| 1.Screening,analysis,validation and clinical study of radiation resistance related genes in cervical cancerObjective:Cervical cancer is the most common malignancy of the female reproductive system and ranks fourth among female cancers in the world.Currently,the incidence and mortality of cervical cancer in China are still increasing and tend to be younger.Although early-stage（IA1,IA2）cervical cancer can be cured by surgery,radiation therapy（RT）remains the cornerstone of cervical cancer treatment.However,radiation resistance is a key influencing factor in the effectiveness of radiotherapy for cervical cancer.Therefore,overcoming radiation resistance is of great significance to achieve better therapeutic outcomes for patients with cervical cancer.There are still no biomarkers for radiation resistance in cervical cancer currently in clinical work.KLK5（kallikrein-related peptidase 5）is an important member of the kallikrein-related peptidases（KLKs）family,plays multiple roles in tumorigenesis and this study initially suggest that KLK5 may be involved in the formation of radiation resistance in cervical cancer.Therefore,the aim of this clinical study was to further confirm the importance of KLK5 for radiation resistance on the basis of screening genes associated with radiation resistance in cervical cancer.To dissect the correlation between KLK5 and the clinical outcome of radiotherapy for uterine cervical cancer,serum SCC values in the patient,the patient’s immune status,and the long-term outcome.Methods:（1）Sample collection:a total of 19 patients with primary squamous cell cervical cancer treated in the second hospital of Jilin University during 2014-2016 were divided into two groups:radiotherapy sensitive group（RS,n=13）and radiotherapy resistant group（RR,n=6）;（2）c DNA library construction;（3）Microarray analysis,scatter plots,volcano plots and heat maps were drawn,GO enrichment and KEGG analysis were performed;（4）The differences in KLK5 expression levels between tissue samples of the RR and RS groups were verified by RT-q PCR and Western blot assay,then statistical analysis was performed;（5）The differences in serum SCC values before radiotherapy and the differences in blood immune parameters（CD3+CD4+/CD3+CD8+）before and 6 months after radiotherapy were statistically analyzed;（6）The differences in tumor volume between patients before radiotherapy were statistically analyzed;（7）The RECIST analysis evaluated the outcome of solid tumors for patients.Results:（1）The results of high-throughput sequencing and bioinformatics analysis revealed that KLK5 were most significantly differentially expressed between RR and RS groups（P<0.001）;（2）The results showed that KLK5 expression in tumor tissues was significantly upregulated in the RR group compared with the RS group by RT-q PCR and Western blot assay（P<0.01）;（3）The serum SCC values in the RR group before radiotherapy was significantly higher than that in the RS group（P<0.05）;（4）Recovery of the immune status of patients in the RS group was significantly better than that of patients in the RR group（P<0.05）;（5）The tumor volume in the RR group before radiotherapy was significantly larger than that in the RS group（P<0.05）;（6）Four patients in the RS group had progressive disease,one had partial response,and one had stable disease;only one partial response was observed in the RS group,and the remaining patients all achieved a complete response.Conclusion:Patients with KLK5 overexpression had higher serum SCC values and larger tumor volumes before radiotherapy and poor recovery of immune status.Meanwhile,patients with high KLK5 expression showed stronger radiation resistance than those with low KLK5 expression,and the lesions persisted,resulting in poor prognosis.2.Study on the mechanism of KLK5 regulating radiation resistance of cervical cancer cellsObjective:Studies have confirmed that KLK5 not only promotes tumor proliferation by activating growth and differentiation signaling pathways,but also cleaves extracellular matrix components,causing cancer cell invasion to adjacent tissues and distant metastasis of cancer cells.In this part,through in vitro cell experiments,we further explore the related mechanisms of the effect of KLK5 on radiation resistance of cervical cancer cells.Methods:（1）Cell lines with high and low KLK5 expression were selected from 5 cervical cancer cell lines,KLK5 stable silencing cell line（sh-KLK5）and overexpression cell line（OE-KLK5）were established;（2）The optimal radiation dose and time point were selected by Western blot and CCK-8 assay;（3）Colony formation assay,cell immunofluorescence assay,Transwell chamber assay,cell scratch assay,and flow cytometry（cell cycle and apoptosis assays）were used to determine the effect of KLK5 on radiation resistance of cervical cancer cells;（4）Western blot was used to detect the changes in the expression of key proteins（PCNA,MMP9,Cyclin B1,Bax,Bcl-2）of cell proliferation,invasion and migration,cycle and apoptosis related pathways.Results:（1）The KLK5 silencing stable cell line MS751-sh-KLK5 and the KLK5overexpression stable cell line ME180-OE-KLK5 were established;（2）The optimal radiation dose and time points for MS751 silencing,blank and negative control cells were as follows:4 Gy-48 h;Overexpression,blank and negative control of ME180 cells were as follows:6 Gy-48 h;（3）The results of colony formation assay,cell