| Part I: The study of Cdc6 in contributing to G1 checkpoint abrogation under hypoxia in HPV E7 expressing cellsObjectiveThe infection of Human papillomavirus （HPV） is the most common sexually transmitted disease in worldwide. At present, more than 170 types of HPV have been found while most of them are not pathogenic. Although HPV vaccine is becoming available in many countries and regions, there is no effective way to the people who has immunosuppressive disease or has already been infected by HPV. Given by this,it is still of importance that finding a reasonable way to contribute the treatment of HPV-associated carcinomas. Many cervical cancer patients are HPV positive. The integration of HPV leads to the instability of human genome. E6 and E7, two critical oncoproteins, leading to the dysfunction of p53 and pRb and promote the formation of tumors.The solid tumor is characterized by irregular blood vessels, low oxygen and deficient of nutrition supplement. The increasing cell proliferation and less oxygen make cells under a hypoxic stress condition. Meanwhile, the dysfunction and overexpression of hypoxia inducible factor 1-alpha （HIF-la） is highly associated with tumor progression. As a transcription factor, HIF-la is implicated in cancer cell survival through up-regulating VEGF, EPO and some kinases in metabolism. On the other hand, HIF-la suppresses cell proliferation. HIF-la induces cell cycle arrest at G1 phase by up-regulating p53 and p27. A new study showed that HIF-1α block cell cycle progression in a non-transcriptional way. HIF-1α binds to cell division cycle 6（Cdc6）, which is very important in DNA replication initiation, to the chromosome.HIF-1α activates Cdc7 to make the dephosphorylation of minichromosome maintenance protein complex （MCM） to stop the cell cycle progression. In cervical cancer, the two cell cycle controlling proteins p53 and pRb are dysfunctional. E7 also increases the transcriptional activity of HIF-1α. Given by this, the responses of cells to hypoxic condition are more complicated, so it is worthwhile to completely study the G1/S transition mechanism under hypoxia in cervical cancer cells.Cdc6 plays an important role in DNA replication of eukaryotes cells. The typical function of Cdc6 is the assembling of pre-replication complex （preRC）, which is contributing to DNA replication initiation. Cdc6 is also known as a biological marker of early diagnosis for cervical cancer. In recent years, it has been reported that Cdc6 plays a role in G1/S phase transition. Cdc6 activates p21/p27-bound cyclin dependent kinase 2 （Cdk2） to regulate cell cycle progression. Our previous study showed that E7 up-regulated the expression of Cdc6. In addition, Cdc6 played a role in serum starvation induced G1 checkpoint abrogation in E7 expressing cells. These clues are leading us to wonder that whether Cdc6 was involved in hypoxic response in E7 cells.To sum up. two goals are needed to be addressed in this study: （1） To explore the cell cycle profile of HPV-16 E7 expressing cells under hypoxia; （2） to study the function of Cdc6 in regulating G1 checkpoint abrogation in E7 expressing cells under hypoxia. This study would be helpful in understanding HPV carcinogenesis and would be informative to clinical treatment of cervical cancer.Methods1. Determination of E7 cells proliferation under hypoxia1.1 The construction of hypoxic model: Using three different methods to mimic the hypoxic condition. Incubating cells in hypoxic chamber with 1% O2; cells were treated with drug DFO or CoCl2 （CoCl2 was abandoned later due to the side effect of DNA damage）.1.2 Cell viability assay of E7 expressing cells under hypoxia: Cells were treated with DFO or CoCl2 at different concentrations. Seventy-two hours later, cell viability was determined by CCK8 assay.1.3 Cell cycle profiling of E7 expressing cells under hypoxia: E7 cells were treated with hypoxic chamber or DFO, and then stained with PI, determined using flow cytometry. E7 cells that treated with the same condition, labeled with BrdU,determined the percentage of S phase using flow cytometry.2. The function of E7 in response to hypoxic condition in cervical cancer cells2.1 siRNA strategy: In cervical cancer, E6*I is the most efficient