| Background The Insulin Growth factor-like Family,also named IGFLs,is a newly discovered secreted protein family.It was found that the members of the family were involved in the regulation of body growth,reproduction,embryonic development,energy metabolism,and closely related to the occurrence and development of various malignancies.The Insulin Growth factor-like Family member 1（IGFL1）is one member of this family and involved in the regulation of the progression of various malignancies.However,the relationship between IGFL1 and LUAD has not been reported till now.Objective To investigate the expression and clinical significance of IGFL1 in LUAD,and to reveal the regulation of IGFL1 on proliferation,apoptosis,migration,invasion and EMT by functional experiments in vivo and vitro.Then,the signal pathway and mechanism of IGFL1 regulating the malignant biological behavior of LUAD were preliminarily explored to provide reliable theoretical and experimental basis for the exploration of effective therapeutic targets.Methods 1.Oncomine and TIMER bioinformatic databases were used to predict the expression of IGFL1 in various human tumors and corresponding normal tissues.The expression of IGFL1 protein in 21 types of human normal tissues and 32 types of common tumor tissues were detected by TMA combined with IHC staining.2.UALCAN and GEPIA bioinformatic databases were used to analyze and predict the expression of IGFL1 in LUAD and normal lung tissues.Kaplan-Meier Plotter was used to predict the relationship between IGFL1 and prognosis of patients in LUAD.Then,a retrospective study about surgically resected LUAD were collected from the Gansu Provincial People’s Hospital,TMAs were constructed and IHC staining was performed to analyze the correlation between the expression of IGFL1 and clinicopathological factors and prognosis of LUAD patients.3.RT-qPCR and Western blot were used to detect the expression of IGFL1 in human normal bronchial epithelial cell line（BEAS-2B）and human LUAD cells（A549,NCI-H1299,H1975）.Lentivirus RNA interference technique was used to construct IGFL1 gene silenced A549 stable cell line.The effects of IGFL1 on the proliferation ability of LUAD cells were detected by plate clonal formation assay and CCK-8 assay.Then,the effect of IGFL1 gene silencing on apoptosis of LUAD was analyzed by flow cytometry.To evaluate the effect of IGFL1 gene on the proliferation capacity of LUAD cells in vivo,a nude mice subcutaneous xenograft model was established and the volume of transplanted tumor was measured and analyzed statistically.Subsequently,HE and Ki-67 staining were used to determine the effect of IGFL1 expression on the growth and proliferation of LUAD cells in vivo.4.The effects of IGFL1 gene silencing on invasion and migration by LUAD cells were investigated by scratch/healing assay and Transwell invasion assay.Western blot and IHC staining were used to detect the effects of IGFL1 gene silencing on key proteins of EMT,including Vimentin,N-cadherin and E-cadherin.To verify the correlation between the expression of IGFL1 and key proteins of EMT.5.The changes of key molecules in AKT/mTOR signaling pathway were analyzed by Western blot.SC-79,an AKT pathway activator,was added to the two groups,and the ability of IGFL1 to regulate the proliferation and metastasis of LUAD cells through AKT/m TOR signaling pathway was verified by CCK-8 assay and Transwell invasion assay.Western blot was used to further verify the changes of key proteins in the AKT/m TOR signaling pathway,and the correlation between IGFL1 and p-AKT protein was analyzed subsequently.Finally,the molecular mechanism of IGFL1 regulating the proliferation and metastasis of LUAD was revealed.Results 1.The distribution and expression of IGFL1 in 21 normal human tissues and32 common tumor tissues were systematically studied by TMA and IHC staining.It showed that in 5 types of normal tissues,including esophageal squamous epithelium,renal tubular epithelial cell,prostate gland,pancreatic and salivary gland ductal epithelial IGFL1 is strongly positive expression,and in the 7 types of common tumor tissues,including hepatocellular carcinoma,papillary thyroid carcinoma,breast intraductal carcinoma and infiltrating ductal carcinoma,Cutaneous squamous cell carcinoma,lung squamous cell carcinoma and LUAD were strong expressed.Comparison with the results of Oncomine and TIMER bioinformatic databases,it showed that IGFL1 was highly expressed in LUAD tissues and low expressed in normal lung tissue,which laying experimental foundation for subsequent histological studies.2.The expression of IGFL1 protein in LUAD tissues was significantly higher than that in normal lung tissues both by UALCAN and GEPIA bioinformatic databases（P<0.05）,and its high expression is associated with poor prognosis in LUAD patients by Kaplan-Meier Plotter（P<0.05）.A total of 208 clinical samples from our center were selected