immunofluorescence assay,Transwell chamber assay,cell scratch assay,as well as flow cytometry（cell cycle and apoptosis assays）demonstrated that KLK5 overexpression significantly enhanced radiation-induced proliferation,invasion and migration,and inhibited radiation-induced G2/M arrest and apoptosis in cervical cancer cells;silencing KLK5significantly inhibited radiation-induced proliferation,invasion and migration,and promoted radiation-induced G2/M arrest and apoptosis in cervical cancer cells;（4）Western blot analysis showed showed that KLK5 overexpression increased the expression of PCNA,MMP9,Bcl-2,and Cyclin B1 protein in radiation-induced cervical cancer cells,which related to cell proliferation,invasion and migration,inhibition of apoptosis,and cell cycle（P<0.01）,while suppressed the expression of Bax protein related to promotion of apoptosis（P<0.01）.However,KLK5 silencing suppressed the protein expression of PCNA,MMP9,Bcl-2,and cyclin B1 in radiation-induced cervical cancer cells（P<0.01）,while increased the expression of Bax（P<0.01）.Conclusion:KLK5 enhanced radiation-induced proliferation,invasion and migration ability of cervical cancer cells,and inhibited radiation-induced G2/M phase arrest and apoptosis of cervical cancer cells,indicating that KLK5 participated in and enhanced radiation resistance of cervical cancer cells.3.Effects of KLK5 combined with X-ray on tumorigenicity of cervical cancer cells in nude miceObjective:In this section,the effect of KLK5 on radiation resistance of tumor cervical cancer cells was further verified by in vivo experiments by establishing a human cervical cancer cell nude mouse xenograft tumor model,and the possible molecular mechanism was further explored.Methods:The human cervical cancer ME180 cell line and its stable overexpression and negative control cell lines,the MS751 cell line and its stable silencing and negative control cell lines were used to construct subcutaneous xenograft tumor models in nude mice,and the efficacy was observed in groups.The grouping was as follows:Ⅰ:（1）ME180-MOCK+0Gy group;（2）ME180-NC+0Gy group;（3）ME180-OE-KLK5+0Gy group;（4）ME180-MOCK+6Gy group;（5）ME180-NC+6Gy group;（6）ME180-OE-KLK5+6Gy group.Ⅱ:（1）MS751-MOCK+0Gy group;（2）MS751-NC+0Gy group;（3）MS751-sh-KLK5+0Gy group;（4）MS751-MOCK+4Gy group;（5）MS751-NC+4Gy group;（6）MS751-sh-KLK5+4Gy group.（1）Ttumor growth curves were plotted against the size of transplanted tumors in each experimental group after irradiation.（2）Changes in the expression levels of KLK5 in transplanted tumor of each experimental group were determined by immunohistochemistry;（3）Changes in the expression levels of Ki67 in transplanted tumor of each experimental group were determined by immunohistochemistry;（4）HE staining was performed on transplanted tumor to evaluate the effect of KLK5 on radiation resistance;（5）The amount of KLK5 protein in transplanted tumor of each experimental group was determined by Western blot assay.Results:（1）The transplanted tumor volume in the ME180-OE-KLK5+6Gy group was significantly larger than that in the ME180-MOCK+6Gy and ME180-NC+6Gy groups（P<0.05）.However,the transplanted tumor volume in the MS751-sh-KLK5+4Gy group was significantly smaller than that in the MS751-MOCK+4Gy and MS751-NC+4Gy groups（P<0.05）;（2）There was no significant change in the expression of KLK5 in ME180-OE-KLK5 cell transplanted tumors before and after irradiation.The expression of KLK5 in ME180-MOCK-KLK5 and ME180-NC-KLK5 cell transplanted tumors decreased significantly after irradiation.The expression of KLK5 in MS751-sh-KLK5 cell transplanted tumor decreased significantly after irradiation,but there was no significant change in the expression of KLK5 in MS751-MOCK-KLK5 and MS751-NC-KLK5cell transplanted tumor before and after irradiation;（3）The levels of Ki67 expression in the ME180-OE-KLK5+6Gy group were significantly higher than those in the ME180-MOCK+6Gy and ME180-NC+6Gy groups.The level of Ki67 expression in the MS751-sh-KLK5+4Gy group were significantly lower than those in the MS751-MOCK+4Gy and MS751-NC+4Gy groups;（4）The extent of transplanted tumor necrosis in the ME180-MOCK+6Gy group was much lower than that in the ME180-MOCK+6Gy and ME180-NC+6Gy groups.However,the extent of transplanted tumor necrosis in the MS751-sh-KLK5+4Gy group was increased compared with that in the MS751-MOCK+4Gy and MS751-NC+4Gy groups;（5）Western blot showed that there was no significant change in the amount of KLK5 protein before and after irradiation in nude mice transplanted with ME180-OE-KLK5 cells,and a significant decrease in the amount of KLK5 protein after irradiation in nude mice transplanted with ME180-MOCK-KLK5 and ME180-NC-KLK5 cells（P<0.05）.The amount of KLK5 protein in nude mice transplanted with MS751-sh-KLK5cells was significantly reduced after irradiation（P<0.01）,whereas the amount of KLK5 protein did not significantly change before and after irradiation in nude mice transplanted with MS751-MOCK-KLK5 and MS751-NC-KLK5 cells.Conclusion:KLK5 promotes the growth of transplanted tumors in nude mice with cervical cancer cells after X-ray irradiation,inhibited the apoptotic and necrosis of transplanted tumors in nude mice with cervical cancer cells after X-ray irradiation,indicating that KLK5 participated in and enhanced the radiation resistance of transplanted tumors in nude mice with cervical cancer cells. |
