A Study On Expression And Mechanism Of The Follistatin-like Protein 1 In Cervical Cancer

BackgroundCervical cancer is one of the common malignant tumors in the female reproductive system.According to global cancer statistics,there are 8.6 million new cancer patients among women worldwide in 2018,of which 47.5% are new cases from Asia,and the incidence and mortality rate of cervical cancer ranks fourth,which seriously threatens women’s physical and mental health.Sustained infection of high-risk human papilloma virus(HPV)is a critical factor in the gradual progression of normal cervix to cervical cancer.With the screening of HPV and the production of HPV vaccine,the incidence of cervical cancer has decreased.However,there are still some new methods for diagnosing and treating cervical cancer for low-income and middle-income countries that lack organized screening and HPV vaccination programmes.Follistatin-like protein 1(FSTL1)is a secreted glycoprotein.Studies have shown that FSTL1 is abnormally expressed in various tumor tissues,and the abnormal expression of FSTL1 is closely related to the clinicopathological parameters of cancer patients.FSTL1 can participate in the occurrence and development of tumor-related diseases by reducing or enhancing the proliferation,migration and metastasis of tumor cells.However,the role of FSTL1 in cervical cancer and its mechanism have not been reported yet.The aim of this study was to investigate the expression of FSTL1 m RNA and protein in cervical cancer,and to analyze the relationship between FSTL1,clinical pathological parameters and high-risk HPV infection in patients with cervical cancer.To investigate the effects of FSTL1 over-expression and knockout on the biological function of cervical cancer cells in vitro and in vivo and the possible regulation mechanism of FSTL1 in cervical cancer.Methods1.From September 2012 to June 2017,127 cervical cancer tissue samples and22 normal cervical tissue samples were collected from the people’s hospital of Guangxi Zhuang Autonomous Region.Quantitative real time polymerase chainreaction(q RT-PCR)and immunohistochemistry were used to detect the expression of FSTL1 m RNA and protein in cervical cancer tissues of 77 patients and 84 patients respectively.Analysis of the relationship between the expression of FSTL1 and clinicopathological parameters of cervical cancer patients.2.DNA was extracted from 66 cases of cervical cancer tissue.The nested multiplex polymerase chain reaction(NMPCR)was used to detect and analyze the classification of high-risk HPV-16,18,31,45,52,58 in the above 66 cervical cancer tissues samples.Analysis of the relationship between the expression of FSTL1 m RNA and high-risk HPV infection.3.The over-expressing FSTL1 lentiviral plasmid with puromycin resistance gene and green fluorescent protein(GFP)were constructed using Lentivirus vector technology.He La and Si Ha cell stable strains over-expressing FSTL1 were screened using puromycin.The effects of over-expressing FSTL1 on the biological functions of proliferation,migration,invasion and apoptosis of cervical cancer cell lines were examined.FSTL1 was knocked out by CRISPR(Clustered regular interspaced short palindromic repeats)/Cas9(CRISPR associated protein 9)knockout technique.Cas9 lentiviral plasmid with puromycin resistance gene and small guide RNA(sg RNA)lentiviral plasmid with GFP were constructed.Cervical cancer He La and Si Ha cell stable strains knocking out FSTL1 were screened using puromycin.The effects of knockout of FSTL1 on the biological functions of proliferation,migration,invasion and apoptosis of cervical cancer cell lines were examined.4.Four to six weeks of BALB/C nude mice were selected.The experimental group was injected subcutaneously with cervical cancer cells with stable over-expressing and knocking out FSTL1.The negative control group was injected subcutaneously with cervical cancer cells with stable empty vector.Each group of 6 to establish cervical cancer xenograft model.In the tumor model,the volume of cervical cancer xenografts in each group of nude mice was measured every 3 days after tumor formation of the subcutaneous transplanted tumor.Nude mice were sacrificed 27 days later,the final volume and weight of the transplanted tumors were measured.The expression of FSTL1 in each group of transplanted tumors was examined.5.Cervical cancer He La cell stable strain over-expressing FSTL1 was detected using genomic microarray technology.The changes of downstream gene expression of FSTL1 were analyzed.Functional annotation and classification of differentially expressed genes and signal pathway enrichment analysis were performed to screenout the most obvious signaling pathways in cervical cancer He La cell stable strain over-expressing FSTL1.Western blotting was used to detect the change of the signal pathway and the expression of downstream molecules.Results1.The relative expression of FSTL1 m RNA in 77 cervical cancer tissue samples was lower than that in 29 the cervical cancer-adjacent tissues samples and 21 normal cervical tissue samples.The difference was statistically significant(P<0.001).The low expression of FSTL1 m RNA was associated with the low differentiation(P <0.001),large tumors(P=0.003),deep infiltration(P=0.042)and advanced FIGO stage(P=0.001)of cervical cancer patients.The expression of FSTL1 protein in 84 cervical cancer tissue samples was lower than that in 22 normal cervical tissues samples(P=0.006).The low expression of FSTL1 protein was associated with deep infiltration(P=0.004)and low differentiation(P=0.001)of cervical cancer patients.2.The relative expression of FSTL1 m RNA in the high-risk HPV positive group was lower than that in the high-risk HPV negative group(P<0.002).The HPV infection rate in the high expression