transcript and is responsible for E7 production. The siRNA targeting E7 not only knock down the E7 but also E6. Given by this, we utilized siRNA198 to interfere E6E7, siRNA209 to specifically knock down E6. The siE6 would be the control of siE6E7 in studying the function of E7 under hypoxia.2.2 Cell cycle profiling of E7 knockdown cells under hypoxia: Knocked down E6E7 or E6 using RNAi. Determine the steady-state level of p53 and pRb to evaluate the knockdown efficiency. Examine the mRNA level of E6 and E7 to assess the knockdown efficiency directly. Using cell cycle profiling method described in 1.4 to assess cell cycle progression and S phase cells under hypoxia.3. Detection of cell cycle related proteins under hypoxia in E7 expressing cells:E7 cells were treated with DFO for 8 hours, proteins were collected and the steady state level of HIF-la, Cdkl, Cdk2, Cdk4, Cdk6, p53 and p27 were determined by western blotting. The mRNA level of selected genes was measured by RT-qPCR.4. Identification of differentially expressed proteins under hypoxia and protein verification4.1 Identification of differentially expressed proteins under hypoxia: Using Label-free mass spectrometry-based protein quantification to identify proteins that differentially expressed in E7 expressing cells under hypoxia.4.2 GO analysis of differentially expressed proteins and verification: Using cut off 1.75 to keep proteins that both up-regulated and down-regulated in E7 expressing cells. Among these proteins. GO functional analysis was done. Cdc6 drew our attention and was setting as a key protein in regulating G1/S transition under hypoxia.Western blotting and RT-qPCR verified the change of Cdc6 in cell line.5. Discovering the mechanism of Cdc6 in hypoxia induced G1 checkpoint abrogation5.1 The function of Cdc6 in response to hypoxia in E7 expressing cells: Knocked down Cdc6 in E7 cells to the level of which in vector control cells using RNAi.Determine the function of Cdc6 in G1/S transition through cell cycle analysis and BrdU assay.5.2 The function of Cdc6 in regulating cell cycle kinases under hypoxia: Knocked down Cdc6 in E7 expressing cells to the level of which in vector cells using RNAi.Determined cell cycle related proteins after Cdc6 knocked down. Using Co-IP assay to detect the binding of p21 bound Cdk1 under hypoxic condition.Results1. HPV-16 E7 abrogates hypoxia induced G1 arrest: After treated with DFO or CoCl2. the cell viability of both E7 expressing cells and vector cells were decreased but E7 cells had a higher rate. At concentration 100 and 200 μM of DFO. 0.4 and 0.6 mM of CoCl2, the differences were significant. Hypoxia also decreased the progression of cell cycle. However. E7 expressing cells could partly bypass G1 checkpoint under hypoxia compared with vector control cells.2. The reduction of E7 impairs the ability of cervical cancer cells bypassing G1 checkpoint under hypoxia: The increased expression of p53 and pRb showed that the knockdown efficiency, which was accordance with the decreased expression of E6 and E7 by RT-qPCR. Under normoxia. knockdown E6E7 or E6 showed similar cell cycle pattern, however, most of CaSki-siE6E7 cells stopped at G1 phase compared with CaSki-siE6 cells under hypoxia. BrdU assay suggested that loss of E7 impaired the percentage of S phase cells under hypoxic conditions.3. Detection of cell cycle related proteins under hypoxia in E7 expressing cells:RPE1 E7 and vector cells treated with DFO or hypoxic chamber for 8 h. The protein level of cell cycle related proteins were detected by WB. The results showed that p53,p21 and p27 were up-regulated in vector cells, but no difference in E7 expressing cells. Both Cdkl and Cdk2 were decreased with the treatment of hypoxia. However,they remain higher in E7 expressing cells. RT-qPCR showed that Cdkl and Cdk2 had a higher level in mRNA.4. Identification of differentially expressed proteins under hypoxia and protein verification: Label-free M.S. showed that 182 differentially expressed proteins were found in E7-expressing cells under hypoxia （at cut off 1.75）. Among the proteins, 99 of them were up-regulated in E7 expressing cells and 83 of them were down-regulated.GO analysis indicated that most of the proteins were belonged