The Role And Mechanism Of Crx In Lung Adenocarcinoma

PART Ⅰ Expression and clinical significance of C rx in lung adenocarcinomaObjective: The molecular mechanism of Lung adenocarcinoma(LUAD)is still unclear,and is important to find new therapeutic targets.The molecular mechanism of LUAD is still unclear,and it is still important to find new molecular therapeutic targets.As an oncogenic transcription factor,cone-rod homeobox(Crx)promotes the development of medulloblastoma.Although bioinformatics analysis has showed that Crx expression is correlated with the LUAD prognosis,its function and molecular mechanism remains unexplored.Therefore,this study aims to investigate the expression and clinical significance of Crx in human LUAD tissues and cells.Methods: 20 cases of LUAD tissue and adjacent tumor-free lung tissue specimens were collected from August 2018 to January 2020 in the Fourth Hospital of Hebei Medical University.All cases were confirmed by the department of Pathology,and all cases were the first onset and the first operation,without chemotherapy,radiotherapy or other treatment before surgery.Half of LUAD and adjacent non-tumor tissue samples were taken and paraffin embedded,while the other half was frozen with liquid nitrogen for total RNA or protein extraction.All samples were stored in the Cancer Institute of the Fourth Hospital of Hebei Medical University.The Ethics Committee of the Fourth Hospital of Hebei Medical University approved this research.The m RNA and protein levels of Crx expression in LUAD and adjacent lung tissues were detected by Real-time PCR and immunohistochemistry staining,respectively.Bioinformatics analyzed the correlation between Crx expression and prognosis of LUAD.Western blot was used to detect Crxxpression in different lung cancer cell lines.Chi-square test was used to compare Crx m RNA expression level in LUAD tissues with clinical parameters of patients,including age,gender,tumor stage,tumor size,lymph node metastasis and occurrence.Results:1.Compared with control,the Crx m RNA expression was significantly increased in LUAD(P<0.05).Consistently,immunohistochemical staining assay showed that Crx expression was also elevated in LUAD tissues.2.Result of Western blot detection showed that the expression of Crx protein was the highest in H1299 cell line.3.The comparison between the Crx m RNA expression and clinical parameters in LUAD tissues showed that the Crx expression level in LUAD patients was positively correlated with tumor size and lymph node metastasis(P< 0.05),but there was no relative to age,sex,BMI,tumor stage and multiple tumor.PART Ⅱ CRX affecting cell proliferation,migration and invasionObjective: To investigate the effects of transcription factor Crx expression on the LUAD cell proliferation,migration and invasion,providing experimental evidence for supporting the therapeutic target of Crx in LUAD through applying the transfected H1299 cells with small interfering RNA(siRNA)and expressing plasmid(pc DNA3.0).Methods: SiRNA(siRNA-Crx)and non-specific control siRNA(si-Con)or recombinant plasmid(pc DNA3.0-Crx)and empty plasmid(pc DNA3.0)were designed and synthesized for transfection in H1299 cells.Western blot was used to detect the transfection efficiency of Crx knockdown or overexpression.Accordingly,CCK-8,scratch test and Transwell test were performed on the H1299 cells treated with inhibition or overexpression of Crx to observe the changes of tumor biological function.Results:1.The results of Western blot showed that compared with si-Con,transfection of si-Crx could effectively interfere with the expression of Crx in H1299 cells,and the interference efficiency was over 75%(P<0.05).In parallel,compared with pc DNA3.0 group,transfection of pc DNA3.0-Crx significantly increased the expression of Crx in H1299 cells,and the overexpression efficiency was over 65%.2.CCK-8 assay showed that the proliferation activity of H1299 cells was effectively inhibited by si-Crx transfection during 96 h compared with si-Con group(P<0.05).Inversely,transfection with pc DNA3.0-Crx significantly enhanced the cell proliferation activity compared with pc DNA3.0 group(P<0.05).3.The