{"id":54144,"date":"2023-12-31T11:32:46","date_gmt":"2023-12-31T11:32:46","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=54144"},"modified":"2024-01-05T06:15:22","modified_gmt":"2024-01-05T06:15:22","slug":"analyzing-the-expression-of-microrna-375-and-its-target-gene-p53-in-oral-squamous-cell-carcinoma-and-its-implication-in-oral-carcinogenesis","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol16no4\/analyzing-the-expression-of-microrna-375-and-its-target-gene-p53-in-oral-squamous-cell-carcinoma-and-its-implication-in-oral-carcinogenesis\/","title":{"rendered":"Analyzing the Expression of MicroRNA-375 and its Target Gene p53 in Oral Squamous Cell Carcinoma and its Implication in Oral Carcinogenesis"},"content":{"rendered":"\n

Introduction<\/strong><\/p>\n\n\n\n

Oral squamous cell carcinoma is the highly prevalent malignancy of\nthe oral mucosa accounting for about 90% of all head and neck cancers1<\/sup>.\nHigh mortality and morbidity rates associated with this disease is attributed\nto late diagnosis and intervention 2<\/sup>. Oral carcinogenesis is a\nmultistep process that disrupts various Oncogenes and tumor suppressor genes3<\/sup>.\nUnderstanding the epi genetic insights of oral cancer would result in the\ndiscovery of a powerful diagnostic and prognostic tool. Results of the research\nstudies conducted during the past several years with similar intentions are yet\nto be clinically translated4<\/sup>.<\/p>\n\n\n\n

 MicroRNAs are endogenous RNAs\nthat possess enormous gene modulatory potential. Based on their sequence and\ncomplementarity, microRNAs combine with specific mRNAs and regulate protein\nsynthesis either by target mRNA degradation or translation repression5, 6<\/sup>.\nIn this manner, the human microRNAs can control about 60% of the human genome.\nEvery cellular process such as cell differentiation, proliferation, mobility,\nand apoptosis are subject to microRNA- dependent regulation7<\/sup>.\nExpression of microRNAs is tightly controlled by various transcription factors.\nMicroRNA expression is heavily dysregulated in human cancers and compelling\nevidence has shown that they affect all the hallmarks of cancer8<\/sup>.  The variations in the expression of many\nmicroRNAs have assisted as diagnostic and prognostic predictors in specific\ntumors and might become potential and promising\ntherapeutic targets in cancer treatment in the future9<\/sup>.<\/p>\n\n\n\n

MicroRNA 375 is a tumor-suppressive microRNA located in 2q3510<\/sup>.  It is an islet-specific miRNA that regulates\ninsulin secretion and glucose homeostasis. Anti-tumor effects of miR-375 are\npotentiated by modulating its oncogenic targets JAK2,\nIGF1R, AEG-1 and YAP1 genes that moderate many processes like apoptosis, invasion,\nand metastasis. Many studies reveal that miR-375 is widely present in\nother human tissues and is significantly downregulated in many malignancies\nsuch as melanoma, glioma, Gastric, Laryngeal, Esophageal, and hepatocellular\ncarcinoma11,12<\/sup>. <\/p>\n\n\n\n

The p53 is a tumor suppressor gene\nwith pro-apoptotic function13<\/sup>. The p53 transcription factor is\nconsidered  the \u201cguardian of the genome\u201d\nas it senses the DNA damage and mediates cell cycle arrest or promotes\napoptosis14<\/sup>. Mutations in p53 are associated with genomic instability and\nincreased susceptibility to cancer15<\/sup>. In normal cells, the half-life of p53\nprotein is very short lasting up to 5-20 minutes making it difficult to be\ndetected in normal tissues16<\/sup>. However, a mutated p53 protein is not\neasily digested and has a prolonged half-life. Therefore, it accumulates inside\nthe cancer cells and can be immunohistochemically detected in premalignant and\nmalignant tissues17, 18<\/sup>.  p53 gene regulates, and in turn, is\nalso regulated by several microRNAs19<\/sup>. p53 has been reported as a\ngene target for miR-37520,21<\/sup>. There are no previous studies in the\nliterature that studied the association between miR375 and p53 expression in\noral cancer. This study analyzed the expression of miR-375 and its gene target\np53 in OSCC tissues and correlated their expression to the clinical and\npathological parameters of the study samples to understand their role in oral\ncarcinogenesis.<\/p>\n\n\n\n

