| BackgroundGastric cancer（GC）and gallbladder cancer（GBC）are common malignant tumors of the digestive system,both of which have obvious differences in gender and geographical location.And the clinical prognosis of early and middle/late stage patients is very different.In recent years,despite the improvement of comprehensive therapy including surgical therapy,radiotherapy,chemotherapy,immunity and targeting,early radical surgical resection is still the most effective method to cure or prolong patients with gastric cancer and gallbladder cancer.Since both GC and GBC have the characteristics of insidious onset,insignificant early symptoms and early lymph node infiltration and metastasis,patients are already in intermediate/advanced stages when they are diagnosed,and often lose the opportunity for surgery,resulting in poor clinical prognosis.Clinically,it can be seen that tumor patients with different genetic backgrounds will have a very different clinical prognosis even if they have exactly the same pathological basis and adopt exactly the same surgical method and postoperative adjuvant therapy,indicating that genetic changes can affect tumor prognosis.Therefore,searching for biomarkers of gene changes that can affect the prognosis of GC and GBC and exploring their mechanisms can help us more accurately understand the occurrence and development mechanism of GC and GBC,which provides a solid theoretical basis for early diagnosis and clinical drug development.PI3K-Akt pathway is involved in normal physiological processes of cells,such as cell proliferation,differentiation,apoptosis,immunity and metabolism.Abnormal activation of PI3K-Akt pathway is closely related to many diseases,especially tumors,and it is involved in various aspects of tumor occurrence,growth,invasion,migration,inflammation and immune regulation,metastasis,and formation of tumor microenvironment.Previous studies have shown that PI3K-Akt pathway plays an important role in both GC and GBC.As an inhibitor of PI3K-Akt pathway,homologous phosphatase and tensin homology（PTEN）plays a role of tumor suppressor gene（TSG）in GC and GBC and participates participates in their clinical prognosis.As another inhibitor of the PI3K-Akt pathway,inositol polyphosphate 4-phosphatase type II（INPP4B）,has not been systematically studied in GC and GBC in the digestive system,but has been studied in colorectal cancer,liver cancer and pancreatic cancer.There is only one foreign report about INPP4B frameshift mutation in GC leading to premature termination of INPP4B protein synthesis and thus affecting the function of INPP4B protein,while it has not been reported in GBC.In addition,it has been reported that PTEN is involved in tumor angiogenesis by inhibiting HIF-1A through PI3K-Akt pathway,HIF-1A is involved in glycolysis metabolism of laryngeal cancer cells by regulating INPP4B,and INPP4B is involved in angiogenesis of prostate cancer,but this has not been studied in GC and GBC.Therefore,this study focused on（1）the expression and clinical significance of INPP4B in GC and GBC;（2）The phenotypic effects and mechanisms of INPP4B on GC and GBC cells;（3）Correlation analysis and clinical significance of INPP4B and HIF-1A with angiogenesis in GC and GBC.一、clinical significance of INPP4B in GC and GBCIn order to detect the expression of INPP4B in GC and GBC and its correlation with clinical prognosis.In this study,real-time quantitative polymerase chain reactive（RT-q PCR）and western blot（WB）were used in fresh paired GC tissues and normal control tissues to detect INPP4B m RNA（messenger RNA,m RNA）and protein levels.Immunohistochemistry（IHC）is applied to detect the location and expression of INPP4B protein in tissues the previously prepared tissue microarray（TMA）and its correlation with clinical significance is analyzed.RT-q PCR results showed that in 37pairs of fresh frozen GC tissues and normal tissues,75.68%（28/37）tumor tissues had lower INPP4B m RNA level than matched normal control tissues,and the average expression level of INPP4B m RNA in GC tissues was lower than that in normal matched tissues（p=0.0204）.WB results showed that the expression level of INPP4B protein in GC tissues was lower than that in normal control tissues.IHC results showed that INPP4B protein was mainly located in cytoplasm.The positive rate of INPP4B in GC tissues was 40.45%（72/178）lower than that in normal control tissues 75.61%（31/41,p<0.001）.The positive rate of INPP4B in GBC tissues was 45.67%（58/127）higher than that in normal control tissues 23.40%（11/47,p=0.008）.Correlation analysis with clinicopathological parameters showed that INPP4B expression was correlated with histological differentiation（p=0.019）and TNM stage（P=0.033）in GC,but weakly correlated with tumor size（p=0.08）.In GBC,INPP4B expression was only correlated with histological differentiation（p=0.026）.Survival analysis found:high INPP4B expression in GC patients with large tumour size/low-undifferentiated/TNM’s III-IV stage was correlated with a poor prognosis but it was correlated with a better prognosis in patients with small tumour size/high-moderate differentiated/TNM’s I-II stage;high INPP4B expression in GBC patients with low-undifferentiated was correlated with a poor prognosis but it was correlated with a better prognosis in patients with high-moderate differentiated.The above experimental results suggest that the expression level of INPP4B is inconsistent in different tumors,and INPP4B may play a contradictory role in different tissue grades and clinical stages.