The Study Of The Relationship Between Virus And Human Breast Cancer And Target Of Gene Therapy

The morbidity of breast cancer was increased annually and breast cancer became the most common female malignancy tumor over the world. Previous research has shown that new cases rate of breast cancer was 29% during 2014 in the United States and it took the first place amoung all kinds of cancers indicating its importance, and its incidence trends to increase by rate of 3.80% per year in China. Breast cancer became the fastest mortality in the city of China. Untill now, the pathogeny of breast cancer is complicated, and there is not any known single or multiple factors which can explained the occurrence and development of breast cancer. The epidemiological study has shown that the occurrence of breast cancer was associated with the environment, lifestyle, diet composition and incretion. The etiologic relationship between virus and human breast cancer has already become the focus field. In 1995 the International Agency for Research into Cancer issued the high risk human papillomavirus (HPV) types 16 and 18-"known to cause human cancer". In vitro studies have shown that the main oncoproteins E6 and E7 from HPV are able to immortalize primary mammary epithelial cell. Reports on the distribution of HPV infection in breast cancer are not limited and the results are controversial. The factors that contribute to this situation include the difference of HPV subtype, pathogenicity, detection method, specimen type and geographical distribution. Mouse mammary tumor virus (MMTV) is a beta-retrovirus. Its genome is an 9 kilobases RNA and contained GAG gene, POL gene and ENV gene encoding envelope protein which played an inmportant role of virus attachment and entry into target cells. Over 90% of mouse mammary tumors had been demonstrated to be related to MMTV. Researches have shown that MMTV resulted in cancers by insertion variation and clonal expansion in mice. Pregnant rats infected by MMTV could generate large amounts of virus and infect to descendants by milk. Studies by scholars such as Indik showed that MMTV could infect human breast cancer cell line, comfirmed MMTV has the capacity to cross barriers between species. But untill now, there was no direct evidence that MMTV could produce human breast cancer. There’s no precedent of using HPV E6 and E7 gene as candidate target for cancer gene therapy. Analysis the existence and expression of virogene in human breast cancer could promote the virogene targeted gene therapy and vaccine research of breast cancer, and help prevent human breast cancer and reduce the risk of dying from human breast cancer. This study had two sections. In the first part, we mainly analyzed the presence of HPV and MMTV in 76 paraffin embedded tissues of breast cancer and SKBR3 cell line. On the basis of screening results, we then mainly analyzed the inhibitory effects of HPV virogene targeted RNA interference on breast cancer cell line in the second part.1. Detection of HPV and MMTV in breast cancerBy setting SKBR3 human breast cancer cell line as control, the primers of HPV L1, HPV16 E6, HPV16 E7, HPV18 E6, HPV18 E7 and MMTV-ENV gene were designed and used to screen 76 pathologically confirmed paraffin embedded tissues of breast cancer by PCR. The percentage of HPV L1, HPV 18 oncogene E6 and E7 was respectively 9.21%,6.58% and 9.21%, while HPV 16 oncogene E6 and E7 was not found in 76 paraffin embedded tissues of breast cancer. And in SKBR3 cell lines, HPV 16 oncogene E6 and E7 was not found, while HPV18 E6 and E7 was existent and accompanied by the expression of HPV18 E6 and E7. Based on clinical-pathological data, the detection rate of HPV18 E6 and E7 was correlated with pathological grade and differentiation degree of breast cancer, but independent on age. HPV18 E6 and E7 was detected in 19 cases of invasive ductal carcinoma, the detection rate of HPV18 E6 and E7 was increased along with the increase of invasive degree. Poorly-differentiated occupy 6 in 19 invasive ductal carcinoma. The detection rate of the enevelop gene of MMTV was 30.26%. This research provides a theoretical basis for the relativity between HPV infection and the occurrence of human breast cancer, and for the virogene targeted gene therapy and vaccine research of breast cancer. The detection rate of HPV 18 E6 and E7 from different types of breast cancer were predicted effectiveness of virogene targeted gene therapy in breast cancer. It offers foundation for clinic curative effect.2. Analysis of the inhibiting effect of virogene targeted RNA interference on breast cancer cell lineOn the basis of screening result, HPV 18 E6 and E7 targeted siRNA were designed using online design tool provided by bioinformatics & research computing. The siRNA was constructed into pSUPER RNAi System, and then the most optimal siRNA was selected by cell viability detection. After the knock down of HPV 18 oncogene E6 and E7 in SKBR3 cells using RNA interference method, we detected the changes of viability, colony forming ability, invasion, cell cycle and the expression of cell cycle regulation genes. In aspect of viability analysis, compared with the control group, the inhibitory rate of HPV 18 E6 and E7 RNA inteference groups was respectively 12% and 9%, and had significant difference (P<0.05). In aspect of colony forming ability analysis, compared with the control group, the inhibitory rate of HPV18 E6 E7 RNA inteference groups was respectively 61% and 64%, and had significant difference (P<0.01). In aspect of invasion analysis, compared with the control group, the inhibitory rate of HPV 18 E6 E7 RNA inteference groups was respectively 59% and 65%, and had significant difference (P<0.01). In aspect of cell cycle analysis, compared with the control group, S% of HPV 18 E6 E7 RNA inteference groups was respectively significantly reduced by 65% and 80% (P<0.01), G0/G1 was respectively significantly increased by 26% and 35% (P<0.01), G2/M was respectively significantly reduced by 50% and 72% (P<0.01). In aspect of cell cycle regulation gene expression analysis, compared with the control group, the E6 inhibitory rate of HPV18 E6 RNA inteference groups was 35%, the E7 inhibitory rate of HPV18 E7 RNA inteference groups was 78%, the TP53-mutant expression of HPV18 E6 E7 RNA inteference groups was respectively reduced by 55% and 39%, the MDM2 was respectively reduced by 63% and 36%, the CCNA1 was respectively reduced by 7% and 15%, the CCND1 was respectively reduced by 18 %and 23%, the BCL-2 was respectively reduced by 39% and 77%, the RB was respectively increased by 94% and 109%, while VEGFA was not apparent changed. HPV18 E6 and E7 targeted RNA inteference has significant inhibiting effect to the human breast cancer cell, offering a possible target for gene therapy of breast cancer.