Materials and Methods<\/strong><\/p>\n\n\n\n

The study was conducted in the Oral Pathology department of the\nUniversity Dental Hospital. The Institutional Review Board approved the study((SRIHER)- IEC-N1\/12\/MAR\/27)\nand all the patients signed informed consent before sample collection. The\nstudy population comprised histopathologically diagnosed cases of squamous cell\ncarcinoma using incisional biopsy samples. Only the patients with primary\ntumors were included in the study. Recurrent tumors and cases with previous\ntreatments such as radiotherapy and chemotherapy were excluded from the study.  After exclusions about 22 cases were included\nin the study. Resected tumors and\nthe corresponding adjacent normal mucosa tissues were obtained from OSCC\npatients. Normal mucosal tissue was obtained from the contralateral side of the\nlesion proper. Fresh tissue samples were preserved in RNAlater<\/em>\n(sigma Aldrich) and stored at -40o<\/sup>C until PCR analysis. Tissue for the histopathological\nstudy was stored in formalin at room temperature and routinely processed.  <\/p>\n\n\n\n

Quantitative Real-time (qRT-PCR) assay<\/strong><\/p>\n\n\n\n

The tumor and normal tissues were homogenized using mortar and pestle and extraction of total DNA was performed by the trizol method (Invitrogen, USA). Stem loop primers (table 1) and primescript RT reagent kit ((Takara Bio Inc) were used for synthesizing the Complementary DNA from the extracted RNA. Reverse transcriptase reactions included 7\u03bcl of RNA and 3 \u03bcl of master mix containing RT buffer, stem loop primers and RT enzyme. 10 \u03bcL aliquots were prepared and incubated for 15 min at 37\u00b0C, and 15 seconds at 85\u00b0C, followed by maintenance at 4\u00b0C. The primeScript SYBR Green PCR kit (Qiagen NV) was used to perform the qPCR analysis. One microliter of cDNA was used as a template in 10\u03bcL reactions. The master mix was prepared separately for the microRNAs and the control gene with the forward and reverse primers (Table 1), SYBR green dye, ROX dye and water. Gene expression was quantified using triplicates in the 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA). The 10 \u03bcL aliquots PCR plates were denatured for 95\u2103 for 30 seconds, annealed at 95\u2103 for 30 seconds, 60\u2103 for 35 seconds (40 cycles), 95\u2103 for 15 seconds and extended for one minute at 60\u2103. <\/p>\n\n\n\n

Table 1: Primer sequence for RT- PCR<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n
\n

GENE<\/strong><\/p>\n<\/td>\n

\n

PRIMER SEQUENCE<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

miR-375 stem loop RT primer<\/p>\n<\/td>\n

\n

5\u2019-GTCGTATCCAGTGGAGGGTCCGAGGTATTCGCACTGG ATACGACTCACGCG-3\u2019<\/p>\n<\/td>\n<\/tr>\n

\n

RNU6 stem loop RT primer<\/p>\n<\/td>\n

\n

5\u2019-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGG ATACGACTATGGAAC-3\u2019<\/p>\n<\/td>\n<\/tr>\n

\n

miR-375 Forward Primer<\/p>\n<\/td>\n

\n

5\u2019-GCCCGCTTTGTTCGTTCGGCT-3\u2019<\/p>\n<\/td>\n<\/tr>\n

\n

RNU6 Forward Primer<\/p>\n<\/td>\n

\n

5\u2019-GTGCTCGCTTCGGCAGCAC-3\u2019<\/p>\n<\/td>\n<\/tr>\n

\n

Universal Reverse Primer   <\/p>\n<\/td>\n

\n

5\u2019-GTGCAGGGTCCGAGGT-3\u2019<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n

