| Background：Oral squamous cell carcinoma (OSCC) is the most common malignant tumor inhead-neck region, accounting for about3%of the all body malignant tumors. Thepredilection sites are the tongue, gum and buccal mucosa. The incidence and mortal-ity of OSCC is among the relatively high malignant tumors. OSCC has a high oc-currence of lymphatic metastasis, especially in the tongue cancer, the cervical lymphnode metastasis rate is60-80%. Local recurrence and invasion along with metastasisof the tumor are the major influencing factors resulting in the low survival rate. It isimperative for us to research the mechanism of tumorigenesis and metastasis of thetumor in order to facilitate the early detection, diagnosis and therapy. Now, the can-cer research has been widely and gradually deepening into the gene, molecular andprotein level. Further researches probing into the detailed mechanism of tumorigene-sis and metastasis of OSCC and tumor marker and suppressor gene are urgentlyneeded, which show great clinical significance.Osteopontin(OPN) is a kind of secreted glycophosphoprotein with a rich blendof sialic acid.OPN is synthesized and excreted in various tissues and cells, which ishighly associated with the metastasis of tumor. OPN can accelerate the degradationof excellular matrix, promote the formation of tumor blood vessels, and eventuallyfaciliate the invasion and metastasis of tumor cell. Researchers found that OPNplayed an important role in many physiologically and pathologically processes withthe help of CD44and integrin which can undertake the function of cell adhesionprotein and cell factor. scholars both at home and abroad verified that OPN ex-pressed highly in many tumors, as well as in head and neck squamous cancer. Withseldom or never expression in normal oral mucosa, OPN shows a high expression inOSCC. In the process of oral carcinogenesis, the expression of OPN mRNA in-creased gradually. Moreover, the expression of OPN showed a positive correlationamong different clinical stages and different lymph node metastasis conditions.TIP30is a newly discovered tumor suppressor gene, with the ability of inhibit- ing the proliferation and invasion of tumor cell, as well as promoting the apoptosisand inhibiting angiogenesis. Currently, the studies show that TIP30can block-up thecells in G0~G1phase, and lead to decline in the cells proportion in S phase.TIP30functions as a inhibitor in the process of tumor cell proliferation.Besides,TIP30caninhibit the genetic transcription and expression of VEGF,thus inhibit the process oftumor invasion and metastasis.What’s more,TIP30can induce the expression ofapoptosis gene such as Bad, which facilitates the apoptosis of tumor cells. The tumorinstitute of the second military medical university certified that TIP30can suppressthe genetic transcription of OPN, down-regulate the expression level of OPN mRNAand OPN protein, thus inhibit the proliferation of hepatocytes and the invasion ofextracellular matrix of tumor cells.At present, the role of OPN and TIP30in OSCC have not been studied prettywell, especially the role of TIP30. This research subject focuses on the expression ofOPN and TIP30in OSCC,as well as their relationship.Objective：To study the expression of OPN and the TIP30in OSCC,as well as their rela-tionship.Methods：The expression of OPN and TIP30mRNA were examined in OSCC and in ad-jacent tissues(n=18) by the method of fluorescence quantitative real-time PCR.Theexpression of OPN and TIP30protein were examined in OSCC(n=70) and in normaloral mucosa(n=20) by the method of En VisionT M.The staining-grade was quantita-tively analyzed by Integrated Optical Density(IOD) using Image Pro Plus6.0imageanalyzer.OPN and TIP30expression with different clinical and pathological indexwere statistically evaluated by means of SPSS17.0software package. The relation-ship of OPN and TIP30were analyzed according to the above data.Results：1、Comparision of expression of OPN mRNA in OSCC and in adjacent tissuesBy the method of fluorescence quantitative real-time PCR, the quantity of ΔCTof OSCC was8.36±2.16, while of adjacent tissues was9.58±1.94, which showedthat the quantity of ΔCT in OSCC was lower comparing with the high quantity inadjacent tissues. According to the following formulas, that is ΔCT=CTtarget gene-CThousekeeping gene，ΔΔCT=ΔCTcancer-ΔCTadjacency, the quantity of ΔCT is inverse proportion to gene copy number, in other words, the expression of OPN mRNA inOSCC was relatively higher than in adjacent tissues. In addition, we can calculatethe ratio of OPN expression in OSCC to OPN expression in adjacent tissues by2-ΔΔCT; OPN can be detected in all the18samples, and the positive rate was100%.Among18samples,15showed a two times ratio of the expression level between inOSCC and in adjacent tissues. Again it indicated that the expression of OPN inOSCC was significantly higher than in adjacent tissues. The results were analyzed bythe statistical software SPSS17.0(P<0.001) and deemed statistically significant.2、Comparison of expression of TIP30mRNA in OSCC and in adjacent tissuesBy the method of fluorescence quantitative real-time PCR, the quantity of ΔCTof OSCC was9.61±1.42, while of adjacent tissues was8.35±1.65, which showedthat the quantity of ΔCT in OSCC was lower comparing with the high quantity inadjacent tissues. According to the following formulas, that is ΔCT=CTtarget gene-CThousekeeping gene，ΔΔCT=ΔCTcancer-ΔCTadjacency, the quantity of ΔCT is in inverseproportion to gene copy number, in other words, the expression of TIP30mRNA inadjacent tissues was relatively higher than in OSCC. In addition, we can calculatethe ratio of TIP30expression in adjacent tissues to expression in OSCC by2-ΔΔCT;Among18samples,14showed a two times ratio of the expression level between inadjacent tissues and in OSCC.Again it indicated that the expression of TIP30in ad-jacent tissues was significantly higher than in