Study On The Function Of PinX1in MCF-7Cells And Its Location In Cell Cycle

Breast cancer is the most common malignant tumor in women,with the highest mortality.There are1.3million people newly diagnosed as breast cancer and500,000died of it each year worldwide. The incidence of breast cancer is rising steadily year by year, there are about470,000breast-cancer patients in china at present. Radiotherapy along with other auxiliary methods such as chemotherapy and endocrinotherapy is used to treat breast cancer. Though technologies and equipments for breast cancer therapy have been greatly advanced in recent years, the5-year survival rate of patients with breast cancer remains low. Moreover, partial and systemic side-effects caused by chemotherapy would greatly harm the patients psychologically and physically. Therefore, it is of importance to study the etiology of breast cancer and explore new and safe modalities for breast cancer therapy.Therefore, it is necessary to further study breast cancer invasion and metastasis mechanism and to explore effective regimen of disease prevention and control,which is helpful to improve the survival and quality of life for breast cancer patients.Telomere is a specialized nucleoprotein complex which contains repetitive G-rich DNA sequences and partner proteins to protect the chromosomal ends from degradation and to prevent the chromosomal end-to-end fusion. Dysfunction of telomere leads to genome instability and higher incidence of cancer in the aged cells. Although the maintenance of telomere length is mainly performed by a specific reverse transcriptase named telomerase in most malignant cancer cells, telomere-associated proteins are essential for adequate maintenance of telomeric DNA. Telomere-associated proteins directly and indirectly interact with telomeric DNA and contribute to telomere structure and function. Moreover, mounting evidence suggests that telomeric associated proteins may play important roles in cell cycle progression. For example, expression level of TRF1is regulated during the cell cycle, besides, it is reported that TRFI can interact with microtubule protein EB1and some spindle checkpoint proteins such as Madl and Nek2.Including breast cancer, most of dangerous malignant tumors have a close relationship with an increase in telomerase activity and mRNA level of telomerase catalytic subunit (hTERT). With the progress of research on telomerase, targeting on telomerase has been considered a promising treatment of tumor-targeted genetic therapy. PinXl was reported in2001in human cervical cancer using the yeast two-hybrid method which is an evolutionally conserved nucleolar protein that has been recently demonstrated as a telomerase/telomere interacting factor in both the humans and the yeasts. The human PinXl gene locates to the chromosome8P23, a region with high frequency of loss-of-heterozygous in multiple human cancers.The PinXl protein possesses328amino acids, with two independent hTERT-bindings identified to either its terminal N-(aal-142)or the C-(aa254-328)terminal polypeptide sequences. PinX1protein is the strongest inhibiting factor to telomerase which was found so far. Suppression of telomerase activity by PinX1is mediated by its direct interaction with hTERT and hTR. However, whether Gno1P, the yeast homolog of human PinX1, can suppress the telomerase ability is controversial. Ectopic overexpression of a PinX1C-terminal polypeptide has been experimentally shown to be able to completely suppress the activity of telomerase, shorten telomere-length and inhibit both invitro growth in a telomerase-positive human cancer cell line as invivo, leading to the suggestion of PinXl an intrinsic telomerase inhibitor and a putative tumor suppressor in human cells. However, the mechanisms of PinXl on telomerase/telomere regulations and its relationship with cancers have not been intensively investigated yet. In addition, PinXl is proved to be capable of interacting with telomeric protein TERF1, which can enhance the location in nucleolus of it in tumor cells with activity of telomerase, but its mechanism is not completely clarified.Although Pinxl is considered as a powerful inhibitor of telomerase and potential tumor suppressor gene, its effect in tumor maintains controversial. Researches in the domestic and overseas have suggested that the expression of Pinxl in hepatic carcinoma and gastric cancer is low but not in prostatic carcinoma. Some scholars found that Pinxl can regulate the length of telomerase in hepatic tumor but do no effect to the formation of cancer. A research even suggests that the length of telomerase is positively associated with the expression of Pinx1in acute leukemia. It seems that Pinxl may be involved in different mechanism so that it would play different roles in diverse tumor. Great majority of researchers tend to believe that Pinxl is a inhibitor in the formation and progress in breast tumors, yet it is not completely accepted worldwide. Based on previous research findings, our current work is intended to elucidate Pinx1expression in breast cancers and healthy human non-tumor breast tissues and it is roles in apoptosis and cell cycle in carcinoma. To study the function of this gene on breast cancers, Pinxl gene was overexpressed in MCF-7cells and the change of proliferation and cell cycle is detected. The effect of Pinxl works in cell cycle was speculated by detecting its varying location and its dynamic expression in different cell cycle phase. This study covers the following four parts:Part I:Primers for amplification the Pinxl CDS sequence was designed and synthesized. The Pinxl ORF was amplified from cDNA from PBMCs and inserted into the clone site of plasmid PDsRed1-C1by double digestion with EcoR I/Xho I and subsequent junction with T4ligase. The positive recombinant plasmids were screened and identified by restriction enzyme digestion, and its sequence fidelity was analyzed by DNA sequencing. The positive constructed recombinant plasmids pdsredl-cl were transfected into the MCF-7cells for expression of Pinxl and screen the transfected cells with culture medium containing G418of proper consistency. Real-time PCR and Western-Blot assay were used to detect the Pinxl expression in the stable expression MCF-7cells from the above step.Part II:MTT, cell colony formation and anaphase-bridge calculation were used to study the proliferation, apoptosis and cell cycle of Pinxl transfected MCF-7cells.Part III:The variation of protein Pinxl in different cell cycle phase was detected by fluorescence microscope after MCF-7cells were fixed onto the cover glass and dined by DAPI. Real-time PCR was used to detect the Pinxl expression of different cell cycle phase after the cells were collected by serum starvation.Combined with both results above, then we speculate the mechanism of regulating cell cycle the Pinxl protein may be involved.Up-regulating Pinxl expression could inhibit proliferation of MCF-7cells and block cell cycle in G0/G1phase. The Pinxl locational survey showed that its site vary in different cycle phase, and the Real-time PCR result revealed that Pinxl expresses dynamically with the cycle phase changed.On conclusion, we had successfully established a Pinxl stable expressional MCF-7cell line, our results that overexpression of Pinxl may inhibit the growth and progression of breast cancer, and induce a cell cycle arrest in G0/G1phase. Combined with previous research, we put forward a speculation that Pinxl may interact with PLK1while it combined with kinetochore, which leads to a Pinxl degradation through ubiquitination pathway mediated by PLK1, it may play a important role in chromosome separation and effect on cell cycle regulation. More studies and evidences are now needed to confirm the speculative path way mentioned above, and give new clues to exploring the innovative cell cycle regulating pathway Pinxl involved.