group of FSTL1 m RNA was lower than that in the low expression group(P=0.021).After He La cells were transfected with HPV18E6 and E7 si RNA,the expression of FSTL1 m RNA was up-regulated compared with the negative control group(NC)and the mock group(MOCK),the difference was statistically significant(P<0.001).3.Cervical cancer He La and Si Ha cell stable strain over-expressing FSTL1 were screened.MTT and clone formation assays showed that the cell proliferation ability of He Le and Si Ha cells in the over-expression group(OE)were lower than that in the NC group and the MOCK group,the difference was statistically significant(P ≤0.007).The proliferation ability of He La cells in OE group on 3 day were lower than that in NC group and MOCK group(P=0.001).The proliferation of Siha cells in OE group were lower than that in NC group and MOCK group on 4 day(P=0.007).The wound healing and Transwell migration assays showed that the wound healing rate of He La and Si Ha cells in the OE group were lower than that in NC group and MOCK group(P ≤ 0.002).The number transcending the basement membrane of He La and Si Ha cells in OE group were less than that in NC group and MOCK group(P ≤ 0.002).Transwell invasion assay showed that the number lining out the matrigel membrane ofHe Le and Si Ha cells in OE group were less than that in NC group and MOCK group(P ≤ 0.001).Annexin V-APC/PI apoptosis assay showed that the apoptosis rate of He La and Si Ha cells in OE group were higher than that in NC group and MOCK group(P≤0.003).In contrast,Cervical cancer He La and Si Ha cell stable strains knocking out FSTL1 were screened.MTT and colony formation assays showed that the cell proliferation ability of He La and Si Ha cells in the knockout(KO)group were higher than that in NC group and MOCK group,the difference was statistically significant(P<0.001).The proliferation ability of the He La cell in KO group on 2 day was higher than that of the NC group and the MOCK group(P<0.001).The proliferation ability of the Si Ha cell in KO group on 2 day were higher than that in NC group and MOCK group(P=0.045).The wound-healing and Transwell migration assays showed that the wound healing rate of He La and Si Ha cells in KO group were higher than that in NC group and MOCK group(P<0.001).The number transcending the basement membrane of He La and Si Ha cells in the KO group was more than that in NC group and MOCK group(P<0.001).Transwell invasion assay showed that the number lining out the matrigel membrane of He La and Si Ha cells in the KO group were more than that in the NC group and the MOCK group(P<0.001).Annexin V-APC/ PI apoptosis experiments showed that the apoptosis rate of He La and Si Ha cells in KO group were lower than that in NC group and MOCK group(P ≤ 0.038).4.Compared with the NC group,the final volume and weight of the subcutaneous xenografts in OE group were lower than those in NC group,the difference was statistically significant(P <0.05).The expressions of FSTL1 m RNA and protein in OE group were higher than those in NC group,the difference was statistically significant(P<0.05).However,the final volume and weight of subcutaneous xenografts in nude mice in KO group were higher than those in NC group,the difference was statistically significant(P<0.05).The expressions of FSTL1 m RNA and protein in KO group were lower than those in NC group(P<0.05).5.Compared with NC group,there were 881 differentially expressed genes in OE group.The differentially expressed genes of FSTL1 were mainly enriched in biological processes such as transcriptional regulation,cell proliferation,apoptosis,embryonic development and hypoxia stress.KEGG and IPA signaling pathway enrichment analysis showed that the differentially expressed genes of FSTL1 were mainly enriched in cancer transcriptional misregulation signaling pathway,mitogen-activated protein kinase(MAPK)signaling pathway,cancer signalingpathway,P53 signaling pathway and IGF-1 signaling pathway,among which IGF-1signaling pathway is significantly activated.Western blotting results showed that the relative expression of IGF-1R,PI3 K,P-AKT and BCL-2 in OE group were lower than those in NC after over-expressing FSTL1 in cervical cancer He La and Si Ha cell lines,while the relative expression of BAX protein was higher than that of the corresponding NC group.The difference was statistically significant(P<0.05).Conversely,the relative expression of IGF-1R,PI3 K,P-AKT and BCL-2 in KO group were higher than those in the corresponding NC group after knocking out FSTL1 in cervical cancer He La and Si Ha cell lines,while the relative expression of BAX protein was lower than that of the corresponding NC group,the difference was statistically significant(P <0.05).Conclusion1.The expression of FSTL1 m RNA and protein was down-regulated in cervical cancer tissues.The low expression of FSTL1 was associated with deep infiltration,low differentiation,large tumor,and advanced FIGO stage of cervical cancer patients.2.The relative expression of FSTL1 m RNA in the high-risk HPV positive group was lower that in the high-risk HPV negative group.The HPV infection rate in the high expression group of FSTL1 m RNA was lower than that in the low expression group.HPV18E6 and E7 oncoprotein genes can inhibit the expression of FSTL1 m RNA.3.FSTL1 can inhibit the proliferation,invasion and migration of cervical cancer cells He La and Si Ha and promote apoptosis.4.FSTL1 can inhibit the growth of subcutaneous xenografts of cervical cancer.5.The differentially expressed genes of FSTL1 were enriched in biological processes such as transcriptional regulation,cell proliferation,apoptosis and adhesion.FSTL1 may inhibits proliferation and promote apoptosis of cervical cancer cells by down-regulating the IGF-1R/PI3K/AKT/BCL-2/BAX signaling pathway.