| Part I: Devolopment and application of a new standing antibody incubationdevice for Western blotting.We developed a new antibody incubation device to overcome the defects of traditionalincubation box for Western blotting experiment. The device consists of an outer boxcontaining with water and a built-in stuff with hydrophobic surface, floatting on the water.This incubator could guarantee stuff surface on horizontal plane, prevent the antibodydiffusing away from the polyvinylidene fluoride (PVDF) or nitro cellulose (NC)membranes, ensure the air humidity and prevent the evaporation of the liquid of antibody inthe box. This device was applied in our Western blotting experiments and authorized for aChina national practical new-type patent in2013(NO. ZL201320144341.7).Part II: The role of inwardly rectifying K+channel Kir2.1in the stemnessmaintenance, invasion and metastasis of gastric cancer stem cells.Gastric cancer is one of the malignant digestive system tumors with high incidenceand mortality in the worldwide. Both invasion and metastasis are main biologicalcharacteristics of gastric cancer and the major cause of death in the patients with gastriccancer. Although progress in gastric cancer treatments, including surgery, chemotherapyand others, has been made in recent years, the theraputic effects are still unsatisfactory and5-year survival remains low. Furthermore the mechanisms underling the invasion andmetastasis of gastric cancer is unclear. Therefore, the intensive study on the invasion andmetastasis mechanisms of gastric cancer is an urgent task.The finding of cancer stem cells (CSCs) is a great progress in the field of cancerresearch in recent years, providing us a new understanding of mechanisms underlyinginvasion and metastasis in gastric cancer. Growing evidence has demonstrated that CSCsserve as "seeds" for invasion and metastasis of tumors. Our group has previouslyisolated/enriched gastric stem cells (GCSCs) by tumorsphere formation in serum-free medium, and finds that the tumorsphere cells possess higher capacities of self-renewal,invasion and metastasis. Thus, our results demonstrate that GCSCs are the cytological basisof invasion and metastasis in gastric cancer. However, the mechanisms underlying theinvasion and metastasis by GCSCs remain unclear.The roles of potassium ion channel in cancer have attracted paied more attention inrecent years. To explore whether potassium ion channels are involved in the pocess ofself-renewal, invasion and metastasis of GCSCs, we applied the whole-cell voltage patchclamp to detect K+current in GCSCs, and found that GCSCs had higher level of inwardlyrectifying K+current as compared with non-GCSCs. There are different kinds of potassiumchannels on cell membrance, which one is the major contributor? We performed an analysisof gene expression microarray and found that potassium inwardly rectifying channel,subfamily J, member (KCNJ-2), was highly expressed in GCSCs, indicating that the K+current could be mainly released by Kir2.1. The result of the microarray was verified usingquantitative real time polymerase chain reaction (qRT-PCR), Western blotting andimmunofluorescence analysis. We also evaluated the expression of Kir2.1in the clinicalspecimens of gastric cancer by immunohistochemical staining, and analyzed the clinicalsignificance of Kir2.1expression. The main results and conclusions are reported as follows:1. High level of inwardly rectifying K+current is closely correlated to Kir2.1expression in GCSCs.(1) We isolated/enriched gastric cancer stem cells from gastric cancer cell lineSGC7901using the method previously established by our group. Then we detected the K+current of GCSCs with whole-cell voltage patch clamp. As compared with monolayer cells(MN), we found that sphere cells (SC), i.e. GCSCs, could release high level of inwardlyrectifying K+current by voltage stimulation.(2) The analysis of gene expression microarray showed that GCSCs highly expressedinwardly rectifying K+channel Kir2.1channel mRNA (Gene: KCNJ-2) as compared withMN. The result was verified by qRT-PCR analysis.(3) Western blotting demonstrated high expression of Kir2.1protein in GCSCs, andimmunofluorescence staining showed that Kir2.1protein was mainly located on the cellmembrane, but was also found in the cytoplasm.(4) Furthermore, the mRNA of Kir2.1could be detected by qRT-PCR in the cells which released inwardly rectifying K+current, but not in the cells without inwardlyrectifying K+current. These results indicate that inwardly rectifying K+current released byGCSCs may be mainly due to high expression of Kir2.1in GCSCs.2. Kir2.1positive (Kir2.1+) cells possess stemness and possess invasion ability.(1) Flow cytometry analysis showed that positive rate of Kir2.1+cells was7.5%inSGC7901cells.(2) As compared to Kir2.1-cells, Kir2.1+cells over-expressed the stemness-relatedtranscription factor Nanog and possessed higher abilities of tumor-sphere formation andcolony formation.(3) Live cell workstation experiment showed that Kir2.1+cells had higher ability ofmovement as compared to Kir2.1-cells. Transwell chamber assay showed Kir2.1+cellspossessed higher capability of migration in vitro as compared with Kir2.1-cell. Meanwhile,qRT-PCR analysis showed that Kir2.1+cells highly expressed matrix metalloproteinase2(MMP2).3. Silencing Kir2.1expression inhibits the capabilities of self-renewal andinvasion.Knockdown of Kir2.1expression with Kir2.1targeting siRNA significantly reducedthe expression of Nanog and MMP2, and decreased invasion of gastric cancer in vitro.4. Kir2.1expression is correlated to the prognosis of patients with gastric cancer.(1) Using immunohistochemical staining, Kir2.1expression in specimens from212cases of gastric cancer was detected. There was no significant relationship between Kir2.1expression and patients’ age (P=0.674), gender (P=0.329), and histological classification(P=0.625), but Kir2.1expression was significantly correlated to the depth of tumorinvasion (P=0.010), TNM stage (P=0.002) and Lymph node metastasis (P=0.028).(2) The survival rate was evaluated by Kaplan-Meier analysis in132gastric cancerpatients who have information of follow-up. Kir2.1highcases had shorter survival span incontrast to Kir2.1lowones, suggesting that Kir2.1could be used as a useful indicator ofprognosis for patients with gastric cancer.The main conclusions are as follows: inwardly rectifying K+channel Kir2.1is highlyexpressed by GCSCs and is closely associated with stemness maintenance and invasion ofGCSCs. Kir2.1expression is positively correlated to the depth of tumor invasion, TNM stage and Lymph node metastasis, and is negtively correlated to survival time in the patientswith gastric cancer, suggesting that Kir2.1may be useful as a potential indicator ofprognosis for gastric cancer patients. |
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