| ObjectiveHuman Macrophage Metalloelastase (HME) , also called MMP-12, is a new member of MMPs (Matrix metalloproteinase) family.In addition to the effect of penetratating basement membranes and invading normal and tumor tissues, it can degrade extracellilar matrix(ECM). However, HME also can change plasminogen into angiostatin that inhibits the proliferation of human vascular endothelial cells and inhibits the growth and invasion of tumors. There are many arguments about it's expression and significance in tumors. Currently, HME expression and significance in tumors is controversial. In this study, we detected the gene expression of HME in three cloned strains of carcinoma of stomach (MGC-803, SGC-7901, AGS) and the specimens of the patients' tissues (including the cancer tissues, the paracancer tissues and the normal tissues) by RT-PCR ,quantitative fluorescence real-time PCR and protein expression by Western Blot, immunohistochemistry . Then, we evaluated apotential role for HME in the carcinogenesis and development of gastric cancer and the value in the judgement of prognosis.Materials and methods1. Clinical data:All cases (43 males and 15 females,aged 3 2-76)were patients operated in the department of gastroenterosurgery and oncosurgery in the First Affiliated Hospital of School of Medicine, Zhejiang University from April 2003 to Aug 2003. Cancer tissues and paraeancer tissues and the normal tissues were collected from each patient. All the specimens were preserved in -200 °C nitrogen in half an hour after ex vivo.2. Cell culture:Human gastric cancer cell lines( MGC-803,SGC-7901,AGS) were purchasedfrom Shanghai cell research institute and maintained in culture in RPMI1640 medium(GIBCO Co. in America) supplement with 10% calf serum (Sijiqing Co. inHangzhou, China) at 37°C> 5%CC<2 They multiplied once 2-3 days. The cellsgrowing in good condition and 90% fusion were selected.3. Expression of HME mRNA by RT-PCR:Total RNA was extracted from cell lines and tissues of gastric cancer using Trizol reagent according to the manufactures instruction. The RNA concentration was quantitated by measuring absorbance at 260nm.The first strand cDNA was synthesized using a first strand cDNA synthesis kitThe amplified first strand was used as template.The cDNA samples were then subjected to PCR analysis using the following primers:HME 5'-TGGCCATTCCTTAGGTCTTG-3' and 5'-AAGCAG CTTCAATGCCAGAT-3'. Conditions used for the PCR amplification were as follow: 30 cycles at 94°C for 20sec, 61 °C for 20sec and 72 °C for 30sec.4. Expression of HME mRNA by quantitative fluorescence real-time PCR assay.Real-time PCR was performed using 4ul of the product of reverse transcription in this 40ul reaction system: 10* Buffer4uK lOmm/L dNTP0.8ul> 25mmol/LMgCl22ul > 2.5U Taq DNA Polymerase0.5ul > template 4ul ^ the reverse and forward-specific primers lul respectively >. specific probe 0.3ul , deionized water26.4ulo The sequences of the primers were 5'-TGAAAGTGACCGGGCAACT -3' and 5'-GGATTGCGTAGTCAACATCCTC-3'. The sequences of the probe was 5'-FAM-ATGCACGCA CCTCGATGTGGAGTCC- TAMRA. Denatured the DNA at 94°C for 3min at first, and then the following procedure for 40 cycles: heating at 94°C for lOsec^ at 60°C for 40sec, at last collected the light at 60°C.5. Expression of HME protein by Western BlotThe cells were washed with PBS and resuspended in 500^x1 cell Lysis on the ice for lOmin, scraped;lOOmg of each specimen were washed with PBS one time and resuspended in 500ul cell Lysis, grinded by glass homogenizer;and then centrifuged at 12, 000 rpm for 10 min at 4°C, the supernatant were collected .Equal amout of protein(80ug)were separated by SDS-PAGE and transferred to PVDF membrane. After blocking, membranes were incubated with rabbit polyclonal antibodies against human MMP-12(dilution 1:2000, Santa Cruz Biotechnologies Inc),followed by detection of specifically bound primary antibodies with peroxidase-conjugated secondary antibodies visualized by enhanced chemiluminesence luminol(ECL). Band intensities were quantified by densitometry with Biosense gel imaging system.6. Expression of HME protein by ImmunohistochemistryThe Envision two-steps immunohistochemical method was applied. 5um Paraffin-embedded sections were deparaffinizedand rehydrated in serial alcohols, blocked endogenous peroxidase( 198ml methanol+2ml 30%H2C>2)at room temperature for 20min, followed by washing with water, washed with PH7.2 buffer solution three times, retrieved with microwave in retrieval buffers for 20min, washed by PH7.2 buffer solution three times, and added with normal serum at 37.0°C for 20min.After removing superfluous serum, it was added with anti-rabbit MMP-12 polyclonal antibody as primary antibody at a dilution of 1:1000 at 37.0°C for 60min, washed with water, and then before adding Envision secondary antibody at 37.0°C for60min, it was washed with PH7.2 buffer solution three times. At last it was washed with PH7.2 buffer solution three times again and followed by being colored with DAB for lOmin , washed by water , less intensed stained with hematoxylin, colored, dehydrated, transparented, mounted.Results1. Analysis of cloned strain of gastric carcinomaHME mRNA was expressed in the cloned strains of gastric MGC-803 .. SGC-7901 -. AGS-by RT-PCR . Using p-actin as an house-keeping gene to correct the sample dose in PCR of the samples above-mentioned detected the 106 copies of p-actin of the samples for real-time PCR, we got similar result. The quantitations of HME mRNA were 8.37 X105, 2.54 XI05 and 1.85 XI04 respectively. The HME protein was also expressed in the three cloned strains by Western Blot and the numerical value by photodensity scan were 3784> 2719 and 3375 respectively.2. Analysis of the patients' tissues(1) RT-PCR HME mRNA positive rates were 24.14%. 