Study On The Expression Mechanism Of ERK1/2 Signaling Pathway Key Factors In HPV16-positive Cervical Cancer

PART Ⅰ The relationship between HPV16 infection and ERK1/2 signaling pathway expression in cervical cancerOBJECTIVE:Cervical cancer is the second most common type of cancer in the world and the sixth leading cause of cancer-related mortality in women worldwide.Cervical cancer infects about 16 women per 100,000 people every year and kills about 8 per 100,000 people every year.Early cervical cancer can be cured by excision or destruction of precancerous or cancerous tissue.However,once cancer cells are transferred to distant organs,the prognosis of patients will be greatly impaired.Standard treatment for cervical cancer includes surgery,chemotherapy and radiotherapy.These treatments cause damage or damage to normal tissues near or far away,and may promote the invasion and metastasis of cancer cells.Metastasis of cervical cancer may lead to the failure of traditional treatments,including surgery,radiotherapy and chemotherapy.Human papillomavirus(HPV)is the most common sexually transmitted infection in the world.Although most strains are relatively harmless,some strains increase women’s risk of cervical cancer.ERK is an extracellular regulated protein kinase involved in many cellular processes.ERK is activated by phosphorylation from cytoplasm to nucleus and participates in the biological reaction of cells.Therefore,exploring the association between human papillomavirus 16(HPV16)infection and the expression of the extracellular signal-regulated kinase 1/2(ERK1/2)signaling pathway in cervical cancer becomes necessary.PATIENTS AND METHODS:In the present study,163 cervical cancer patients admitted to our hospital(Second Hospital Affiliated to Suzhou University and Fengcheng Branch of Ninth People’s Hospital Affiliated to Shanghai Jiaotong University)from March 2016 to March 2018 were selected as subjects of study as cervical cancer group,while the healthy subjects during the same period were selected as control group.The HPV16 positive rate in specimens in control group and experimental group was detected via HPV DNA type Expression of ERK1/2 protein in the cervical cancer and non-cervical cancer groups via IHC staining,Two groups were selected according to the inclusion criteria and exclusion criteria as cervical cancer specimens(30 cases as cervical cancer group)and healthy cervical specimens(23 cases as non-cervical cancer group),the transcriptional and translational levels of ERK(the key gene in the ERK1/2 signaling pathway)were detected via quantitative real-time polymerase chain reaction(q RT-PCR)and Western blotting.RESULTS:(1)The HPV16 positive rate in cervical cancer group was 81.5%,The positive rate of HPV in control group was 14.1%.The positive rate of HPV 16 in cervical cancer specimens was significantly higher than that in healthy cervical specimens(P < 0.05).ERK1/2 was strongly positive in cervical cancer group,the positive rate was significantly higher than that in healthy cervical specimens(P < 0.05).(2)The results of q RT-PCR revealed that the transcriptional and translational levels of ERK1/2 in HPV16-positive cervical cancer specimens were significantly higher than those in control group,and there were significant differences(P <0.05).(3)The results of Western blotting revealed that the transcriptional and translational levels of ERK1/2 in HPV16-positive cervical cancer specimens were significantly higher than those in control group,and there were significant differences(P <0.05).CONCLUSIONS:HPV16 can facilitate the proliferation of cervical cancer cells through activating the ERK1/2 signaling pathway,thereby promoting the deterioration of cervical cancer.Moreover,activating the ERK1/2 signaling pathway can also promote the proliferation of cervical cancer cells,displaying a mutual-promoting relation.PART Ⅱ The relationship between inhibition of HPV16 infection and ERK1/2 signaling pathway expression in cervical cancerOBJECTIVE: From the first part of the experiment,it is concluded that HPV16 can promote the proliferation of cervical cancer cells by activating ERK1/2 signaling pathway,thereby promoting the deterioration of cervical cancer.Therefore,we need to explore the relationship between inhibition of human papillomavirus 16(HPV16)infection and expression of extracellular signal-regulated kinase 1/2(ERK1/2)signaling pathway in cervical cancer.We try to find a new method to treat cervical cancer with ERK1/2 as the entry point,so as to achieve precise medical treatment.PATIENTS AND METHODS:Moreover,the HPV16-positive Si Ha cells were selected as objects of the in vitro study.Then the HPV16 gene expression was inhibited via RNA interference assay,Thus forming the intervention group and the control group.the transcription and translation levels of ERK1/2 were detected via fluorescence q RT-PCR and Western blotting,and the cell viability was determined via cell counting kit-8(CCK-8)assay.Then the ERK1/2 inhibitor U0126 was added into Si Ha cells,U0126 was used in intervention group,while U0126 was not used in control group.and the proliferation and morphology of Si Ha cells were observed.RESULTS:(1)According to the detection of transcriptional and translational levels of ERK1/2 in HPV16-positive Si Ha cells before and after RNA interference via q RT-PCR and Western blotting,they significantly declined after RNA interference in HPV16 expression,and there were significant differences(P <0.05).