二、Study on the functional mechanism of INPP4B on GC and GBCCombined with the above experimental results,we speculated that INPP4B might play an important functional role in GC cells and GBC cells.In order to further verify our hypothesis,we selected two GC cells（AGS and BGC-823）and two GBC cells（GBC-SD and SGC996）respectively to study the cell functional phenotypes.Lentiviral infection was used to construct cell lines with stable overexpression and knockdown of INPP4B.Cell proliferation assay and clonogenesis assay were used to analyzed the effect of INPP4B on the growth and proliferation of tumor cells.Apoptosis assay was used to analyze the effect of INPP4B on apoptosis of tumor cells.Scratch assay and Transwell assay were used to detect the effect of INPP4B on tumor cell motility.WB detected changes in the levels of key factors in PI3K-Akt and SGK3 pathways including Akt,p-Akt,SGK3 and p-SGK3,and analyzed the possible functional roles of INPP4B in different tumor cells through which pathway.Cell proliferation and clonal formation experiments showed that in GC cells,overexpression of INPP4B did not significantly change the proliferation ability of AGS cells,but interference with INPP4B could significantly reduce the proliferation ability of BGC-823 cells.Similar results were also observed in GBC-SD and SGC996 cells of GBC.Interference with INPP4B significantly reduced the proliferation of these two cell lines,while overexpression of INPP4B had little effect on the proliferation of these two cell lines.Apoptosis experiments showed that in GBC cells,overexpression of INPP4B could reduce the apoptosis rate of AGS cells,and interference with INPP4B could increase the apoptosis rate of BGC-823 cells.Similar results were observed in SGC996 cells of GBC,while in GBC-SD cells,both overexpression and interference with INPP4B increased the apoptosis rate,although the interference changes were more significant.Scratch assay and Transwell assay showed that overexpression of INPP4B could enhance the migration and motility ability of GC cells（AGS and BGC-823）and GBC cells（GBC-SD and SGC996）,and interference with INPP4B could weaken the migration and motility ability of these tumor cells.WB results showed that INPP4B overexpression enhanced the phosphorylation level of SGK3（p-SGK3）in AGS cells,but did not affect the phosphorylation level of Akt（p-Akt）;INPP4B interference enhanced the level of p-Akt in BGC-823 cells,but had no effect on the level of p-SGK3.In GBC-SD cells,overexpression of INPP4B increased the expression levels of p-SGK3 and p-Akt,and interference with INPP4B decreased the expression levels of p-SGK3 and p-Akt;In SGC996 cells,overexpression of INPP4B enhanced the expression level of p-SGK3,but had no effect on the expression level of p-Akt;interference with inpp4b had no effect on the expression levels of p-SGK3 and p-Akt.These results suggest that INPP4B plays an oncogenic role in GC cells and GBC cells,and may affect their biological functions through different signaling pathways in different tumor cells.三、Correlation analysis and clinical significance of INPP4B,HIF-1A and angiogenesis in GC and GBCThe expression and clinical significance of INPP4B in GC and GBC have been described in detail in the first part.This part mainly analyzes the relationship between inpp4b,angiogenesis and HIF-1A.CD34 was expressed in vascular endothelial cells and microvascular density（MVD）was analyzed by CD34 immunohistochemistry.In GC tissues,MVD of INPP4B+group（86.24±5.596）was lower than that of INPP4B-group（108.1±6.337,p=0.016）.In normal gastric tissue,there was no significant difference in MVD between INPP4B+group（80.65±10.64）and INPP4B-group（74.10±11.18,p=0.744）.In GBC tissues,MVD of INPP4B+group（139.5±9.910）was slightly higher than that of INPP4B-group（127.8±11.36,p=0.447）,but the difference was not statistically significant.In normal gallbladder samples,MVD of INPP4B+group（225.4±15.04）was significantly higher than that of INPP4B-group（168.4±11.01,p=0.012）.HIF-1A plays an important role in tumor angiogenesis and clinical prognosis.In the tissue microarray（TMA）of GC,it was found that HIF-1A