Experimental Study On CRISPR-Cas9 Gene Editing And Gene Treatment Of HPV58 Positive Cervical Cancer C33A Cells

BackgroundCervical cancer is one of the most common malignant tumors in women.According to the statistical calculation,the number of new cases of cervical cancer among women in the world in 2018 was 570,000,and the number of deaths was 311,000,ranking the fourth in both the number of cases and the proportion of deaths among female malignant tumors.Nearly 90 percent of cervical cancer deaths worldwide occur in developing countries.In China there are about 140,000 new cases of cervical cancer and about 37,000 deaths every year.Persistent infection with Human Papillomavirus(HPV)is currently recognized as a major risk factor for cervical cancer.It takes about 10-20 years from the initial HPV infection to the development of cervical cancer.which means the development of cervical cancer is a complex process with multiple factors.Most cervical cancers are caused by HPV 16 and HPV 18 infections.In China,HPV58 is the third important type of cervical cancer,and the infection rate has increased in recent years.In the past 20 years,the 5-year survival rate of cervical cancer has not changed,and the efficacy of all current treatment strategies(radiotherapy,chemotherapy and surgery)has leveled off,which are harsh on normal cells and not targeted enough on cervical malignant tissues.The positive measure to effectively prevent HPV infection is to vaccinate HPV vaccine,but current vaccines can neither prevent all types of HPV infection nor treat existing diseases.On the basis of studying the pathogenesis of HPV,how to effectively eliminate HPV infection and control the pathogenicity of HPV has always been the key link in the prevention and treatment of cervical cancer.Currently,there is an urgent need for an effective method for the treatment of HPV infection and a new and more effective way of treating cervical cancer.From the late 1980s to the early 1990s,researchers observed a DNA sequence in the genomes of bacteria and archaea called Clustered Regularly Interspaced Short Palindromic Repeat squences(CRISPR).With the development of research,it has been recognized that CRISPR-Cas system is an acquired immune system in bacteria and archaea.Through gRNA(guide RNA)mediation,it specifically cleaves exogenous genetic material to resist invasive viruses and plasmids.In recent years,the research based on CRISPR-Cas has developed in blowout mode.CRISPR-Cas9 system can edit cell genome DNA of higher organisms accurately and efficiently.The CRISPR-Cas9 system has shown broad application prospects in gene therapy for various human diseases because of the abnormal gene expression that causes many human diseases.CRISPR-Cas9 system is a very effective method for eliminating viral infections in organisms.So far,there have been many studies on the application of CRISPR-Cas9 system in the treatment of viral driven cancer,such as hepatitis B virus and liver cancer,Epstein barr virus and Burkitt lymphoma and nasopharyngeal carcinoma,human herpes virus and Kaposi’s sarcoma,HPV and cervical cancer.At present,CRISPR-Cas9 gene editing technology is mainly applied in the research of gene therapy of cervical cancer related to HPV 16 and ls,but there is no report on the application of it in the treatment of cervical cancer related to HPV58.This project is to study the gene editing and gene treatment of CR1SPR-Cas9 system in HPV58 positive cervical cancer cells and tissues.The specific research contents include the following two parts:Part Ⅰ:Experimental study on the effect and mechanism of CRISPR-Cas9 system on proliferation and apoptosis of HPV58-positive cervical cancer