Analysis of relative gene expression<\/strong> <\/p>\n\n\n\n

The relative gene expression in the tumor and normal tissues was calculated\nusing the 2-\u0394\u0394Cq<\/sup> method (Livak method) by normalizing it to the U6\nhousekeeping gene22<\/sup>. The first step in the analysis involved the\ndetermination of the threshold cycle (Ct) value of individual samples. Using\nthe threshold cycle value of each microRNA and control gene the \u0394Ct value was\ncalculated using the formula \u0394Ct= Ct miR375 – Ct U6. The \u0394\u0394Ct value was\ncalculated from the from the \u0394Ct values using the formula (\u0394\u0394Ct= \u0394Ct Cancer –\n\u0394Ct Normal). Finally, the fold value (2-\u0394\u0394Ct<\/sup> value) was calculated.  A Fold value more than 1, is considered as an upregulation\nof the gene and a fold value of less than 1 signifies a downregulation of the\ngene. <\/p>\n\n\n\n

Immunohistochemistry (IHC)<\/strong><\/p>\n\n\n\n

The tumor and control tissues were routinely processed, and 4-um paraffin sections were taken on charged slides. The sections were dewaxed using xylene, dehydrated in graded alcohols, and subsequently rinsed thoroughly using distilled water. Endogenous peroxidase activity was blocked by applying peroxide block to the tissue sections for 5 minutes at 37\u2103. The pressure cooker method was used for antigen retrieval. Further p53 primary antibody (Mouse monoclonal, clone DO7, class-IgG1 -BioGenex Life sciences Pvt Ltd) was applied to the tissue sections for 1 hour.  Further, the sections were thoroughly rinsed with citrate buffer and incubated with universal secondary antibody for 30 minutes (Super SensitiveTM<\/sup> Polymer HRP Kit Manual- BioGenex life sciences Pvt Ltd). Finally, the sections were thoroughly rinsed with distilled water and stained with the chromogen diaminobenzidine (DAB), counterstained with Harris hematoxylin, air dried, cleared and mounted with dibutyl phthalate xylene (DPX). Breast cancer tissues were used as a positive control for mutant p53. Internal negative controls were used to validate the staining. <\/p>\n\n\n\n

All the slides were scored by two certified pathologists. Mutant\np53 positivity was confirmed by the presence of brown staining of the nucleus\nof the cells. The sections were considered negative when <5% of the cells were\nstained for p53. Further positive sections were graded as mild positive\n(5-25%), Moderate positive (25-50%) and intense positive (> 50%) based on\nthe number of cells that were immune-positive for mutant p53.<\/p>\n\n\n\n

Data collection\/statistical analysis<\/strong><\/p>\n\n\n\n

The clinical parameters of the study samples were obtained from the\nmedical records session. The study sample comprised 16 men and 6 women. The mean\nage of the patients was 54.5. Eleven cases were excised from buccal mucosa, 5\nfrom the tongue, 3 from alveolar mucosa, 2 from the lip, and one from the floor\nof the mouth. The revised International TMN Staging System was used to assess the\ntumor stage. 15\/22 cases had nodal metastasis of the disease. However, none of\nthe patients had distant organ metastasis confirmed by a PET scan. The date of\nthe surgery was considered the beginning of the disease. Follow-up data was\nobtained for 5 years. Recurrence was noted in 2 of the 22 cases. The statistical analysis of the study results was performed using\nversion 18 of SPSS software. Descriptive statistics were presented as numbers\nand percentages. Mean and SD were calculated for all the data.  Statistical differences between\nclinicopathological parameters and the miRNA375 levels were evaluated using\nnon-parametric tests. The difference in microRNA expression between the\nlesion and control tissues was calculated student T-test. A chi-square test was used for\ncomparison between two attributes. Inter-observer reliability was calculated\nusing Cohens Kappa. A p-value less than 0.05 was considered statistically\nsignificant.<\/p>\n\n\n\n

