The Role And Mechanism Of PIK3CA Gene In Regulating The Migration And Invasion Of Gastric Cancer

Objective:Migration, invasion and matastasis is the most important malignant characteristics of tumors and are the leading cause of gastric cancer-related death. But the underlying mechanism remains unknown. PIK3CA is an oncogene and located at3q26.3. What the mutation of PIK3CA is closely associated with the development and progression of many solid tumors including colon, brain, breast and lung cancer has been widespreadly accepted in recent research. However, some documents have indicated that lower frequency of PIK3CA mutation from4.6%to16%in gastric cancer was irrelevant with clinically pathological characteristics of gastric cancer, which suggests that mutation of PIK3CA have little effect on the development and progression in gastric cancer. But whether the abnormality of PIK3CA expression associated with invasion and matastasis of gastric cancer is unrecorded as far as we know in current literature. Thus, we first detected the expression level of PIK3CA in gastric cancer cell lines and gastric cancer tissues, and analyzed the relationship between its expression and clinically pathological characteristics of gastric cancer. The chemically synthesized siRNA targeting PIK3CA was transfected into either PIK3CA mutation or wild type gastric cancer cell line to explore the role and possible mechanism in regulating the proliferation and invasion of gastric cancer cells. Furthermore, effect of PIK3CA/mTOR dural inhibitor PI-103on invasion of gastric cancer cells was assayed. Finally, miRNAs targeting PIK3CA gene were predicted through bioinformatics and the expression of these candidate miRNAs was detected in gastric cancer cells and serum. The aim of the study is to explore the role and possible mechanism of PIK3CA gene in regulating the metastasis of gastric cancer and to provide a scientific evidence for early diagnosis and therapy of gastric cancer targeting metastasis. Methods:1. RT-PCR and Western blot were respectively explored to detect the mRNA and protein expression levels of PIK3CA in three differently differentiated gastric cancer cells including HGC-27, BGC-823and SGC-7901.Transwell chamber assay was used to observe the invasive ability of the gastric cancer cells mentioned above in order to evaluate the effects of PIK3CA expression on the invasion of gastric cancer cells.2. Real-time quantitative polymerase chain reaction (PCR) and immunohistochemistry was respectively used to detect the mRNA and protein levels of PIK3CA in specimens of normal gastric mucosa, primary foci,lymph node metastasis and distant metastasis of gastric cancer. Western blot was explored to examine Akt and phosphorylated Akt protein in the tissues mentioned above in order to analyze the effect of PIK3CA expression on the activation of PI3K/Akt signalings.3. The point mutation of PIK3CA in exons9and20was screened by PCR and followed by sequencing in some gastric cancer cell lines including HGC-27, SGC-7901, BGC-823, MGC-803and MKN-45cell line. siRNA targeting PIK3CA was chemically synthesized and transfected into these two cell lines including both BGC-823cells, which harbor no mutations in either of the PIK3CA exons, and HGC-27cells, which contain PIK3CA mutation in vitro. mRNA and protein expression of PIK3CA were detected by real time PCR and western blotting, respectively. The phosphorylation of a serine/threonine protein kinase (Akt) was analyzed by western blotting. The proliferation, migration and invasion of these cells were examined by MTT, cell wound healing and Transwell chambers assay, respectively.4. Gastric cancer cell line BGC-823and control were treated with either PI-103at a concentration of0.1,0.5,1,1.5and2.5μM or an equal volume culture medium. MTT, cell wound healing assay and Transwell assay was used to detect the effect of PI-103on the proliferation, migration and invasion of BGC-823cells. 