as the study subjects.The expression of IGFL1 in LUAD tissues were significantly higher than that in corresponding normal lung tissues（P<0.05）.By retrospective analysis of clinical informations,it was found that IGFL1 high expression was closely correlated with clinicopathological factors,such as pleural invasion,tumor size,LNM,TNM stage,histological type and histopathological grade（P<0.05）.The results of Kaplan-Meier survival curve showed that the expression of IGFL1 was negatively correlated with overall survival（P<0.05）.Univariate and Multivariate Cox hazard regression model analysis showed that IGFL1 was an independent harzard factor for prognosis of LUAD patients（P<0.001）.And ROC curve proved that IGFL1 had high sensitivity and specificity as a diagnostic indicator for distinguishing LUAD tissues from normal lung tissues（AUC=0.7239;P<0.0001）.3.RT-q PCR and Western blot showed that IGFL1 was highly expressed in LUAD cell lines,and there was statistical difference between IGFL1 and normal bronchial epithelial cells（P<0.05）.A549 cells with IGFL1 gene silenced were constructed by lentivirus,interference efficiency were verified by immunofluorescence,RT-q PCR and Western blot.The effect of IGFL1 on the proliferation and apoptosis of LUAD was detected in vitro assay.The results of CCK-8 assay showed that number of cell viability was significantly lower in sh IGFL1 group than in sh Ctrl group（P<0.05）.The ability of the clone formation in sh IGFL1 group was significantly weaker than in sh Ctrl group（P<0.05）.The apoptosis rate of sh Ctrl group was 3.5%,sh IGFL1-1 and sh IGFL1-2groups was 9.03% and 6.4%,respectively.The apoptosis rate of sh IGFL1 group was significantly higher than in sh Ctrl group（P<0.05）.IGFL1 gene silencing can promote apoptosis of LUAD cells.Finally,xenografts model of nude mice further demonstrated that IGFL1 gene silencing inhibited that the tumorigenic ability of LUAD A549 cells.It showed that the volume of tumors in sh IGFL1 group were significantly lower than those in sh Ctrl group（P<0.05）.The results of HE staining showed that the tumor cells in sh IGFL1 group were less heteromorphic,more differentiated,less pathological mitotic and less areas of neoplastic necrosis.And the results of IHC staining showed that the Ki-67 Index was lower in sh IGFL1 group than sh Ctrl group（P<0.01）.4.The results of scratch/healing assay showed that LUAD cells migration ability of sh IGFL1 group was significantly weaker than sh Ctrl group（P<0.05）.Transwell assay showed that silencing IGFL1 could inhibit invasion of LUAD cells.Western blot showed that silencing IGFL1 gene could change the expression of key proteins in the EMT pathway,and the expression of E-cadherin was increased,while Vimentin and Ncadherin were decreased.The IHC staining of nude mouse xenograft showed Vimentin and N-cadherin were low expressed in sh IGFL1 group,while E-cadherin was high expressed.The result was confirmed that IGFL1 can promote EMT of LUAD.5.Western blot was used to detect the changes of related protein in AKT/m TOR signaling pathway and showed that the level of p-AKT and p-m TOR were downregulated after IGFL1 gene silencing.Subsequently,the results of functional assays showed that SC-79 can effectively deaden the inhibitory effects of sh IGFL1 on proliferation and invasion ability of A549 cell lines（P<0.05）.Moreover,it showed that SC-79 could up-regulate the expression of p-AKT and p-m TOR in sh IGFL1 group by Western blot.Spearman rank correlation method was used to analyze the correlation between IGFL1 and p-AKT protein expression levels in LUAD samples.And it showed that IGFL1 was significantly positively correlated with p-AKT（r=0.335,P<0.001）.It preliminarily demonstrated that IGFL1 promoted the proliferation and metastasis of LUAD cells by activating p-AKT,a key protein in the AKT/m TOR signaling pathway.Conclusion1.The distribution and expression of IGFL1 protein in normal human tissues and common tumor tissues were systematically revealed.2.The high expression of IGFL1 in LUAD tissue suggests that IGFL1 plays a role of oncogenic gene in the development and progression of LUAD.3.The expression of IGFL1 is closely related to clinicopathological factors and prognosis of patients with LUAD,and could be used as a potential independent harzard factor for prognosis of patients.4.IGFL1 was facilitated the ability of proliferation,clone formation,invasion and migration,inhibited apoptosis,and inducd EMT of LUAD cells,suggesting that it may be a key molecule in the malignant progression of LUAD.5.IGFL1 promotes the proliferation and metastasis of LUAD cells by activating AKT/m TOR signaling pathway.In conclusion,IGFL1 is closely related to the occurrence and development of LUAD,which can be used as a biological indicator to verdict the prognosis of patients and a potential target for diagnosis and treatment. |
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