Results\n<\/strong><\/strong><\/p>\n\n\n\n

The study analyzed the MicroRNA375 expression in OSCC and its\npaired normal tissues by using qRT-PCR. The tissues were also analyzed for the\nexpression of p53 using immunohistochemistry. The microRNA and p53 expression\nwere also compared to the clinical and pathological attributes of the tumor. <\/p>\n\n\n\n

Expression of miR-375 in oral cancer and normal tissues<\/strong><\/p>\n\n\n\n

A significant difference was noted in the expression level of miR-375 between the tumor and adjacent normal tissues. Among 22 OSCC cases, 15(68.1%) tumor samples demonstrated downregulation of miR-375(p<0.05) <\/em>(Figure1). A mean fold change of 83.96 was noted in the tumor tissues compared to the controls. <\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 1: Downregulation of miR-375 in OSCC.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

 MicroRNA-375 expression correlation to clinico-pathological\nparameters<\/strong><\/p>\n\n\n\n

 Table 2 displays miR-375 expression based on the\nclinicopathological parameters. All the cases below 40 years, 50% of cases\nbetween 40-60 years and 77.8% of cases above 60 years of age demonstrated\nmiR-375 downregulation. Men showed significant downregulation of miR-375\ncompared to women (p <0.05).<\/em> Cases with pan\/gutkha chewing habits\n(77.8%) showed significant (p <0.05)<\/em> miR-375 downregulation compared\nto cases without habits. Smokers and non-smokers did not exhibit any\nsignificant difference in miR-375 expression(P>0.05)<\/em>. 80% of the cases with N2 nodal\ninvolvement exhibited downregulation of \nmiR-375. However, this finding was not statistically significant.\nThe miR-375 expression levels did not reveal any statistically significant\nrelationship with patient age, tumor size, location, nodal metastasis, histopathological\ngrade, and survival of the patient. The Kaplan \u2013Meier analysis could not\nyield a meaningful interpretation.<\/p>\n\n\n\n

p53 Expression in OSCC<\/strong><\/p>\n\n\n\n

The positive control breast cancer tissue stained intensely with\np53 antibody (Figure 2), and the internal negative controls did not exhibit any\np53 positivity. The\ninterobserver variation or the measurement of agreement between the observes\nwas statistically analyzed and was highly significant (p <0.00)<\/em> The\nkappa score was calculated to analyze the overall significance of p53\nexpression between the normal and OSCC sections and was statistically\nsignificant (p <0.00)<\/em><\/p>\n\n\n\n

 In our study mutant p53 expression was observed in the nuclei of the dysplastic epithelial cells of OSCC. Staining ranged from focal positivity in the tumor islands to intense positivity in sheets of dysplastic squamous cells in the underlying connective tissue (Figure 3-5). Among 22 tumor cases, 14 tissues (63.6%) exhibited immunopositivity for mutant p53 (4 – mild positive; 5-Moderate positive & 5- intense positive). <\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 2: Intense expression of mutant p53 in the positive control tissue (Breast cancer) (IHC x 40X), Inset -(IHC x 10X).<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Figure 3: Mild immunopositivity of mutant p53 in OSCC (IHC <\/strong>X<\/strong> 40X).<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Figure 4: Moderate immunopositivity of mutant in p53 OSCC (IHC <\/strong>X<\/strong> 40X).<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Figure 5: Intense immunopositivity of mutant in p53 OSCC (IHC <\/strong>X<\/strong> 40X), Inset -(IHC x 10X).<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Mutant p53 expression correlation to clinico\u2011 pathological parameters of OSCC<\/strong> <\/p>\n\n\n\n

The expression\nof mutated p53 in the study samples is presented in Table 2. All the smokers\nexhibited mutant p53 expression compared to the nonsmokers (p < 0.05).<\/em>\nRegarding the tumor size, the T1 tumors exhibited mild to moderate staining\ncompared to the intense immunopositivity noted among T2 and T4 tumors (p\n<0.05).<\/em> Intense mutant p53 staining was observed in moderately\ndifferentiated and poorly differentiated tumors compared to the well-differentiated\nOSCC tissues (p <0.05).<\/em> Tumor cases with N2 nodal\nmetastasis showed intense mutant p53 staining followed by the N1 cases. Whereas patients without nodal\nmetastasis showed weak to moderate immunostaining (p <0.05).<\/em>  There was no significant association between\nthe mutant p53 expression with age, gender, or the clinical location of the\ntumor (P>0.05).<\/em> <\/p>\n\n\n\n