cellsObjective:To study the molecular mechanism of CRISPR-Cas9 system targeting the inactivation of E6 and E7 genes in HPV58-positive cervical cancer C33A cells(C33A/LV-HPV58E6E7)and the inhibition of proliferation and apoptosis of cancer cells,and to verify whether CRISPR-Cas9 system can be developed into a new generation of gene therapy technology for HPV58 infection and related cervical cancer.Methods:1.Four pairs of different gRNA sequences were designed and synthesized by using double gRNA method(paired gRNA/Cas9 D10A strategy),and linked into the corresponding expression box of the vector paio-mcherry with their respective enzymes.Thus,the expression plasmids pAIO-mCherry/gRNAI,pAIO-mCherry/gRNA2,pAIO-mCherry/gRNA3 and pAIO-mCherry/gRNA4 of the CRISPR-Cas9 system targeting the two genes of E6 and E7 of HPV58 were constructed and verified by Sanger sequencing respectively.2.T7 endonuclease I(T7 EI)digestion test was used.To verify the cutting efficiency of CRISPR-Cas9 system on target genome.Target fragments with Cas9 cutting efficiency were selected,sequenced and analyzed.3.Expression plasmid of CRISPR-Cas9 system was transfected into human C33A cervical cancer cell Iine(C33A/LV-HPV58E6E7)with stable expression of HPV58E6E7 fusion gene.4.The effects of CRISPR-Cas9 system on the expression of C33A/LV-HPV58E6E7 cell lines E6,E7,p53 and pRb protein as well as cell proliferation and apoptosis were detected by Western-Blot assay,CCK-8 proliferation assay,AnnexinV-FITC/PI staining and flow cytometry assay,respectively.Results:1.Detection of cleavage efficiency of CRISPR-Cas9/gRNA by T7 endonuclease I(T7EI):The results of T7 endonuclease I(T7EI)digestion test showed that the CRISPR-Cas9 system constructed by us could successfully and efficiently cut and edit gene target fragments of cells.The cutting rates of Cas/gRNAI,Cas/gRNA2,Cas/gRNA3 and Cas/gRNA4 were 41.5%,54.6%,62.4%and 49.7%,respectively.2.CRISPR-Cas9 system can inhibit the expression of E6 and E7 in C33A/LV-HPV58E6E7 cells,and up-regulate the corresponding p53 and pRb proteins:C33A and C33A/LV-HPV58E6E7 cells were transfected with Cas/gRNAI,Cas/gRNA2,Cas/gRNA3 and Cas/gRNA4 plasmids,respectively,48 hours after transfection.Western-Blot detection of the expression of E6,E7,p53 and pRb proteins in cells showed that Cas/gRNA1 and Cas/gRNA2 could inhibit the expression of E6 gene in C33A/LV-HPV58E6E7 cells,without affecting the expression of E7 gene,and the corresponding increase of p53 protein was caused.Cas/gRNA3 and Cas/gRNA4 can inhibit the expression of E7 gene in C33A/LV-HPV58E6E7 cells,but have no effect on the expression of E6 gene,and accordingly cause the increase of pRb protein.3.CRISPR-Cas9 system can inhibit the proliferation and growth of C33A/LV-HPV58E6E7 tumor cells:C33A/LV-HPV58E6E7 cells,C33A/HPV58 and C33A cells transfected with Cas/gRNA1,Cas/gRNA2,Cas/gRNA3 and Cas/gRNA4 were tested by cck-8 cell proliferation assay.The results showed that the CRISPR-Cas9 expression system could effectively slow down the growth of OD value ofC33A/HPV58+gRNA1,C33A/HPV58+gRNA2,C33A/HPV58+gRNA3 and C33A/HPV58+gRNA4 cells respectively,that is,the CRISPR-Cas9 system could inhibit the proliferation activity of C33A/LV-HPV58E6E7 cells.This trend was obvious from the monitoring point on day 1 and could be maintained until day 5.The proliferation rate of C33A cells transfected with only the blank plasmid vector was not affected by the CRISPR-Cas9 system.The results also showed that HPV58 promoted the growth of C33A tumor cells.4.CRISPR-Cas9 system can promot the apoptosis of C33A/LV-HPV58E6E7 tumor cells:Cas9/gRNA1,Cas9/gRNA2,Cas9/gRNA3 and Cas9/gRNA4 were respectively used to treat C33A/LV-HPV58E6E7 tumor