Clinical Significance And Mechanism Of KHSRP Expression In Esophageal Squamous Cell Carcinoma

Background and objective:Esophageal carcinoma is the eighth most common cancer and the sixth leading cause of cancer-related deaths worldwide.The incidence rates among different regions vary widely up to 21 times,with the highest rates found in China,and the lowest rates found in the western Africa.The two main subtypes of esophageal carcinoma are esophageal squamous cell carcinoma(ESCC)and esophageal adenocarcinoma(EAC).While at high-incidence area for esophageal cancer in China,ESCC comprises up to 90% of esophageal cancer cases.Recently,the radical resection,radiotherapy and chemotherapy are the main treatment for ESCC.However,there are still a lot of problems,including the low five year survival rate,esophago-tracheal fistula after radiotherapy andthe intolerance to additional toxicity of chemotherapeutic drugs in some patients.With the development of molecular biology,target therapy has gradually become a new method for cancer treatment.Target therapy usually needs a pivotal target,which is often the tumor related oncogene.Therefore,a new oncogene is needed to be found,which provides a new strategy for target therapy of ESCC.KHSRP(KH-type splicing regulator protrin,KHSRP)is a single-stranded nucleic acid-binding protein located in both the nucleus and cytoplasm.KHSRP exerts its posttranscriptional regulation by combining with unstable mRNA to promote its degradation and promoting the maturation of microRNA.KHSRP was first reported as an enhancer element upstream of the c-myc oncogene promoter in 1996.So with the research deep-going,KHSRP is not only involved in cell proliferation,differentiation,inflammationand natural immune lipid metabolism,but also its expression and protein structure are closely related to the development of cancer.It has reported that glioblastoma multiforme(GBM)patients with KHSRP overexpression had high survival rate.The further analysis of RNA sequencing results in GBM patients showed that KHSRP was significantly associated with cefotaxime and netilmicin,implying KHSRP might be a new therapeutic target for GBM.KHSRP promoted cell proliferation and migration in human hepatocellular carcinoma.KHSRP also played an important role in the development of small cell lung cancer and non-small cell lung cancer.KHSRP resulted in tumorigenesis by causing abnormal expression of epithelialto-mesenchymal transition related genes in human breast cancer.Meanwhile,it has reported that PI3K/AKT pathway activation phosphorylated KHSRP and prompted KHSRP to shuttle into the nucleus from the cytoplasm,affecting the cell proliferation and chemoresistance.It was demonstrated that KHSRP was highly expressed in osteosarcoma,and inhibited cell proliferation and migration in vitro,suggesting KHSRP plays a key role in pathogenesis of osteosarcoma and might be a potential therapeutic target for osteosarcoma.However,the role of KHSRP in ESCC is still unclear.Therefore,these findings prompted us to investigate the expression and mechanism of KHSRP in ESCC,providing a new target for the treatment of ESCC.In this study,the relationship between KHSRP expression and prognosis of ESCC was predicted by bioinformatics,and the KHSRP expression was verified in ESCC patients.Then,the correlation between KHSRP expression and clinicopathological parameters was analyzed.The role of KHSRP inhibition on cell proliferation,migration and invasion of ESCC cells was investigated in in vitro expermients and then confirmed in mouse models.Moreover,the target drug for KHSRP protein was predicted.Finally,the mechanism of the action of KHSRP and immunization related cytokines in tumor microenvironment of ESCC was further studied.Part 1 The expression of KHSRP in ESCC patients Methods1)TCGAand GEO data were used to analyze the expression of KHSRP in ESCC.2)The expression of KHSRP in ESCC tumor tissues adjacent non-tumor specimens were detected by qRT-PCR and IHC.3)The relationship between KHSRP mRNA/protein expression and clinicopathological parameters was analyzed by chi-squared test.4)The correlation between KHSRP mRNA/protein expression and prognosis was investigated.Results1)The results of TCGA/GEO data analysis showed that the KHSRP mRNA expression in esophageal carcinoma was significantly higher than that in adjacent nontumor specimens.2)KHSRP mRNA and protein expression in ESCC specimens was significantly higher than that in adjacent non-tumor specimens.3)KHSRP mRNA and protein expression level was significantly associated with TNM stage,differentiation,tumor invasion and lymph node metastasis.4)The survival time of patients with higher KHSRP expression was shorter than that of patients with lower KHSRP expression.Summary1)KHSRP mRNA and protein expression in ESCC specimens was significantly higher than that in adjacent non-tumor specimens.There was a significant correlation between KHSRP expression and TNM stage,differentiation,tumor invasion and lymph node metastasis.These data suggest that KHSRP may play a critical role in the development of ESCC.2)KHSRP expression level was related with the survival time of ESCC patients,suggesting that KHSRP could be a prognostic biomarker for ESCC.Part 2 The Biological