The relative expression of mutant p53 was compared with miR-375 expression in OSCC cases and about 73.3% (11\/15) of miR-375 down regulated cases showed mutant p53 expression in OSCC demonstrating a very significant association within the genes. (p < 0.05).<\/em><\/p>\n\n\n\n

Table 2: Clinco-pathological Correlation of OSCC patients to miR-375 and p53 expression<\/strong>.<\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

 <\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

miR375<\/strong><\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

p53 expression<\/strong><\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n<\/tr>\n

\n

Characteristics<\/strong><\/p>\n<\/td>\n

\n

n<\/strong><\/p>\n<\/td>\n

\n

Downregulation<\/strong><\/p>\n<\/td>\n

\n

p-value<\/strong><\/p>\n<\/td>\n

\n

Weak <\/strong><\/p>\n<\/td>\n

\n

Moderate <\/strong><\/p>\n<\/td>\n

\n

Intense<\/strong><\/p>\n<\/td>\n

\n

Negative  <\/strong><\/p>\n<\/td>\n

\n

p-value<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Age in years<\/strong> <\/p>\n<\/td>\n

 <\/td>\n<\/tr>\n
\n

< = 40<\/p>\n<\/td>\n

\n

3<\/p>\n<\/td>\n

\n

3<\/p>\n<\/td>\n

\n

0.191<\/p>\n<\/td>\n

\n

1<\/p>\n<\/td>\n

\n

2<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

0.411<\/p>\n<\/td>\n<\/tr>\n

\n

41 – 60<\/p>\n<\/td>\n

\n

10<\/p>\n<\/td>\n

\n

5<\/p>\n<\/td>\n

\n

2<\/p>\n<\/td>\n

\n

1<\/p>\n<\/td>\n

\n

3<\/p>\n<\/td>\n

\n

4<\/p>\n<\/td>\n<\/tr>\n

\n

> 60<\/p>\n<\/td>\n

\n

9<\/p>\n<\/td>\n

\n

7<\/p>\n<\/td>\n

\n

1<\/p>\n<\/td>\n

\n

2<\/p>\n<\/td>\n

\n

2<\/p>\n<\/td>\n

\n

4<\/p>\n<\/td>\n<\/tr>\n

\n

Gender<\/strong><\/p>\n<\/td>\n

 <\/td>\n<\/tr>\n
\n

Male<\/p>\n<\/td>\n

\n

16<\/p>\n<\/td>\n

\n

13<\/p>\n<\/td>\n

\n

*0.032<\/p>\n<\/td>\n

\n

3<\/p>\n<\/td>\n

\n

4<\/p>\n<\/td>\n

\n

5<\/p>\n<\/td>\n

\n

4<\/p>\n<\/td>\n

\n

0.158<\/p>\n<\/td>\n<\/tr>\n

\n

Female<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

2<\/p>\n<\/td>\n

\n

1<\/p>\n<\/td>\n

\n

1<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

4<\/p>\n<\/td>\n<\/tr>\n

\n

Location<\/strong><\/p>\n<\/td>\n

 <\/td>\n<\/tr>\n
\n

Buccal mucosa<\/p>\n<\/td>\n

\n

11<\/p>\n<\/td>\n

\n

9<\/p>\n<\/td>\n

\n

 <\/p>\n

 <\/p>\n

0.471<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

4<\/p>\n<\/td>\n

\n

3<\/p>\n<\/td>\n

\n

4<\/p>\n<\/td>\n

\n

 <\/p>\n

 <\/p>\n

0.27<\/p>\n<\/td>\n<\/tr>\n

\n

Tongue<\/p>\n<\/td>\n

\n

5<\/p>\n<\/td>\n

\n

3<\/p>\n<\/td>\n

\n

2<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

2<\/p>\n<\/td>\n

\n

1<\/p>\n<\/td>\n<\/tr>\n

\n

Lip<\/p>\n<\/td>\n

\n

2<\/p>\n<\/td>\n

\n

1<\/p>\n<\/td>\n

\n

1<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

1<\/p>\n<\/td>\n<\/tr>\n

\n

Alveolus<\/p>\n<\/td>\n

\n

3<\/p>\n<\/td>\n

\n

1<\/p>\n<\/td>\n

\n

1<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