protein was localized in the cytoplasm or/and nucleus of GC tissues and normal gastric tissues.The positive rate of cytoplasmic HIF-1A in GC tissues（35.39%,63/178）was lower than that in normal gastric tissues（51.22%,21/41,p=0.06）,although the data did not reach statistical significance.The positive rate of nuclear HIF-1A in GC tissues（38.20%,68/178）was significantly higher than that in normal gastric tissue（2.44%,1/41,p<0.001）.Correlation analysis showed that there was a negative correlation between the expression of HIF-1A in cytoplasm and nucleus of GC tissues（r=-0.324,p<0.001）.In GC tissues,MVD（114.4±8.073）in nuclear HIF-1A positive（nuclear HIF-1A+）group was higher than that in nuclear HIF-1A negative（nuclear HIF-1A-）group（89.87±5.046,p=0.007）,and there was no significant difference between MVD（95.52±7.755）in cytoplasmic HIF-1A+group and that in cytoplasmic HIF-1A-group（101.3±5.463,p=0.538）.In normal gastric tissues,MVD of cytoplasmic HIF-1A+group（88.38±14.79）was slightly higher than that of cytoplasmic HIF-1A-group（69.25±7.473,p=0.262）,but the difference was not statistically significant.Cox proportional hazards regression and Kaplan Meier survival analysis found that the clinical prognosis of GC patients in nuclear HIF-1A+group was worse than that in nuclear HIF-1A-group,and the clinical prognosis of cytoplasmic HIF-1A+GC patients was better than that in cytoplasmic HIF-1A-patients.These findings suggest that HIF-1A regulates angiogenesis in GC and participates in clinical prognosis.In the TMA of GBC,it was found that HIF-1A protein was localized in the cytoplasm or/and nucleus of GBC tissues and normal gallbladder tissues.The cytoplasmic HIF-1A+（65.96%,31/47）and nuclear HIF-1A+（36.17%,17/47）in normal gallbladder tissues were higher than those in GBC tissues（40.94%,52/127,p=0.003;21.26%,27/127,p=0.045）.Correlation analysis showed that there was no correlation between cytoplasmic HIF-1A and HIF-1A nuclear expression in GBC tissues（r=0.137,p=0.124）,while there was negative correlation between cytoplasmic HIF-1A and HIF-1A nuclear expression in normal gallbladder tissues（r=-0.370,p=0.010）.In GBC tissues,the MVD of nuclear HIF-1A+group（175±17.020）was higher than that of nuclear HIF-1A-group（121±7.540,p=0.002）,and there was no significant difference between cytoplasmic HIF-1A+group（138±10.360）and cytoplasmic HIF-1A-group（129±9.867,p=0.520）.In normal gallbladder tissue,the MVD of nuclear HIF-1A+group（146±20.910）was lower than that of nuclear HIF-1A-group（201±7.834,p=0.005）,and the MVD of cytoplasmic HIF-1A+group（197±8.836）was higher than that of cytoplasmic HIF-1A-group（151±21.370,p=0.022）.Cox proportional hazards regression and Kaplan Meier survival analysis found that the clinical prognosis of GBC patients in nuclear HIF-1A+group was worse than that in nuclear HIF-1A-group,and cytoplasmic HIF-1A had little effect on the clinical prognosis of patients with GBC.These findings suggest that HIF-1A regulates angiogenesis in GBC and participates in clinical prognosis.Correlation analysis showed that INPP4B was positively correlated with cytoplasmic HIF-1A（r=0.219,p=0.003）,but not with nuclear HIF-1A（r=-0.011,p=0.885）in GC tissues.INPP4B was positively correlated with cytoplasmic HIF-1A in normal gastric tissues（r=0.783,p<0.0001）.There was no correlation between INPP4B and cytoplasmic HIF-1A（r=0.139,p=0.120）and nuclear HIF-1A（r=0.037,p=0.679）in GBC tissues.INPP4B was positively correlated with cytoplasmic HIF-1A（r=0.412,p=0.004）and negatively correlated with nuclear HIF-1A（r=-0.319,p=0.029）in normal gallbladder tissues.These results indicate that INPP4B was correlated with HIF-1A,but the correlation was different in different tissues.Conclusions:We first found that INPP4B was low expressed in GC tissues and high expressed in GBC tissues,indicating that the expression level of INPP4B is different in different tumors.In the stratified analysis of INPP4B and clinical prognosis,it is found that INPP4B participates in the clinical prognosis of GC and GBC patients,but plays a dual role in promoting and inhibiting tumor progression at different pathological tissue levels,indicating that INPP4B plays different roles at different pathological tissue levels.In addition,cell function experiments in vitro have found that INPP4B plays a functional role of oncogene by influencing different signaling pathways to promote the growth and migration of tumor cells and inhibit the apoptosis of tumor cells.Finally,a complex relationship network among INPP4B,HIF-1A and CD34 was found in GC tissues and GBC tissues.This study first elaborated the clinical significance,function role and preliminary mechanism of INPP4B in GC and GBC,providing new ideas and solid theoretical basis for the clinical treatment of INPP4B gene in GC and GBC. |
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