| Colorectal cancer (CRC) is one of the major malignancies in the world. The prognosis of CRCs is poor, due to frequent metastasis and tumor recurrence. Worldwide almost one million new cases occur annually, amounting to492 000 related deaths. With the many changes having taken place in people's diet and lifestyle, CRCs has become the third most common type of digestive tumor in China, and the number of new cases arising each year is still increasing. The overall incidence is identical in men and women, with the risk beginning at age 40 and increasing with age. Thus colorectal cancer ranks as the frequent cause of cancer deaths among China. Despite the rate of improvements in surgery, radiotherapy and chemotherapy, the prognosis of CRCs has not been gained. progress over the past decades, with an overall five-year survival rate of around 40%- 50%.The local treatment of surgery and radiotherapy are powerless to control tumor recurrence and metastasis, while the conventional chemotherapy regiments for the treatment of colorectal cancer have limited efficacy and are associated with significant toxicity unfortunately. Hence, it is urgent need to find a new treatment strategy. The method of gene therapy is one of hot spots in cancer research.Tumor invasion and metastasis are very complicated multi-step process, in which a number of structural barriers composed with extracellular matrix and basement membrane must be broken through, and then a new cell colony with exuberant proliferation in distant parts is established. The whole process is precisely controlled by a number of signal transduction pathways and mutual cross-linking of signal transduction network in which each of signal transduction pathways is quite complex. Invasion and metastasis of tumor cells require the capacity of migration of tumor cell itself apart from extracellular protease degradation of surrounding tumor.Phosphatidylinositol-3-kinase (PI3K) is a lipid kinase and is responsible for the phosphorylation of 3 position of the inositol ring of PⅠ(4,5)P2, to generate PⅠ(3,4,5) P3, a potent second-messenger required for fundamental cellular functions such as transcription, translation, proliferation, growth, and survival. There are three members in PI3K family. Signaling pathway composed of Class-ⅠA PI3K and serine/threonine kinase Akt/PKB has close relationship with tumor progression. This pathway regulates proliferation and survival of cancer cells. The disturbed activation of PI3K signaling leads to not only neoplastic transformation of normal cells, but also correlation with tumor cell migration, adhesion, tumor angiogenesis, as well as the degradation of extracellular matrix. Class-ⅠA PI3K is heterodimers composed of a catalytic subunit (P110) and an adapter/regulatory subunit (P85). P85 regulatory subunit is essential for stabilization and collection of P110 catalytic subunit and for activation of Class-ⅠA PI3K. P85αis the most abundant regulatory subunit of PI3K family and is encoded by pik3rl gene. It has been reporetd that mutation of PI3K p85αcould activate PI3K/Akt signaling pathway. Evidences have showed that PI3K p85αis a kind of oncogene. Most studies focused on the mutation and the mutation form of PI3K p85αin cancer. Reports about the invasion and metastasis of PI3K p85αin tumor are rare.RNA interference is to import a section of double-stranded RNA (dsRNA) into cell through experimental means and the host cell respond quickly to the dsRNA so that the dsRNA will be cutted by their endonuclease Dicer in the cytoplasm into much small fragment of RNA with specific cut length and structure which is called siRNA. The siRNA can prevent efficiently particular gene from expression in vivo through promote mRNA degradation of specific genes so that cell-specific gene phenotype is deleted, which meet certain experimental or therapeutic purposes. Our laboratory has constructed stable transfected CRC cells Lovo and SW480 cells (L324 and SW1124), which were tranfected with shRNA vector constructed ourselves. In this study, we investigate the expression and significance of PI3Kp85a in the progression of colorectal cancer, including normal colorectal tissue, colorectal adenoma, primary colorectal carcinoma, and metastatic colorectal carcinoma. Then we observed the effect of PI3Kp85αdepletion on colorectal cancer cell invasion and metastasis, and investigate its molecular mechanism。