Results
- Expression of integrin in isolated proteins
During the experiment, the extraction of alpha 6 ( 6) protein was done on two cell lines marked A and B using western plotting. The use of the BCA essay was to assist in computing protein content of the two samples. 0.0mg/ml, 0.03mg/ml, 0.06mg/ml, 0.125mg/ml, 0.25mg/ml, 0.5mg/ml, 1.0mg/ml, and 2mg/ml are the main protein concentrations that were used during the experiment.
So as to be in the position of constructing a standard curve, the average absorbance of the 8 BSA concentrations were plotted against their known concentrations using the excel program. In order to determine the unknown protein concentration, the standard curve equation was also used.
Figure 1
After computing the protein concentrations, the SDS PAGE gel was used to run the eight samples before transferring them to nitrocellulose membranes. After that, specific antibodies were used to incubate them so as to obtain 6 proteins. During this study, Chemiluminasecence system was used taking into consideration the effect of horseradish peroxidase (HRP) on the substrates, which in return lead to the creation of light. The result obtained was compared with the expression of the -actin protein in the samples. Although it was found out that OSCC B cell line is that one that contained bands closer to the anticipated molecular weight of the IGB6 protein (95 KDa), the OSCC A cell did not express it. This then implies that it is only the OSCC B that was transfected with the IGB6. So as to be able to confirm the results obtained, the OSCC A1, A1, A2, B1, and B2 cell lines were duplicated
The result obtained indicates that the IGB6 expression in OSCC at protein level is associated with the literature findings that proves the fact that integrin 6 is linked with carcinogenesis. Therefore, the existing literature suggests that the expression of IGB6 occurs both in oral carcinomas and cervical carcinoma. Despite that, although the expression of integrin 6 occurs at varying levels, at a higher level, such an expression is linked with more migratory and more invasive phenotype. On the other hand, the up regulation of the IGB6 in the SCC proves the fact that such an integrin plays a crucial role when it comes to tumor progression.
- The expression of IGB6 at gene level
During the experiment, real time quantitative PCR was used for the purpose of evaluating the transfection effectiveness of the gene expression using the integrin 6. Ideally, this was to assist in the identification and quantification specific RNA sequence. In order to determine that, one of the OSC cell lines was used to assess the integrin 6 expression level. After isolating RNA from the cell lines, duplication of the test was done using A1, A2, B1, and B2.
Nevertheless, TAE Agarose electrophoresis was used to conduct RNA analysis, with the SDS PAGE gel demonstrating ribosomal RNA bands. The reason for that is because it is the one that was in return to aid in indicating the high quality of RNA. The purity and the concentration of the RNA were done using the Nanodrop. The concentration results obtained is shown on table 1 below.
To study the quality of RNA, Agarose gel was used. Before using the G-box to visualize it, TAE Agarose Gel Electrophoresis was used to load the RNA with markers. The reason for that was to ensure that RNA with high quality has been separated and shown as ribosomal RNA bands. After preparing the cDNA, at the mRNA level, real-time-qPCR was used to show the expression of IGB6. The results obtained were to be compared that concerning the expressions of the unireverse-transcriptase control from the same sample.
Protein concentrations
Table 1
|
Concentration in mg/ml |
OSCC cell lines samples |
|
3.642034mg/ml |
A |
|
3.96893mg/ml |
B |
|
3.078864mg/ml |
C |
|
5.656337mg/ml |
D |
|
3.612568mg/ml |
E |
|
Expression of IGB6 |
Figure 2
So as to be in the position of assessing the expression of integrin 6 in the OSCC cell lines, RT-PCR was used to aid in synthesizing cDNA from extracted RNA. Next, qPCR maintained at 6U was used to aid in the amplification of mRNA together with the housekeeping gene that was expressed as x-folds. Although the data collected showed significant expression on the cell lines of sample OSCC A, there was minimal expression on the cell lines of sample OSCC B (mean ). Likewise, the negative RT control did not show the same expression.
In order to be in the position of assessing the IGB6 expression at mRNA, it was importat to prepare cDNA. On the same samples, the real-time qPCR was used to determine its expression level before comparing the results obtained with un-reverse or negative transcriptase control expression. The up-regulation of the integrin 6 was done on OSCC A cell lines and not in the OSCC B cell lines. In these lines, it was found out that 16-folds were induced at the 6 mRNA level as compared to the 28-fold up-regulated in the OSCC A1 cell lines as shown in figure 4 below. On the gene level, the results of this study suggest that IGB6 was transfected with the OSCC A cell lines. Furthermore, these findings also aid in conforming the results obtained at the protein level.
