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Objective The shRNA library was used to screen the tumor suppressor genes related to lung epithelial cells,and its function of inhibiting malignant transformation in lung cells was preliminarily verified,which provided a theoretical basis and a new therapeutic target for tumor prevention and treatment. Methods The shRNA retrovirus library was constructed by GP2 -293 virus packaging cell line to infect the immortalized lung bronchial epithelial BEAS-2B cells. The transfected epithelial cell clones were screened by soft agar colony formation,and a single transformed cell clone with a diameter greater than 1. 0 mm was selected. The in-serted shRNA fragment was amplified by PCR,and the target candidate gene corresponding to shRNA was determined by the conven-tional DNA sequencing and blast alignment. A candidate tumor suppressor gene INPP4B was verified by soft agar cloning and tumor formation in nude mice. MTT assay was used to detect the cell proliferation. Results Six lung epithelial malignant transformation in-hibitory factors were screened by soft agar colony formation. The candidate INPP4B gene was selected for functional experiments. Si-lencing INPP4B gene in BEAS-2B cells promoted the formation of clones in the soft agar plates,and the cell proliferation rate was accelerated. The silencing cell line showed the enhanced tumorigenicity in nude mice,indicating that INPP4B was involved in tumor formation. Conclusion shRNA library and soft agar colony formation assays are a powerful tool for screening tumor suppressor genes, and INPP4B is a malignant transformation inhibitor of lung epithelial cells.
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Objective To investigate the expression of INPP4B in gastric cancer and its relationship with clinicopathological features and prognosis. Methods The expressions of INPP4B mRNA in fresh cancer tissues of 36 patients with gastric cancer and the paracancerous normal gastric mucosa tissues in the Affiliated Cancer Hospital of Shanxi Medical University between July 2014 and December 2014 were detected by using real-time quantitative polymerase chain reaction (RT-qPCR). The expressions of INPP4B protein and its downstream molecule phosphorylation AKT (p-AKT) in paraffin-embedded tumor tissues and the corresponding margin tissues of 49 gastric cancer patients between January 2010 and December 2010 were detected by using immunohistochemistry. The relationship between the expression of INPP4B and clinicopathological features and prognosis was analyzed. Results RT-qPCR results showed that the expression level of INPP4B mRNA was 0.21 ±0.04 compared with adjacent cancer normal tissues (t= -2.208, P< 0.05). Immunohistochemistry showed that INPP4B protein was highly expressed in normal margin tissues and lowly expressed in tumor tissues. There was a statistical difference in the positive intensity score between the two groups (u=4.70, P<0.01). However, p-AKT protein was overexpressed in tumor tissues and underexpressed in normal margin tissues. There was a statistical difference in the positive intensity score between the two groups (u=5.77, P<0.01). The expression of INPP4B and p-AKT protein was negatively correlated (r= -0.644, P< 0.01). The positive expression rate of INPP4B protein in gastric cancer patients was 34.7％ (17/49). There were no statistical differences of the positive expression rate of INPP4B protein in gender, age, pathological type, depth of invasion and lymph node metastasis (all P > 0.05). The median overall survival time of patients with INPP4B negative expression was 47 months, and that of patients with INPP4B positive expression was 48 months, and there was no statistical difference (P> 0.05). Conclusion The expression of INPP4B in gastric cancer tissue is low, which may play a role of tumor suppressor in the occurrence and development of gastric cancer by affecting the activity of AKT.
Résumé
Objective@#To investigate the expression of INPP4B in gastric cancer and its relationship with clinicopathological features and prognosis.@*Methods@#The expressions of INPP4B mRNA in fresh cancer tissues of 36 patients with gastric cancer and the paracancerous normal gastric mucosa tissues in the Affiliated Cancer Hospital of Shanxi Medical University between July 2014 and December 2014 were detected by using real-time quantitative polymerase chain reaction (RT-qPCR). The expressions of INPP4B protein and its downstream molecule phosphorylation AKT (p-AKT) in paraffin-embedded tumor tissues and the corresponding margin tissues of 49 gastric cancer patients between January 2010 and December 2010 were detected by using immunohistochemistry. The relationship between the expression of INPP4B and clinicopathological features and prognosis was analyzed.@*Results@#RT-qPCR results showed that the expression level of INPP4B mRNA was 0.21±0.04 compared with adjacent cancer normal tissues (t = -2.208, P < 0.05). Immunohistochemistry showed that INPP4B protein was highly expressed in normal margin tissues and lowly expressed in tumor tissues. There was a statistical difference in the positive intensity score between the two groups (u = 4.70, P < 0.01). However, p-AKT protein was overexpressed in tumor tissues and underexpressed in normal margin tissues. There was a statistical difference in the positive intensity score between the two groups (u = 5.77, P < 0.01). The expression of INPP4B and p-AKT protein was negatively correlated (r = -0.644, P < 0.01). The positive expression rate of INPP4B protein in gastric cancer patients was 34.7% (17/49). There were no statistical differences of the positive expression rate of INPP4B protein in gender, age, pathological type, depth of invasion and lymph node metastasis (all P > 0.05). The median overall survival time of patients with INPP4B negative expression was 47 months, and that of patients with INPP4B positive expression was 48 months, and there was no statistical difference (P > 0.05).@*Conclusion@#The expression of INPP4B in gastric cancer tissue is low, which may play a role of tumor suppressor in the occurrence and development of gastric cancer by affecting the activity of AKT.
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BACKGROUND: The present study aimed to investigate the underlying role of interferon-regulatory factor 2 (IRF2)-inositol polyphosphate-4-phosphatase, type-II (INPP4B) axis in the regulation of autophagy in acute myeloid leukemia (AML) cells. METHODS: Quantitative real time PCR (QRT-PCR) and western blot were performed to determine the expression levels of IRF2, INPP4B and autophagy-related markers in AML cell lines. Autophagy was assessed by elevated Beclin-1 expression, the conversion of light chain 3 (LC3)-I to LC3-II, downregulated p62 expression and green fluorescent protein (GFP)-LC3 puncta formation. The colony formation and apoptosis assays were performed to determine the effects of IRF2 and INPP4B on the growth of AML cells. RESULTS: IRF2 and INPP4B were highly expressed in AML cell lines, and were positively correlated with autophagy-related proteins. Overexpression of IRF2 or INPP4B stimulated autophagy of AML cells, whereas inhibition of IRF2 or INPP4B resulted in the attenuation of autophagy. More importantly, IRF2 or INPP4B overexpression reversed autophagy inhibitor, 3-methyladenine (3-MA)-induced proliferation-inhibitory and pro-apoptotic effects, while IRF2 or INPP4B silencing overturned the proliferation-promoting and anti-apoptotic effects of autophagy activator rapamycin. CONCLUSION: IRF2-INPP4B signaling axis attenuated apoptosis through induction of autophagy in AML cells.