The SW480 cell line, overexpressing PIWIL2 gene, maintains the expression of stemness and proliferation genes in the mice xenografts.

Aim: This study aims to confirm previous fundamental in vitro findings about the PIWIL2 gene by investigating the effects of its overexpression on cell cycle, proliferation, apoptosis, and stem cell expression markers in colorectal cancer cells (CRC cells) at in vivo level. Background: PIWIL2 has a critical role in maintaining cellular stemness and proliferation. PIWIL2 is an oncogene whose expression in CRC is associated with the occurrence, metastasis, and poor prognosis. Methods: SW480 cells harboring expression vectors with/without PIWIL2 were cultured and inoculated in BALB/c nude mice. Tumor formation and growth were monitored every 3 days. On the 28th day after inoculation, the tumors were harvested for their total RNA extraction, and the expression profiling of the candidate genes was performed by Real-time PCR. Results: Our results for the expression profiling of the xenografted tumors showed a significant increase in the expression of cancer stem cell markers, including CD24, CD133, and pluripotency marker SOX2 in the PIWIL2 over-expressing xenografts, compared to the control cell line. Moreover, PIWIL2 dramatically promoted the anti-apoptotic pathway by inducing STAT3 and BCL2-L1 genes in the PIWIL2 over-expressing xenografts, along with the up-regulation of Cyclin D1 and Ki-67 genes. Conclusion: This research supports our prior in vitro findings, highlighting the critical role that PIWIL2 plays in the development of CRC and its substantial promise as a leading candidate for CRC-targeted therapy.

PIWIL2 Regulates the Proliferation, Apoptosis and Colony Formation of Colorectal Cancer Cell Line.

Background: Tumor cells proliferation and apoptosis inhibition are the mechanisms through which the Colorectal Cancer (CRC) progression, metastasis and chemoresistance are promoted pathologically, offering clinical advantages for characterizing their molecular regulators. Objectives: In this study, to unravel the role of PIWIL2 as a potential CRC oncogenic regulator, we examined the effect of its overexpression on proliferation, apoptosis and colony formation of SW480 colon cancer cell line. Material and Methods: Established SW480-P (overexpression of PIWIL2) and SW480-control (SW480-empty vector) cell lines were cultured in DMEM containing 10% FBS with 1% penicillin-streptomycin. The total DNA and RNA was extracted for further experiments. Real-Time PCR and western blotting assay were performed to measure the differential expression of proliferation associated genes including the expression of cell cycle and anti-apoptotic genes as well as Ki-67 and PIWIL2 in both cell lines. Cell proliferation was determined using MTT assay, doubling time assay and the colony formation rate of transfected cells was measured with the 2D colony formation assay. Results: At the molecular level, PIWIL2 overexpression was associated with significant up-regulation of cyclin D1, STAT3, BCL2-L1, BCL2-L2 and Ki-67 genes. MTT and doubling time assay showed that PIWIL2 expression induced time-related effects on proliferation rate of SW480 cells. Moreover, SW480-P cells had markedly greater capacity to form colonies. Conclusions: PIWIL2 plays important roles to promote cancer cell proliferation and colonization via the cell cycle acceleration and inhibition of apoptosis, the mechanisms through which this gene seems to contribute to CRC development, metastasis and chemoresistance, hence potentially highlighting PIWIL2 targeted therapy as a valuable tool for CRC treatment.

Prognostic Value of BRAF and KRAS Mutation in Relation to Colorectal Cancer Survival in Iranian Patients: Correlated to Microsatellite Instability.

PURPOSE: To evaluate the prognostic role of BRAF and KRAS mutations after adjustment for microsatellite instability (MSI) in Iranian colorectal cancer (CRC) patients. METHODS: BRAF and KRAS mutations and MSI status were assessed in 258 Iranian subjects with CRC. Two hundred fifty-eight consecutive stages I-IV CRC patients, who underwent surgical resection of adenocarcinoma from 2012 to 2016, were enrolled in the research. Pyrosequencing and Cast-PCR methods were used to the detection of KRAS and BRAF mutations. Kaplan-Meier and Cox regression were employed to estimate hazard ratios (HR) for the association between BRAF and KRAS mutation and overall survival (OS). RESULTS: KRAS and BRAF mutations were detected in 36 (14%) and 15 (5.8%) cases of 258 patients with CRC, respectively. BRAF mutations that all comprised V600E and KRAS mutations was found in codon 12 and 13 (80.6% and 19.4%), respectively. KRAS mutations were detected in 19 (15.4%) patients of 123 microsatellite stable (MSS) CRC and it is significantly associated with tumor location and metastasis. BRAF and KRAS mutant vs. wild type of BRAF and KRAS, 5-year OS was 73.3% vs. 82.3% and 83.3% vs. 81.5% (long-rank P > 0.05), respectively. KRAS mutant vs. KRAS-wild-type tumors in MSS/MSI-L status CRC patients, 5-year OS was 78.9% vs. 90.4% (long-rank p = 0.046). CONCLUSION: The present study revealed that BRAF and KRAS mutations were not related to the worse overall survival, while KRAS mutation can be a prognostic factor for overall survival in sporadic microsatellite-stable (MSS) status in Iranian CRC patients.