The results of this study shows huge variations taking into account the amount of IGB6 expression amongst the OSCC B cell lines. Although significant differences were found between the OSCC A cell lines and OSCC B cell lines, it was found out that there were no significant differences in the expression levels of mRNA among the OSCC B1 cell lines and OSCC B2 cell lines. On the hand, the differences obtained means that sample B2 had a high integrity and quality as compared to sample B2. This was also contributed by its high concentration efficiency. The absence of the IGB6 gene expression is as a result of the negative reverse transcriptase control of the mRNA.
According to the present studies, at mRNA level, the IGB6 expression was not ultimately is limited to carcinomas, for instance, cervical cancer, liver cancer, and colon cancer. Despite that, the findings of this study indicate that, at mRNA level, the IGB6 expression was not ultimately limited to oral squamous cell carcinoma. From the vitro research that was initially done, it was found out the general IGB6 expression is extremely associated with aggressive colon carcinoma (Wang eta al., 2008. On the same note, the inhibition of the ECM degradation is contributed by the suppression of the IGB6 expression. The up-regualtion of the integrin in cervical squamous cell carcinoma is linked with fabronectin rich stroma. It is these effects that have the likelihood of facilitating the invasion of cancerous cells.
Discussion and conclusion
The results of this study suggest assists in showing the fact that the expression of integrin 6 in the OSCC cell lines with respect to its protein level results in the production of bands of about 95 KD together with IGB6 maintained at the same range. In the OSCC, the up-regulation of the mRNA of the integrin the number of folds produced was found to increase in association to the housekeeping gene (Scully & Bagan, 2009). The result obtained corresponds with the data obtained from the existing literature. The same results demonstrate the fact that although integrin cannot be expresses using epithelial cells, the same expression can be experienced in different carcinomas (Agada et al., 2009).
According to research, the increase in IGB6 expression in carcinomas is brought about as a result of poor prognosis which in return decreases their survival rates. This is to imply that higher IGB6 expression have the potential of mediating nodal metastases as well as deeper invasion. Despite that, the real biological role of the integrin alpha ( ) have not been discovered hence the need of ensuring that the manner in which they mediate cancer progression in human have been addressed (Thomas et al., 2006). The reason for that is because other studies suggests that before invasion in connective tissues, integrin 6 expressions in dysplastic lesions have also be realized to one of the factors that proven to initiate cancer development (Ramos et al., 200).
The findings of this research prove the fact that IGB6 plays a crucial role in promoting as well as progressing OSCC. In addition to that, inhibiting or tampering with these receptors has been noted to have the ability of preventing the degradation of extracellular matrix as well as cell invasion. Therefore, in order to fight oral squamous cell carnoma, it is important to target the expressions of integrin alpha 6 ( 6) (Hazelbag et al., 2007).
References
Agada, F.O, Patmore, H., Alhamarneh, O., Stafford, N.D, & Greenman J. (2009). Genetic profile of head and neck squamous cell carcinoma: clinical implications. J Laryngol Otol 2009; 123:266- 272.
Hazelbag, S., Kenter, G.G., Gorter, A., Dreef, E.J., Koopman, L.A., Violette, S.M., Weinreb, P.H., & Fleuren,G.J. (2007). Overexpression of the alpha v beta 6 integrin in cervical squamous cell carcinoma is a prognostic factor for decreased survival. The Journal of Pathology 212, 316 -324.
Ramos, D.M., But, M., Regezi, J., Schmidt, B.L., Atakilit, A., Dang, D., Ellis, D., & Jordan ,R, L. (2002). Expression of integrin beta 6 enhances invasive behavior in oral squamous cell carcinoma. Matrix Biol. 2002 Apr;21(3):297-307.
Scully, C & Bagan, J. (2009). Oral squamous cell carcinoma overview. Oral Oncol. 45:301–308. 2009. View Article : Google Scholar : PubMed/NCBI
Thomas, G. J., Nystrom, M. L., & Marshall, J.F. (2006). αvβ6 integrin in wound healing and cancer of the oral cavity. Journal oral pathology 35, 1-10.
Wang, J., Zhang, Z., Xu, K., Yang, G., Niu, E., Peng, C., Lin, P., Chen, R., Agrez, M., & Niu, J. (2008). Supression of integrin avβ6 by RNA interference in colon cancer cell inhibits extracellular matrix degradation through the MAPK pathway. International Journal of Cancer 123, 1311-1317