5. PIK3CA-tageting miRNAs were predicted through bioinformatics combined with previous literature screening PIK3CA-tageting miRNAs. The expression of these miRNAs was detected by real-time PCR in gastric cancer cell and serum.Results:1. The mRNA and protein expression level of PIK3CA in in undifferentiated HGC-27cells is higher than that in either poorly differentiated BGC-823cell or moderately differentiated SGC-7901cell, Which is dependent on the differentiation of gastric cancer.The cell number migrating into lower chamber in undifferentiated HGC-27cell (45.24±3.16cells in lower chamber) is more (p<0.05) than that in either poorly differentiated BGC-823cell(22.28±1.65cells in lower chamber) or moderately differentiated SGC-7901cell (14.42±1.02cells in lower chamber). Which suggest that invasive ability of gastric cancer cells is negatively associated with differentiation of gastric cancer, but positively with expression level of PIK3CA in three cell lines indicated above.2. The mRNA and protein expression level of PIK3CA in tissues from primary gastric cancer, gastric cancer with lymph node metastasis or distant metastasis is markedly higher than that in tissues from normal gastric mucosa (p<0.05). The mRNA and protein expression level of PIK3CA in gastric cancer tissues with metastasis including lymph node metastasis or distant metastasis is significantly higher than that in primary gastric cancer tissues. PIK3CA mRNA in gastric cancer tissues with lymph node metastasis is5-fold higher or2-fold than that in tissues from normal gastric mucosa or primary gastric cancer (p<0.05).There is litlle diferance for PIK3CA mRNA between gastric cancer tissues with lymph node metastasis and distant metastasis(p>0.05). The results form immunohistochemistry indicate that staining rate of PIK3CA protein in tissues from primary gastric cancer, gastric cancer with lymph node metastasis or distant metastasis is35%,67%和61%, respectively. There is remarkably diferance for PIK3CA protein between primary gastric cancer tissues and gastric cancer tissues with lymph node metastasis(35%vs67%,p=0.015<0.05) or distant metastasis(35%vs61%, p=0.044<0.05). The up-regulation of PIK3CA protein is followed by enhanced expression of hosphorylated Akt, and has little effect on total Akt. Protein. The high expression of PIK3CA protein in gastric cancer tissues is significantly related to lymph node metastasis(p<0.05), but not to sex, age of patients and clinical stage of gastric cancer(p>0.05).3. The siRNA directed against PIK3CA down-regulates both mRNA and protein expression of PIK3CA and thereby significantly reduces phosphorylation of Akt (p<0.05) in both HGC-27cells and BGC-823cells. Furthermore, simultaneous silencing of PIK3CA results in a significant reduction of tumor cell proliferation activity, migration and invasion potential (p<0.01). HGC-27cells containing PIK3CA mutation exhibite a stronger invasive ability than that of wide-typte BGC-823cells harboring no mutations in either of the PIK3CA exons. Knockdown of PIK3CA in mutant HGC-27cells are contributed to reduce invasion to a greater extent than in non-mutant BGC-823cells.4. PI-103markedly inhibits the proliferation inhibition of gastric cancer BGC-823cells in a dose-dependent manner. BGC-823cells treated with an indicated concentration of PI-103have slower healing and less cells migrating into lower chamber compared with contro(p<0.05).5. There is little difference for miR-203expresion between both SGC-7901and MGC-803gastric cancer cell line. miR-203expresion in serum from gastric cancer patients is significantly higher than that from normal healthy person. There is little difference for miR-506expression between SGC-7901and MGC-803gastric cancer cell line, and between serum from gastric cancer patients and from normal healthy person.Conclusion:1. PIK3CA is higherly expressed in gastri cancer cell lines and gastri cancer tissues.The PIK3CA expression is negatively associated with differentiation of gastric cancer, but positively with the invasive ability of gastric cancer, which suggest that PIK3CA overexpression is acted as a biomarker for evaluating the potential matastasis of gastric cancer.2. The siRNA directed against PIK3CA down-regulates both mRNA and protein expression of PIK3CA and reduces the invasive ability of gastric cancer HGC-27cells and BGC-823cells. PI-103, a dual inhibitor for PI3K and mTOR, inhibits the proliferation and reduces the migration and invasive ability of gastric cancer BGC-823cells, which suggest that PI3K/Akt/mTOR signallings play an important role in regulating the migration and invasive ability of gastric cancer and act as a target for gene-therapy for metastasis of gastric cancer.3. Increased expression of PIK3CA is related to decresed expression of miR-203, but not to miR-506. The expression level of miR-203in serum could used as a biomarker for evaluating matastasis of gastric cancer.