to translation, cell viability, protein complex assembly, DNA replication and redox balancing. Protein Cdc6 remained a higher level in E7 expressing cells and verified by WB and RT-qPCR.5. Cdc6 plays a role in E7-mediated G1 checkpoint abrogation under hypoxia:Using siRNA to examine the function of Cdc6 in G1/S transition. To. ensure the integrity of cell cycle, we knocked down Cdc6 in E7 expressing cells to the level of Cdc6 in vector cells. Cell cycle analysis showed that the G1 phase in E7-siCdc6 was 74.2% while in E7-siCon cells was 63.9%, the difference was significant. BrdU labeling showed that the S phase in E7-siCdc6 was 5.43 （compared with E7-siCon,19.12%）. Knocked down Cdc6 impaired the ability of E7 cell in abrogating G1 checkpoint and less cells were entranced to S phase, and this function was independent with its role in DNA replication initiation.6. Cdc6 was contributing to the release of Cdkl bounded by p21: We recently showed that Cdkl plays a role in DNA induced G1 arrest in E7 expressing cells. WB results showed that Cdc6 knocked down had no influence to the expression of p53,p21 and Cdkl, Cdk2, indicating that Cdc6 was not able to convert cell cycle related proteins directly. Co-IP showed that after Cdc6 knocked down, the association of p21 with Cdcl was enhanced, illustrating that Cdc6 was controlling the release of Cdkl bounded by p21, and the higher level of Cdcl would be able to accelerate cell cycle progression under hypoxic conditions.Conclusion1. HPV-16 E7 plays a role in G1 checkpoint abrogation under hypoxia in cervical cancer cells.2. The differentially expressed proteins in E7 expressing cells are examined using M.S. quantification. Cdc6 is up-regulated in E7 cells and is verified by WB.3. Cdc6 is contributing to G1/S transition in E7 expressing cells under hypoxia, and this role is independent with DNA replication initiation.4. Cdc6 decreases the association of Cdkl and p21, which could possibly accelerate cell cycle progression under hypoxia.In summary, our study examined the E7 cell cycle profiles under hypoxia, provided evidences that Cdc6 was contributing to HPV-associated cervical cell progression under hypoxia, and shed light to clinical treatment of cervical cancer.Part Ⅱ: The Construction of a Novel Method in Determining HPV-16 Integration Sites in Human GenomesObjectiveHead and neck cancer is a group of cancers that starts from the mouth, nose,throat, larynx, sinuses or salivary glands. Basically it can be divided into three parts:Neck cancers, ENT cancers and oral and maxillofacial tumors. More than 90% of head and neck cancers are squamous cell carcinoma. The head and neck cancers are dominant among all the other cancers in primary sites and pathological types. Head and neck cancer is highly related with alcohol or tobacco. Infection of human papillomavirus （HPV） is also a major cause of head and neck cancer, especially oropharyngeal squamous cell carcinoma （OPSCC）, which has over 80% HPV positive rate. According to an epidemiological survey, OPSCC is closely related with the people that have multiple sexual partners, frequent oral sex but with low smoking rate.Although the incidence of head and neck cancer is decreased, OPSCC has an increasing rate worldwide. Given by these, a deep study in pathogenesis of OPSCC is very important.HPV is a small DNA virus from the papillomavirus family, and can be classified as high-risk or low-risk based on the carcinogenesis. The E6 and E7 of high-risk HPV are closely related with tumor development. In addition to degrading tumor suppresser p53 and pRb, E6 or E7 binds or inactivates other cellular proteins and makes them dysfunctional, such as hTERT. E6 and E7 are not enough for cells malignant transformation, further genome behavior is more closely with tumor development.Therefore, it is believed that the infection and integration of HPV exacerbates the genomic instability, and E6 or E7 increases the mutation rate of tumor suppressor and adjacent genes of virus integration cites is the key factor leading to cell carcinogenesis.Increased genomic instability is an important sign for tumor development, followed by polyploidy formation, which led to the emergence of aneuploidy cells. Persistent high-risk HPV