OSCC. The results were analyzed bythe statistical software SPSS17.0(P<0.001) and deemed statistically significant.3、Comparison of protein expression level of OPN in OSCC and in normal oralmucosaThe expression of OPN in OSCC paraffin specimens and in normal oral mucosawere investigated by immunohistochemistry. OPN rarely expressed in normal oralmucosa, while expressed significantly in OSCC. Every section was observed underhigh magnification microscope (400times), which was randomly selected withoutoverlapping. Using the Image Pro Plus6.0software, accumulated optical densityvalue(IOD) of each high-power field were calculated. The quantity of IOD in OSCCwas66096.0±10921.0,while in normal oral mucosa was46748.0±9513.3.By statis-tical analysis, it show statistically significance(P<0.001).According to the clinicalstages, the70OSCC paraffin specimens can be divided into three groups, that werestage I(40), stageII (12) and stage III～IV(18),and the corresponding quantity of IODof each group were60695.0±8948.5、68780.0±5825.2、76308.0±9714.8. By statis- tical analysis, the results showed statistically significant, which also indicated thatOPN expressed differently in different clinical stages. With the increase of the clini-cal stage, the expression of OPN was enhanced. According to different metastasiscondition of lymph nodes,70OSCC paraffin specimens can be divided into twogroups, that were cervical lymph metastasis group(12) and noncervical lymph me-tastasis group(58),and the corresponding quantity of IOD of each groupwere78484.0±8780、2,63532.0±9514.6.By statistical analysis, the results showedstatistically significant, which also indicated that the expression of OPN in cervicallymph metastasis group was significantly higher than in noncervical lymph metasta-sis group.70OSCC paraffin specimens were divided respectively into groups ac-cording to the age, gender, pathological grading and primary site. All the results wereinvestigated by statistical analysis without finding statistical difference.4、Comparison of protein expression level of TIP30in OSCC and in normal oralmucosaThe expression of TIP30in OSCC paraffin specimens and in normal oral mu-cosa was investigated by immunohistochemistry. TIP30expressed significantlyhigher in normal oral mucosa than in OSCC. Every section was observed under highmagnification microscope (400times), which was randomly selected without over-lapping. Using the Image Pro Plus6.0software, accumulated optical density value(IOD) of each high-power field were calculated. The quantity of IOD in OSCC was28566.0±6887.4, while in normal oral mucosa was47486.0±11625.0. By statisticalanalysis, it show statistically significant (P<0.001). According to the clinical stages,the70OSCC paraffin specimens can be divided into three groups, that were stageI(40), stage II (12) and stage III～IV(18), and the corresponding quantity of IOD ofeach group were33036.0±4510.3、25930.0±3896.3、21304.0±5225.4. By statisticalanalysis, the results showed statistically significant, which also indicated that TIP30expressed differently in different clinical stages. With the increase of the clinicalstage, the expression of TIP30was decreased. According to different metastasis con-dition of lymph nodes,70OSCC paraffin specimens can be divided into twogroups,that were cervical lymph metastasis group(12) and noncervical lymph me-tastasis group(58), and the corresponding quantity of IOD of each group were19590.0±3368.5,30423.0±5896.0. By statistical analysis, the results showed statis-tically significant, which also indicated that the expression of OPN in cervical lymphmetastasis group was significantly higher than in noncervical lymph metastasis group.Furthermore,70OSCC paraffin specimens were divided respectively intogroups according to the age, gender, pathological grading and primary site. All theresults were investigated by statistical analysis without finding statistical difference.5、 Correlation analysis of OPN and TIP30in18OSCC specimensThe experimental data of RT-PCR were analyzed by Spearman rank correlationanalysis.In18OSCC specimens,the correlation coefficient of OPN and TIP30was-0.246(P=0.040).It showed a negative relationship,but not significantly.6、Correlation analysis of OPN and TIP30in18adjacent tissue specimensThe experimental data of RT-PCR were analyzed by Spearman rank correlationanalysis.In18adjacent tissue specimens,the correlation coefficient of OPN andTIP30was-0.477(P=0.034).It showed a significantly negative relationship.7、Correlation analysis of OPN and TIP30in70OSCC paraffin specimensThe experimental data of immunohistochemistry were analyzed by Spearmanrank correlation analysis.In70OSCC paraffin specimens,the correlation coefficientof OPN and TIP30was-0.255(P=0.033).It showed a significantly negative relation-ship.8、Correlation analysis of OPN and TIP30in20normal tissue paraffin specimensThe experimental data of immunohistochemistry were analyzed by Spearmanrank correlation analysis.In20normal tissue paraffin specimens,the correlation coef-ficient of OPN and TIP30was-0.489(P=0.029).It showed a significantly negativerelationship.Conclusions:1、In OSCC,OPN mRNA showed a high expression,while TIP30mRNA showed alow expression.It showed a significant difference with adjacent tissues.The resultsindicated that tumor can promote proliferation.2、In70OSCC paraffin specimens,the protein expression of OPN and TIP30showed a accordance with mRNA expression in OSCC.There were significant dif-ferences among different clinical stages and between different cervical lymph me-tastasis.OPN and TIP30may have some relationship with the proliferation and me-tastasis of OSCC.3、By the method of statistics,the relationship of OPN and TIP30(from two differ-ent samples) were analyzed.It showed a negative relationship. The negative relation-ship in70OSCC paraffin specimens was more obvious... |
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