2<\/p>\n<\/td>\n<\/tr>\n

\n

Floor of mouth<\/p>\n<\/td>\n

\n

1<\/p>\n<\/td>\n

\n

1<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

1<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

Habits- Pan chewing<\/strong><\/p>\n<\/td>\n

 <\/td>\n<\/tr>\n
\n

Present<\/p>\n<\/td>\n

\n

18<\/p>\n<\/td>\n

\n

14<\/p>\n<\/td>\n

\n

*0.040<\/p>\n<\/td>\n

\n

4<\/p>\n<\/td>\n

\n

4<\/p>\n<\/td>\n

\n

4<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

0.762<\/p>\n<\/td>\n<\/tr>\n

\n

Absent<\/p>\n<\/td>\n

\n

4<\/p>\n<\/td>\n

\n

1<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

1<\/p>\n<\/td>\n

\n

1<\/p>\n<\/td>\n

\n

2<\/p>\n<\/td>\n<\/tr>\n

\n

Smoking<\/strong><\/p>\n<\/td>\n

 <\/td>\n<\/tr>\n
\n

Smokers<\/p>\n<\/td>\n

\n

9<\/p>\n<\/td>\n

\n

8<\/p>\n<\/td>\n

\n

0.083<\/p>\n<\/td>\n

\n

1<\/p>\n<\/td>\n

\n

3<\/p>\n<\/td>\n

\n

5<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

0.003*<\/p>\n<\/td>\n<\/tr>\n

\n

Non-Smokers<\/p>\n<\/td>\n

\n

13<\/p>\n<\/td>\n

\n

7<\/p>\n<\/td>\n

\n

3<\/p>\n<\/td>\n

\n

2<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

8<\/p>\n<\/td>\n<\/tr>\n

\n

Tumor size<\/strong><\/p>\n<\/td>\n

 <\/td>\n<\/tr>\n
\n

T1<\/p>\n<\/td>\n

\n

11<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

0.323<\/p>\n<\/td>\n

\n

3<\/p>\n<\/td>\n

\n

4<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

4<\/p>\n<\/td>\n

\n

0.05*<\/p>\n<\/td>\n<\/tr>\n

\n

T2<\/p>\n<\/td>\n

\n

9<\/p>\n<\/td>\n

\n

7<\/p>\n<\/td>\n

\n

1<\/p>\n<\/td>\n

\n

1<\/p>\n<\/td>\n

\n

3<\/p>\n<\/td>\n

\n

4<\/p>\n<\/td>\n<\/tr>\n

\n

T4<\/p>\n<\/td>\n

\n

2<\/p>\n<\/td>\n

\n

2<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

2<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

Nodal status<\/strong><\/p>\n<\/td>\n

 <\/td>\n<\/tr>\n
\n

N0<\/p>\n<\/td>\n

\n

7<\/p>\n<\/td>\n

\n

5<\/p>\n<\/td>\n

\n

0.717<\/p>\n<\/td>\n

\n

2<\/p>\n<\/td>\n

\n

2<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

3<\/p>\n<\/td>\n

\n

0.037*<\/p>\n<\/td>\n<\/tr>\n

\n

N1<\/p>\n<\/td>\n

\n

10<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

2<\/p>\n<\/td>\n

\n

2<\/p>\n<\/td>\n

\n

1<\/p>\n<\/td>\n

\n

5<\/p>\n<\/td>\n<\/tr>\n

\n

N2<\/p>\n<\/td>\n

\n

5<\/p>\n<\/td>\n

\n

4<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

1<\/p>\n<\/td>\n

\n

4<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

Tumor grade<\/strong><\/p>\n<\/td>\n

 <\/td>\n<\/tr>\n
\n

WDOSCC<\/p>\n<\/td>\n

\n

12<\/p>\n<\/td>\n

\n

7<\/p>\n<\/td>\n

\n

0.33<\/p>\n<\/td>\n

\n

4<\/p>\n<\/td>\n

\n

4<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

4<\/p>\n<\/td>\n

\n

0.035*<\/p>\n<\/td>\n<\/tr>\n

\n

MDOSCC<\/p>\n<\/td>\n

\n

8<\/p>\n<\/td>\n

\n

7<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

4<\/p>\n<\/td>\n

\n

4<\/p>\n<\/td>\n<\/tr>\n

\n

PDOSCC<\/p>\n<\/td>\n