infection leads to DNA damage in host cells, leading to polyploidy formation by destroying microtubules, activating spindle assembly checkpoint.Therefore, comprehensive detection of HPV integration sites in OPSCC, analyze the integration frequency and diversity is informative for understanding the mechanism of HPV infection to OPSCC in clinical treatment.Early detection of HPV is using molecular biology methods, such as polymerase chain reaction （PCR）. Assuming that the integration of HPV leads to the absence of early gene E2 in the host chromosome, the primers are specifically designed for E6 and E2 to evaluate HPV integration level. With the development of new technologies,SPF-LiPA assay and APOT analysis based on hybridization were becoming more efficient in determining HPV integration sites. These technologies can specifically combine and partly amplify the virus fusion sites adjacent fragments to get the integration information. However, the above methods have a great limitation in primer （or probe） design, so they are biased and can only detect a small amount of HPV types and cannot be widely used in clinical. In recent years, with the rapid development of next generation sequencing （NGS）, many HPV integration detection method based on this technology have been established. At present. the most comprehensive detection for HPV integration sits was done by Huada team using whole genome sequencing method. They detected 5 different cervical cancer cell lines and 104 clinical samples of cervical cancer patients. This method covers a wide range of HPV sequences and with lower bias, but also costly and time consuming. It is not flexible in research model conversion, which is not conducive to the popularization of technology and the extraction and detection of individual patient information. To solve these problems, we created a PCR-based sequencing technology. determined HPV integration sites in formalin-fixed, paraffin-embedded （FFPE） OPSCC patient samples. This method requires small amount of specimen, with short time and high sensitivity. It also provides an accurate detection mean for exogenous DNA detection.Methods1. Determination of HPV rate in OPSCC FFPE samples1.1 RNA extraction in OPSCC FFPE samples: We extracted RNA from American OPSCC FFPE slides （p16 positive） based on QIAGEN extraction kit.1.2 Design of specific HPV-16 primers: According to our paper published previously, we designed the E6/E7 primers that were specific aiming to HPV type 16,18, 31,33, 35, 39, 45, 52, 56, 58, 59, 66 and 68. GAPDH and β-actin were used as controls.1.3 Plate preparation of HPV profiling: In 384 well plate, each sample was equally divided into 32 wells, and 12 samples in each plate. Diluted the primers mentioned above using TE buffer to 0.5μM and aliquot 5 μL to each well. Vacuumed the primers for 1 hour and stored in -20℃.1.4 Examine HPV level in OPSCC FFPE sample by RT-qPCR: Reverse transcription of RNA to cDNA, aliquot cDNA to prepared HPV profiling plate. RT-qPCR to examine the HPV rate in OPSCC FFPE samples.2. Construction of the new PCR-based sequencing method and library construction2.1 DNA extraction in OPSCC FFPE samples: We extracted DNA from American OPSCC FFPE slides （p16 positive） based on QIAGEN extraction kit.2.2 Design of HPV-16 sequencing primers based on Illumina platform: In this process, we used dual-sequencing method with two indexed and 1 molecular index. In adapter part, the sequences were the same with OligoA and have a short tail for annealing. Oligo B was introduced by PCR. As for primer design. we programmed primers using Perl language. HPV-16 has more than 7900 bases, primers were designed according to the following principles: a, the length is between 21 and 25; b,the Tm is between 61.5℃ and 63℃; c, GC content is in the middle of 40% to 60%; d,primers are with high complexity and have no repeated sequence; e. no self-complimentary sequences. At last, 19 primers of sense strand or anti-sense strand were chosen with a gap of 400 bases between each primer in same direction. These primers are able to cover the whole length of HPV in two directions.2.3 Optimization of library construction methods in cell line: HPV-16 positive CaSki cells were used as positive