and up to one-third of the total number of human mRNAs are targets of more than one miRNA [81]. So, the actions of miRNAs exert profound effects on gene expression at the posttranscriptional level in almost every biological process. However, miRNA expression itself, similar to any other transcription mediated by pol II, is regulated both at the transcrip‐ tional and posttranscriptional levels.

Many cellular transcription factors, including c-Myc, p53, and E2F, have been described to regulate miRNA transcription. Oncogenic HPV E6 induces degradation of p53 and E7 mediates degradation of pRB to release E2F from the pRB–E2F complex, it is conceivable that oncogenic HPV infection causes aberrant expression of cellular miRNAs [81]. Other factors involved in miRNA maturation and processing after transcription are Drosha (an RNase-II endonuclease that produces pre-miRNA from pri-miRNA), DGCR8 (DiGeorge syndrome critical region gene 8, a double-stranded RNA-binding protein needed for Drosha activity), exportin 5 (for premiRNA export), Dicer (an RNase-III enzyme that produces mature miRNA from pre-miRNA), TRBP (a Dicer partner), and Ago2 (a major component for RISC) [81,83-84].

results. By this, we have demonstrated the subcomponents of a complete integrated in vitro diagnosticsystem:fromclinicalsampleinputtosamplepreparationtoamplificationtodetection, thus advancingtowards a "sample-in, answer-out"diagnosticplatform.TheprototypeNASBA platform combined extraction and amplification in a microfluidic device consisting of extrac‐ tion and NASBA chips. In addition, the adopted NASBA method offers the unique characteris‐ tics of isothermal amplification, greatly simplifying the thermocycling requirements for the system: effectively allowing a "blackbox" technology to bedevelopedencompassing amplifica‐ tion and detection simultaneously in a real-time format. The Authors described this technolo‐ gy platform is not limited to diagnostics of cervical precancer and cancer, but it has enormous potential in the monitoring and diagnosis of gene activity in areas such as infectious disease,

38 Human Papillomavirus and Related Diseases – From Bench to Bedside A Diagnostic and Preventive Perspective

oncology, immune response to allergens, immunotherapies, and chemotherapies [77].

**3.1. MicroRNA analysis in Human Papillomavirus (HPV)**

tional and posttranscriptional levels.

The mRNA assays with real-time PCR may be a useful tool in investigation of as well as in primary screening for cervical neoplasias, and it might be worthwhile to consider which genotypes to include in further investigations to optimize sensitivity and specificity, especially in a post vaccine era, when it may be necessary to reconsider HPV testing strategies [66].

MicroRNAs (miRNAs) are noncoding regulatory RNAs 18-25 nucleotides in size that are derived from RNA polymerase II (pol II) transcripts of coding or noncoding genes. MiRNAs most often function by binding to the 3´ UTRs of target messenger RNAs, whereby they induce mRNA degradation or translational repression [78]. Many miRNAs are tissue- or differentia‐ tion-specific, and their temporal or short-lived expression modulates gene expression at the posttranscriptional level by base-pairing with complementary nucleotide sequences of target mRNAs [79-80]. The functions of miRNAs are still largely unknown, but it seems that they are

As of August 2012, the miRBase database (http://www.mirbase.org/) [112] had collected 21,264 entries representing hairpin precursor pre-miRNAs. Human genome contains ~ 416 miRNA genes encoding 1048 distinct mature miRNAs from every chromosome except Y. Approxi‐ mately 113 miRNA genes encode a cluster of miRNAs and produce ~ 390 miRNA sequences. Bioinformatics prediction shows that each miRNA targets ~ 200 RNA transcripts directly or indirectly, and up to one-third of the total number of human mRNAs are targets of more than one miRNA [81]. So, the actions of miRNAs exert profound effects on gene expression at the posttranscriptional level in almost every biological process. However, miRNA expression itself, similar to any other transcription mediated by pol II, is regulated both at the transcrip‐

Many cellular transcription factors, including c-Myc, p53, and E2F, have been described to regulate miRNA transcription. Oncogenic HPV E6 induces degradation of p53 and E7 mediates degradation of pRB to release E2F from the pRB–E2F complex, it is conceivable that oncogenic HPV infection causes aberrant expression of cellular miRNAs [81]. Other factors involved in miRNA maturation and processing after transcription are Drosha (an RNase-II endonuclease that produces pre-miRNA from pri-miRNA), DGCR8 (DiGeorge syndrome critical region gene 8, a double-stranded RNA-binding protein needed for Drosha activity), exportin 5 (for pre-

important in the regulation of cellular gene expression and behavior [81,82].