Significance of KHSRP in Esophageal Cancer Cell Lines TE1 and EC1 Methods1)KHSRP gene enrichment analysis was carried out through TCGA,and the correlation analysis was used to analyze the related markers.database.2)The expression of KHSRP in ESCC cell lines and esophageal epithelial cell line was analyzed by qRT-PCR.3)ESCC cell lines EC1 and TE7 with stable downregulation of KHSRP were constructed.4)The colony formation assay and CCK-8 assay were emplied to analyze the cell proliferation after KHSRP downregulation in ESCC cells.5)Cell apoptosis and cell cycle were investigated by flow cytometry after KHSRP downregulation in ESCC cells.6)The effect of KHSRP downregulation on the sphere formation ability was observed.7)The wound healing assay was adopted to confirm the effect of KHSRP downregulation on migration ability of ESCC cells.8)Transwell assay was utilized to confirm the effect of KHSRP downregulation on invasion ability of ESCC cells.9)The effect of KHSRP downregulation on the angiogenesis ability was observed.10)The effect of KHSRP downregulation on the xenograft tumor was observed in vivo.11)The epression levels of markers for proliferation and invasion in the tumor tissues of mouse were detected by IHC and qRT-PCR.12)Bioinformatics and molecular simulation methods were implied for KHSRP protein analysis,modeling and molecular docking,predicting target drugs for KHSRP.Results1)The gene enrichment analysis showed that KHSRP was associated with the biological process of ESCC cell division and cell cycle.Meanwhile,the KHSRP had a positive correlation with Ki-67 and CDK1,implying KHSRP was related with cell proliferation and cell cycle in esophageal cancer.2)The KHSRP downregulation was confirmed by qRT-PCR and western blot.3)KHSRP downregulation induced G0/G1 arrest,inhibited cell proliferation and decreased the sphere formation ability in ESCC cells.4)KHSRP downregulation decreased the migration and invasion abilityof ESCC cells.5)There was no significant difference in the apoptosis and angiogenesis after KHSRP downregulation in ESCC cells.6)KHSRP downregulation inhibited tumorigenic ability in vivo.7)KHSRP downregulation decreased the expression levels of Ki-67 and MMP9 in tumor tissue of nude mice8)Results of molecular docking showed that rifabutin could be used as an inhibitor for KHSRP.Summary1)KHSRP downregulation inhibited cell proliferation and self-renew ability,blocked cell cycle and decreased migration and invasion ability in vitro.However,there was on siginificant influence on cell apoptosis and angiogenesis.These data suggest that KHSRP play a role in the malignant behaviors of ESCC.2)KHSRP downregulation inhibited the development of xenograft tumor in vivo.Part 3 KHSRP suppress the recruitment of mononuclear macrophage by regulating the activity of NF-?B paththway Methods1)Cytokine array,ELISA and qRT-PCR were used to analyze the expression of chemokines after KHSRP downregulation in tumor microenvironment of ESCC.2)IHC and qRT-PCR assay were used to detected the expression of CCL2 in the tumor tissues of mouse.3)Transwell assay was utilized to investigate the effect of KHSRP downregulation on chemotaxis to mononuclear macrophages.4)The effect of KHSRP downregulation on the activation of NF-kB paththway was analyzed by western blot.The CCL2 expression was further detected after the treatment of NF-kB inhibitor by ELISA and qRT-PCR.5)qRT-PCR was employed to detect the expression of KHSRP,CCL2,CD14 and CD163 in ESCC specimens and the correlation analysis was carried out.6)Kaplan-Meier survival curve assay was utilized to analyze the correlation between the CCL2 mRNA expression and the prognosis of patients with ESCC.Results1)CCL2 expression decreased in ESCC cells with KHSRP downregulation.2)KHSRP and CCL2 downregulation of ESCC cells weakened the chemotaxis to mononuclear macrophages.3)The supernatant of esophageal squamous carcinoma cells low expressed KHSRP and CCL2 attenuated monocyte macrophage chemotaxis.4)KHSRP downregulation inhibited the activation of NF-kB paththway.CCL2 expression was repressed by NF-kB inhibitor.5)KHSRP expression was positively associated with CCL2,CD14 and CD163 expression in ESCC specimens.6)The survival time of ESCC patients with CCL2 high expression was significantly lower than that of patients with low expression.Summary1)KHSRP downregulation decreased the CCL2 expression in tumor microenvironment and suppressed the recruitment of mononuclear macrophages byinhibiting the NF-kB paththway activation,2)KHSRP expression was positively associated with CCL2,CD14 and CD163 expression in ESCC specimens.And the CCL2 mRNA expression was correlated with prognosis of ESCC patients.Conclusion1)KHSRP expression was significantly increased and correlated with clinicopathological parameters and prognosis in ESCC patients.2)KHSRP downregulation blocked cell cycle,inhibited cell proliferation and sphere formation ability,and suppressed migration and invasion in ESCC cells.KHSRP downregulation inhibited the development of xenograft tumor in vivo.3)The NF-kB paththway activation was inhibited and the CCL2 expression was decreased in tumor microenvironment by downregulating KHSPR,which suppressing the recruitment of mononuclear macrophages in ESCC.