cells and C33A tumor cells.Cas9gRNA1 and Cas9gRNA2 caused the apoptosis rate of C33A/LV-HPV58E6E7 cells to reach 60%,and Cas9gRNA3 and Cas9gRNA4 caused the apoptosis rate of C33A/LV-HPV58E6E7 cells to reach 40-50%.However9 the apoptosis rate of C33A cells treated with Cas9/gRNA was basically the same as that of the blank group,with no significant change.Conclusions:1.CRISPR-Cas9 system constructed by double-gRNA method can cut the genome of HPV58 tumor cells accurately and effectively,which 1s an efficient and convenient gene knockout technology.2.CRISPR-Cas9 system can inhibit the expression of E6 and E7 in C33A/LV-HPV58E6E7 cells,up-regulate the expression of p53 and pRb proteins,inhibit the proliferation of C33A/LV-HPV58E6E7 cells and promote the apoptosis of C33A/LV-HPV58E6E7 cells.CRISPR-Cas9 system could be developed as a gene therapy technique for adjuvant treatment of HPV58 infection and associated cervical cancer in the future.Part Ⅱ:Experimental study of nano-drugs synthesized by PBAE and Michelle with CRISPR-Cas9 plasmid for HPV58-related cervical cancerObjective:To validate that poly beta-aminoacid(PBAE)nanoparticles and F127/PPO-NMe3/pCas9 nanomicelles(Micelle)synthesized with CRISPR-Cas9 plasmid are novel small molecular targeted gene drugs for HPV58 infection and cervical cancer.Methods:1.PBAE,nanoparticles and Micelle nanomicelles were synthesized and characterized.2.PBAE nanoparticles and Micelle nanomicelles were synthesized with the CRISPR-Cas9 plasmids respectively.HPV58-positive fresh cervical cancer tissues and C33A/LV-HPV58E6E7 cell lines were treated with nano-drugs.3.Western-Blot was used to detect the expression of E6,E7,p53 and pRb proteins in the HPV58 positive cervical cancer tissues,and CCK-8 proliferation assay was used to detect the influence on the proliferation of C33A/LV-HPV58E6E7 cell lines.Results:1.The new nano drug can inhibit the expression of E6 and E7 in cells,and up－regulate p53 and pRb proteins in HPV58 positive cervical cancer tissues:Nanoparticles made with PBAE and Micelle were respectively bound to Cas9/gRNAl,Cas9/gRNA2,Cas9/gRNA3 and Cas9/gRNA4 functional plasmids and acted on the above-mentioned two patients with HPV58 positive cervical cancer tissues.Western-Blot analysis showed that gRNA1 and gRNA2 could inhibit the expression of E6 gene in cells and correspondingly cause the increase of p53 protein.GRNA3 and gRNA4 can inhibit the expression of E7 gene in C33AJLV-HPV58E6E7 cells,and correspondingly cause the increase of pRb protein.2.The new nano drug can inhibit the growth of C33A/LV-HPV58E6E7 cells:The C33A/LV-HPV58E6E7 cell lines were significantly inhibited by C33A/LV-HPV58E6E7 cell proliferation assay after C33A/LV-HPV58E6E7 cell lines wererespectively bound to the c9/gRNA1,Cas9/gRNA2,Cas9/gRNA3,and Cas9/gRNA4 functional plasmids using nanoparticles made from PBAE and Micelle.This trend was obvious at the monitoring site on day 1 and maintained until day 5.The proliferation rate of C33A cells transfected with only the blank plasmid vector was not affected by the CRISPR-Cas9 system.Conclusion:Using PBAE nanoparticles and Micelle Micelle as drug carriers,CRISPR-Cas9 plasmid can be transported into cells.The proliferation of C33A/LV-HPV58E6E7 cells was inhibited by inhibiting the expression of E6 and E7 and up-regulating p53 and pRb proteins in HPV58-positive cervical cancer cells.By inhibiting the expression of E6 and E7 and up-regulating the expression of p53 and pRb protein in HPV58-positive cervical cancer cells,the proliferation of C33A/LV-HPV58E6E7 cells can be inhibited,which is expected to become a new small molecular targeted gene drug for HPV58 infection and related cervical cancer.