The Functional Mechanism Of HCINAP In Cervical Cancer Under Hypoxia And The Clinical Study Of Endostar Combined With Radiotherapy And Chemotherapy In The Treatment Of Cervical Cancer

Background and objective:Cervical cancer is the most common malignant tumor of reproductive system in women.In 2018,the number of deaths of cervical cancer worldwide reached 310000,in which recurrence and metastasis are the important causes of death of patients with cervical cancer.Therefore,there is an urgent need to find new treatment targets or develop new treatment models to improve the prognosis of patients with advanced cervical cancer.The progression of cervical cancer involves many factors,among which tumor hypoxia i s an important factor in the enhancement of invasiveness and treatment tolerance of cervical cancer.hCINAP（also known as Adenylate Kinase6,AK6）is a kind of adenylate kinase.Current studies have shown that hCINAP can enhance the glycolysis ability of ca ncer cells under hypoxic conditions to promote cancer cell invasion and treatment resistance,but the function and mechanism of hCINAP in cervical cancer is not clear.In addition,endostar（recombinant human endostatin）is a vascular targeting drug independently developed in China.Recent studies have confirmed that endostar can normalize tumor blood vessels and improve the hypoxia state of the tumor,so as to enhance the effect of radiotherapy and chemotherapy.However,whether e ndostar can improve the oxygen supply of cervical cancer tissue and improve the clinical efficacy of patients with locally advanced cervical cancer needs further clinical observation.The purpose of this study was to explore:（1）the effect and mechanism of hCINAP on the invasion a nd metastasis of cervical cancer cells under hypoxia.（2）The efficacy and side effects of endostar combined with simultaneous radiotherapy and chemotherapy in patients with locally advanced cervical cancer.The effect of endostar on vascular aberration,angiogenesis and blood flow changes of cervical tumor was evaluated by transrectal contrast-enhanced ultrasound,so as to indirectly evaluate the effect of endostar on the hypoxia state of cervical cancer.Methods:（1）After up-regulating or down-regulating the expression of hCINAP in cervical cancer cells,scratch test and Transwell test were used to observe the changes of migration and invasion ability of cervical cancer cells under hypoxia,and after intervention of hCINAP expression,the expression of（EMT）marker protein of epithelial-mesenchymal transformation,key protein of Akt/m TOR pathway,expression of p53-dependent apoptosis-related factors and the proportion of apoptosis were detected by Western Blot and flow cytometry.The mechanism of HIF-1α/Arnt regulating hCINAP expression was analyzed based on bioinformatics method,and the transcriptional activity of HIF-1α/Arnt on hCINAP under hypoxia was further verified by Ch IP.（2）120 patients with locally advanced cervical cancer were randomly divide d into two groups:simultaneous radiotherapy and chemotherapy combined with endostar group（CRT+E group,n=60）and simultaneous radiotherapy and chemotherapy group（CRT group,n=60）.The treatment regimen in CRT+E group was given intravenously 5 days b efore radiotherapy,with a dose of 7.5mg/m² for 10 days and repeated administration for 5 days for a total of 4 cycles.All patients received cisplatin alone with a dose of 40mg/m² once a week for 4-6 times.All patients were treated with6MV-X external irradiation intensity modulated radiotherapy with a dose of 45-50Gy/25f.Intracavitary brachytherapy was performed with^{1 9 2}Ir high dose rate afterloading radiotherapy with a dose of 28-30Gy/4-5f.At the same time,some patients were examined by transrectal contrast-enhanced ultrasonography before radiotherapy,15 times and 25times,respectively.The tumor vascular malformation rate was calculated by the vascular reconstruction image o f the largest cross section of the tumor,and quantitatively analyzed by contrast-enhanced ultrasound quantitative analysis software.the peak intensity（peak intensity,PI）,peak time（time to peak,TTP）and mean transit time（mean transit time,MTT）of the tumor vessels were recorded before and after treatment,and then the changes of oxygen supply of the tumor were evaluated.Results:（1）Down-regulation of hCINAP can inhibit,however,up-regulation of hCINAP can promote the migration and invasion of cervical cancer cells under hypoxia.Under hypoxia hCINAP can promote EMT in cervical cancer cells by activating Akt/m TOR signal pathway,and HIF-1α/Arnt expression can promote hCINAP expression.High expression of hCINAP can inhibit p53-dependent apoptosis under hypoxia.（2）Follow-up to January 2021,the median follow-up time in CRT+E group was 30.67 months（26.40-34.93 months）.The median follow-up time in CRT group was 32.40 months（19.96-44.84 months）.The results are as follows:（1）CRT+E group compared with CRT group,complete remission rate CR was（68.3%vs 35%）,objective remission rate ORR（98.3%vs 100%）,1-year OS（100%vs 98.3%）,1-year PFS（94.3%vs 93.3%）1-year DMFS（98.0%vs 94.7%）,2-year OS（95.3%vs 91.9%）,2-year PFS（89.5%vs 84.0%）,2-year DMFS（90.7%vs84.6%）,and the 3-year OS was（85.1%vs 80.2%）,3-year PFS（82.0%vs 81.0%）,3-year DMFS（84.3%vs 81.6%）.The difference in CR rate was statistically significant（P=0.038）.Although the 1,2 and 3-year OS,PFS and DMFS in the CRT+E group were higher than those in the CRT group,the difference was not statistically significant（P>0.05）.（2）The acute toxicity reaction of CRT+E group and CRT group was mainly gradeⅠ-Ⅱ,and the most common adverse reactions were hematological toxicity and gastrointestinal toxicity,the incidence of which were91.6%vs90%（P=0.945）and76.6%vs75%（P=0.937）,respectively.Among them,the toxicity reaction of gradeⅠ-Ⅱwas51.6%vs43.3%（P=0.585）,70.0%vs66.7%（P=0.865）,and the toxicity reaction of the gradeⅢ-IV was 40.0%vs 46.7%（P=0.643）,6.7%vs 8.3%（P=0.784）,respectively.There was no significant difference in acute toxicity between the two groups（P>0.05）.（3）Contrast-enhanced ultrasound showed that there was signific ant difference in vascular malformation rate between CRT+E group and CRT group（0.235 vs0.309）at 25 times of radiotherapy,but there was no significant difference between PI,TTP and MTT groups（P>0.05）.In CRT+E group,the MTT of pre-radiotherapy,15 times and 25 times of radiotherapy was40.10s vs 84.35s vs 71.12s respectively.The results showed that there was significant differences in MTT changes before radiotherapy,15times of radiotherapy and 25 times of radiotherapy（P=0.028）,but there was no significant difference in other parameters（P>0.05）.Conclusion:（1）hCINAP promotes the migration and invasion of cervical cancer cells,hCINAP can regulate the malignant phenotype of cervical cancer by activating Akt/m TOR pathway and inhibiting p53-dependent apoptosis,and HIF-1α/Arnt can promote the expression of hCINAP in cervical cancer cells under hypoxia.（2）The short-term efficacy of endostar combined with simultaneous radiotherapy and chemotherapy in the treatment of locally advanced cervical cancer is better than that of simultaneous radiotherapy and chemotherapy alo ne,but the long-term effect needs further follow-up observation.The acute side effects of endostar combined with simultaneous radiotherapy and chemotherapy in the treatment of locally advanced cervical cancer are tolerable and safe,while late toxicity needs further follow-up.Endostar can reduce the rate of vascular malformation and shorten the mean transit time of blood flow in cervical cancer,which may normalize blood vessels and improve the hypoxic state of cervical cancer so as to increas e the sensitivity of radiotherapy and chemotherapy and improve the therapeutic effect.