\n

2<\/p>\n<\/td>\n

\n

1<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

1<\/p>\n<\/td>\n

\n

1<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

   <\/strong>*p < 0.05 Statistically Significant, p > 0.05 Non Significant<\/p>\n\n\n

Discussion <\/strong><\/strong><\/p>\n\n\n\n

MicroRNAs\nare molecular regulators of physiological cellular processes. MicroRNA\ndysregulation has been noted in various diseases including cancer8<\/sup>.\nIn current study analyzed miR-375 expression in 22 OSCC tissues and the\ncorresponding normal tissues. The results reveal that MicroRNA-375 expression is\nconsiderably downregulated in OSCC compared to non-tumor tissue which is\nsupported by the previous studies23-29<\/sup>. In the current study miR-375 was 83.9 %-fold downregulated\ncompared to the adjacent paired normal tissues. MicroRNA studies have shown a\n10 to 22-fold downregulation of miR-375 in OSCC and a 32-fold downregulation in\nHNSCCs24,26,87,30<\/sup>. This very high level of downregulation of\nmiR-375 in our study is attributed to a single case of poorly differentiated\nsquamous cell carcinoma which showed a very high Ct value in the PCR analysis.<\/p>\n\n\n\n

Identified as a\ntumor suppressor gene, miR375 suppresses the malignant properties of cancer\ncells. miR-375 loss has been noted\nin various human malignancies including gastric, Pharyngeal, esophageal,\nhepatic and breast cancers 11<\/sup>. Lian et al demonstrated that miR-375\ntargets tyrosine kinase Janus kinase 2 (JAK2) and inhibits cell proliferation in gastric\ncancer31<\/sup>. Further miR375 could suppress rapid cancer cell\ngrowth by inhibiting aerobic glycolysis via the PI3K-Akt signaling pathway. On the other hand, miR-375 upregulation\nresults in cell cycle arrest at the G0 <\/sub>\/ G1 <\/sub>phase\nin oral cancer cell lines thereby inhibiting tumor growth26<\/sup>.\nMicroRNA-375 downregulation is also a contributing factor for the progression\nof potentially malignant disorders to oral cancer32, 33<\/sup>. Our study\nresults show that miR-375 downregulation in oral cancer showed a positive\nassociation with paan chewing habits. Our findings along with the literature\nsupport provide evidence that miR-375 loss in oral cancer could contribute to\noral carcinogenesis.<\/p>\n\n\n\n

Men\ndemonstrated significant downregulation of miR-375 versus women in our study.\nThis distribution could be attributed to a higher number of men involved in the\nstudy compared to the women. miR-375\nexpression did not correlate to the oral cancer disease progression in our\nstudy.  By contrast,\nSiow MY et al revealed that\nmiR\u2010375 downregulation is associated with tumor size and disease progression in\noral cancer27<\/sup>. Zhang\nB et al reported reduction in miR-375 levels correlates with increased\nlymph node metastasis and reduced survival rate in OSCC patients26<\/sup>. <\/p>\n\n\n\n