control. We established the sequencing method in OPSCC cell line UM-SCC-47 and optimized the library construction, including initial DNA amount, ligation condition, the chosen of two-step PCR or one-step PCR in library construction and PCR condition.2.4 Determination of HPV integration sites in OPSCC FFPE samples: 102 HPV-16 positive samples were divided into 2 batches and with 1 ovarian control. Whole lane sequencing was used for all samples at reading 2 x 250.3. Data output and bioinformatics analysisData analysis consists of 3 parts; the reading of raw data, data processing and data output. We programmed 3 scripts using Perl language for data analysis.3.1 Processing of raw data: Run the first script and completed the tasks as follows: a,abandoning incompletely data or incorrect data; b, shaving data that containing adapter or primer sequences; c, data clustering. Although data was collected by dual directions, each cluster shared the same name so we could easily combine two reads by using 10 "L".3.2 Data comparison with HPV-16 sequence: Run the second script to accomplish the comparison between the data and HPV-16 sequence. The minimum length was setting as 30 bases. To completely search HPV sequences, we used a strategy that starting from the first read to 30 read to compare with HPV sequences, such as 1-30,2-31,3-32, et al. Abandoned reads with matching bases below 30.3.3 Data comparison with human genome: Run the third script to accomplish the comparison between the data output from last step with human genome sequences.Similar, we used the same strategy and the minimum base was setting as 30.Abandoned reads with matching bases below 30 and output data with chromosome information.Results1. More than 90% OPSCC patients are HPV positive: The quality of RNA extracted from OPSCC FFPE samples （p16 positive） passed the HPV profiling quality check. Among 135 samples, 13 patients were HPV negative, 3 were HPV-18 positive,3 patients were HPV-33 positive, 1 was HPV-59 positive and 115 patients were HPV-16 positive. HPV-16 positive patients were chosen for further integration sites detection.2. HPV integration sites detection method is established and applied in OPSCC patients: The quality of DNA that extracted from HPV-16 positive OPSCC FFPE samples passed the library construction quality check. Using CaSki as the positive control, 293T cells as the negative control, we successfully established the new PCR-based sequencing method in UM-SCC-47 cells. We then implemented this method to 102 HPV-16 positive OPSCC patients and determined the HPV integration sites. We used the sequencing data of sample 0450R0461F as a template to show the data reading, DNA library analysis and the HPV-16 integrated sites. We made a comparison of this method to targeted sequencing and whole genome sequencing,showing the advantages in convenient, and quite informative to OPSCC research that infected by HPV.ConclusionThe samples we chose in this study were p16 positive in immunohistochemistry.Protein p16 is commonly used to predict HPV-positive cancers. Here. we firstly used HPV profiling to measure 13 common types of HPV level in OPSCC patients, and we found that the HPV positive rate was more than 90%, and mostly was type 16.Moreover, HPV 18, 33 and 59 types were also found, which is similar with previous report. This method provides an accurate and effective mean for clinical HPV screening of either head and neck cancer or cervical cancer patients.We estabalished a novel method which is Multiplex PCR based and 96-well prepared, to determine the HPV-16 integration sites in HPV-16 positive OPSCC FFPE samples. We performed the sequencing in UM-SCC-47 cell line and showed data analysis in TP63 gene. We examined 102 HPV-16 positive OPSCC FFPE samples the HPV integration sites, and took 0450R0461F as an example to show library control and human genome integration sites.This method is highly innovative, unbiased with time-saving and shows more tolerant to samples. We firstly examined the HPV integration sites in OPSCC patients using NGS sequencing method and this method could be potentially used in exogenous DNA detection and very informative to clinical treatment. |
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