The Clinical Significance Of SALL4 Expression In Non-small Cell Lung Cancer And Its Molecular Mechanism

Objectives:Lung cancer has become one of the most malignant tumors with the highest morbidity and mortality in China.Small cell lung cancer and non-small cell lung cancer are the two main pathological types of lung cancer,of which non-small cell lung cancer accounts for more than 85%.At present,the traditional treatment methods for lung cancer include surgical treatment,radiotherapy and chemotherapy.However,for patients with advanced lung cancer,due to the metastasis of cancer cells and low sensitivity to chemotherapy,the treatment effect is not ideal,and the five-year survival rate is still low.The occurrence of lung cancer is a process involving the high expression of oncogenes and the inactivation of tumor suppressor genes.In recent years,molecular targeted therapy on lung cancer has made great progress,and improved the survival of many lung cancer patients.Therefore,it is of great clinical significance to explore the molecular mechanism of the occurrence and metastasis of lung cancer and provide new ideas and intervention targets for the clinical treatment of lung cancer.Spalt like transcription factor 4(SALL4)gene is an oncogene discovered in recent years.It encodes a kind of zinc finger protein transcription factor and plays an important role in maintaining the self-renewal and pluripotency of embryonic stem cells.SALL4 participates in the occurrence and progression of many malignant tumors such as liver cancer and gastric cancer by regulating different signaling pathways,transcription factors and epigenetic modifications,and its expression level is related to the prognosis of tumor patients.At present,reports about the regulatory role and clinical significance of SALL4 in lung cancer are still rare.Therefore,this study aims to explore the expression of SALL4 in lung cancer and analyze the correlation between SALL4 espression and the clinicopathological parameters and prognosis of lung cancer patients.Then to explore the regulatory effect of SALL4 on the biological function of lung cancer cells and its possible mechanism.Methods:(1)RT-PCR method was used to detect the expression levels of SALL4 mRNA in tumor tissues of 30 patients with lung cancer.Immunohistochemistry was used to detect the expression of SALL4 in tumor tissues(62 patients)and the staining results were identified.The correlation analysis were conducted between SALL4 expression and clinical pathological parameters of patients.The Kaplan-Meier survival curve was drawn according to the return visits,then we analyzed the effect of SALL4 expression on the overall survival and disease-free survival of patients.The univariate and multivariate regression analysis were conducted to identify the clinical significance of SALL4 expression in the prognosis.(2)RT-PCR and Western blot methods were used to detect the expression levels of SALL4 mRNA and protein in five lung cancer cell lines.Cell transfection method was used to establish lung cancer cell lines with SALL4 expression stably silenced.Clone formation assay and MTT methods were used to detect the lung cancer cell proliferation capacity.The effects of SALL4 on the cell cycle and apoptosis of lung cancer cells were detected by flow cytometry,and the cell cycle and apoptosis-related proteins were detected by Western blot method.Cell scratch test was used to detect the migration of lung cancer cells.Transwell method was used to detect cell migration and invasion ability.Western blot method was used to detect the expression of EMT-related proteins in lung cancer cells.Furthermore,subcutaneous xenograft model of nude mice were established,and the effect of silencing SALL4 expression on the growth of xenografts in vivo was observed.Western blot method was used to detect the expression of related proteins in the tumor tissues.(3)Western blot was used to detect the expression of STAT3 and p-STAT3 in lung cancer cells after transfection.The expression of STAT3 was further up-regulated in cells with SALL4 expression inhibited.MTT,flow cytometry and Transwell methods were used to detect the changes in cell proliferation,apoptosis,and invasion abilities after co-transfection of siRNA-SALL4 and STAT3.Results:(1)The expression of S ALL4 mRNA in lung cancer tumor tissues(30 cases)were significantly higher than those in adjacent normal tissues,and the difference was statistically significant(P<0.05).The immunohistochemical(Lung cancer tissue chip,62 cases)results showed that the positive rate of SALL4 in tumor tissue was 67.7%(42/62),while the positive rate of SALL4 expression in normal adjacent tissues was 19.4%(12/62),with the significant difference(P<0.05),indicating that SALL4 expression is up-regulated in lung cancer tissues.Correlation analysis showed that SALL4 expression was closely related to TNM stage and lymph node metastasis(P<0.05),but had no significant correlation with patient gender,age,smoking status,tumor size and degree of differentiation(P>0.05),of which The SALL4 high expression group had more lymph node metastasis cases and higher TNM stages than the SALL4 low expression group.The prognostic analysis showed that patients with low SALL4 expression had longer overall survival and disease-free survival than those with high SALL4 expression,and SALL4 expression was an independent risk factor for the prognosis of patients with lung cancer.(2)The expression of SALL4 mRNA and protein was significantly up-regulated in lung cancer cell lines,which was consistent with the expression in tissues.The expression of SALL4 were stably silenced in lung cancer cell line A549 and H358.RT-PCR results showed that after transfection of siRNA-SALL4,the expression of SALL4 in A549 and H358 cells was significantly reduced,indicating successful transfection.The results of plate cloning formation assay showed that the number of colony formation decreased significantly after the inhibition of SALL4 expression.The MTT results showed that the growth activity of cells in the siRNA-SALL4 group was significantly lower than that in the control group from the second day,indicating that silencing SALL4 can inhibit the proliferation of lung cancer cells.The results of flow cytometry showed that after inhibiting the expression of SALL4,the proportion of lung cancer cells undergoing apoptosis increased significantly,and lung cancer cells could be blocked in the G0/G1 phase,thereby inhibiting the progress of the cell cycle.The results of cell wound scratch assay and Transwell results showed that inhibition of SALL4 expression significantly reduced the ability of migration and invasion of lung cancer cells.Western blot results showed that inhibition of SALL4 expression increased the expression of Bax,Caspase3 and Caspase9 protein in lung cancer cells,while the expression of Bcl-2 protein decreased.Similarly,the expression of cell cycle related proteins Cyclin B,Cyclin E,and Cyclin D1 were also significantly reduced,and the EMT process was inhibited.The results of animal experiments showed that after silencing SALL4 expression,the growth rate of tumor volume and weight was significantly reduced,indicating that knock-down SALL4 expression in vivo can inhibit the growth of lung cancer xenografts.(3)Western blot results showed that after silencing the expression of SALL4,the expression levels of STAT3 and p-STAT3 decreased significantly,indicating that SALL4 can simultaneously inhibit the expression of STAT3 and the phosphorylation of STAT3.After co-transfection of siRNA-SALL4 and STAT3,the lung cancer cells in the si-SALL4+STAT3 group had significantly enhanced growth and invasion ability compared with the siRNA-SALL4 group,while the apoptosis ability was significantly reduced,indicating that overexpression of STAT3 can reverse the silencing SALL4 expression effects on proliferation,apoptosis and invasion of lung cancer cells.Conclusion:SALL4 is up-regulated expression Oin lung cancer tissues and cell lines,and its expression is significantly related to TNM stage,lymph node metastasis and prognosis of lung cancer patients.Silencing SALL4 expression can significantly inhibit the proliferation activity,migration and invasion ability and EMT process of lung cancer cells,and the possible mechanism is related to the targeted activation of STAT3 signaling pathway.This study provides new ideas for clarifying the molecular mechanism of the occurrence and development of lung cancer,and provides a theoretical basis for finding new targets for the diagnosis and treatment of lung cancer.