In the study [81] was profiled 455 miRNAs via miRNA microarray in 4 cervical cancer tissues and 4 normal cervical cancer tissues and they found significant overexpression of 18 miRNAs and underexpression of 15 miRNAs cervical cancer tissues compared to normal cervical cancer tissues [85]. In the analysis from157 cellular miRNAs via the TaqMan MicroRNA Human Early Panel Kit (Applied Biosystems) in 10 early stage squamous cell carcinomas and 10 normal cervical samples. The authors found overpression of 68 miRNAs and underexpression of two in the cervical carcinomas versus normal cervical samples [86].

A recent review published [81] presented that cervical cancer represents a unique tumor model forunderstandinghow viralE6 andE7 oncoproteinsderegulate the expressionofthemiR-15/16 cluster, miR-17-92 family, miR-21, miR-23b, miR-34a, and miR-10b/93/25 cluster via the E6 p53 and E7-pRb pathways. miRNAs may influence the expression of papillomavirus gene in a differentiation-dependent manner by targeting viral RNA transcripts. Genome-wide profiling of miRNA signatures has indicated that aberrant, increased or decreased, miRNA expression is common in most human tumors [83,86]. The table 2 [81] presented a summary of miRNA expression profiling studies in cervical cancer, as illustrated below, in three distinct studies.

In the study conducted in 2011 was found significant overexpression of 18 miRNAs and underexpression of 2 miRNAs in cervical cancertissues comparedto normal cervicaltissues via TaqMan MicroRNA arrays. Those authors demonstrated, via individual qRT-PCR assays, significantoverexpressionofmiR-16,miR-21,miR-106b,miR135b,miR-141,miR-223,miR-301b and miR-449a, and underexpression of miR-218 and miR-433 in cervical cancer and dysplasia from normal cervical compared to normal cervical tissue. Their results showed that miR-21, miR-135b, miR-223 and miR-301b were overexpressed in the cervical cancer tissue compared to both normal and cervical dysplasia tissue. So, those authors concluded that such miRNAs are good candidates for markers of progression from normal to dysplasia to cancer [85].

Another recent study published in 2011 was analyzed six paired normal and cervical cancer tissues by using the same miRNA array plataform and showed an increased expression of 12 miRNAs and decreased expression of 11 miRNAs, as presented in table 2 [87]. The study presented by [88] found increased expression of miR-21 in cervical cancer and a decreased expression of both miR-143 and miR-145. Those data together indicated that cervical cancer expresses no or very little of the miR-143/145 cluster. A PCR-based miRNA assay was used to analyze 102 cervical cancer samples [89] and the authors identified miR-200a and miR-9 as two molecular markers that could be used to predict cervical cancer survival.

In the analysis [90] was investigated 802 unknown and 122 predicted human miRNAs via CaptialBio mammalian miRNA arrays V 3.0 in 13 HPV-16 and HPV-18 positive cervical cancers and their adjacent normal tissues. The authors found that miR-141 is overexpressed and miR-218 is underexpressed in cervical cancer compared to normal cervical tissue. In the study [111] analyzed miRNA expression via microarray in 5 cervical squamous cell carcinomas that appeared to be HPV-negative and 5 normal cervical tissues and they found that miR-21 to be overexpressed in the cervical cancer samples.

MiR-21 is the most highly overexpressed miRNA in numerous cancers and it down regulates the tumor suppressor PTEN in non-small cell lung cancer and in hepatocellular cancer, in which PTEN regulation leads to overexpression of matrix metalloproteinases MMP2 and MMP9 [85]. These proteins promote cellular migration and invasion. MiR-21 also targets the tumor suppressor genes tropomyosin I (TPM1), programmed cell death 4 (PDCD4) and maspin, and the latter two have been implicated in tumor invasion and metastasis [91]. Increased miR-21 expression in cervical cancer may be attributable to both E6 and E7. Such viral oncoproteins play important roles in regulating cellular miRNA expression and their function can be reduced due to epigenetic modification of miRNA genes. Identification of a panel of miRNAs that can be used as early biomarkers in cervical cancer is potentially useful to determine disease behavior and prognosis. They may also provide new targets for anticancer therapy [85].