Materials and methodsThe expression and significance of PI3K p85αin progression of colorectal cancerImmunohistochemical staining was used to detect the expression and significance of PI3K p85αin the progression of colorectal cancer, including normal colorectal tissue, colorectal adenoma, primary colorectal carcinoma and metastatic colorectal carcinoma. The relationship between the expression of PI3K p85αprotein and clinicopathological factors was also analyzed.The effect of PI3K p85αdepletion on invasion and metastasis in colorectal cancer cells1. Transwell invasion assays The effect of PI3K p85αdepletion on invasion in colorectal cancer cells was detected by Transwell invasion assay.2.Transwell migration assays and wound healing test The changes of migration in colorectal cancer cells were observed by Transwell and Wound healing test in Lovo, sw480 andsw1124 cells espectively.3. Gelatin zymography assays The expression and activity of MMP-2 in colorectal cancer cells were detected by Gelatin zymography.4. Flat plate clone formation assays The independent growth abilities of cancers cells were detected by Flat plate clone formation assays.Statistical AnalysisAll experiments results were from at least three separate experiments. Dates are expressed as the mean±SD. Student's t test were used in group comparison. A value of P<0.05 was considered statistically significant. ResultsThe expression and significance of PI3K p85αin progression of colorectal cancerImmunohistochemistry was performed to examine PI3K p85αexpression levels in paraffin-embeded tissue from colorectal mucosa, adenomas and primary colorectal cancers to metastasis colon cancers. PI3K p85αexpression was highest in surface epithelium of colorectal mucosa, and expression in the stroma was limited to inflammatory cells, with a predominantly cytoplasmic distribution. Representative shows that PI3K p85αexpression is negative in normal colorectal mucosa, begins to increase in adenoma,over-expressed in primary colorectal adenocarcinoma and extremely much higher expressed in lymphnode metastasis (P=0.000). Furthermore, there was a significant difference in the positive rates of the PI3K p85αexpression duiring different Dukes' stage (P=0.015). No obvious correlation was found between expression of PI3K p85αand pathological diagnosis (P=0.058).The effect of silencing PI3K p85αexpression by RNA interference on the invasion and metastatisis of colorectal cancer cells1. Transwell invasion essays showed a significant decrease in the invasive capacity of L324and sw1124 cells, The numbers of cell invasion experiments in each group were counted as follow:405.80±7.11 in Lovo group,108.33±6.05 in L324 group, 147.93±6.51 inSw480 group and 74.67±6.50 in sw1124 group, There is significant statistically difference in the number of cell invasion.2. Using transwell migration assays we observed a significant decrease in migration capacity of L324and sw1124 cells.The numbers of cell migration experiments in each group separately are 562.93±5.15 in Lovo group,114.87±5.45 in L324 group,306.40±11.82 in sw480 group and 155.47±9.20 in sw1124 group。There is significant statistically difference in the number of cell migration by Independent-samples T Test.3.A monolayer wound-healing assay revealed almost no migration inL324 cells compared with Lovo cells 12h later, and after 24h, Lovo cells were full of the scratch, but L324cells spreaded very little. Monolayer scratch after 24 h, compared with the control group, the number of cell migration was obviously diminished in sw1124 group.4. A gelatin zymogram for MMP activation demonstrated a significantly decrease in MMP-2 activity in L324and swl 124 cells compared with Lovo and sw480cells.5. Using Flat plate clone formation assays, we observed a significant decrease in the independent growth ability of L324and sw1124 cells compared with Lovo and sw480cells.Conclusions1. Immunohistochemical results show that the expression levels of PI3K p85αgradually increases from normal colorectal mucosa, adenoma, and primary colorectal adenocarcinoma to metastasis colon cancers, indicating that PI3K p85αplays an important role in the progression of colorectal cancer metastasis.2. Depletion of P13K p85αcould inhibit Lovo and SW480 cells invasion.3. Depletion of PI3K p85αcould inhibit Lovo and SW480 cells migration.4. Depletion of PI3K p85αcould significantly decrease the activity of MMP-2 in Lovo and sw480cells, indicating that PI3K p85αmay control metastasis of colon cancers through decreasing the activity of MMP-2.5. Depletion of PI3K p85αcould decrease the clone formation ability of colon cancer cells. |
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