Study On The Effect Of B7-H6 On The Progression Of Cervical Cancer

Objective: Cervical cancer is the fourth most common female malignant tumor in the world and the main cause of cancer death in developing countries.At present,the initial treatment of cervical cancer patients can obtain effective clinical remission,including surgical treatment of early cervical cancer patients ± postoperative chemoradiotherapy,and concurrent chemoradiotherapy in patients who could not tolerate surgery or advanced cervical cancer of stage IIB or above.However,for patients with persistent,recurrent and distant metastasis of cervical cancer,the clinical options are very limited.Only the combination chemotherapy including bevacizumab,or the selection of pamumab / rotrototinib according to the results of genetic testing,but the patients who benefit from it are not many,and the clinical benefits are not optimistic.Therefore,to find effective immunotherapy and targeted therapy for patients with persistent,recurrent and metastatic cervical cancer has become an urgent problem in the clinical treatment of cervical cancer.This study aims to find new molecules involved in the occurrence and development of cervical cancer,explore the role of new molecule in the immune regulation of cervical cancer,and explore the possibility of new molecular as molecular and immunotherapy target in the treatment of cervical cancer.Research content: B7 homologue 6(B7-H6),also known as natural killer cytotoxic receptor 3 ligand 1(NCR3LG1),is a new costimulatory molecule discovered by bioinformatics and mass spectrometry in recent years.The specific expression of B7-H6 can be detected in a variety of tumor cells.The specific expression of B7-H6 in tumor cells can promote the occurrence and development of tumors;On the other hand,it can participate in the immune regulation of tumors by affecting the activity of NK cells.Through related experiments,this study explored the expression of B7-H6 in cervical lesions,the effect of B7-H6 specific expression on the occurrence and development of cervical cancer,and the effect of B7-H6 on the function of NK cells in the microenvironment of cervical cancer.Methods: In this study,the expression of B7-H6 and NK cell surface activating receptor NKp30 in normal cervical tissue,cervical precancerous lesions and cervical cancer tissues were detected by immunohistochemistry,and the specific expression and difference of B7-H6 in cervical precancerous lesions and cervical cancer tissues were determined,and the relationship between B7-H6 and NKp30 was also determined;Secondly,in the cell experiment,we constructed He La cells with low B7-H6 expression and compared He La cells: sh B7-H6-1,sh B7-H6-2 and sh B7-H6-NC,and we also constructed He La cells with high B7-H6 expression and compared He La cells: h B7-H6,h NC.The CCK-8 experiment,the Transwell experiment,the scratch experiment,the cell cloning experiment,the apoptosis and cell cycle detection were completed,so as to determine the effect of B7-H6 on the development of cervical cancer.Finally,the He La cells with different expression levels of B7-H6 were co-cultured with NK-92 cells.The expression of NKp30,the degranulation ability of NK cells and the release of the Granomycin B(GZMB)and the perforin(PFP)from NK cells were detected by flow cytometry.The release of the Interferon-γ(INF-γ)was detected by ELISA.The effect of B7-H6 on NKp30 expression and cytotoxicity of NK-92 cells was investigated.The phosphorylation of ERK in NK-92 cells was detected by Western Blot to analyze the possible mechanism of B7-H6 regulating NKp30 expression and NK-92 cell killing ability.Results: In the first part,immunohistochemical results showed that B7-H6 was positively expressed in different levels of cervical intraepithelial neoplasia(CIN)and cervical cancer tissue.The expression of B7-H6 was not found in normal cervical tissues.And the H-score of B7-H6 expression in cells increased with the progression of cervical disease stage.The H-score of B7-H6 expression in the CINⅡ lesions was significantly higher than that of CIN I lesions.The H-score of B7-H6 expression in CINⅢ lesions was significantly higher than that in CINⅡlesions.The H-score of B7-H6 expression in cervical cancer tissue was significantly higher than CINⅢlesions.The NKp30 was expressed on the surface of NK cells in normal cervical tissue and lesion cervical tissue.There was no significant difference between the H-score of NKp30 expression on NK cells in normal cervical tissues and in CINⅠ lesions.The H-score of NKp30 expression on NK cell surface in cervical lesion tissue also increased with disease progression.The H-score of NKp30 expression on NK cell surface in CINⅡ lesions was significantly higher than that in CINⅠlesions.The H-score of NKp30 expression on NK cell surface in CINⅢ lesions was significantly higher than that in CIN Ⅱlesions.The H-score of NKp30 expression on NK cell surface in cervical cancer tissues was significantly higher than that in CINⅢ lesions.There was no significant correlation between the H-score of B7-H6 and NKp30 expression in CINⅠlesions,and the H-score of B7-H6 and NKp30 expression in CINⅡlesions or above was positively correlated.In the second part,CCK-8 test and cell cloning test suggested that B7-H6 could promote the proliferation activity of the He La cells.The Transwell test and the scratch test suggested that B7-H6 could promote the invasion and migration ability of the He La cells.Flow cytometry results showed that after knockdown of B7-H6,the He La cells underwent late apoptosis and G2/M cell arrest.In the third part,NK-92 cells were co-cultured with cervical He La cells with different B7-H6 expression levels,the expression of NKp30 and the killing ability of NK-92 cells was detected.Compared with NK-92 Only,the expression of NKp30 on the surface of NK-92 cells was significantly up-regulated with the increase of B7-H6 expression level in He La cells;the killing rate,the role of degranulation and the release ability of INF-γ of NK-92 cells were significantly enhanced with the increase of B7-H6 expression level in He La cells;the PFP releasing ability of NK-92 cells decreased significantly,but correspondingly increased after the increase of B7-H6 expression level in He La cells.The GZMB releasing ability of all the NK-92 cells co-cultured did not change significantly.Conclusion: The differential expression of B7-H6 in cervical precancerous lesions and cervical cancer suggests that B7-H6 may be a new tumor biomarker for cervical cancer;B7-H6 can promote the occurrence and development of cervical cancer cells,suggesting that it may be a cervical cancer oncogene,and is expected to become a potential target for molecular therapy of cervical cancer;The positive regulation of B7-H6 on NKp30 and NK cell killing function suggests that B7-H6 is involved in the immune regulation of cervical cancer and is expected to become a new immune target in the treatment of cervical cancer.