(2)At the same time,it was found in the CCK-8 cell viability assay that the cell proliferation was obviously decreased after HPV16 was inhibited,and there were significant differences(P <0.05).(3)After the ERK1/2 inhibitor was added,both proliferation and adherent growth ability of Si Ha cells were remarkably weakened,and there were significant differences(P <0.05)..CONCLUSIONS: Inhibiting HPV16 can inhibit ERK 1/2 signaling pathway,and inhibiting HPV16 and ERK1/2 can reduce cell viability,and there were significant differences(P <0.05).Part Ⅲ Effectors of ERK1/2 on the biological function of cervical cancer cellsOBJECTIVE:Cervical cancer is one of the major cancer diseases affecting women’s health.Among them,HPV infection is the main cause of cervical cancer.A large number of studies have found that there are many biological signals found in cervical cancer.Studies have shown that the mitogen-activated protein kinases(MAPKs)signaling pathway also exists in cancer cells,and has an important impact on the development of cancer cells.In the MAPK family,extracellular signal-regulated kinase(ERK)is an important factor that can transmit extracellular signals to the nucleus,and also has the function of transducting extracellular signals to the nucleus.The role of phosphorylated nuclear transcription factors affects the expression of tumor cell-related factors.A large number of studies have pointed out that the expression of ERK in cervical cancer cells is obvious and has a positive trend with the degree of lesion,but the reason is still unknown.Therefore,this study further explored the regulatory effect of ERK on nuclear transcription factors in cervical cancer cells by extracellular experiments,in order to clarify the possible mechanism of ERK in the occurrence and development of cervical cancer.METHODS:HPV positive Si Ha cervical cancer cells were taken as the research object.ERK inhibitor U0126 was used to intervene cervical cancer cells,thus forming intervention group and control group.U0126 was used in intervention group,while U0126 was not used in control group.Cell growth was measured by cell counting method,cell viability was measured by CCK-8 method,cell cycle and apoptosis were detected by flow cytometry,expression of relevant nuclear transcription factors was detected by q RT-PCR method,expression of related nuclear transcription factors was detected by Western-blot method,and the expression of related nuclear transcription factors was detected by SPSS soft.The collected data were processed and analyzed statistically.RESULTS:(1)In the control group,Siha cervical cancer cells grew in epithelial adherence,with faster cell proliferation,clear cell outline and fewer suspended cells.Compared with the control group,the cell proliferation in the intervention group was slower,and there were more suspended cells,and the cells showed irregular changes;(2)Compared with the control group,the number of cells in the intervention group was constant.In addition,the results also showed that the inhibition rate of Si Ha cervical cancer cells was 98.54% after the addition of ERK inhibitors;(3)Compared with the control group,the cell proliferation index of the intervention group was significantly lower,and the difference was significant(P < 0.05).There was significant difference(P < 0.001);in addition,compared with the control group,the number of cells in the Si Ha cervical cancer intervention group was higher in G0/G1 ratio,lower in S phase ratio,and the difference was statistically significant(P < 0.001);(4)Compared with the control group,the apoptotic rate of Si Ha cervical cancer intervention group increased significantly,the difference was significant.Significant,statistically significant(P < 0.05).In addition,compared with the control group,the apoptotic rate of the intervention group increased significantly(P < 0.05);(5)Compared with the control group,the expression level of hn RNP E1 and hn RNP E1 in the intervention group decreased significantly(P <0.05);(6)Compared with the control group,the expression of c-Fos and c-Fos、 p-c-Fos in the intervention group were significantly lower(P <0.05);(7)Compared with the control group,the expression of c-Jun in the intervention group was significantly lower(P <0.05).The expression levels of c-Jun protein and p-c-Jun protein were significantly decreased(P < 0.05).CONCLUSION:(1)ERK activation can reduce the apoptosis of cervical cancer cells and promote the proliferation and differentiation of cervical cancer cells;(2)ERK plays an important role in regulating the expression of hn RNP E1 in cervical cancer cells,which may also involve the co-participation of HPV infection;(3)ERK can activate c-Fos in cervical cancer cells.And c-Jun factor,thus affecting the occurrence and development of cervical cancer cells,in this process,HPV infection may also participate in a synergistic manner.