20.69% and 6.90% in the cancer tissues, the paracancer tissues and the normal tissues by RT-PCR respectively. The positive rate of the cancer tissues was higher than that of the normal tissues(P<0.05), the positive rate of the paracancer tissues was higher than that of the normal tissues (P<0.05), the positive rate of the cancer tissues was higher than that of the paracancer tissues, but there was no statistical significance(P>0.05). Image analysis of the PCR amplified product showed that the level of HME mRNA in cancer tissues was highest and the level in normal tissues was lowest.(2) Real-time PCR Using 106(3-actin copies in amplificated template as an internal standard to correct the detection in Real-time PCR of all the samples of the three kinds of tissues . HME mRNA positive rates were 31.03%> 25.87%, 18.97% respectively in the cancer tissues > the paracancer tissues and the normal tissues.There was no significant difference in the three groups(P>0.05). The logarithm values of the copies in positive ones were 6.06±0.76> 3.97±1.42 and 3.38±0.80 respectively. Therewas significant difference in the three groups(P<0.01).The expression of HME mRNA in the cancer tissues was higher than that in the paracancer tissues (P<0.01);the expression of HME mRNA in the cancer tissues was higher than that in the normal tissues(P<0.01);although the expression of HME mRNA in the paracancer tissues was higher than that in the normal tissues,there was no statistical significance (P>0.05).(3 )The relationship between the expression of HME mRNA and the prognosis in patients of gastric carcinoma Divided the patients into positive group and negative group according to the result of Real-time PCR and analyzed the relationship between the expression of HME mRNA and the prognosis. 18 cases were positive and 40 cases were negative. We found there were no correlation between the expression of HME mRNA and the tumor's location, size , depth of invasion, degree of malignancy , drug resistance gene top and PGP. The rate of lymph node metastasis in HME mRNA positive group was lower than that in HME mRNA negative group(P<0.05);less cases in HME mRNA positive group expressed GST than in HME mRNA negative group significantly(P<0.05);two-year survival rate in HME mRNA positive group was higher than that in HME mRNA negative group(P<0.05).(4) Western Blot Applied Western Blot to detect the expression of HME protein in the three kinds of the tissues and quantitated the expressions by photodensity scan. HME protein positive rates were 27.58%% 25.87% and 24.14% in the cancer tissues > the paracancer tissues and the normal tissues respectively. The positive rate of the cancer tissues was higher than that of the normal tissues,but there was no statistical significance(P>0.05).The quantitation of the positive ones were 3358±1053 % 2891±974 and 2305±915 in the cancer tissues> the paracancer tissues and the normal tissues respectively. There was significant difference in HME expression between the cancer tissues and the normal tissues (P<0.01),but no significant difference was observed in HME expression between the cancer tissues and the paracancer tissues (P>0.05) .Similarly ,no significant difference was observed inHME expression between paracancer tissues and the normal tissues(P>0.05).(5 ) Immunohistochemistry HME was localized in cytoplasm of the tumor cells and matrix cells nearby. Staining was grated based on the method as follows.negative :positive cells accounted for <10% of the total;positive+: 10-50% of cells were positive;positive++:>50% of cells were positive. The results showed that there were 54 cases negative and 4 cases positive+(6.9%)in the normal tissues;there were 49 cases negative, 4 cases positive+ (6.9%)and 5 cases positive++(8.6%)in the paracancer tissues;there were 20 cases negative , 8 cases positive+(13.8%)and 30 cases positive-H-(51.8%)in the cancer tissues. The HME protein expression in the cancer tissues was increased compared with the paracancer tissues and the normal tissues(P<0.01).Conclusions1. Both HME mRNA and HME protein were positive in cloned strains of gastric cancer MGC-803, SGC-7901, AGS, which have potential value in clinical research.2. The expression of gene and protein of HME was highest in the cancer tissue group and next in the paracancer tissue group and lowest in the normal tissue group ,which indicated that HME may be a potential tumor marker for gastric carcinoma.3. The rate of lymph node metastasis in HME mRNA positive group was lower than that in HME mRNA negative group(P<0.05), the expression of gene GST was lower in HME mRNA positive group than that in HME mRNA negative group (P<0.05)and two-year survival rate in HME mRNA positive group was higher than that in HME mRNA negative group(P<0.05).It indicates that there is negative correlation between the expression of HME mRNA and the metastasis of gastric carcinoma and HME may be a good prognostic marker in gastric carcinoma.
|
PDF Full Text Request