The Mechanism Of RLF-Mediated Paclitaxel Chemoresistance In Squamous Cervical Carcinoma

Objective:Cervical cancer is still the gynecological malignant tumor with the highest incidence and mortality in China.Squamous cervical carcinoma is the most common type of cervical cancer,which seriously threatens women’s health.Chemotherapy is an effective means of treating squamous cervical cancer,but chemoresistance is a challenge for squamous cervical cancer treatment.Sequencing technology can effectively identify genes that are closely related to tumorigenesis,cancerous development,and chemoresistance.Through the wholeexome sequencing(WES),the RLF(Rearranged L-myc Fusion)was identified to be closely related to chemoresistance in cervical cancer.The study of the molecular mechanisms of RLF-mediated paclitaxel chemoresistance in cervical cancer could enrich the paclitaxel chemoresistance regulatory network of cervical cancer and provided a theoretical basis for the clinical reversal of paclitaxel chemoresistance.Methods:(1)A retrospective study was conducted on 156 patients with stage IB to IIB Squamous cervical carcinoma who underwent neoadjuvant chemotherapy in Tongji Hospital and Central Hospital in Wuhan from January 2016 to December 2020,and the chemotherapy sensitivity of these patients was judged according to the WHO clinical response criteria.Squamous cervical cancer tissue specimens and peripheral blood samples of these patients were subjected to WES to map the characteristic chemoresistance genes of cervical cancer;(2)For the chemoresistance-related gene RLF screened through WES,immunohistochemical staining(IHC)was used to analyzed the expression level of RLF in patients with different chemotherapy responses;(3)In order to explore the biological function of RLF in paclitaxel chemotherapy resistance in cervical cancer,the phenotypic differences of the cervical cancer cells whose RLF expression was knocked-down by si RNA or sh RNA and negative control cervical cancer cells under the action of paclitaxel were compared by CCK8,colony formation,apoptotic flow cytometry and cell cycle flow cytometry;(4)To discover the underlying molecular mechanism of which RLF caused paclitaxel chemoresistance in cervical cancer,q PCR,luciferase reporter gene assay,cellular immunofluorescence and Western blot(WB)were utilized to explore the changes in molecular pathways associated with paclitaxel chemoresistance after the downregulation of RLF.And the mechanism accuracy was verified by reversing key molecules of downstream pathways by the small molecule inhibitor;(5)To further explore the effect of RLF on chemotherapy of cervical cancer under in vivo conditions,a nude mouse transplantation tumor model was established to analyze the effect of RLF downregulation on tumor growth in mice and to verify the molecular mechanism of paclitaxel chemoresistance through IHC of nude mouse transplantation tumor tissue;(6)The correlation of RLF and the downstream gene in the study was analyzed through IHC of cervical cancer tissues of chemotherapy patients.Results:(1)Combined with the WES results and the comprehensive analysis of clinical data of squamous cervical cancer patients,a higher frequency of RLF missense mutations(chisquare test,P = 0.029)was found in chemoresistance cervical cancer patients,and the results of IHC of cervical cancer tissue showed that the expression of RLF was lower in chemoresistance patients(chi-square test,P = 0.0066 for pre-treatment;P = 0.0013 for posttreatment);(2)Downregulation of RLF expression in cervical cancer cell lines,increased tolerance of cervical cancer cells to paclitaxel toxicity,significantly decreased the apoptosis of cancer cells,and reduced G2/M phase arrest of cervical cancer cells caused by paclitaxel;(3)Further analysis showed that the expression of RLF and the cell cycle-related protein CLSPN were positively correlated,and the transcriptional activity of the CLSPN promoter was decreased after the downregulation of RLF.Immunofluorescence and WB showed that the downregulation of RLF facilitated the activity of CDK1 by inhibiting the CLSPN-CHK1 signaling pathway,eventually inducing paclitaxel chemoresistance of cervical cancer;(4)The paclitaxel chemoresistance of downregulated RLF nude mouse cervical cancer transplantation tumors increased significantly,and further IHC analysis of transplantation tumors confirmed that the downregulation of RLF inhibited the activity of CLSPN-CHK1 signaling pathway,improved the activity of CDK1,and suppressed the level of apoptotic indicators with paclitaxel treatment.And after inhibiting CDK1 activity by CDK1 inhibitor Ro3306,the chemoresistance of paclitaxel was reversed;(6)The IHC of squamous cervical cancer tissue specimens of cervical cancer patients exhibited the expression of RLF and CLSPN downregulated in chemoresistance patients compared with chemosensitive patients whether it was pre-treatment or post-treatment,and the expression of CLSPN and RLF were positively correlated(Spearman’s coefficient R = 0.72,P < 0.05 for pre-treatment;R = 0.73,P < 0.05 for post-treatment).Conclusions:The downregulation of RLF facilitated the activity of CDK1 by inhibiting the CLSPNCHK1 signaling pathway,reduced the G2/M arrest caused by paclitaxel,and ultimately induced paclitaxel chemoresistance in squamous cervical cancer.