The p53 gene is one of the most\nfrequently mutated genes in human cancers13<\/sup>. About 63.3% of the tumor tissues demonstrated mutant p53\nimmunopositivity in this study which is consistent with the results of Patil NN et al (61%) Dave KV et al (62%)\nand Ghanghoria S et al (63%) in OSCC cases34-36<\/sup>. Further\nprevious immunohistochemical studies have documented significant variability in\nthe expression of mutant p53 in oral cancer, spanning from 36% to 80% 37-39<\/sup>.\nDetection of p53 in oral cancer confirms the mutation of p53 in oral cancer\ntissues. However, the presence of p53 beyond the basal layer of epithelium\ndenotes an early sign of initiation of oral carcinogenesis, thereby demonstrating the fact that the\ngenomic mutations take place well in advance of the observable morphological\nalterations in the affected tissue40<\/sup>. Thus, the deactivation of the\np53 protein or alteration in its coding gene might have a significant impact on\nthe development of oral cancer. <\/p>\n\n\n\n

Mutant P53 expression was noted in all the smokers\ninvolved in this study. Santos FD et al also recorded an association between p53\nexpression and smoking41<\/sup>. A wide spectrum of p53 mutations were diagnosed\nin cancers reported among both active and former smokers. Exposure to cigarette carcinogens causse a variety of\ngene mutations that are closely related to tumorigenesis42,43<\/sup>. Langdon\nJD\u2019s findings indicate that p53 mutations were frequently detected in tumors\nfrom individuals who had a history of heavy smoking. This suggests that genetic\ndamage to p53 could be a contributing factor in this patient group38<\/sup>. \nAmong 9 smokers included in the study 8 cases (88.9%) showed miR-375\ndownregulation. However, this finding was not statistically significant. It can\nbe hypothesized that nicotine associated carcinogens might downregulate\nmiR-375. Further, miR-375 dysregulation might initiate carcinogenesis by\ninactivating the p53 pathway. Further molecular studies are required to\nsubstantiate the above statement.<\/p>\n\n\n\n

The current study results also demonstrated a greater intensity of expression of mutant p53 with increasing tumor size, tumor grade and nodal metastasis. This finding indicates the role of p53 in tumor progression. Mutated p53 gene forfeits its ability to inhibit cancer and behaves like an oncogene and promotes tumor growth by stimulating cell division44<\/sup>. The findings of the present study show that expression of mutant p53 was noted in 66.7% of miR-375 down regulated cases of OSCC demonstrating a very significant association within the genes. Liu Y et al study results revealed that microRNA-375 directly binds to the 3\u2032-UTR regions of p53 and mediates down-regulation of the gene. Studies have shown that miR-375 overexpression leads to inactivation of the p53 pathway and reduces the p53 protein levels in gastric cancer cells21<\/sup>. Further, it aids in the evasion of apoptosis after damage to the DNA20<\/sup>. Song L et al demonstrated that miR-375 regulates radio resistance of cervical cancer cells through the p53 pathway45<\/sup>. Based on our findings, and literature evidence miR-375 downregulation may induce mutation in the p53 gene which might further result in the initiation of oral carcinogenesis.<\/p>\n\n\n\n

Conclusion<\/strong><\/p>\n\n\n\n

In summary, MicroRNA-375 is a tumor-suppressive microRNA and its significant down-regulation in oral cancer highlights its association with oral carcinogenesis. miR-375 favors oral carcinogenesis by targeting the p53 gene, which is the frequently mutated gene in oral cancer. Further studies are required to analyze the molecular pathways involved in the interaction between these genes. Loss of miR-375 in oral cancer holds diagnostic implications and should be evaluated further as early diagnostic markers for OSCC. Targeting miR-375 could provide a promising strategy for oral cancer intervention in the future.<\/p>\n\n\n\n

Acknowledgments<\/strong><\/p>\n\n\n\n

We extend our sincere thanks and gratitude to Prof. Ganesh Venkatraman and Prof Rayala Suresh Kumar for providing laboratory access to conduct our PCR experiments.<\/p>\n\n\n\n

Conflict of Interest<\/strong><\/p>\n\n\n\n

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest regarding the publication of this paper.<\/p>\n\n\n\n

Funding Sources<\/strong><\/p>\n\n\n\n

No funding resources to be declared.<\/p>\n\n\n\n

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