Lack of BRAFV600E mutation in stage I and II of colorectal cancer.

AIM: We aimed to explore the frequency of BRAFV600E mutation in Iranian patients with colorectal cancer (CRC) as well as its association with clinic pathological characteristic of patients. BACKGROUND: CRC is the third leading cause of cancer related death. There is a growing body of data showing the association of BRAFV600E mutation with malignant transformation and clinical outcome of different tumors, including CRC. These findings suggest that BRAFV600E mutation can be used as diagnostic and/or prognostic biomarker for management of cancer patients. PATIENTS AND METHODS: A total of 85 patients with sporadic tumor were recruited. BRAFV600E mutation was investigated using sequencing of extracted DNAs from formalin-fixed paraffin-embedded (FFPE) tumor tissues. Electropherograms were analyzed using Laser-gene 6 software. RESULTS: More than 95% of patients were in stage I and II and none of them were in stage IV. Patients were mostly below 55 years old and tumors were dominantly located in the distal colon. Of note, no BRAFV600E mutations were detected in our population. CONCLUSION: Our results showed no V600E mutation in the BRAF gene in stage I and II of CRC patients. Further studies in multi-center settings are warranted to examine the prognostic and/or predictive value of this marker in different stages of colorectal cancer patients.

Gastric atrophy: use of OLGA staging system in practice.

AIM: This study used the OLGA system to characterize the histology pattern of gastritis in dyspeptic outpatients with a mean age of 45 years from regions with different gastric cancer risks. BACKGROUND: Several classification systems have been purposed for understanding the status of the gastric mucosa. Currently, the Sydney system is the most widely employed. Nevertheless, the applicability of the Sydney system in therapeutic and prognostic areas is a matter of debate. Given this shortcoming an international group of gastroenterologists and pathologists developed a new system named Operative Link on Gastritis Assessment (OLGA). PATIENTS AND METHODS: In this cross-sectional comparative study the OLGA system was used to characterize the histology pattern of gastritis in 685 dyspeptic patients referring to the department of gastroenterology of a training hospital. RESULTS: No significant correlation was found between active inflammation and total OLGA score (P > 0.05). Also, no statistically significant correlation was found between activity and intestinal metaplasia, dysplasia, atrophy, and cancer (P > 0.05). Even though, there is a positive correlation between mild chronic inflammation and total OLGA score, no correlation has been identified between chronicity and dysplasia or cancer (P > 0.05). Nearly, In all cases with no dysplasia OLGA score was zero but all patients with gastric cancer OLGA score was more than two. CONCLUSION: Generally, the activity is not a useful factor in predicting prognosis and its loss of relation with total OLGA score does not make OLGA score any less predictable.

Prognostic Significance of Nuclear ß-Catenin Expression in Patients with Colorectal Cancer from Iran.

BACKGROUND: Beta catenin plays a key role in cancer tumorigenesis. However, its prognostic significance in patients with colorectal cancer (CRC) remains controversial. It has been demonstrated that 90% of all tumors have a mutation in individual components of multiple oncogenes in Wnt/ß-catenin pathway. Accumulation of nuclear ß-catenin in cytoplasm leads to uncontrolled cell proliferation. Thus, nuclear ß-catenin accumulation may be a valuable biomarker associated with invasion, metastasis and poor prognosis of CRC. OBJECTIVES: In this study the prognostic value of beta catenin expression in 165 Iranian CRC patients was evaluated. PATIENTS AND METHODS: In this cross sectional retrospective study immunohistochemistry analyses of formalin-fixed paraffin-embedded (FFPE) tumor tissues were performed to characterize the expression of nuclear ß-catenin in a series of 165 Iranian patients with colorectal carcinoma. Heat-induced antigen retrieval using the microwave method was applied for all staining procedures. Staining was scored independently by two observers, and a high level of concordance (90%) was achieved. Statistical analysis was done using the SPSS software for Windows, version 13.0.0 (SPSS Inc., Chicago, IL). Two-tailed P < 0.05 was considered statistically significant. RESULTS: The patients consisted of 85 males and 80 females. Eighty-eight patients had primary tumor of the rectum and sigmoid, while 77 patients had primary tumor of the colon. The mean period of follow-up was 47.2 ± 10 months and the median period of follow-up was 38 months (range 6 - 58) for each patient. Of 165 tumors, 32 tumors (19.39 %) showed expression of ß-catenin and 133 (80.6 %) were negative for ß-catenin expression. Based on our findings the distribution of Microsatellite Instability (MSI) status differed between patients with nuclear ß-catenin positive and negative tumors and this difference was significant (P = 0.001). Patients with nuclear ß-catenin positive expression profile were found to be younger than patients with negative nuclear ß-catenin expression (P = 0.010). Univariate and multivariate analysis showed that tumors with ß-catenin expression had a poorer prognosis compared to tumors without ß-catenin expression. CONCLUSIONS: According to our findings, the distribution of nuclear b-catenin expression is a poor prognostic marker in patients with colon cancer.