results of scratch test showed that the migration ability was effectively inhibited by si-Crx transfection in H1299 cells compared with si-Con group(P<0.05).Inversely,compared with pc DNA3.0 group,transfection of pc DNA3.0-Crx significantly enhanced the cell migration ability(P<0.05).4.Transwell assay showed that transfection with si-Crx could effectively inhibit the invasion ability in H1299 cells compared with si-Con group(P<0.05).Inversely,transfection with pc DNA3.0-Crx significantly enhanced the cell invasion ability compared with pc DNA3.0 group(P<0.05).PART Ⅲ Molecular mechanism of Crx regulating cell proliferation,migration and invasion in LUADObjective: To investigate the molecular mechanism by which Crx regulating circRNA＿0072088 transcription,and circRNA＿0072088 inhibiting miR-1261 expression through sponge adsorption and up-regulating PIK3 CA expression,which promote proliferation,migration and invasion in H1299 cells.Methods: Bioinformatics predicted the potential binding sites between circ＿0072088 and the ZFR promoter region,miR-1261 and PIK3 CA,as well as Crx and circRNA＿0072088.Online analysis was performed to indentify the relationship between the expression of Crx and PIK3 CA and the prognostic of LUAD patients.The expression of circRNA＿0072088 in LUAD tissues and in the transfected H1299 cells with knockdown or overexpression of Crx by the above method were detected by q TR-PCR.CircRNA＿0072088 was confirmed by RNase R treatment in lung cancer cells.The construction of circ＿0072088was identified by Sanger sequencing.FISH assay verified the expression and localization of circ＿0072088 in cells and tissues.The PIK3 CA was detected by immunohistochemical staining.The double luciferase reporter expression gene assay verified the interaction between Crx and circRNA＿0072088 from ZFR gene promoter,circRNA＿0072088 and miR-1261,and miR-1261 and 3’UTR of PIK3 CA.RIP experiment was used to verify the interaction between circ＿0072088 and miR-1261.The expressions of circ＿0072088,miR-1261 and PIK3 CA were detected by q RT-PCR and Western blot.The effect of circ＿0072088 and its downstream miR-1261 and PIK3 CA on proliferation,migration and invasion of H1299 cells were confirmed by CCK-8,scratch and Transwell assay.Results:1.The results of database prediction showed that there were some potential binding sites between Crx and circ＿0072088 on ZFR promoter.The prediction results of Targetscan database showed that the binding site of miR-1261 in circ＿0072088 sequence got the highest score,and there exists a possible binding between miR-1261 and 3’UTR of PIK3 CA m RNA.2.QRT-PCR and FISH assay showed that the increased and decreased circ＿0072088 was consistent with overexpression and knockdown of Crx,circ＿0072088 and miR-1261 were co-located in the cytoplasm.The expression of Mir-1261 in LUAD was significantly lower in LUAD tissues than that in control(P<0.05).3.RIP,Ch IP-PCR and luciferase reporter gene assay showed that there were direct interactions between Crx and ZFR promoter,circ＿0072088 and miR-1261,miR-1261 and PIK3 CA m RNA 3’UTR,respectively.4.The results of q RT-PCR and immunohistochemistry showed that PIK3 CA expression in LUAD tissues was significantly higher than that in the para-cancer tissues(P<0.05).The high expression of Crx and PIK3 CA was correlated with poor prognosis in LUAD patients(P<0.05).5.CCK-8,scratch,and Transwell assay confirmed that the increased and decreased expression of circ＿0072088 promoted or inhibited the proliferation,migration and invasion of H1299 cells,respectively.The results of biological function enrichment analysis showed that the changes of tumor cell function were related to the expression of miR-1261.Conclusions:1.Crx expression was up-regulated in LUAD and it correlated with tumor size and metastasis.2.Crx activated the transcription of circ＿0072088,which promoting the cell proliferation,migration and invasion in LUAD.3.Circ＿0072088-miR-1261-PIK3 CA pathway mediates Crx promoting the proliferation,migration and invasion in LUAD cells;Silencing Crx gene or inhibiting circ＿0072088-miR-1261-PIK3 CA signaling axis would be the targeted therapy of LUAD.