**miRNA Chromosome locationWang et al [108] Li et al [109] Witten et al [110]**

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41

miR-10b 2q31.1 Down Down

miR-125a 19q13.41 Down Down miR-125b 11q24.1 Down Down

miR-133b 6p12.2 Down Down miR-143 5q32 Down Down Down

miR-424 Xq26.3 Down Down Down miR-450 Xq26.3 Down Down Down

The incidence of cervical cancer in the decades to come might in fact increase in developing countries due to the aging of the population and the persistent absence of adequate screening programs [95]. In countries with screening, the target of preventive efforts has shifted from cervical cancer detection to the diagnosis and treatment of cancer precursor lesions. However,

miR-183 7q32.2 Up

miR-223 Xq12 Up

miR-324-5p 17p13.1 Up

miR-30b 8q24.22 Down

miR-133a 18q11.2 Down

miR-145 5q32 Down miR-191 3p21.31 Down

miR-378 (422b) 5q32 Down miR-422a 15q22.31 Down

miR-574 4p14 Down

**Table 2.** Summary of miRNA signatures in cervical cancer.

miR-185 22q11.21 Up Up

miR-224 Xq28 Up Up

miR-29a 7q32.3 Down Down

miR-34a 1p36.22 Down Down

miR-126 9q34.3 Down Down miR-127 14q32.2 Down Down

miR-218 4p15.31;5q34 Down Down

miR-455 9q32 Down Down

Up: upregulated in cervical cancer; down, downregulated in cervical cancer.[81].

**4. Clinical utility of molecular HPV diagnosis**

Until now, there are evidence indicating that HPV regulation of cellular miRNA expression most likely occurs through viral E6 and E7 [81], although oncogenic HPVs do not produce viral miRNAs, they are responsible for the aberrant expression of oncogenic or tumor suppressive miRNAs. High-risk E6 and E7 interact separately with several dozens or even hundreds of cellular factors and these interactions could lead to increased or decreased expression cellular miRNAs. Some of the altered miRNA expression could be a result from both E6 and E7. It is known that the main function of oncogenic HPV E6 is to target p53 for degradation [81-93]. As a transcription factor, p53 plays an important role in the transcription of numerous coding and noncoding genes [92-94] and it seems that oncogenic HPV is capable of regulating the expression of many cellular miRNAs via p53 [81], and its downstream targets are miR-34a and miR-23b, increasing cell growth and migration in cervical tissues.



Up: upregulated in cervical cancer; down, downregulated in cervical cancer.[81].

**Table 2.** Summary of miRNA signatures in cervical cancer.

MiR-21 is the most highly overexpressed miRNA in numerous cancers and it down regulates the tumor suppressor PTEN in non-small cell lung cancer and in hepatocellular cancer, in which PTEN regulation leads to overexpression of matrix metalloproteinases MMP2 and MMP9 [85]. These proteins promote cellular migration and invasion. MiR-21 also targets the tumor suppressor genes tropomyosin I (TPM1), programmed cell death 4 (PDCD4) and maspin, and the latter two have been implicated in tumor invasion and metastasis [91]. Increased miR-21 expression in cervical cancer may be attributable to both E6 and E7. Such viral oncoproteins play important roles in regulating cellular miRNA expression and their function can be reduced due to epigenetic modification of miRNA genes. Identification of a panel of miRNAs that can be used as early biomarkers in cervical cancer is potentially useful to determine disease behavior and prognosis. They may also provide new targets for anticancer

40 Human Papillomavirus and Related Diseases – From Bench to Bedside A Diagnostic and Preventive Perspective

Until now, there are evidence indicating that HPV regulation of cellular miRNA expression most likely occurs through viral E6 and E7 [81], although oncogenic HPVs do not produce viral miRNAs, they are responsible for the aberrant expression of oncogenic or tumor suppressive miRNAs. High-risk E6 and E7 interact separately with several dozens or even hundreds of cellular factors and these interactions could lead to increased or decreased expression cellular miRNAs. Some of the altered miRNA expression could be a result from both E6 and E7. It is known that the main function of oncogenic HPV E6 is to target p53 for degradation [81-93]. As a transcription factor, p53 plays an important role in the transcription of numerous coding and noncoding genes [92-94] and it seems that oncogenic HPV is capable of regulating the expression of many cellular miRNAs via p53 [81], and its downstream targets are miR-34a and

**miRNA Chromosome locationWang et al [108] Li et al [109] Witten et al [110]**

miR-21 17q23.1 Up Up Up

miR-155 21q21.3 Up Up

miR-182 7q32.2 Up Up Up

miR-23b, increasing cell growth and migration in cervical tissues.

miR-15b 3q25.33 Up Up miR-16 13q14.2 Up Up miR-17-5p 13q31.3 Up Up miR-20a 13q31.3 Up Up miR-20b Xq26.2 Up Up

miR-93 7q22.1 Up Up miR-106a Xq26.2 Up Up

miR-15ª 13q14.2 Up

miR-146a 5q34 Up miR-148a 7p15.2 Up

miR-181c 19p13.13 Up

therapy [85].
