Exploring Studies On Gene Expression Profile Of Cells With Long-term Low Expression Of Kras And Potential Therapeutic Targets Of Pancreatic Cancer

Pancreatic cancer (PC) is exceptionally aggressive. There is no effective treatment for PC owing to lack of diagnostic symptoms in the early stages and rapid progression in PC. Such as other malignancies, PC is a genetic disease. The molecular mechanisms involved in PC development are extremely complex. Many of cancer-related genes (such as proto-oncogenes and tumor suppressor genes, etc.) interact with each other and involve in the progress of PC. KRAS is the most important proto-oncogene in PC, and is an attractive therapeutic target for PC. However, clinical trials have shown that KRAS as a molecular target for PC treatment is not ideal. Intracellular signaling pathway is cross linked with each other to form a complex signaling network. Specific inhibition of activated KRAS in PC may cause genetic changes that involve in changing expression of many genes, which could develop resistance to various treatments. In order to explore which genes would respond to KRAS inhibition in PC cells, as well as to explore whether these genes could be used as the potential therapeutic target for PC, we focus following aspects to research in this paper:1. Three stable transfected cell lines (P-M, P-W and P-H) were constructed through lentivirus-mediated shRNA targeting KRAS of human pancreatic cancer PANC-1cells. Compared with untreated PANC-1cells, the KRAS mRNA levels in P-M, P-W and P-H cells were54.4%,54%and59%. KRAS protein levels also reduced in P-M, P-W and P-H cells compared with PANC-1cells.2. As an experimental model, P-M and P-W cells were used to study the gene expression profile about PANC-1cells with long-term silencing of KRAS through human whole genome microarray experiments. Compared with PANC-1cells,43genes were up regulated and182genes were down regulated in both P-M and P-W cell lines. Microarray results were validated by qPCR assays. Fourteen genes chosen for qPCR validation were RPL21, RPL26, RPL10A, RPL24, RPL39, RPL29, HAPLN3, RPL31, PTPLAD2, ITPRIPL2, N0L7, FBXO27, TMEM106A, PRAGMIN. qPCR assays showed the same trend as microarray analysis.3. Five ribosomal protein genes (RPL26, RPL29, RPL31, RPL21and RPL39) were selected out from up-regulated43genes for further research. A series of siRNAs targeting these five ribosomal protein genes were designed and synthesized. Silencing the expression of RPL26, RPL29, RPL31, RPL21and RPL39respectively with siRNAs (40nmol/L):(1) suppressed PANC-1cells proliferation and colony formation.(2) induced G0/G1phase arrest in PNAC-1cells. Compared with untreated PANC-1cells (47.31%), silencing RPL26, RPL29, RPL31, RPL21and RPL39with siRNAs (40nmol/L,72h) enhanced up to74.35%,70.8%,71.85%,76.72%and76.18%, respectively.4. Silencing the expression of RPL26, RPL29, RPL21and RPL39respectively with siRNAs enhanced apoptosis of PNAC-1cells. The proportion of early apoptotic cells were3.45%in untreated PANC-1cells. Compared with untreated PANC-1cells, silencing RPL26, RPL29, RPL21and RPL39with siRNAs (40nmol/L,72h) enhanced up to11.15%,8.65%,14.39%and10.2%. Silencing RPL26and RPL29with siRNAs (40nmol/L,48h) significantly decreased the intracellular ROS generation in PANC-1cells. Silencing RPL31with siRNAs in PANC-1cells significantly reduced the cell migration and VEGF expression in culture supernatant. For VEGF expression, compared with untreated PANC-1cells (2804.62pg/106cells/24h), silencing the expression of RPL31reduced to1563.45pg/106cells/24h.5. Human pancreatic cancer BxPC-3cells were used to subcutaneously inoculate into BALB/c nude mice to develop a xenograft tumor model of human pancreatic cancer. Intratumoral injection of siRNAs silencing RPL31, RPL21and RPL39suppressed the xenograft tumor growth, reduced the angiogenesis of xenograft tumor cells, and induced xenograft tumor cell apoptosis. Immunohistochemistry and TUNEL assays indicated that:(1) the expression proportion of Ki-67was55.78%in solvent control group. Compared with solvent control, silencing RPL31, RPL21and RPL39in xenograft tumor reduced to11.08%,7.36%and9.28%.(2) the expression proportion of CD31was56.35%in solvent control group. Compared with solvent control, silencing RPL31, RPL21and RPL39in xenograft tumor reduced to9.67%,5.19%and17.73%.(3) the apoptosis index was2.92%in solvent control group. Compared with solvent control, silencing RPL31, RPL21and RPL39in xenograft tumor enhanced up to39.58%,35.61%and51.37%.