III. REVIEW OF A PRIMARY SOURCE ARTICLE

HPV’s viral oncoproteins E6 & E7 are multifunctional

As introduced in my previous post about the mechanism of HPV infection in the molecular level, researches have reveal HPV’s E6 and E7 proteins interact with p53 and retinoblastoma protein (pRb) in host cells.  By attacking host cell’s guardian proteins like p53 and pRb, E6/E7 proteins deregulate normal cellular processes of cell cycle control, apoptosis, migration, and immune evasion.   But it is not the end of the story of viral proteins.  Like all viral oncoproteins, viral E6/E7 proteins are found to be multifunctional.  In this post, I would like to feature another aspect of viral E6/E7 proteins’ interaction with host micro RNAs, reported by Xiaohong Wang et al. in their article entitled “microRNAs are biomarkers of oncogenic human papillomavirus infections” published in PNAS in 2014.

First of all, what is miRNA?

MicroRNAs (miRNA) are non-coding single strand RNA consisting of about 21 nucleotides.  miRNA are formed through the transcription by RNA polymerases II and III and a series of cleavages events. miRNA is known to regulate gene expressions of certain proteins by forming RISC (RNA-induced silencing complex) and binds to the ‘3 untranslated region of the target mRNAs’ complementary sequence.  This way, miRNA interrupts host cell’s protein synthesis and often causes destruction of the target mRNA.  The exact functions of many miRNA are still unknown and numerous studies are currently conducted to identify their mechanism and function. 

Does HPV infection affect the level of miRNA?

This is a basic research to observe any changes in the level of different miRNAs amount in host cells in presence of E6/E7 proteins using miRNA microarray analysis and miRNA sequencing technique. 

First, researchers determined if HPV infection changes the level of miRNA profiles in (1) human foreskin and vaginal keratinocyte raft cultures infected with cancer-causing type HPV16, (2) cultures infected with cancer-causing types HPV 18, and (3) untreated cultures (i.e., control).  The expression level of miRNA in both HPV-infected cultures (1) & (2) was found decreased in some miRNA and increased in others, compared to the control cultures. 

Further, researchers identified 13 miRNA have altered expression after HPV infection through miRNA array and miRNA sequencing: 8 of miRNAs increased the expression, and 5 decreased the expression after the HPV infection.  Because this result might have been affected by the model system but not solely by the HPV infection, researchers further observed miRNA profiles of HPV18-infected raft cultures at different times over the course of the infection.  Researchers eliminated those miRNA that were associated with cell proliferation and skin differentiation, as their expression naturally vary according to cells’ multiplication.  Researchers verified that 9 of 13 miRNA’s expression were in fact affected by HPV infection.  Of 8 miRNA were selected for the further study: miR-16, miR-22, miR-25, miR-27a, miR-29a, miR-92a, miR-100, and miR-378.

Do E6 and E7 oncoproteins changed the miRNA expression level?

In order to confirm whether it was E6/E7 oncoproteins that changed miRNA expression, researchers infected raft cultures with (1) a retrovirus (as a control), (2) retroviruses expressing E6, (3) retroviruses expressing E7, or (4) retroviruses expressing both E6/E7, and examined miRNA levels using reverse transcription quantitative PCR.  The results confirm that E6/E7 could alter the expression miRNA level, with an exception of miR25 with E6. 

Any difference found in the miRNA expression level as cancer progresses?

Researchers further evaluated the 8 miRNA expression level in 158 cervical tissue samples.  Prepared were: 38 from normal cervix without HPV infection (i.e., control), 13 cervical intraepithelial neoplasia (CIN) 1 and 2, 39 CIN3, and 68 cervical cancer samples.  The results indicate the increased level of miR-25, miR-92a, and miR-378 in the HPV infected tissues, but no significant change in in miR-22, miR-29a, or miR-100 in the HPV infected tissues.  Samples with miR-16 expression showed increased trend in CIN3 and cancer samples, but inconclusive due to too many expressions in all samples. miR-27a showed no change in CIN tissues but significant increase in cancer samples.  

What does the statistics tell you?

Researchers now specifically identified which miRNAs increase or do not change after the infection.  In order for the statistical analysis, researchers results in two groups: a group of miR-25, miR-92a and a group of miR-22, miR-29a.  The expression levels of each miRNA are averaged in each group.  As shown in the graph, the mean value of miR-25/92a increased as the cell’s abnormality increased from normal to CIN1, 2, 3, to cervical cancer, where the mean value of miR-22/29a did not exhibit the trend.  Researchers took the expression ratio between miR-25/92a and miR-22/29a groups, then set the value of 1.5 as the threshold ratio for the future diagnosis of HVP-induced cancer or CINs.  If the miR-25/92a expression ratio is equal to or higher than 1.5, 88.2% of chance that the tissue has cervical cancer, and 48.7% of chance, the tissue developed to CIN3, and 38% of chance, the tissue developed to CIN1/2.

Expression ratio of two miRNA groups can be used for diagnosis of CIN and CC
		miR-25/92a vs. miR-22/29a
		<1.5		≥1.5
Pathology	Total samples	Samples	%	Samples	%
Normal	38	33	86.8	5	13.2*
CIN1+2	13	8	61.5	5	38.5
CIN3	39	20	51.3	19	48.7
CC	68	8	11.8	60	88.2
*P < 0.0001 (F test) among four groups with a ratio equal to or greater than 1.5.

What is the significance of this study?

The significance of the study is that the change of specific miRNA expression level can be used as a marker to detect and diagnose to determine if a person is infected with the cancer-causing type of HPV, and how far it has been progressed.  Currently, the Pap-smear test is the major screening test, which involves microscopic observation of abnormal cell growth.  The result of this study could offer additional screening or monitoring means of the cervical cancer caused by the HVP infection in the future.  

My recommendations for future research
In this study, the last experiment was conducted using only 158 cervical tissues.  If their study were to be used in the future diagnostic standard, more cervical tissues from different individuals be tested.  Also, HPV is known to cause neck and throat cancers in men and women as well.  Applications of this study result to diagnosis of HPV-derived cancer in other parts of body than cervix should be considered and studied.         

Xiaohong Wang et al. “microRNAs are biomarkers of oncogenic human papillomavirus infections”.Proc Natl Acad Sci U S A. 2014 Mar 18; 111(11): 4262–4267.